MICROBIOLOGY

Laboratory Practicals Journal

For II M.B.B.S.

NAME:_______________________________

BATCH:___________ROLL NO.:___________

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 DEPARTMENT OF MICROBIOLOGY
P.D.U. MEDICAL COLLEGE
RAJKOT

CERTIFICATE

ROLL No: -.................. University No: -.................

This is to certify that Shri / Kumari

____________________________________________

of the Second M.B.B.S. Class Academic

Year______________, Batch __________, has attended

the required number of practical classes as prescribed in

GMER - 2019 under CBME and He / She is eligible to

appear for the University summative examinations.

Professor & Head, Dept.

Date: of Microbiology, P.D.U.
Medical College, Rajkot

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 GENERAL INSTRUCTIONS

1. Practical classes must be attended punctually.

2. Listen attentively to instructions given orally in the practical class

3. Wearing an apron is compulsory.

4. Practical journal, logbook, color pencilsand scale are essential

requirements.

5. Use color pencils only for drawing diagrams.

6. Answer the questions which accompany the exercise.

7. Entries in the journal must be completed and signed at regular intervals.

8. Discard all infected materials like slides in the appropriate containers.

9. Any injury or spillage of material provided should be reported

immediately.

10. Please be careful as you are working with infectious microorganisms.

Kindly follow Covid appropriate precautions.

11. Clean objective lens of your microscope before leaving the class.

12. Wash your hands before leaving the laboratory.

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 INDEX

FIRST TERM

Sr. Page
Date Practical Signature
No. No.
General Microbiology
1 Microbial World 9

Infection control practices: Biosafety, Universal

2 13
precautions, Hand hygiene, PPE.

3 Collection & Transport of Specimens 23

4 Basic Microscopy 31

5 Gram`s Staining 35

6 Z. N. Staining 39

7 Sterilization & Disinfection 41

Morphology & Physiology of bacteria

8 51
includingAnaerobiosis
Laboratory Diagnosis of Bacterial Infections-1
9 59
(Culture media)
Laboratory Diagnosis of Bacterial Infections-2
10 67
(Isolation and identification)
Laboratory Diagnosis of Bacterial Infections-3
11 79
(Antibiotic sensitivity test)

12 Laboratory Diagnosis of Fungal Infections 85

Laboratory Diagnosis of Parasitic Infections

13 91
(Stool Microscopy)

14 Laboratory Diagnosis of Viral Infections 99

15 Hospital Waste Management 103

Immunology
16 Immunology and Serology 107

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 SECOND TERM

Sr. Page
Date Practical Signature
No. No.
Gastrointestinal tract Infections
Laboratory Diagnosis Of Diarrhea And
17 117
Dysentery

18 Laboratory Diagnosis Of Enteric Fever 125

Hepatobiliary system infections

19 Laboratory Diagnosis Of Viral Hepatitis 127

Cardiovascular system &Bloodstream infections

Laboratory Diagnosis Infective Endocarditis
20 131
And Rheumatic Fever

21 Laboratory Diagnosis Of Malaria And Filaria 139

22 Laboratory Diagnosis of HIV infection 145

Respiratory tract Infection

Laboratory diagnosis of Respiratory tract
23 151
infections

24 Laboratory Diagnosis Of Tuberculosis 159

Musculoskeletal & Soft Tissue infections

Laboratory Diagnosis of Skin, soft tissue and
25 165
musculoskeletal infections

26 Laboratory diagnosis of Anaerobic infections 171

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 THIRD TERM

Sr. Page
Date Practical Signature
No. No.
Central nervous system infections
27 Laboratory Diagnosis of CNS infections 173

Genitourinary infections
Laboratory diagnosis of Sexually transmitted
28 177
infections

29 Laboratory diagnosis of Urinary tract infections 181

Miscellaneous
30 Microbiology of Water, Air and Food 185

31 Confidentiality In Laboratory Results 189

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 PRACTICAL NO-1.
MICROBIAL WORLD

MEDICAL MICROBIOLOGY:
It is the study of interactions between humans and the microorganisms with which they
coexist. The microorganisms involved are classified, according to the nature of their
interactions with humans, on a spectrum that varies from beneficial to harmful.
Classification of microorganisms:
● Saprophytes: Free living microbes that live on dead or decaying organic matter. They are
found on soil and water. They are generally unable to invade the living body.
● Parasites: Microorganism which lives on living host and derives nutrition and shelter from
the host, without any benefit to it and may cause harm.
● Commensals: Organisms that routinely colonize body surfaces without doing harm and
are often referred to as the normal microbial flora.
● Pathogens: Organisms that damage the human host either by direct invasion and injury or
by the production of harmful toxic products.
● Opportunistic Pathogen: Commensals and saprophytes can produce disease when host
resistance is lowered.
Host-microbe interactions.
The pathogenic potential of many organisms is variable. It is influenced by both the
intrinsic properties of the microorganism and the state of health of the human host.
● Host defenses and natural immunity: It is a multifactor system of protective mechanisms
that prevent entry of microorganisms into normally sterile areas and limit the spread of
those invaders that overcome the first line of defense.
These mechanisms may be weakened by a variety of insults, including direct physical
trauma, systemic diseases, drugs, and toxins.
When normal defenses are impaired, the person loses the ability to combat infection and the
injury caused by pathogens, even those with low intrinsic virulence. In such cases, the
compromised host often succumbs to infection.
● Pathogenicity: It is the ability of microbes to produce disease.
● Virulence: It is the relative intrinsic ability of a microorganism to cause disease.
Organisms of high virulence have evolved efficient mechanism for circumventing normal
host defenses. Virulent organisms are adept at gaining entry and doing damage even when
the inoculum is small.
CLASSES OF PATHOGENIC MICROORGANISMS:
Pathogens vary in size and biologic complexity. Some are able to extract sufficient nutrients from
an inanimate environment and, hence may be cultured on artificial media. Others are incapable of
growth outside living host cells and are referred to as obligate intracellular parasites.
Pathogens are divided into four major groups.
● Viruses: They are the smallest intact organisms with demonstrated pathogenic potential.
They are too small to be seen with a light microscope. Viruses are obligate intracellular
parasites that depend entirely upon the host cell‘s synthetic machinery for reproduction.
Viruses contain only one type of nucleic acid, either DNA or RNA, but not both. After
entering a host cell, a virus sheds its coat and releases viral nucleic acid into the cell. Under
the direction of the viral genes, the host cell diverts its activities toward producing new
viral components, which are then assembled within the cell into new virus particles
(virions). The virions are released, additional cells are infected, and the cycle is repeated.

● Bacteria: They are larger and more complex than viruses. Most bacteria are visible under
the light microscope. Bacteria are termed prokaryotes because, unlike higher organisms,
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 they lack a true cell nucleus. Since no nuclear membrane is present, the genetic material, in
the form of a nucleoid, lies within the cytoplasm. Unlike viruses, bacteria possess both
DNA and RNA. Bacteria reproduced by binary fission. Many pathogenic bacteria are
capable of independent growth and thus, may be cultured on artificial media. Some
bacteria, however, lack the ability to produce important metabolites and are obligate
intracellular parasites. These organisms must be grown in tissue culture if their recovery is
necessary.

● Fungi: They are larger than bacteria and have a more advanced cell structure. As
eukaryotic organisms, their genetic material is separated from the cytoplasm by a nuclear
membrane. Some fungi reproduce by budding (yeasts), whereas others form growing
colonies of attached organisms (molds). Most pathogenic fungi exist in the nature as
environmental saprophytes, and human infection does not appear to be necessary for their
life cycle.

● Parasite: It is a general term often used in a narrow sense to refer to a variety of protozoan
and multicellular eukaryotic organisms capable of causing disease. Many parasites undergo
complex life cycle that may involve several host species, including humans. The parasitic
diseases remain major health problem and source of economic drain.

WORK:
1. Demonstrations:
● Bacteria – Staphylococci
● Virus-HIV
● Fungus – Candida
● Parasite – Roundworm
2. Draw figures of various demonstrations.
3. Questions.
A. Write in brief about five kingdom classification.
B. Briefly describe the usefulness of microorganisms in human life.

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Staphylococci HIV-Virus

Candida
Round Worm

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PRACTICAL NO- 2.
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 INFECTION CONTROL PRACTICES: BIOSAFETY, UNIVERSAL
PRECAUTIONS,HAND HYGIENE, PPE

Biosafety: Biosafety is the measures employed to avoid infecting oneself, others or the
environment when handling biohazard materials.
Biohazard: An agent of biological origin that has the capacity to produce deleterious effects on
humans, i.e. microorganisms, toxins and allergens derived from those organisms.
Examples;
• Microorganisms such as viruses, bacteria, fungi, and parasites.
• Blood and body fluids, as well as tissues from humans and animals.
Biosafety Levels
Four biosafety levels provide increasing degrees of protection against various pathogenic
microorganisms.
Level 1: Suitable for work involving well-characterized agents not known to cause disease in
healthy adult humans and of minimal potential hazard to laboratory personnel and the
environment.
Examples: Bacillus subtilis, E. coli.
Level 2: Suitable for work involving agents of moderate potential hazard to personnel and the
environment.
Examples: Measles virus, Salmonellae, Toxoplasma species, Hepatitis B virus.
Level 3: Suitable for work with infectious agents which may cause serious or potentially lethal
disease as a result of exposure by the inhalation route.
Example: Mycobacterium tuberculosis.
Level 4: Suitable for work with dangerous and exotic agents that pose a high individual risk of
aerosol transmitted laboratory infections and life threatening disease.
Example: Ebola Zaire virus.

Safety Equipment: (Primary Barriers)

These are designed to remove or minimize exposures to hazardous biological materials.
Examples: Biological Safety Cabinets, safety centrifuge cup, personal protective equipment
(PPE), such as gloves, coats, gowns, shoe covers, boots, respirators, face shields, safety glasses, or
goggles.
Biosafety Cabinets:
3 Classes
Exhaust - HEPA (High Efficiency Particulate Air)
• Class I: Do not protect the work from contamination Air entering cabinet is not filtered
• Class II: Each type recirculates different amount of air Some are hard ducted, and some
exhaust into the room
• Class III: Totally enclosed, ventilated cabinets - Work through portals with attached glove

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Standard Precautions
Standard precautions are the minimum infection prevention practices that apply to, all
patient care regardless of suspected or confirmed infection status of the patient, in any
setting where health care is delivered.
Objectives of Standard Precautions
1. To prevent cross infection.
2. To protect HCW from blood borne as well as other contagious infection.
Component ofStandard Precautions
1. Universal precautions.
2. Hand hygiene
3. Personal protective equipment.
4. Spillage cleaning.
5. Respiratory hygiene.
6. Transmission based precautions.
7. Biomedical waste including sharp handling.
8. Disinfection.

Hand Washing:
Hand washing is the MOST IMPORTANT STEP in preventing the spread of disease!
Method: (According to WHO)
• Wet hands with water;
• Apply enough soap to cover all hand surfaces
• Rub hands palm to palm
• Right palm over left dorsum with interlaced fingers and vice versa.
• Palm to palm with fingers interlaced
• Backs of fingers to opposing palms with fingers interlocked
• Rotational rubbing of left thumb clasped in right palm and vice versa
• Rotational rubbing, backwards and forwards with clasped fingers of right
hand in left palm and vice versa
• Rinse hands with water
• Duration 20-40 seconds.
• Dry hands thoroughly with a single use towel
• Use towel to turn off faucet
• Your hands are now safe.
• Alcohol based hand rub can be used in place of soap when hands are not visibly dirty
or soiled.

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 HOW TO HAND WASH ?

Personal Protective Equipments (PPE)

Used to protect the skin and mucous membranes of Health Care Workers (HCW) from exposure to
blood and/or body fluids; & from the HCW’s hands to the patient during sterile and invasive
procedures.

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A1. Used when there is a risk of infection to HCWs (e.g. while touching
Gloves (non-sterile) blood, body fluids, secretions, excretions of patients, items/equipment
or environment).
A2.Gloves (sterile) Used when there is a risk of infection to HCWs as well as to the
patients (during surgeries /invasive procedures).
B.Plastic apron Used during surgeries
C.Gown Used during surgeries and when soiling is likely to be expected.
D. Surgical mask Used during surgeries and while handling patients on droplet
precautions
E.N95 mask Used while handling patients on airborne precaution (tuberculosis,
Covid-19).
F.Cap, G. face Used when spillage of blood is suspected, e.g. during major cardiac
shield, H. Goggles surgeries etc.& while handling patients on airborne precaution
I.Surgical shoes Used mainly in ICUs and operation theatres to protect HCWs and
environment from transmission of organisms.

Gloves:When to wear gloves?

• Anytime you may come in contact with blood or other body fluids such as
urine, saliva, vomit, or the mucous membranes of the mouth or nose.
• When touching skin that may have sores, open wounds, cuts, or scratches.
• When handling any object that may have been soiled with blood or body fluids.
• When you have a cut or scratch on your hands.
• Gloves are not substitute of Hand hygiene.

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Donning of Gloves:Perform hand hygiene by rubbing with an alcohol-based hand rub or by
washing with soap and water

Doffing of Gloves:

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PPE DONNING: To be performed only in the designated area.

• Remove all jewelry, watch, wallet, mobile phone etc..

• Perform hand hygiene with soap and water.

Wearing 1st pair of gloves:

• Perform hand hygiene with alcohol rub
• Wear 1st pair of gloves.

Wearing coverall suit:

• Examine coverall suit for any damage including tears.
• Star by wearing the leg sleeves, then the arm sleeves and zip up. Cover the zip with
flap attached over the zip

Wearing shoe cover:

• Sit on clean chair
• Wear shoe cover and pull them uptill your calves.

Wearing N-95 mask and wearing hood:

• Hold the N-95 mask like a cup in your hand with both the strap hanging out in the
front.
• Place the mask on your face, pull and wear lower strap first placing it below your
ears, then pull and wear upper strap placing above the ears.
• Wear the hood covering the forehead and cheeks.

Wearing face shield / eye goggles:

• Wear the face shield over the hood and ensure the fit. Tighten or loosen the face
shield with help of screw at the back.

Wearing 2ndpair of gloves:

• Wear 2nd pair of gloves, pull them to cover the sleeve cuff and forearms as much as
possible.

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PPE DOFFING: To be performed only in the designated area.

• Inspect your coverall suit for any gross contamination which could be disinfected with
alcohol based wipes.
• Disinfect your gloves with an alcohol based hand rub ( hand hygiene)

Removing shoe cover:

• Sit on dirty chair with legs apart. Remove the shoe covers by slowly pulling the outer
surface, starting from the top and then pulling from toes end. Discard them in red bin.

Removing outer pair of gloves:

• Disinfect the outer gloves with an alcohol based hand rub ( hand hygiene)
• Remove the outer pair of gloves. Discard them in red bin.

• Disinfect inner gloves with an alcohol based hand rub ( hand hygiene)

Removing face shield / eye goggle:

• Loosing the face shield by adjusting the screw and remove by holding it from back
handle. Discard them in red bin.

• Disinfect inner gloves with an alcohol based hand rub ( hand hygiene)

Removing hood and coverall suit:

• Slide back the hood by holding it from the top.
• Separate the zip flap and unzip the coverall suit. Holding suit at the arms slide it off the
shoulder slightly, then pull the suit from the side of waist and roll your arms out one by
one. By touching inner surface of the suit carefully roll out each leg, discard it in red bin.

Removing inner pair of gloves:

• Disinfect the inner gloves with an alcohol based hand rub ( hand hygiene)
• Remove the inner pair of gloves. Discard them in red bin.

Removing N-95 mask:

• Perform hand hygiene.
• Now wear a new pair of gloves.
• Do not touch the exposed surface of the mask. Stood forward and first remove the lower
strap, then carefully remove the mask by pulling out the upper strap. Discard in red bin.

• Disinfect gloves with an alcohol based hand rub ( hand hygiene)

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 Disinfecting shoes:
• Sit on clean chair.
• Using alcohol wipes, clean the outer surface of shoes while sitting on a clean chair.

Removing last pair of glove:

• Disinfect gloves with an alcohol based hand rub (hand hygiene).
• Now remove the last pair of gloves.
• Perform hand hygiene again.
• Exit doffing area and take shower.
• Wipe your shoes on sodium hypochlorite soaked doormat at exit.

Summary:
• Standard Precautions recommend that you treat all body fluids as if they
are infected with a blood-borne disease.
• Personal protective equipment includes gloves, face shields or eyewear,
gowns, aprons and masks.
• Wash hands before putting on gloves and immediately after removing gloves.

WORK:
1. Demonstration:
● Biosafety cabinets
● Personal protective equipment
● Hand washing (Certifiable skill in Logbook)
● PPE- Donning (Certifiable skill in Logbook)
● PPE- Doffing (Certifiable skill in Logbook)
2. Draw figures of various personal protective equipments.
3. Questions:
a. Describe in brief various Biosafety levels.
b. What is HEPA?
c. Define Standard precautions.
d. Enumerate important Standard precautions.
e. Write the steps of hand washing.
f. Write steps of PPE donning.
g. Write the steps of PPE doffing.

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Biohazard Symbol Biosafety Cabinet

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 PRACTICAL NO-3.
COLLECTION & TRANSPORT OF SPECIMENS

The proper collection of sample is the most important step in the ultimate confirmation that a
microorganism is responsible for the infectious disease process.
Failure to isolate the causative organism in an infectious process is not necessarily the fault of
inadequate cultural methods; it frequently results from faulty collecting or transport techniques.
So there are general considerations regarding the collection and transport of material for culture.
1. Whenever possible specimen should be collected before antimicrobial agents have been
administered.
2. The material should be collected where the suspected organisms is most likely to be found
with as little external contaminations as possible.
3. Specimen should be of a sufficient quantity to permit complete examination.
5. The specimen should be placed in a sterile leak proof container to prevent hazard to nurse
&laboratory staff.
6. Provision should be made for the prompt delivery of specimen to the laboratory.
7. One important thing is that clinicians should given a sufficient clinical information to guide
the microbiologist in selection of suitable media and appropriate technique.
8. The clinicians must appreciate the limitations and potentials of the bacteriology laboratory
andrealize that a negative result does not necessarily invalidate the diagnosis.
9. Laboratory personnel should reject specimens not obtained in a proper manner.
10. Each specimen must be clearly labeled with the date and time of collection, patient's name,
number, ward or health centre.
11. Each specimen must be accompanied by a request form which gives:
(i) Patient's name, age, number and ward or health center with name of consulting doctor
&Patients address.
(ii) Type of specimen and date and time of its collection,
(iii) Clinical diagnosis with patient's history
(iv) Patient's immune status.
(v) Any antimicrobial treatment that may be started at home or in the hospital.

(1) Collection and transport of blood (For Culture):

Bacteraemia: The presence of bacteria in the blood is called bacteraemia,
Septicemia : The term septicemia refers to a severe and often fatal infection in the blood in which
bacteria multiply and release toxins into the blood stream.
> Indications for blood culture:
(i) Septicemia an often life threatening' microbial invasion from an infected focus
accompanied by increase in temperature, increase in pulse rate or chilis followed by fever.
(ii) Bacterimia which accompanies chronic infections such as disseminated severe
infections exemplified by meningitis, pneumonia or deep seated abscesses.
(iii) Intravascular infections such as endocarditis, thrombosed blood vessels or those due to
intravenous catheters.
(iv) Bacteremia of multisystem infections such as enteric fever, leptosprirosis, brucellosis.
(v)Bacterima secondary to traumatic insults and instrumention such as puncture wounds,
urinary tract catherierization, contaminated intravenous medication.
(vi) Patients having fever of unknown origin (PUO)

> Timing for collection of blood for culture:

(i) Blood should be collected at the time of the patient's temperature beginning to rise &
before antimicrobialadmistration.
(ii) Bacterial shedding is intermittent in subacute bacterial endocarditis, so three samples
should be collected at 1 hours interval or two samples collected at 12 hours interval.

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> Media for blood culture:
- Blood culture media should contain a nutrient broth and an anticoagulant.
❖ Glucose broth
❖ Taurocholate broth (Salmonella)
❖ Brain heart infusion broth
❖ Trypticase soya broth
❖Hartley's Broth
❖Thioglycollate broth
❖ Now a days resin-containing medium are available that inactivates most antibiotics
nonselectively by adsorbing them to the surface of the resin particles.
> Collection of blood:
Blood is collected by proper phlebotomy. Skin should be cleared properly before collecting
blood. It is necessary to reduce risk of introducing contaminants into blood culture media. It is
less desirable to draw blood through a vascular shunt or catheter since these prosthetic devices
are difficult to it intravenous line of possible, since, blood above the line will be diluted with
the fluid being infused.
❖Method:
➢ Using a pressure cuff locate a suitable vein in the arm.
➢ Cleanse thoroughly the skin over the vein using tincture of iodine followed by Alcohol.
➢ Remove the protective cap from the top of the culture bottle and cleanse the top of each
bottle using a Alcohol swab.
➢ Using a sterile syringe and 21 gauge needle withdraw 5-10 ml of blood. (Pediatric 2-5 ml)
➢ With care remove the needle from the syringe and dispense the blood into culture bottle in
the vicinity of a flame.
➢ Using an Alcohol swab wipe the top of culture bottle and replace the cap. Gently mix the
blood with the broth(Blood/ broth volumes should be at least 1:10).
➢ Label the bottle with name, number of patient, ward, unit and date and time of collection.
➢ As early as possible, incubate media at 35⁰-37°C.

► Signs of bacterial growth in blood culture bottle:

Examine bottle daily for upto 7 days (for slow growers like Brucella upto 14 days) for
1. Turbidity,2. Haemolysis of the red blood cells, 3. Gas bubbles. Pellicle formation
A sterile culture usually remains clear. If there aresigns of bacterial growth, subculture the
broth on solid media and examine a Gram stained smear for bacteria and then do standard
follow up for identification of organism.

Automated blood culture systems:

After inoculation of culture bottles with blood sample, they are incubated in specialized
instruments which have automated systems for bacterial growth detection. e.g.
a. BACTEC: Bacterial growth is detected by fluorometric method
b. BacT Alert: Bacterial growth is detected by colorimetric method
Advantage:
(i) It has more rapid detection time.
(ii) Ability to monitor growth without visual inspection or subculture.
(iii)Automated handling of large number of blood culture bottles.
Disadvantage:
(i) There are more chances of false positive results.
(ii) It is more costly

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(2) Collection and transport of various body fluids:
(Like Synovial, Pleural, Pericardial, Ascitic and Hydrocele fluid).
An effusion is fluid which collects in a body cavity. Fluids which are collected due to an
inflammatory process are referred as an exudates and that which form due to a non-inflammatory
process are referred as a transudate.
❖Collection and transport of effusions
(i) After aspiration, aseptically dispense the fluid as follows:
➢ 2-3 ml into dry sterile screw-capped tube or bottle for microscopy and culture .
➢ 2-3 ml into a screw-cap tube or bottle which contains sodium citrate for cell count &
protein estimation
(ii)If there is delay in transport of specimen then dispense into a bottle of sterile thioglycollate
broth.
❖Collection and transport of CSF:
Inflammation of the meningies (membranes that cover the brain and spinal cord) is called
meningitis.
Pathogens reach the meningies from blood stream or occasionally by spreading from nearby
sites such as the middle ear or nasal sinuses.
Pyogenic meningitis, when the CSF contains mainly polymorphonuclear neutrophils (pus
cells), as in meningitis caused by N. Meningitides, 'H. Influenzae, 'S. Pneumoniae. And also
in acute amoebic menningoencephalitis.
Lymphocytic meningitis when the CSF contains mainly lymphocytes as in meningitis caused
by viruses, M. Tuberculosis and C. Neoformans. Lymphocytes also found in trypanosomiasis
, meninogoencephalitis and neurosyphilis.
Cerebrospinal fluid must be collected with aseptic procedure to prevent organisms being
introduced into the central nervous system.
Fluid usually collected from arachnoid space. A sterile wide bore needle is inserted between
the L4 & L5 vertebrae & the CSF is allowed to drip into a dry sterile container.
CSF should be collected in two dry sterile container labeled as No.:1 (about 1 ml CSF for
culture) and No.:2 (about 2 ml CSF required for cell count and biochemistry)
Deliver immediately the samples with a request form to the laboratory.
If there is delay in transport then transfer CSF sample No.:2 to a screw-cap bottlecontaining
Sodium fluoride oxalate (this will preserve the cells & prevent the breakdown of glucose).
CSF-in No.:1 container should be inoculated immediately on appropriate culture media and
incubate in a CO2 jar. It should not be refrigerated.

(3) Collection and transport of throat and mouth swabs:

Streptococcus pyogene is the commonest cause of streptococcal pharyngitis (sore throat)
especiallyin young children and is also associated with rheumatic heart disease.
Corynebacterium diptheriae cause a serious disease diphtheria producing a powerful and often
fatalexotoxin.
C. albicans infections of the mouth (oral thrush) is often found in neonates & in patient with
HIV infection.
❖Collection of Swabs:
For 8 hours before swabbing the patient must not be treated with antibioitics or antiseptic
mouth washes.
1. In a good light and using the handle of a spoon to depress the tongue examine the inside of
the mouth.Look for- inflammation & Presence of any membrane exudates or pus.
In diphtheria - grayish - yellow membrane
In streptococcal sore throat - tonsils are inflamed and covered with yellow spots
In infectious mononucleosis - tonsil may be covered with a white exudates

25
 In C. albicans infection - patches of white exudates
2. Swab the affected area using a sterile cotton or alginate wool swab. Taking care not to
contaminate the swab with saliva, return it to its sterile container.
3. Within 2 hours of collection, deliver the swab with a request form to the laboratory.
4. If there is delay in transport then transfer the swab in tubes containing 3-5 gm of desiccated
silica gel (Here, C. diphtheriae, S. Pyogenes & S. aureus remains viable for at least 3 days).

(4) Collection and transport of Nasal swabs and Naopharyngeal aspirates:

❖Collection of pernasal swabs:
Infection with B. pertussis and B. parapertussis (Whooping cough) usually occurs in very young
children who have not been immunized against the disease. The organisms are best isolated from
pernasal swabs.
1. Using a sterile cotton or alginate wool swab attached to an easily bent piece of wire,
gently pass the swab along the floor of one nostril directing the swab downwards and
backwards as far as thenasopharynx
2. Taking care not to contaminate the swab, replace it in its sterile container. Label and
deliver immediately to the laboratory with a request form (B. pertussis does not survive
on a swab.)
3. If there is delay in transport, then insert the swab in a biju bottle containing special
bordetella transport medium.
❖Collection of anterior nasal swabs:
Mostly anterior nasal swabs are collected to detect carriers of pathogens like S. aureus. S.
pyogenes, N. meningitides. H. influenzae.
1. Using a sterile cotton wool swab moistened with sterile peptone water, gently swab the
inside surface of the nose.
2. Put it in its sterile container label and transport within 2 hours to the laboratory.
3. If there is delay in transport then transfer it in Amies transport medium.
❖Collection of Nasopharyngeal aspirates:
When it is not possible to obtain sputum from children with suspected pneumonia or
bronchopneumonia, pathogens can often be isolated from a specimen of mucous aspirated from
thenasopharynx.
Gently pass a sterile catheter through one nostril as far as the nasopharynx. Attach a sterile
syringe inthe catheter and aspirate a specimen of mucous. Dispense the specimen into a small
sterile container. Label and deliver with a request form to the laboratory as early as possible.
If there is delay in transport then make a swab of the aspirate and transfer in amies transport
medium.
West's Postnasal swab is used for collection of nasopharyngeal swab for culture to
detectmeningococcal carriers.
(5) Collection and transport of urogential swabs:
Diseases caused by organisms that are transmitted by sexual intercourse are referred as veneral
disease.
The most serious of these are syphilis cause by T. pallidum and gonorrhoea caused by N.
gonorrhoeae.
U. urealyticum is associated with preterm labour spontaneous abortion and still birth.
C. trachomatis and U. urelyticum are associated with non-gonococcal urethritis in men and PID
in women.
C. trachomatis (Serotypes L1,L2,L3) cause lymphogranuloma venereum (LGW).
T..vaginalis (Protozoa) cause acute vaginitis with a purulent and frothy discharge in women.
C. albicans cause vaginiitis producing white discharge.
G. Vaginalis. C. granulomatis also causes genital infection.
H. ducreyi causes chancroid (Soft sore).
S.pyogenes is the most frequently isolated pathogen in hospital acquired puerperal sepsis.

❖ Collection of urogential specimen:

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 (i) Specimens required for the diagnosis of gonorrhoea.
In male patient →
• Smear of urethral discharge.
• Swab of urethral discharge in Amies medium or inoculated directly on a selective culture
medium.
• Swab from rectum in case of homosexuals
In female patient →
• Smears of mucous from the cervix and urethra.
• Swab of mucous from cervix in Amies medium or inoculated directly on a selective culture
medium.
• Urethral swab in Amies medium or inoculated directly on selective culture medium.
(ii) If Trichomonas vaginalis or Gardnerella vaginalis infection is suspected
Collect a small sample of discharge on a slide, add a drop of fresh physiological saline and
cover with a cover glass avoid making the preparation too thick. Examine it immediately.
Appearance of the vaginal discharge:
T. Vaginalis - Yellow - green purulent discharge with pH > 4.5
C. albicans - White discharge with ph< 4.5
G. vaginalis - Gray, offensive, thin discharge with pH > 4.5
(iii) Specimens for detection of T. pallidum, C. granulomatis, H. ducreyi& C. trachomatis

ORGANISM SPECIMEN COLLECTION EXAMINATION

T. Pallidum Wearing rubber gloves cleanse around the Dark field microscopy
ulcer (chancre) using a swab moistened
with physiological saline. Collect a sample
of serous exudates on a cover glass and
invert in on slide.
Deliver immediately to laboratory
C. Cleans the ulcerated area using swab Stain by Giemsa
granulomatis moistened with physiological saline. Make technique
a smear of exudates on a slide and allow to
air dry and deliver to laboratory within 2
hours (otherwise fix smear with methanol
H. duceryi Make a smear from ulcerated area Gram's stain
C. trachomatis Make a smear of exudates from a bubo, (i) Giemsa Stain
urethra or cervix (ii) lodine stain

(6) Collection & transport of Urine:

The presence of bacteria in urine is called bacteriuria. Significant bacteriuria is usually
accompanied by pyuria (pus cells in urine).
Bacteriuria without pyuria may occur in early UTI, diabetes, enteric fever, bacterial endocarditis.
Pyuria with sterile urine culture may be found with renal tuberculosis, gonococcal urethritis,
leptospirosis or when patient treated with antimicrobiasis.
E.coli is the commonest cause of urinary infection. Women are more frequently infected than
men.
Pseudomonas, Proteus, Klebsiella infections often follow catheterization and gynecological
surgery.
Urethritis (infection of the anterior urinary tract ) is mainly caused by N. gonorrhea,
staphylococci,streptococci &chlamydiae.
M.tuberculosis is usually carried in the blood to the kidney from another site of infection.
S. typhi & S. paratyphi can be found in the urine of about 25% of patients with enteric fever
from the third week of infection. Excretion of bacteria is not associated with pyuria. Typhoid
carriers may excrete S. typhi in their urine for many years. In Leptospirosis L.interrogans can be
found in the urine from about second week of infection.
27
 Urinary tract is normally sterile except for the urethra which may contain a few commonsells
such as Acinetobacter species and Diptheroids.
❖Collection:
1. Midstream urine is collected for microbiological examination.
2. Give the patient a sterile dry' wide-necked, leak proof container collecting a specimen with as
little contamination as possible.
3. About 20 ml of urine should be collected.
4. Label the container with the date, name and number of patient and the time
5. Deliver the specimen to the laboratory as early as possible. If not possible: if delay > 2 hours,
refrigerate the urine at 4-6°C or add boric acid preservative (0.1gm/10 ml urine)
6.If renal tuberculosis is suspected then collect the first urine passed (entire specimen) on three
successive morning. All specimens should be stored 4°C until pcocessed.
❖The following changes occur when unpreserved urine is left at room temperature:
Any bacteria in the urine will multiply so that the bacterial count will be unreliable.

(7) Collection and Transport of Sputum:

Sputum is collected in sterile wide mouthed container.
Avoid contamination with saliva. First morning sample is preferable.
Transfer to Laboratory as early as possible.
(8) Collection and Transport of Stool (Feaces):
Stool is collected in sterile wide mouthed container. Avoid contamination with urine.
Transfer to Laboratory as early as possible. If delay, use Transport Media - Cary Blair
medium, Glycerol Buffered Saline Medium ( Salmonalla, Shigella) and V.R. Medium (V.
cholerae).
(9) Collection and Transport of Blood (For serology):
2-5 ml blood sample is collected in plain bulb or vaccutainer. Transfer to Laboratory as
early as possible in cold chain. Store in refrigerator at 4°C.
(10) Collection and Transport of Pus (Swab):
Sample is collected on moistened sterile cotton wood swab or directly into sterile container.
Transfer toLaboratory' as early as possible. Store in refrigerator at 4°C in case of delay.

(11) Collection and transport of virological specimen:

The type of specimen required will depend on the viral infection suspected and the laboratory
techniques to be used.
❖ When to collect samples:
➢Specimen for detection of viruses should be collected as soon as possible after the appearance
of symptoms that is when concentration of virus is at its highest.
➢ Blood for antibody testing:
Paired samples of serum are necessary to detect rise in antibody titre. A four fold rise in
titre between paired sera established a confirmed diagnosis.
❖ Transport of specimens:
The use of viral transport medium (VTM) will prevent specimen from drying out and help to
preserve viral activity
All most all virological specimens are best transported at the temperature of melting ice, 4°C.
This can be achieved by packing specimens in an insulated box or thermos flask which contain
frozen ice packs or pieces of ice sealed in plastic bags or bottles. Dry crumpled newspaper can
act as insulation material inside a cold box and help to reduce fluctuations in temperature. The
lid of an insulated box must by airtight and the box should not be opened during transport.

❖ Collection:
28
 (I) Collection of anticoagulated blood for culture:
Collect 7-10 ml of blood into a sterile screw-cap tube or bottle which contain heparin (1 IU/ml
blood). Gently mix the blood with the anticoagulant.
viruses that can be cultured from blood in living cell systems include dengue viruses and some other
arboviruses and many of the viral haemorrhagic fever viruses.
(II) Collections of specimen for isolation of viruses:
a. Faeces:
Place about 4-8 gm of faeces in a clean, dry leak-proof container. Deliver to the laboratory
immediately. If there is likely to be delay of more than four hours then suspend about 1 gm of
faeces in 9 ml of phosphate buffered saline if possible, centrifuge at 2000 rpm for 15 min and then
transfer the supernatant fluid into a clean, sterile leak proof container and keep at -20°C send to the
laboratory in a cold box.
Faecal specimens are required to diagnose viral gastroenteritis especially that caused by rotaviruses
and to investigate poliomyelitis and other diseases caused by enteroviruses.
b. Nasopharyngeal specimens:
Nasopharyogeal specimens are required to diagnose respiratory syncytial viral infection and also to
investigate infection caused by influenze, parainfluenza virus, common cold viruses, measles virus,
rubella virus adeno viruses.
c. Cereborspinalfluid(CSF):
CSF is required mainly to investigate some arboviral infections.
Collect about 0.5-1.0 ml of CSF in a dry, leak proof, sterile container. Unlike CSF collected for
bacterial culture, CSF for viral culture should be refrigerated immediately at 4°C. Transport it to the
laboratory in an insulated cold box.
d.Skin and ulcer specimens:
Skin specimens are required to investigate infections caused by herpes simplex viruses, papilloma
viruses, pox viruses and rabies virus.
Collect skin scrapping in a dry , sterile container, Refrigerate immediately at 4°C and transport in
an insulated cold box.
Fluid from pustules and vesicles can be collected in a glass capillaries, seal and placed in a secure
airtight tube or bottle. If a needle or syringe are used to collect the fluid the specimen should be
transferred to a small sterile leak proof container and VTM should be used to wash any remaining
specimen from the syringe and needle into the container. Place biopsies and postmortem material in
a leak proof container and add VTM to preserve the specimen. NEVER USE FORMAL SALINE.
(III) Collection of blood for serological tests:
Two serum specimens are required to diagnose an infection serologically. Collect first sample
within 5 days of the onset of symptoms and the second sample 5-10 days later. Collect 5-10 ml of
various blood in a dry sterilescrew-cap glass tube or bottle avoiding spillage forming aerosols and
haemolysing the blood.
After allowing the blood to clot refrigerate it at 4°C until it can be transported in a cold box to the
virology laboratory. If there is likely to be more than 2 days delay in transport aseptically separate
the serum from the clotted red cells.
→ INFORMATION TO ACCOMPANY SPECIMENS:
(a) A request from must have following information will each specimen.
(b) Patient's name, age, number, place of residence and details of any recent travel.
(c) Type of specimen and the date and time of its collections.
(d) Investigation required
(e) Full clinical information, including the nature of illness its possible source (e.g. arthropod bit
orcontact with animals such as rodents)
(f) Details of antimicrobial therapy and relevant immunizations.
(g) Name and address of hospital or health center and medical officer attending the patient
If the specimen is HIGH RISK this should be indicated on the request form as well as on the
specimen using a red pencil or markers.

(12) Collection and transport of Parasitological specimen:

Sample is collected depending on the site of lesion. (Refer to Lab diagnosis of
29
 Parasitological practicals)
(13) Collection and transport of Mycological specimen:
Sample is collected depending on the site of lesion. (Refer to Lab diagnosi of Mycology
practical)

DEMONSTRATION:
1. Specimen containers.
2. Sterile swab.
3. Transport media
QUESTIONS:
1. Why urine sample to be transported to the laboratory immediately?
2. Why first morning mid-stream urine sample for culture is considered an ideal?
3. Why CSF sample for culture should not be refrigerated ?
4. How much amount of blood will you collect for blood culture ?

Container Vial

Swab Blood culture bottle

PRACTICAL NO-4.

30
 BASIC MICROSCOPY

Microscope is an instrument to view objects so small that are not visible to the naked eye. Invented
by Antony Van Leeuwenhoek.
Use: To make very small, i.e., microscopic objects clearly visible to human eyes.
There are two characteristics of microscopes, which contribute to its magnifying property.
1. Magnification power (MP): It is the ability of a microscope to magnify the object with respect
to their size and make them visible to our eyes.In routine laboratory microscope, two kinds of
lenses are used.
i. Eye piece: Usually it has magnification power of 10X
ii. Object lenses: They have magnification power ranging from 4X to 100X
Following different object lens are used in routine microscopes
• Scanning field (4X)
• Low power field (10X)
• High power field (40X)
• Oil immersion field (100X)
The total MP of a microscope is the product of MP of object lens and MP of eye piece.
e.g. During examination under microscope, if we use the object lens with MP of 100X, the overall
magnification will be, 10X100 = 1000X. This means that the image of the object will be
magnified by 1000 times than its original size.

2. Resolution (resolving) power (RP): It is the ability of microscope to visualize the magnified
images of very small objects clearly and separately. RP of naked eye is 0.1 to 0.2 mm. RP of
laboratory microscope is 0.1 to 0.2 µm. It means, with 1000X magnification, microscope can
make an object as small as 0.1 to 0.2 µm sized objects clearly and separately visible.

The size of majority of medically important bacteria range from 0.2 µm to 20 µm and can be
visualized with light microscopes routinely used in laboratory. However, their detailed internal
structure cannot be visualized clearly.

Types of Microscopes:
1. Bright field microscope:It is a routinely used light microscope in clinical laboratory.
Principle:
The light coming from the light source below passes through the object on the stage and then
enters the objective lens, which magnifies the original object size. This magnified image serves as
the object for the eye piece lens which further magnifies it. Hence, the object on the stage is
magnified at two levels in the process.

31
According to the objective lens used, the intensity of light should be changed with diaphragm and
condenser. This is because if more than or less than required light is allowed to pass through the
object, the image may appear blurred. While examining under oil 100X objective, the condenser
should be highest in its position; while examining under 40X or below magnification objective lens,
condenser should be lowered.

While examining under 100X objectives, the light rays pass from the object and before entering in
the objective lens (100X), get deviated due to air in between the glass of slide and objective lens.
This makes the vision blurred. The immersion oil has the same refractory index as the glass. So, if
the oil is placed on the slide and 100X objective touches the oil, the intermediate air would be
replaced, the light rays would now not be deviated and make the vision more clear

2. Dark field microscope:

Principle: It is similar to light microscope, except a different type of condenser is used which has a
central stop through which light cannot pass directly in the objective. The light rays falling on the
outer area of the condenser will converge and fall on object present on the slide. Only the rays
which reflect from the object enter the objective lens. So the object appears self illuminated against
dark background. This mechanism would increase the resolution power. So, it is used to see thin
and delicate bacteria like Spirochaetes. It is also used to demonstrate bacterial motility.

3. Fluorescent microscope:
Principle: Here source of light is an ultraviolet lamp. The stains used are fluorescent dyes. The
organism stained with these fluorescent dyes absorbs the light of lower wavelength and emit light of
a longer wavelength. So the objects stained with dyes would be brightly fluoresced and clearly
visible against dark background.
Uses: - Auramine is used to stain Mycobacteria.
- Acridine orange is used to stain malarial parasites.
32
 - Various immuno-fluorescent techniques are used to demonstrate antibodies in patient’s
serum andfor detection of antigens in the specimen.
4. Phase contrast microscope:
Principle: Different cells and their parts have different refractory index. The light focused on the
objects is with special devices having two different sets of source. One set of light comes directly
from the light source and the second set from the reflected or defracted light from the object. The
interplay of these two sets of lights brings out the differences in the refractory indices of the cell and
its constituents. According to the intensity of light, few parts would appear darker and few lighter.
This makes the internal structure of tissue cells and organisms to be visualized distinctly. When the
two sets of light rays are ‘in phase’ (elevations & depressions of waves coincide), bright images are
formed, when they are ‘out of phase’ (elevations & depressions of waves don’t coincide), dark
image (background) is formed.
5. Electron microscope:
An electron beam instead of light is allowed to pass through the object. Instead of glass lens,
electromagnetic lenses are used to capture the electrons getting scattered from the object. These
produce a magnified image in the fluorescent viewing screen. Due to very short wavelength of
electrons, the resolving power of this microscope is much superior.
There are two types of electron microscope:
i. Transmission electron microscope
ii. Scanning electron microscope
Use: - To demonstrate, detailed structure of bacteria, parasites
- Demonstration of viruses.

Points to remember during microscopy:

1) Cleaning the stage and the lens of the microscopes with a linen cloth or tissue paper can
enhance the image quality.
2) The slide after staining should be allowed to AIR DRY properly because slightest amount of
moisture on the smear will lead to bubble formation under 100X lens, & hence cause difficulty
in reporting the smear.
3) Bubbles appear as thick black bordered circular structures flowing in the oil.
4) If you have mistakenly focused the wrong side of the smear, the image will be blurred in spite
of all proper and adequate adjustments.
5) Apart from condenser and diaphragm, light can be adjusted with a knob present on the base of
the microscope.

Micrometry
Micrometry: The measurement of objects using a calibrated eyepiece scale (micrometer) is called
micrometry. It is done by specialized micrometry eyepiece and slide
An ocular micrometer is a small glass disk on which uniformly spaced lines of unknown
distance, ranging from 0 to 100. The ocular micrometer is inserted into the ocular of the
microscope and then calibrated against a stage micrometer, which has uniformly spaced lines of
known distance etched on it. The stage micrometer is usually divided into 100 divisions, one
division measuring 0.01 mm.
Mehod:
1. Place the ocular micrometer in eye piece area of microscope & stage micrometer in microscope
stage.
2. Calculate the actual distance between the lines of the ocular micrometer by observing how
manyspaces of thestage micrometer are included within a given number of spaces on the ocular
micrometer.
3. Because the smallest space on the stage micrometer equals 0.01 millimeter or 10 µm, we can
calibrate the ocular micrometer using the following:

10 spaces on the ocular micrometer = Y spaces on the stage micrometer.

33
Since the smallest space on a stage micrometer = 0.01 mm,
then 10 spaces on the ocular micrometer = Y spaces on the stage micrometer x 0.01 mm
1 space on the ocular micrometer = Y spaces on the stage micrometer x 0.01/10 mm.
For example, if 10 spaces on the ocular micrometer = 7 spaces on the stage micrometer,
then 1 ocular space = 7 x 0.01/10 mm ,
1 ocular space = 0.007 mm or 7.0 µm.

Now remove the stage micrometer and focus the wanted smear in which we have to find out the
size of a bacterial or polymorph or any other structure and measure the particular size with the
help of ocular micrometer.

WORK:
1. Demonstration:
● Microscopes and its components.
2. Question:
a). Enumerate the types of microscopy
b). Define resolution power of microscope
c). What is micron (µm), nanometer (nm) and angstrom unit (A0 ) ?
d). What is the advantage as keeping oil when oil immersion lens is used?

Exercise: Label the parts of Microscope in below diagram

34
 PRACTICAL NO: 5
GRAM STAIN
This is the most important staining method in bacteriology. It is the first and the most common
method employed for the diagnostic identification of bacteria in clinical specimens.

Bacteria are semi transparent and hence staining is required for their visualisation. On a clean
grease free slide the smear is prepared & is fixed by passing 3-4 times over the flame of a burner.
Heat can damages leucocytes, so it is unsuitable for fixing smear which contains intracellular
organism such as N. gonorrhoeaand N. meningitides, in that cases chemical like methanol or
ethanol can be used for fixation of smear. The fixation kills vegetative bacteria, render them
permeable to stain, make them stick to the surface of the slide and prevent autolytic changes. One
of the most widely used staining method is that of Gram's stain There are many different
modifications of the original Gram's Stain.
Importance of Gram staining:
● Distinguishes two categories of organisms 1) Gram-positive, which stains dark purple
2) Gram-negative, which stains light red.
● Demonstrating the morphology of bacteria, whether cocci, bacilli, diplococci or
coccobacilli
● Demonstrating the arrangements of bacteria, whether in clusters, short chains, pairs
or single.
● Presence of leucocytes (Pus cells) and epithelial cells in smears of clinical specimens
● Provisional clinical diagnosis can be made in for clinical conditions, like
meningitis by Meningococci, and gonorrhea by N. gonorrhea.
Principle of Gram's Stain: This reaction depends on the fact that when certain bacteria are
stained with certain aniline dyes, such as Methyl Violet, Gential Violetor other and are
subsequently treated with a solution of iodine, mordanting action occurs which prevents the
subsequent decolourisation of the bacteria on treatment with acetone or alcohol. Other bacteria
after similar treatment are readily decolourised.
➢Kopeloff's and Beerman's modification of Gram Stain:

Solution
No Procedure Time
required
1 Methly violet Cover the smear with Methly violet and allow to 5 min
remain for
2 lodine solution Tip off the methyl violet stain hold the steep 2 min
slope and wash. off the residual stain with iodine
solution, cover the whole slide with fresh iodine
solution and leave it for
3 Acetone 100% Decolourise with acetone. Tip off the iodine and 2-3
hold the slide at steep slope pour acetone from seconds
its upper end. .Decolourisation is very rapid and
is completed in
4 Tap water The acetone must be at once removed by
washing thoroughly with water under a running
tap
5 Basic Fuchsin Cover the smear with Basic Fuchsin stain and 30 Sec.
allow to remain for
6 Tap Water Wash thoroughly with water. Blot and allow to
air dry

Examine under oil immersion lens.

35
➢ Result:
• Gram Positive organisms - Violet in colour
. • Gram Negative Organism - Pink in Colour

➢ Actions of Ingredients:
1. Methyl Violet: It is primary stain. Stains both gram positive as well as gram negative
organism
2. lodine Solution: Acts as a chemical mordant. Mordant means any agent which helps in
fixing the dye in thecell
3. Acetone: Decolourises only gram negative organism. Gram positive retain the dye.
4. Basic Fuchsin :Those organisms which are decolourised by adding acetone are counter
stained by Basic Fuchsin.
➢ Theories of Gram stain:
1. Magnesium ribonucleate: Gram positive organisms possess magnesium
ribonucleate while gram negative organisms lack this substance. So, dye iodo
complexes are formed in gram positive organisms but not in gram negative
organisms. In gram negative organisms dye remains free inside the cytoplasm. The
complexes which are formed in gram positive organisms are larger than the pores on
the cell wall so they cannot come out on treatment with acetone.
2. Iso-electric pH Theory: This is the most accepted theory. Gram positive organism
has more acidic cytoplasm than gram negative organism. Further it is enhanced by
treatment with iodine. So gram positive organisms retain basic dye.
3. Peptidoglycan Theory: The difference between the two types of bacteria is that the
Gram- positive have thicker and denser peptidoglycan layers in their cell walls, which
makes them less permeable to the stain than those of the Gram-negative bacteria. The
iodine has a critical role in enhancing this difference. It seems to bind temporarily to
the peptidoglycan and make it even less permeable to the dye.
4. Lipid content Theory: Lipid content in gram negative bacteria is higher, which gets
dissolved in organic solvents like alcohol/acetone and causing decolorization. While
Gram positive bacteria has negligible amount of lipid so it resists the decolorization
with alcohol/acetone.

➢ Examples:
Gram Positive Gram Negative
Cocci: Staphylococci (in cluster) N. meningitides(Meningococcus) (in pair)
Streptococci (in chain) N.gonorrhoea (Gonococcus) (in pair)
Pneumococci (in pair, Veillonella
casulated)
Peptostreptococcus
Peptococcus
Bacilli: Corynebacteria Escherichia Pseudomonas
Clostridia (bulging spore) Klebsiella V. Cholerae
Bacillius(non bulging spore) (capsulated) Haemophilus
Mycobacteria Proteus Bordetella Brucella
Lactobacilli Salmonella Yersinia pestis
Shigella

36
Work:
Demonstration:
● Gram staining procedure.
● Gram-positive & Gram-negative organisms.

Exercise:
A fixed smear is given to you. Perform Gram Stain and examined under oil immersion lens.
Take guidance from instructor of exact method to focus slide under oil Immersion lens.

Gram`s staining (Certifiable skill in Logbook)

Questions:
1. Why heat fixation of smear is required before staining ?
2. What is mordant? Which are the types of mordant ?, Give examples.
3. When can a gram positive bacteria be seen as gram negative?
4. Difference between Gram-positive and Gram-negative cell wall.

37
 GRAM STAIN

Gram Positive Cocci Gram Positive Bacilli

Gram Negative Cocci Gram Negative Bacilli

38
 PRACTICAL NO: 6
ZIEHL NEELSEN STAIN

The acid fast bacilli are more difficult to stain than other bacteria. Simple or Gram Stains do not
usually give satisfactory results.
Principle: They can be stained with basic dyes like carbol fuchsin containing phenol and
application of heat is required for penetration of even more intense stains. Once stained,
however the organisms do not give up the stain readily, they are "fast" to acid decolorisation,
hence their popular name. Most bacteria possessing this property belong to the genus
Mycobacterium and a few to the genus Actinomycetes.
Procedure:
1. The already fixed slide with sputum smear is flooded with Carbol Fuchsin and heated gently
for five minutes. Keep the stain steaming, but do not boil. Add more stain if needed. Do not
let the stain evaporate to dryness. Here phenol present in Carbol Fuchsin acts as chemical
mordants & heating acts as physical mordants.
2. Wash with water.
3. Decolorise with 25% sulphuric acid. When treated with acid the smear turns yellow but the
pink colour returns when washed with water. This process of treating with acid and washing
is repeated every 3 minutes until faint pink colour persists after washing with water.
Decolorization generally requires 3-10 minutes contact of sulphuric acid, depending on
thickness of smear. Acid plus Alcohol can be used instead of Acid only.
4. Wash the slide well with water to remove all traces of acid.
5. Counterstain with Loeffler‘s methylene blue for one minute.
6. Wash with water and blot dry and examine under oil immersion lens.
Result:Acid-fast bacilli are stained bright red, while other organisms, tissue cells and debris are
stained blue.
Acid fastness:
1. Acid fast bacteria are rich in lipids, fatty acids and higher alcohol. Acid fastness is mainly due
to the presence of unsaponifiable wax of the nature of hydroxy acid known as mycolic acid.
2. Acid fastness also depends on the structural integrity of the cell because when the cell is
ruptured by mechanical means OR autolysis its acid fastness is lost.
3. Acid fastnessis lost or decreases:
❖ Acid fastness decreases when organisms are treated with fat solvents
❖ Acid fastness is lost by mechanical means or autolysis
❖ Tubercle bacilli when exposed to INH acid fastness lost.

Sputum Grading: (NTEP)

Bacilli Result Grading No. of fields examined
> 10 AFB / OIF Positive 3+ 20
1-10 AFB / OIF Positive 2+ 50
10-99 AFB /100 OIF Positive 1+ 100
1-9 AFB / 100 OIF Scanty Record actual number 100
No AFB Negative 0 100
OIF = Oil Immersion Field
Importance of Grading:
● For quantitative assessment of number of bacilli in sputum
● To estimate the infectiousness of the patient
● To monitor effectiveness of Tuberculosis treatment using anti-TB drugs

39
Examples:
Acid-fast Organisms: Mycobacterium tuberculosis
Mycobacterium leprae
Atypical mycobacteria
Nocardia
Cyst of certain parasites: Cryptosporidium, Cyclospora, Isospora
Modification of Ziehl'- Neelsen Method:
➢ M. leprae: 5% acid fast and alcohol fast
➢ M. smegmatis: 1% acid fast but NOT alcohol fast.
➢ Nocardia, isospora&microspore: 1% acid fast
➢ Clostridial spores: 0.25 to 0.50 % acid fast.
Cold ZN Stain ( Kinyoun Stain):
By increasing the concentration of basic fuchsin and phenol, the need for heating the slide is
avoided. This method is used for staining Nocardia, isospora and microspora. Here heat is not
applied while 0.5 – 1% sulphuric acid is used.

Work:
Demonstration:
● ZN staining procedure.
● Acid-fast bacilli.

Excercise:
A smear already fixed is given to you. Perform the Ziehl Neelsen stain. Record your observation.

ZN staining (Certifiable skill in Logbook)

Questions:
1. Why mycobacteria show acid fastness ?
2. Why heat is required for acid fast staining ?
3. Which are the mordants used in Z. N. Stain ?
4. How will you differentiate M. tuberculosis. M leprae and M. smegmatis by staining
procedures?
5. What is Kinyoun stain?

Mycobacterium tuberculosis Mycobacterium leprae

40
 PRACTICAL NO: 7
STERILISATION & DISINFECTION

Microorganisms are the causative agents of infection. They include bacteria, viruses, fungi, and
parasites. Infection preventions often rely on placing barriers between the host and
microorganisms. Protective barriers are physical, mechanical or chemical processes, which help to
prevent the spread of infectious microorganisms from client to client, staff to client or vice versa
due to lack of infection prevention practices or from contaminated instruments or equipment.

IMPORTANT TERMINOLOGIES
1. Sterilisation: It is a process by means of which an article, surface or medium is freed from all
living microorganisms, including spores.
2. Disinfection: It is defined as a method of destruction of all vegetative forms of pathogenic
organisms, spore may survive.
3. Antisepsis (against infection): It is a collective term used to denote all methods employed for
the prevention of infection, usually by inhibiting the growth of bacteria in wounds or tissues.
4. Asepsis (no infection): It indicates the techniques used to prevent infections from gaining
access to a sterile place, surface or tissue.
5. Cleaning: It is just a soil removing process, which may remove many microbes. It is an
important pre-requisite to sterilisation& disinfection.
6. Decontamination: It is a method of making a surface or area free from any kind of
contaminants, microbial, chemical, radioactive, etc.
Various methods of sterilization and disinfection
A. Physical Methods
1. Sunlight
2. Dry heat - Flaming, red heat, hot air oven, incinerator
3. Moist heat: Pasteurization, inspissation, boiling, tyndalization, autoclave
4. Filtration: Membrane filter, sand filter, Asbestos, Sintered glass filter, HEPA filter
5. Radiation: Gamma radiation, UV radiation
B. Chemical disinfectants
1. Alcohol: Ethanol, Methanol, Isopropanol
2. Aldehyde: Formaldehyde, Gluteraldehyde,
3. Phenol derivatives: Cresol, chloroxylenol, chlorohexidine, hexachlorophane.
4. Quaternary ammonium compounds: Benzalkonium chloride, cetylpyridium chloride.
5. Halogens :Chlorine: Chlorine dioxide (Cl2) gas, Chlorine releasing compound like
sodium hypochlorite
Iodine: Aqueous iodine, tincture iodine, Iodophores.
6. Hydrogen peroxide (H2O2)
7. Heavy metal salts: Mercuric chloride, silver nitrate, sodium ethyl mercurio-
thiosalicylate (merthiolate).
8. Surface active agents &Quaternary ammonium compound:
Cetrimide, Benzalkonium chloride
9. Dyes: Acridine dyes (Gentian violet ,Acriflavine)
10. Gas sterilization:
- Formalin Gas
- Ethylene - oxide (ETO)
- Plasma gas

41
 A. Physical Methods:
Type Mechanism of action
Sunlight Ultraviolet rays
Dry Heat The killing effect is due to protein denaturation of bacterialprotein.
Moist heat It denatures bacterial protein & coagulates enzyme & protein.
Ionising radiation Formation of radiation tracks in the bacterial DNA, leading to its
death.
Non ionising radiation Denaturation of bacterial protein, DNA damage

Sunlight: Possesses appreciable bactericidal activity.

Dry heat:
1.Flaming-Inoculating loop or wire, the tip of forceps, needles are held in a bunsen flame till
they become red hot.
2. Incineration-Excellent method for safely destroying materials such as contaminated cloth,
animal carcasses and pathological materials.
3.Hot air oven-Heated by electricity, with heating elements in the wall of the chamber. A holding
period of 1600 for two hour is used. It is used for the sterilization ofGlassware, Syringes, Petri
dish, Test tube etc., Surgical Instrument like ,Forceps, Scissors etc. Oily fluids, Powders.
Sterilization control:
 Thermocouples.
 The spores of a nontoxigenic strain of Clostridium tetani 106 spores are used as a
microbiological test of dry heat efficiency.
 Browne's tube after proper sterilization green color is produced.
Moist Heat: Temperatures below 100⁰C:
 Pasteurization of milk:
 Holder Method: Milk is heated at 63 °C for 30 minutes.
 Flash Method: Milk is heated at 72 °C for 15-20 seconds.
Followed by cooling quickly to 13 °C or lower.
Destroy most of pathogenic vegetative bacteria like Mycobacteria, Brucellae, Salmonellae and
Co. burnetti. (Co. burnetti may survive in Holder method)
 Vaccines bath:
 Bacterial vaccines at 60 °C x 1 hour.
 Serum or body fluids at 56 °C x 1 hour on several successive days.
 Inspissation:
 It is a fractional sterilization.
 It is a chamber made of copper plates fitted with water jacket around it. Chamber is closed with
glass lid.
 Serum containing media are kept in slanting position.
 Temperature:
85 oC x 1 hour (1st day)
75 oC x 1 hour (2nd & 3rd day)
 Use: Sterilization of Loeffler’s serum, Dorset egg, LJ Medium

Moist Heat: Temperatures at 100⁰C:

 Boiling water bath:
 Vegetative bacteria are killed immediately at 90-100 °C.
 Spore require prolonged periods of boiling.
 Boiling is not recommended for sterilization of surgical instruments.
 Hard water should not be used.
 Sterilization may be promoted by the addition of 2% sodium bicarbonate to the water.
 Material should be immersed and boiled for 20-30 minutes.
 Lid should not be opened.

42
  Tyndalization or Intermittent Sterilization:
 Used to sterilize culture media which may decompose if subjected to higher temperatures.
 Used for media containing sugars or gelatin. An exposure of 100 °C for 20 minutes on three
successive days is used.
 First exposure kills all vegetative bacteria & the spores, since they are in a favorable medium,
will germinate and be killed on subsequent occasions.
Moist Heat: Temperatures above 100⁰C:
 Autoclave:
 Principal: Water boils when its vapor pressure equals that of the surrounding atmosphere. Hence
when pressure inside a closed vessel increases, the temperature at which water boils also
increases.
 Saturated steam has penetrative power. When steam comes into contact with a cooler surface it
condenses to water and gives up its latent heat to that surface.
 The large reduction in volume sucks in more steam to the area and the process continues till the
temperature of that surface is raised to that of the steam. The condensed water ensures moist
conditions for killing the microbes present.
 Made up of thick walled on jacketed chamber from stainless steel or gunmetal.
Type: Horizontal, Vertical.
Use: Sterilization of Culture media, Glass ware, dressings, instruments, laboratory ware, media
and pharmaceutical products. Surest way of destruction of pathogenic organisms.

essure Temperature Holding Time Remark

(lbs) 109⁰C 60 (min)
(lbs) 115⁰C 45 (min)
(lbs) 121⁰C 18 (min) commonly used
(lbs) 134⁰C 3 (min)
Sterilization Control:
 Spores of Geobacillus stearothermophilus require an exposure of 12 minutes at 121 °C to be
killed. Paper strips impregnated with 106 spores of Bacillus stearothermophilus are dried at
room temperature and placed in paper envelope.
 Chemical indicators, autoclave tapes.
 Thermocouples
Filtration:
Use: Pore size not more than 0.75 µm.
 Remove bacteria from heat labile liquids such as serum and solutions of sugars or antibiotics
used for preparation of culture media.
 Obtain bacteria-free filtrates of clinical samples for virus isolation.
 Measure size of viruses.
 Concentrate bacteria from liquids as, for example, in testing water samples for cholera vibrios or
typhoid bacilli.
 Bacterial toxins & bacteriocin can be obtained by passing cultures through filters.

Type of filters: There are FOUR types of filters.

Type I : Earthenware filters : They are made up of special type of earth.
Berkefeld filter: made up of diatomaceous earth.
V = Coarsest. N = Normal. W = Finest.
Chamberland filter: made up of unglazed porcelain
Type II:Asbestos and Asbestos pad filter:
Seitz filter – made up of purified asbestos pressed in disc form.

43
 Type III :Sintered glass filter – made up of finely ground glass fused together
without destroying porosity and fitted in a glass funnel.
Type IV : Cellulose membrane filter : Made up of cellulose esters or other polymers
have largely replaced other types of filters. They are routinely used in
water purification and analysis, sterilization and sterility testing and for the
preparation of solutions for parenteral use.
HEPA: Filtration of air is accomplished with the use of high-efficiency particulate air
(HEPA) filters. These filters are used in laboratory hoods, biosafety cabinet and in rooms
of immunocompromised patients.

Radiation:
Ionizing radiation in the form of gamma rays or electron beams, is of short wavelength
and high energy. This method of sterilization is used by the medical industry for the
sterilization of syringes, catheters, gloves, surgical catgut, tissue grafts, dressing etc.Since
there is no appreciable increase in temperature in this method, it is referred to as cold
sterilization. A dose of 2.5 M rad used.
Nonionizing radiation in the form of ultraviolet rays is of long wavelength and low
energy. Because of its poor penetrability, usefulness of nonionizing radiation is limited; it
is commonly used to disinfect surfaces.

B. Chemical methods:
Just as physical methods are used mainly to achieve sterilization, chemical agents are used
mainly as disinfectants. Some chemical agents, however, may be used to sterilize. These are
known as chemosterilizers.
Chemical agents act in various ways:
 Protein coagulation: Alcohols.
 Disruption of cell membrane: Phenols, Surface active agents.
 Destroy / modify functional group of protein / enzymes: Halogens, Aldehydes /
Alkylating agents, Dyes, Heavy metals.

1. Alcohol
Members: Ethanol, Isopropanol, Methanol.
Spectrum: Vegetative bacteria, fungi, viruses, fungal spores (methanol).
Concentration: 60% - 70%. Antimicrobial activity is increased in presence of water.
Uses:i) Skin disinfection.
ii) Disinfection of biological cabinets and incubators. (Inner surfaces of incubator
cleaned with cotton soaked with methanol)
Disadvantages:i) Inactive against bacterial spores.
ii) Vapors of methanol are toxic & inflammable.

2. Aldehydes
Members:Glutaraldeyde, formaldehyde, betapropriolactone (BPL).
Spectrum: Active against bacteria (including M. tuberculosis), viruses, fungi, spores.

i) Formaldehyde
Concentration:
Liquid form: For preservation of anatomical speciemen, 10 % formal saline is needed.
Gaseous form: 250 gm of KMnO4 is added to 500 ml formalin for every 1000 cu. ft. for
formation of formaldehyde gas. Exposure time – 4 hours. (Done after
closing all doors & windows)

44
 Uses: - Fumigation of wards & operation theatres.
- Preservation of anatomical specimen.
- Sterilisation of bacterial vaccines.
- Preparation of toxoid from toxin.
ii) Glutaraldehyde:
Concentration: 2% buffered solution. It has to be activated before use. Shelf life after
activation is 14 days. Exposure time 4 to 6 Hours.
Uses:
i) Sterilisation of scopes (glutaraldehyde does not cause erosion of lenses of scopes)
ii) Sterilisation of corrugated rubber anesthetic tubes, plastic endotracheal tubes
iii) Disinfecting baby incubators, anaesthetic machines.

Disadvantages of aldehydes:
i) Irritant to eyes, skin and respiratory mucosa. Less toxic and irritant than
formaldehyde
ii) Carcinogenic
iii) Can not kill saprophytic mycobacteria like M. fortuitum and M. Cheloni
3. Phenol derivatives:
Lister, the father of antiseptic surgery 1st used (1865) as antiseptic.
Members: Cresol, chloroxylenol, chlorohexidine, hexachlorophane.
Spectrum: Active against gram positive &gram negative organisms, milder activity
against Mycobacteria& decreased activity against spores & viruses.
Concentration:i) 1% phenol is bactericidal.
ii) 1% hexachlorophene is bacteriostatic.
Uses:i) Cresol: Ingredient of soaps. Widely used to clean floors in hospital premises.
ii) Chloroxylenol: Active ingredient of Dettol. Used as hand washes, mouth wash
gargle. Less effective against Pseudomonas.
iii) Chlorohexidine: Specific action against Pseudomonas aeruginosa. Effective in
presence of blood, pus. Used to decontaminate surgical instrument, mouth wash
gargle
iv) chlorhexidine + cetrimide is an active ingredient Savlon: Commonly used for
wound dressing, preop. Prep. Of skin, PV examination.
v) 10% Phenol is used for disinfecting pts excreta & secreta
Disadvantages:Phenol is toxic & irritant to skin & MM, so its derivatives is commonly
used.
4. Quaternary ammonium compounds
Members: Benzalkonium chloride, cetylpyridium chloride.
Spectrum: Effective against many bacteria. But ineffective against some bacteria like
Pseudomonas, Mycobacteria, fungi.
Concentration: Bacteriostatic at low concentrations &bacteriocidal at high
concentrations.
Uses : Excellent cleaning agents especially for floors due to their germicidal detergent
action.
Disadvantages: i) Neutralised by organic matter.
ii) Resistant strains of Pseudomonas easily grow & sustain in
their solutions.

45
5. Halogens -
a). Chlorine:
Members:Chlorine dioxide (Cl2) gas, Chlorine releasing compound like sodium
hypochlorite.
Spectrum: Effective against bacteria, viruses, fungi; It is bactericidal. Hypochlorite is
specifically effective against certain viruses like Hepatitis B virus.
Concentration:i) Aqueous solution of sodium hypochlorite at a concentration of 5.25 %
used as household bleach.
ii) For blood spillage, 10,000 ppm of available chlorine is used. (1:5 dilution of
household bleach)
Uses:i) Chlorine dioxide (Cl2) gas is used to disinfect Water supplies, swimming pools.
ii) Chlorine releasing compound like sodium hypochlorite is for cleaning sufaces
(0.1%), as disinfectant (0.5%), for treating blood spillage(1%).
Disadvantages:i) Hypochlorites are inactivated by organic matter.
ii) Hypochlorites corrode metal; hence contact with metallic equipments
should be avoided.
iii) Hypochlorite solution is unstable, has to be prepared fresh for daily
use.
b). Iodine
Members: Aqueous and alcoholic solution iodine, Iodophores.
Spectrum: Effective against bacteria, viruses, fungi.
Concentration: Commercial preparations ranging from 0.1% to 10% of povidone-iodine
(commonest iodophore used) are available.
Uses: Iodine producing staining & irritation. Its derivatives (iodophores) like Povidone
iodine (0.1-7.5%) (Non irritant) commonly used for disinfection of skin & Mucus
Membrane, wound dressing, preop.Prep. Of skin, Bladder irrigation.
6. Hydrogen peroxide (H2O2)
Mechanism of action: H2O2 releases free hydroxyl radical on decomposition. These
radical damages Microbial cell.
Spectrum: All organisms including spores are killed.
Concentration: 3% - 6% for majority microbes; 10% - 25% for spores.
Uses:i) Disinfection of plastic implants, surgical prostheses, contact lenses.
ii) Used as a vapor phase disinfectant to disinfect Operation theatres and intensive
care units.
Disadvantages:i) May be toxic.

COLD FOGGING OF OPERATION THEATRE (OT):

5% Hydrogen Peroxide with 40 ppm colloidal silver (Ecoshield) have tremendous
antimicrobial property & harmless for human being. With the help of fogging machine,
droplets of 7-20 u are released. It traps suspended particulate matters & kills the
microorganisms. OT can be used 2 hours after cold fogging.

7. Heavy metal salts

Members: Mercuric chloride, silver nitrate, sodium ethyl mercurio-thiosalicylate
(merthiolate).
Spectrum: Generally bacteriostatic with weak bactericidal & fungicidal activity.
Uses:i) Used as a preservative for serum, urine – Merthiolate (1 in 10,000)
ii) Used as antiseptics in patients with burn wounds – Silver nitrate (specific
effectivity against P. aeruginosa)
Disadvantages: i) May be toxic. (Mercuric chloride)

46
4. Surface active agents & Quaternary ammonium compound:
A. Surface active agents
Members: Cetrimide (Active ingredient of Savlon).
Groups: Anionic, cationic, non-ionic, amphoteric.
Spectrum:Cationic agents have limited antibacterial activity;
Anionic disinfectants (present in soaps) are more effective against gram
negative bacteria.
Amphoteric (Tego) compounds have detergent properties of anionic &
antimicrobial character of cationic agents, also active against some viruses.
Concentration: Amphoteric agents – active against some viruses at a concentration of
1% in water.
Uses:i) Used as wetting agents, detergents & emulsifiers.
ii) Used as skin & wound antiseptics.
Disadvantages:i) Limited spectrum of activity.

B. Quaternary ammonium compound:

Members: Benzalkonium chloride
Use: Antiseptic and disinfectant actions similar to that of cationic surfactants. Mainly it
is used as preservative in medicinal product in concentration of 0.01-0.02%.

9. Dyes:
Members:Aniline dyes (brilliant green, malachite green, crystal violet, Gentian violet),
Acridine dyes (proflavine, acriflavine, euflavine, aminacrine)
Spectrum: Both groups of dyes are more effective against gram positive bacteria than
gram negative.
Uses: i) Used as selective agents in culture media. (Malachite green in Lowenstein-
Jensen medium)
ii) Used for bacterial staining, (Crystal violet in Gram stain)
iii) Disinfection of skin. (Gentian violet, acriflavine)
Disadvantages: i) Action of aniline dyes inhibited in the presence of organic matter.
ii) Lesser activity against gram negative bacteria, hence limited use.

10. Gas sterilization:

A. Ethylene oxide (ETO/EO)
• Effective chemisterilant, active against all bacteria & spores. Requires humidity for
effective killing.
• Gas at room temperature (boiling point of 10.7 °C.)
• Highly inflammable. (Non inflammable with 10% CO2) & carcinogenic.
• Can be used for sterilisation of fiber optic scopes, Plastic goods, Bone tissue grafts,
Vaccine, Culture media.
B. Plasma sterilisation
This is a sterilisation process that usually is done at room temperature and hence poses
no dangers associated with high temperatures (unlike autoclaves). It doesn’t involve
any chemicals and hence is non-toxic (unlike EO). Also, the time of treatment is fast
and of the order of 1 min or less. It is versatile and can sterilize almost any material.

Plasma sterilization uses a tri- phasic technique. Plasma is basically ionized gas. When
you apply an electric field to a gas, it gets ionized into electrons and ions. When the
plasma is turned on, it generates a whole lot of particles- UV photons, electrons, ions
and neutral particles. The UV photons and radicals are the main working population
amongst these.

47
 The spores are basically made up of simple atoms like C, O, N, H and the like. The
radicals react with these atoms to form simple compounds like CO2, which can
subsequently be flushed out. When the organism loses such atoms that are intrinsic to
its survival, it dies. Plasma sterilisation is effectively used with vapor phase
disinfectants.

C. Formaldehyde gas / Glutaraldehyde

Sterilization or Disinfection is done at three levels according to the type

of instrument / item involved. This is known as Spaulding’s classification.

Classification Type of instrument Level of Appropriate process

infection
Critical items Equipments that enter Most Sterilisation :
sterile tissue, vascular serious i) Use of single use sterile
system, body cavities product.
& non intact mucous ii) Steam sterilisation
membranes. iii) Low temperature methods
e.g. surgical (hydrogen peroxide plasma,
instruments. ethylene oxide, peracetic acid)
Semi critical Equipments that Moderate High level disinfection
items develop direct or i) Thermal disinfection.
indirect contact with ii) Chemical disinfection
intact mucous (glutaraldehyde, hydrogen
membranes or non peroxide, chlorine dioxide)
intact skin.
e.g. endoscopes,
anaesthetic
instruments.
Non critical Equipments that come Mild Low level disinfection :
items in contact with intact i) Manual cleaning.
skin but not mucous ii) Mechanical cleaning.
membranes.
e.g. Stethoscopes, BP
cuffs.

Central Sterile Supply Department (CSSD):

• It is an integrated place in hospitals that performs sterilization of medical devices,
equipment and consumables; that are used in the operating theatre (OT) of the hospital
and also for other aseptic procedures.
• The processing area of CSSD consists of four unidirectional zones starting from an
unsterile area to a sterile area separated by physical barrier.
• Decontamination area → Packaging area → Sterilization area → Sterile storage area

48
 WORK:
Demonstrations: Various instruments and chemicals used for sterilization and disinfection.
● Instruments: Waterbath, Serum Inspissator, Autoclave, Hot air oven.
● Filters: Candle filter, Seitz filter, Sintered glass filter, Membrane filter
● Radiation: UV light.
● Chemicals.
● Controls of sterilization.
Draw figures of: Autoclave, Sintered glass filter, candle filter and Seitz filter.
Questions.
1. What is the difference between sterilization and disinfection?
2. What are the difference between sterilization by dry heat & moist heat?
3. What is pasteurization ?
4. Describe in brief the principle of autoclave.

Field Visit: CSSD

Activities: Visit a section of the hospital assigned to you; and note down antiseptic /
Disinfectantsused in that section as per the performa.
PERFORMA

NAME OF EQUIPMENT USE

1.

2.

NAME OF
USE
ANTISEPTIC/DISINFECTANTS

1.

2.

3.

4.

49
 Autoclave Sintered Glass filter

Candle filter Seitz filter

50
 PRACTICAL NO-8
MORPHOLOGY AND PHYSIOLOGY OF BACTERIA (including Anaerobiosis)

MORPHOLOGY OF BACTERIA:
Bacteria are prokaryotic micro-organisms.

Morphology of bacteria can be described under the following headings:

1. Size: The size of bacteria is measured by micrometry. The unit of measurment

used is micrometer (µm). The size is variable:
Cocci : 0.75 µm to 2 µm
Bacilli : 0.7 µm to 8 µm
Spirochaetes : 12 µm to 20 µm
2. Shape: According to shape bacteria have been classified into following types.
1. Spherical - Cocci round in shape
2. Bacillar - Bacilli rod shapped cell
3. Comma - Curved appearance
4. Spirilla - Rigid spiral form
5. Spirochetes - Flexuous spiral form
6. Branching - Filamentous form e.g. Actinomycetes
3. Arrangement:
Cocci arranged in: Clusters Staphylococci
Chains Streptococci
Pairs Pneumococci, Neisseria
Group of four Tetrads
Packets of eight Sarcina.
Bacilli arranged in: Singly Esch. Coli
Chinese letter C. Diptheriae.
Chains Strptobacilli.

Routine staining techniques:

Gram stain: Divides bacteria in to two group: Gram positive & Gram negative
ZN stain: Identify Acid fast bacilli & other acid fast structures.

Special staining techniques:

a). Fontana stain for spirochaetes: Organisms which are thickened by impregnation of silver on
the surface. Fontana’s method for staining spirochaetes involve the use for ammoniated silver
nitrate solution. The silver is deposited on the organisms which stain brownish black on a
brownish yellow background.
b).Capsule: Many bacteria are surrounded by a covering layer of a relatively firm gelatinous
material which is visible with light microscope. It is called a capsule. Capsulated organisms are
Pneumococci. Klebsiella pnuemoniae. etc.
1. Manveal’s Stain: is allowed to act upon cells. Capsules are decolourized, while cells retain
the stain congored. Thus congored functions both as a decolorizer and as a negative stain.
Thus the capsules remain unstained. bacteria are reddish brown ( orange colour) and
the background is also become reddish brown (orange colour).

2. India ink or nigrosin preparation is used for the direct microscopic examination of the capsules
of many microorganisms. The fine granules of the India ink or nigrosin give a semiopaque
background against which the clear capsules can easily be seen. This technique is particularly

51
 useful in visualizing the large capsules of Cryptococcus neoformans in CSF, Peumococci in
sputum and other secretions. This is also called as negative staining.
c). Metachromatic granules: Some bacteria contain granules composed of polymetaphosphate.
They are known as Metachromatic granules. They are also called as volutin or Babes–Ernst
granules. They are situated at the poles of the bacilli and are called polar bodies eg. C. diptheriae
and Lactobacilli.
Albert stain: Albert’s stain contains Toludine blue and Malachite green so by this stain
metachromatic granules are stained bluish black and the protoplasm green.
d). The Spore: Spore is resistant, refractile, metabolically, inactive state of micro organisms. The
situation of the spore may be terminal, sub terminal or central. The spores may be bulging or non-
bulging. The spore is seen in members of genera Bacillus, Clostridium. The change from
vegetative to spore formation in adverse condition is known as sporulation. The change from spore
form to vegetative form which occur in favorable condition is known as germination.
Modified Acid fast stain for Spores: Spores appear as unstained portion in Gram Stain. But the
dye can be driven into them by application of heat. They can be stained by modified Ziehl Neelsen
staining. (decolourisation by only one half percent sulphuric acid). Nigrosin is used for counter
stain instead of methylene blue so spores stained bright red and the protoplasm of the bacilli
colourless. Nigrosin provides a dark background which outlines the unstained bacillary bodies.
e). Fluorochrome: This fluorochrome dye stains mycobacteria selectively by binding to the mycolic
acid in the cell wall. This stain demonstrates mycobacteria better than conventional acid-fast stains
and permits screening of smears at lower magnification because organisms are more easily seen.
Acridine orange is a stain particularly well adapted for the demonstration of bacteria in blood
culture broth, CSF, urethral smears, or other smears where they may be present in relatively small
numbers or when they are obscured by a heavy background of leukocytes or other debris. At pH
below 4.0, bacteria and yeast cells stain brilliant orange against black, light green or yellow
background.

f). Lactophenol aniline blue: Currently it is most commonly used for the staining of fungal
mycelium and fruiting structures, which take on a delicate light bluecolor.
PHYSIOLOGY OF BACTERIA
Bacteria derive their nutrition from the substance in which they grow but food requirements of
pathogenic bacteria vary widely from organism to organism. Bacteria require Carbon, Nitrogen,
Oxygen, Mineral Salts etc. for their growth and multiplication. All the nutritional requirements are
supplied in the form of artificial media.
Some pathogens can easily grow under wider range of nutrition and environment. Whereas others
which are strictly parasitic can grow under limited nutritional and environmental conditions and
require growth factor, optimum temperature and adjustment of pH of the media.

 Lovibond Comparator: Media are always standardized for their pH i.e. H-ion Concentration.
Lovibond comparator consists of a bakelite case with two holes at the top for tubes of standard
bore and of colorless glass. The hinged door of the comparator holds a rotating disc containing a
series of standard coloured glasses corresponding to various pH vales (For each indicator a disc is
available to match the colour). After matching the colour, the pH of the solution is automatically
noted in the disc.

 The pH of the nutrient media can also be adjusted by the use of pH strips or by the use of pH
meter.

 Incubator: After the inoculation of organism on the culture media, they are kept for proper
incubation. Most of the pathogenic organisms grow best at about body temperature and cultures
are kept at this temperature in a special chamber known as the incubator which maintains a
constant temperature of 37°C.It is a chamber having double walls. The front part of the chamber is
52
 covered with a glass door inside and a wood or copper door outside. The interior of the chamber
contains some adjustable shelves over which cultures are placed. It is heated with electricity.
Whenever bacteria grow in the media some physiological factors also affect the growth of
bacteria. These are,
1. Influence of Oxygen:
According to oxygen requirement, bacteria are classified into the following categories
a. Obligate aerobes
- They strictly require oxygen for their growth. E.g. Vibrio cholerae
b. Obligate anaerobes
- They can grow only in absence of oxygen.
- They may even die on exposure to oxygen e.g. Clostridium tetani
c. Facultative anaerobes
- They are normally aerobic, but can also grow in absence of oxygen.
- Majority of medically important bacteria are facultative anaerobes e.g. Escherichia coli
d. Aero tolerant
- They are anaerobic bacteria but can tolerate the presence of very low level of oxygen
(0.1 to 1%)
e. Microaerophilic
- They grow best at low oxygen tension (4-5%) e.g. Helicobacter sp.

2. Carbon dioxide level in environment

Majority pathogenic bacteria can grow at the usual carbon dioxide level in the environment.
Some bacteria require higher CO2concentration. They are called capnophilic bacteria.
E.g. Meningococci and Brucella sp. need 5-10 % of CO2
3. Influence of Temperature:
The optimum temperature required by majority of pathogenic bacteria is 370C (human body
temperature). According to the temperature range at which they can grow, bacteria are classified
in the following categories,
a. Mesophilic:
- Temperature range: 20-400C
- Majority pathogenic bacteria are mesophilic
b. Thermophilic:
- Temperature range: 40-600C or above
- e.g. Bacillus stearothermophilus
c. Psychrophilic:
- Temp range: 10-200C or below
5. Influence of H – ion concentration: Majority of bacteria prefers to grow at neutral or slightly
alkaline pH, but some like lactobacillus acidophilus require acidic pH while certain organisms
require alkaline pH, for their growth e.g. V. Cholerae.
6. Influence of light and other radiation: Darkness is favorable for the growth. Ultraviolet rays
are bactericidal. Bacteria are also killed by ionizing radiations. Exposure to light may influence
pigment production. Photochromogenic mycobacteria form pigment only on exposure to light and
not when incubated in the dark.
7. Influence of osmotic effect: As the cytoplasmic membrane is semi permeable, bacteria are
subjected to osmotic pressure.
8. Influence of mechanical and sonic stresses: Although the bacterial cell wall have considerable
strength and elasticity, it is possible to rupture and kill the organisms by exposing them to
mechanical stresses. Bacteria can also be disintegrated by supersonic and ultrasonic vibrations.

METHODS OF ANAEROBIOSIS:
Obligate anaerobes grow only in absence of free oxygen. These bacteria lack mechanism of
oxidation through respiratory enzymes like cytochrome oxidase, catalase and peroxidase resulting
in H2O2 accumulation, which is toxic for the growth of anaerobic bacteria.

53
 Techniques:
Various methods are available for achieving Anaerobiosis based on following principles.
● Displacement of oxygen.
● Absorption of oxygen by chemical or biological means.
● Reduction of oxygen.

1. GasPak system:
It is the method of choice for preparing anaerobic jars. It is available as a disposable envelope,
containing chemicals, which generate hydrogen and carbon dioxide on the addition of water.
After the inoculated plates are kept in the jar, the GasPak envelope, with water added, is
placed inside and the lid screwed tight. Hydrogen and carbon dioxide are liberated and the
presence of a cold catalyst in the envelope permits the combination of hydrogen and oxygen
to produce an anaerobic environment. The Gaspak is simple and effective.

2. McIntosh and Fildes anaerobic jar:

It is the most reliable and widely used anaerobic method. It consists of glass or metal jar with
metal lid, which can be clamped airtight with screw. The lid has two tubes, one act as a gas
inlet and the other one as outlet. Additionally lid has two terminals, which can be connected
to electrical supply. Inoculated culture plates are inside jar Outlet tube is connected to vacuum
pump and air inside is evacuated. The outlet tap is closed and inlet tube is connected with
hydrogen, electric terminals are connected so that palladinised asbestos is heated. This act as
catalyst for combination of hydrogen and residual oxygen. It ensures complete anaerobiosis. It
carries the risk of explosion.

3. Robertson’s cooked meat medium:

It is probably most widely used fluid medium for culture of anaerobes. It consists of fat-free
minced cooked meat in broth, with a layer of sterile vaseline over it. It permits the growth of
even strict anaerobes and indicates their saccharolytic or proteolytic activities, by the meat
being turned red or black, respectively.

4. Anaerobic chamber:
It is used for fastidious anaerobes, particularly for quantitative cultures. The anaerobic
chamber is an airtight, glass-fronted cabinet filled with inert gas, with an entry lock for the
introduction and removal of materials, and gloves for the hands. Pre-reduced media are
required for isolation of anaerobes.An indicator should be employed for verifying anaerobic
conditions in various anaerobic techniques. Reduced methylene blue is generally used. It
remains colorless anaerobically but turns blue on exposure to oxygen.

Work:
DEMONSTRATIONS:
1. Gram stain: Staphylococci, Streptococci, Pneumoccoci, Niesseria, Gram negative bacilli,
C. diptheriae, Actinomycetes, Cl. Tetani.
2. Special stain: Fontana, Manveal’s, Albert, Modified Z-N stain
3. Instrument: Incubator, Lovibond comparator, carbon dioxide jar, McIntosh Filde’s Jar,
Gas Pak method, Robertson’s Cooked meat media.

DRAW FIGURE: for various demonstration morphology, physiology and anaerobiosis.

QUESTIONS:
a). Enumerate special stains used in microbiology.
b). Define the:(i) Microaerophilic organisms. (ii) Obligate anaerobes. (iii) Facultative
anaerobes.
c). Describe the methods of anaerobiosis.
54
 MORPHOLOGY OF BACTERIA

Gram positive cocci Gram positive cocci

in clusters - Staphylococci in chains - Streptococci

Gram positive cocci Gram positive cocci

in pairs - Pneumococci in pairs - Neisseria group

Gram positive bacilli

Gram negative bacilli
Chainese letter arrangement
C. diptheriae

55
 MORPHOLOGY OF BACTERIA

Spiral shaped organism Actinomycetes Showing

Spirochaetes - Fontana's stain branching Filaments
Gram Stain

Pneumococci showing
Meta chromatic granules
Capsule - Manveal's stain
of C. diphtheria - Albert'sstain

Cl. tetani - showing Cl. tetani - Spore stained by

terminal spore: Gram Stain modified Zn Stain

56
 PHYSIOLOGY OF BACTERIA

Incubator Lovibond
comparator

Carbon dioxide Pellicle

jar

57
 METHOD OF ANAEROBIOSIS

McIntosh and Fildes GasPak System

Anaerobic Jar

RCMM

58
 PRACTICAL NO: 9
LABORATORY DIAGNOSIS OF BACTERIAL INFECTIONS-1
CULTURE MEDIA

ISOLATION CULTURE MEDIA:

Culture media gives artificial environment simulating natural conditions necessary for
growth of bacteria. The basic requirements for culture media are:
 Energy source.
 Carbon source.
 Nitrogen source.
 Salts.
 Optimum pH.
 Adequate oxidation-reduction potential.
 Growth factors.
Components of culture media:
 Water- Source of hydrogen and oxygen.
 Electrolytes- Sodium chloride or other electrolytes.
 Peptone- It is a complex mixture of partially digested proteins prepared from animal or
vegetable proteins by enzymatic actions. Commercial peptone contains peptones,
proteases, polypeptides, amino acids and inorganic salts including phosphates, minerals
and necessary growth factors like riboflavin.
 Meat extract, yeast extract (Lab lemco) - These contains protein degradation products,
carbohydrates, inorganic salts and certain growth factors.
 Blood- It enriches media. Usually 5-10 % defibrinated horse or sheep blood is used,
sometimes serum is also used.
 Agar- It is derived from sea-weed (Algae-geledium species) contains mainly
carbohydrates, a small amount of protein-like material and traces of long chain fatty acids
and variety of impurities. It is used in 2-3 % concentration. It melts at 950 C and solidifies
at 420 C. Agar acts merely as solidifying agent of culture medium and does not provide
any nutrition to the bacteria.

Classification of media:
Media have been classified in many ways:
A: Solid media, Liquid media and Semisolid media
B: Simple media and Complex media/Special media
C: Aerobic & Anaerobic media.
Simple media: It is also called basal media. It consists of meat extract, peptone, sodium
chloride, Agar agar and water
Complex media/Special media: There are ingredients for special purposes or for bringing out
certaincharacteristics or providing special nutrients required for the growth of certain
organisms.

● Enriched media: In enriched media, growth enhancers like blood, serum or egg is
added to basal medium to allow fastidious organisms to flourish.
● Enrichment broth: Enrichment broth is designed to encourage the growth of small
number of a particular organism while suppressing other flora present.
● Selective media: Selective media support the growth of one type of microbe over
another. A selective medium may contain inhibitory substances to prevent the growth of
microbes. Such media may be created by adding antibiotics or other inhibitory
chemicals to nonselective media.
● Indicator media: The media contain an indicator, which changes color when bacteria
grow in them.
59
 ● Transport media: Delicate organisms like gonococci which may not survive the time
taken for transporting the specimen to the laboratory or may be overgrown by non-
pathogen. A special media is required for transport of such delicate microorganisms,
which is called as transport media. Stuart‘s medium is a transport medium.
● Differential media: Differential media allow grouping of microbes on the basis of
different characters displayed on the medium. Media may be differential and
nonselective (blood agar) or differential and selective (MacConkey agar).
● Anaerobic media: These media are used to grow anaerobic organisms. Robertson‘s
cooked meat medium is an anaerobic medium. Methylene blue is used as an indicator
for anaerobic condition.

SIMPLE MEDIA
Action of
Important Method of
Media Important Uses
Ingredients Sterilization
Ingredients
Peptone, Basic requirement 1. Used as a base for other media
Peptone Autoclaving 2. Used for the growth of
Water Salt, for growth of
Water microorganisms non- exacting microorganism
Peptone water
Nutrient & --------- Do ------- ------ Do --- --------------- Do -----------------
Broth Meat extract --

Nutrient Nutrient broth ------ Do ---

Agar & --------- Do ------- - --------------- Do -----------------
agar agar
Semisolid ------ Do --- 1. Used for detecting motility of
Agar ------ Do ----- --------- Do ------- - bacteria

ENRICHED MEDIA:
Action of
Media Important Method of Uses
Ingredients Important Sterilization
Ingredients
1. Used for the growth of
Sterile blood
Staphylococci, Streptococci,
Nutrient Blood is for added to
Blood Pneumococci & Neisseria
Agar agar & enriching the Autoclaved
group
blood medium nutrient agar
2. To differentiate the
at<55 0 C
hemolytic properties
When blood is Blood agar
heated Plates
Blood agar 1. For growth of H. influenzae
heated factors X, Y and are kept in
Chocolate 2. For growth of pyogenic
at 560 C for 1 V are released hot air oven
Agar Streptococci
hour from RBC, which at 56 0 C for 3. For growth of Neisseria group
are required for 30 minutes
microorganism
Nutrient Serum and glucose
Loeffler‘s
serum broth, for Inspissation 1. For growth of C. diphtheriae
agar glucose, enriching the
serum medium
Nutrient
Dorset Egg foe
broth, egg 1. Used for growth of
egg enriching the Inspissation
white & Mycobacterium tuberculosis
medium medium
yellow

60
ENRICHMENT MEDIA:

Media Important Action of Important Method of Uses

Ingredients Ingredients Sterilization
Glucose Nutrient broth Glucose is enriching Blood culture of pyogenic
& for Tyndallisation organisms
Broth glucose pyogenic organisms
Peptone water Selenite enriches the
growth of Enriching Salmonella
Selenite & sodium
Salmonella and Tyndallisation species from faecal
F broth hydrogen
inhibits growth of specimens
Selenite
coliform organisms

Alkaline High pH inhibits

Peptone water commensals other Used for growth of Vibrio
peptone Autoclaving
pH: 8.2 than cholerae
water
Vibrio species

SELECTIVE MEDIA

Media Important Action of Important Method of Uses

Ingredients Ingredients Sterilization
Blood is for enriching the
Nutrient medium
Potassium
agar, blood & Potassium tellurite is less Used for the growth of C.
tellurite Autoclaving
potassium inhibitory to C. diphtheriae
Medium
tellurite diphtheriae compared to
other throat commensals
Sodium Taurocholate is
1. For growth of
inhibitory to non intestinal
Peptone water, intestinal
organisms
agar agar, organisms
Lactose will be fermented
MacConke sodium 2. For differentiating
differently by different Autoclaving
y‘sAgar Taurocholate, lactose fermenting
Enterobacteria
lactose, organisms from non-
Neutral red will give pink
neutral red lactose fermenting
color along with lactose
fermentation organisms

Glucose,
Wilson & sodium Brilliant green and
sulphite, bismuth sulphite inhibit 1. For growth
Blair Autoclaving Salmonella
Medium brilliant green, intestinal organisms
bismuth Produces black colonies
sulphite
Egg to enrich the medium
Egg, Malachite green is least 1. Used for the
Lowenstei Malachite inhibitory for growth of M.
n Jensen green, mineral Mycobacterium Inspissation
tuberculosis, human
Medium salts and tuberculosis
Glycerol allows the variety
glycerol
growth of human variety

1. For cultivation of
Cooked Meat pieces are for
Nutrient broth anaerobic organisms
Meat producing Autoclaving
& Meat pieces 2. For maintaining
Medium anaerobic condition
the stock culture

61
 Sabouraud Glucose,
Low and high sugar
peptone, Autoclaving 1. For cultivation of
‘s content make it selective fungi
Medium, agar for fungus
pH : 5.4

INDICATOR MEDIA
Media Important Action of Important Method of Uses
Ingredients Ingredients Sterilization
Sugar Peptone water, Sugar fermentation gives Tyndallisation 1. Used for the study
Media 1% sugar, acid and gas production of sugar
Andrade‘s Acid production is fermentation
indicator indicated by pink color Gas
production is seen as a
bubble formation in
Durham‘s tube

TRANSPORT MEDIA:

Media Important Action of Important Method of Uses

Ingredients Ingredients Sterilization
Charcoal, Charcoal absorbs the
inorganic bacterial inhibitory
Stuart‘s phosphates, substances 1. Provides viability of
Reducing agents Autoclaving
Media buffer, Gonococci and aerobes
prevents oxidation
sodium during the
thioglycolate transportation
Crude salt pH High alkaline condition 1. For transpiration of Vibrio
V. R.
preserves viability of Autoclaving
Medium 8.6 Cholerae
Vibrio species
Glycerol,
Glycerol sodium and 1. Preserves Salmonella,
Prevents growth of
saline potassium Autoclaving Shigella from faecal
intestinal commensals
Medium hydrogen specimens
phosphate

62
Nutrient agar

63
 Enriched Media

Blood Agar Chocolate agar

Loeffer's Serum Dorset's egg medium

64
 Selective media

MacConkey’s medium
L.J. Medium

Sabouraud Dextrose Agar

R.C.M.M.

65
66
 PRACTICAL NO-10
LABORATORY DIAGNOSIS OF BACTERIAL INFECTIONS-2
ISOLATION AND IDENTIFICATION OF BACTERIA

In any bacterial infection, causative agent may be present at the infected site. If a sample is taken
from this site and cultured in suitable culture media, the bacteria can be recovered (isolated). The
isolated bacteria is identified and further characterized, which helps in proper management of the
infectious diseases.
Isolation of bacteria can be done by following methods.
1. Bacteria culture: By growing the organism on artificial culture media at suitable
temperature, pH .
2. Isolating Microorganism In Animals (Animal inoculation)
In clinical laboratory indications for culture are:
● Isolation of bacteria in pure culture.
● To demonstrate their properties.
● To obtain sufficient pure growth for preparation of antigen and for other tests.
● For typing bacterial strain, isolate by method like bacteriophage bacteriocin susceptibility.
● To determine sensitivity to antibiotics.
● To estimate viable counts.
● To maintain stock culture.

Culture of bacteria includes:

1. Inoculation: Seeding of the sample in culture media is known as inoculation.
2. Incubation: After inoculation, the inoculated culture media are kept in suitable environment
which favors the growth of bacteria

CULTURE METHODS:
A.Methods of inoculation:

67
1. Streak culture:Most common method
Materials required:
a. Wire loop
- Made up of nichrome or platinum.
- Thickness of wire is 23 or 24 SWG.(Standard Wire Gauge)
- Length of wire is 6.5 cm.
- Internal diameter of loop is 2-4 mm.
b. Culture plates
- It is a glass or plastic petridish with diameter of 90 mm or 100mm.
- It contains suitable culture media which are solidified with agar.
c. Bunsen burner
- It is utilized for sterilization of wire loop
Procedure:
Usually employed method is known as the four flame method
a. Wire loop is first sterilized with Bunsen burner.
b. One loopful of specimen is taken & smeared on dried surface of culture plates on an
area of approximately 1 to 2 cm diameter (known as well)
c. Loop is re-sterilized and used to draw 3 to 4 parallel lines coming out from well
d. Loop is sterilized again and similar lines are drawn at right angle to initial set of lines
e. Step “d” repeated twice. Care should be taken to sterilize the loop between each
sequence.
f. Plates are closed and incubated in inverted position in the incubator.

Streak lines
Well

Use:
It is a routinely employed method for isolation of bacteria in pure culture from clinical
specimen.

2. Stroke culture
Procedure:
It is done in tubes containing agar slope or slant. The surface of agar slope is seeded with
wire loop in a zig-zag manner.
Use:
- To obtain pure culture of bacteria.
- For performing biochemical reactions.

68
3. Stab culture
Procedure:
It is done in tubes containing solid or semisolid culture medium. It is done with a straight
wire by stabbing the center of the medium from the top. Afterwards, the wire is
withdrawn in the same line to avoid splitting of medium.
Use:
- For performing biochemical reactions.
- To demonstrate motility of bacteria in semisolid agar
- To prepare bacterial stock culture for their preservation and future study.

4. Lawn culture/Carpet culture

Procedure:
It is done on the surface of agar plates. A liquid culture medium containing bacteria or a
bacterial suspension is flooded on entire surface of agar plate. The inoculum may be
evenly spread on the surface with the help of a sterile swab.
Use:
- Antibiotic susceptibility testing
- Bacteriophage typing
- To obtain large amount of bacterial growth for preparation of bacterial antigens and
vaccine

5. Pour plate method/Shake culture

Procedure:
a. Pre-sterilized 19 ml of nutrient agar is kept in a molten condition at 45-500C in a flask
b. One ml of bacterial suspension is mixed with the above medium and shaken properly
for even distribution
c. This mixture of medium and bacterial suspension is poured in a petriplate and allowed
to settle and solidify.
d. The settled plate is incubated and on next day number of bacterial colonies is counted,
which grow within and on the surface of medium.

Use:
This method is used for counting viable bacterial number in given liquid culture or
suspension.
69
 6. Exposed plate method
Use:
- For bacteriological analysis of air in operation theater and ICU: A culture plate is kept
open in an upright position so that the surface of the medium is exposed for a specific
period of time. The suspended particles in the air will get settled on the plate. The
plate’s lid is closed and then it is incubated.
- Used for direct seeding of culture plates where sampling is very difficult. i.e. In case of
Pertussis, bacteria are shredded in very less amount in sputum. In this case patient is
asked to cough directly on exposed surface of plate.

B. Incubation:
The inoculated culture media are kept in an environment according to the requirement of
the bacteria to be isolated and which are suspected to be present in the sample.
Environmental conditions vary with different bacteria & their growth requirements which
are as under:
1. Temperature:
The optimum temperature required by majority of pathogenic bacteria is 370C (human
body temperature). According to the temperature range at which they can grow,
bacteria are classified in Mesophilic, Thermophilic, Psychrophilic,
2. Oxygen tension in environment: Incubated in atmosphere as per type of bacteria
whetherObligate aerobes, Obligate anaerobes, aero tolerant or Microaerophilic.
3. Carbon dioxide level in environment: Majority pathogenic bacteria can grow at the
usualcarbon dioxide level in the environment. capnophilic bacteria.E.g. Meningococci
and Brucella sp. need 5-10 % of CO2
Equipments used for incubation.
1. Incubator
- It is a closed chamber of internal size 2x2x2 Cu ft or 3x3x3 Cu ft.
- Wall of the chamber is insulated to avoid effect of the surrounding temperature on
the temperature of chamber.
- It has heating elements and thermo regulator to maintain the temperature of inner
chamber at a fixed and desired level.
- Use: General purpose incubator. For isolation of obligate aerobic and facultative
anaerobic bacteria, media can be directly incubated in the incubator.
2. BOD incubator
- This is superior in temperature accuracy
- Contains both heating and cooling elements with thermostat to prevent temperature
overshoot
3. Anaerobic jar with gaspak
- The gaspak is a disposable packet of aluminum foil.
- After opening the gaspak is immediately placed in the jar & the jar is tightly closed.
- The reaction within gaspak takes place in a closed jar, which releases hydrogen and
carbon dioxide and absorbs oxygen present within the air of jar
- Use: for growth of anaerobic bacteria
4. McIntosh and filde’s anaerobic jar
- The lid of jar contains an inlet cork, an outlet cork and a catalyst
- In an air tight jar, air is evacuated with vacuum pump through outlet cork
- Through the inlet cork, hydrogen & nitrogen is supplied
- The catalyst facilitates the reaction between remaining oxygen with supplied
hydrogen and forms water.
- Use: For the growth of anaerobic bacteria
70
 5. Candle jar
- The inoculated media is placed in this airtight jar
- A candle is burnt and then the lid of jar is closed tightly.
- The burning candle will utilize most of the available oxygen inside jar and release
carbon dioxide.
- Use: For growth of bacteria that need high CO2.
- This is not a good method for anaerobiosis.
6. Water bath
- This is a chamber with heating elements at bottom.
- It is filled with water and water temperature is maintained at a desired level
- Incubation in water bath would avoid temperature fluctuation.

Selections of temperature and environmental conditions:Once medium is selected,

temperature and environmental conditions must also be considered.
● Environmental conditions are chosen according to the growth requirements of the
indigenous flora or pathogens suspected for the body site from which the specimen
is taken.
● Fastidious organisms may require increased CO2 or an anaerobic environment for
growth.
● Most routine bacterial culture plates are incubated at 35 C to 37 C for 48 hours.
0 0

● Broth cultures when included are routinely held 5 to 7 days.

Primary isolation media for unusual and fastidious bacteria:Temperature

requirements and length of incubation vary for individual organisms. Unusual organisms
may require special processing and selections of medium beyond the routine.

Bacteria multiply so rapidly, that the single cell deposited on a medium produces a mass
of cells within 24 to 48 hours. This mass of cells can be seen with the naked eye and is
called a “colony”.

Every colony is a pure growth of one single kind of bacteria. Colonies that are different in
appearance represent pure growth of different bacteria. Colony morphology helps in
identification of organisms. If a measured quantity of proper dilutions is inoculated, it can
help in quantitation of sample also.

IDENTIFICATION:
Pure bacterial culture is processed to study systematically. It includes the following steps.
1. Microscopic morphology:
Smears prepared from the bacterial colony or liquid culture are examined by staining
methods. Gram‘s staining divides bacteria into Gram-positive and Gram-negative;
Albert‘s staining shows morphological characteristics of diphtheria bacilli and Ziehl-
Neelsen staining differentiates acid-fast and non acid-fastbacilli. A motility preparation
in normal saline is helpful in distinguishing motile from non-motile ones.

2. Cultural characteristics:
(a) In liquid media – nutrient broth is commonly used, the points to be noticed are
(1) Deposits at the bottom and its nature.
(2) Presence or absence of turbidity and
(3) Surface growth such as pellicle formation.
(b) On solid media – Colony morphology: size, shape edge, elevation, surface,
transparency,color etc..of the colonies are noted.

71
3. Biochemical reactions:
1. Sugar fermentation: Different sugars are used in 1% concentration. Acid production
is shown bychange in the colour of the medium to pink and the gas production in
Durham’s tube. Andrade’s indicator is used.
2. IMViC tests: Indole production, Methyl red test (MR), Voges – Proskauer test (VP),
Citrate utilization test are very useful in identification of Gram negative bacteria.
a) Indole Production:-

b)Methyl red test:-

This test shows the production of sufficient acid during carbohydrate fermentation
of pH below 4.5. Organism is grown in glucose phosphate peptone water to 48 hour
at 37 0C & methyl red indicater’s 5 drops are added to culture.
Positive reaction - Bright red colour. (E.coli)
Negative reaction - Yellow colour. (Klebsiella)

72
 c) Voges Proskauer Test:-

d)Citrate Utilization Test:-

This test indicates the ability of organism to utilize citrate as sole source of carbon.
Koser’s citrate.(liquid form) or simmon’s citrate ( solid form) can be used.
Method: -Citrate is incorporated in the medium, inoculate the organism to be the
tested in the citrate medium& incubate it for 16-24 hour.

Simmon’s cilrate medium: -Colour changes due to alkaline condition.

Indicator used is bromothymol blue.
Positive: Blue colour (Klebsiella) .
Negative: Green colour (E-coli)

e) Urease test: are useful for proteus and providencia organisms.

The bacteria which contain urease, decompose urea into ammonia. Ammonia
production is tested by means of a suitable pH indicator.
Method :-Inoculate urease medium which contains urea with organism & incubate
it for 24 hour. See for colour change after 48 hour.
Positive Reaction :- Pink colour ( Klebsiella, Proteus)
Negative Reaction :- No colour.
f) Phenylalanine Deaminase (PPA) test: are useful for proteus and providencia
organisms.It is to determine the ability of an organism to deaminate phenylanine to
phenylpyruvicacid enzymetically with resulting acidity.
Method :Inoculate the organism on phenylanine medium. Incubate for 18-24 hours.
Add 4-5 drops of 10% Fecl3directly to an 18-24 hour in incubated tube. A positive
green colour occurs within 1-5 min. This is due to formation of phenylpyruvic acid.
Positive test is produced by proteus, providencia&morganella group.
g) Oxidase test: Neisseria, Pseudomonas, V.cholarae are oxidase positive organisms.
This test detects the presence of oxidase in bacteria which catalyses the transport of
electrons between electron doner in bacteria & redox dye Tetra methyl
paraphenylenediaimine dihydrochloride. The dye is reduced and gives a deep
purple colour.
Method :- Soak the filter paper with solution of the dye with a glass rod or
wooden rod. Pick a part of bacterial colony & touch the filter paper.
Positive Reaction: -A purple colour develops within 10 seconds.
( Vibrio, Pseudomonas, Neisseria)
Negative Reaction: No colour.

73
h). Catalase test: This test demonstrates the presence of catalase which catalyses the
release of O2 from H2 O2
2H2 O2 +catalase = 2H2O+ O2
O2 is detected by bubbiling in the tube
Method :- Take 1 ml 3% of solution of H2 O2 (Hydrogen peroxide).
Put small inoculum of bacteriun by platinum loop / glass rod. See
for bubbling or effervesence.
Positive : Bubbling / effervescence (Staph)
Negative: No bubbling (Strepto, Pneumococcus)
i). TSI test (Triple sugar iron test) : It is to determine the ability of an organism to
attack a specific carbohydrate incorporated in a basal growth medium, with or
without the production of gas, along with the determination of possible hydrogen
sulfide (H2S ) production
This agar contains:-
1. Yeast
2. Peptone
3. Glucose (1%)
4. Sucrose (10%)
5. Lactose (10%)
6. Ferric Citrate
7. Nacl
8. Sodium thiosulphate
9. Agar
10. Phenol red
11. Distilled water

The medium is distributed in tubes, with a butt & slant. The TSI medium facilitates
preliminary identification of gram negative bacilli. The test must be read &
interpreted within an 18-24 hour incubation period.

Butt Slant Interpretation

Acid Alkaline
only glucose attacked
Acid Acid Glucose and lactose / Sucrose attacked.
No change Alkaline
Neither glucose nor lactose nor sucrose attacked,
peptones used.
Blackening Blackening formation of H2S
Bubbles - indicate gas production

74
Biochemical tests for identification for bacteria:
Tests for metabolism of Substrate Observation
Carbohydrate
Break down of sugars
Glucose, Sucrose, Acid production sometimes
a. Oxidative (aerobic)
Lactose, Mannitol, with gas.
b. Fermentative
Glucose
Voges-Proskaur (VP) Acetyl methyl carbinol
(Glucose phosphate broth)
Glucose
Methyl red (MR) Sufficient acid (<pH 4.5)
(Glucose phosphate broth)
Growth
Citrate utilization Sodium citrate
(Sole carbon source)
Protein
Indole Tryptophan (Peptone water) Indole
Hydrogen sulphide Sulphur containing
Hydrogen sulphide
Production amino acids (TSI agar)
Phenylalanine deaminase Phenylalanine Phenylpyruvic acid (PPA)
Gelatin Liquefaction Gelatin Liquefaction
Amino acid Decarboxylase Arginine, lysine, Ornithine Carbon dioxide
Tests for detection of Enzymes
Coagulase Plasma Coagulum
Catalase Hydrogen peroxide Oxygen
Nitrate reduction Potassium nitrate Nitrite
Urease Urea (Urea broth) Ammonia

4. Agglutination reaction :
To confirm the species of bacteria, agglutination (clumping) reaction with polyvalent and
monovalent antisera is observed.
Monovalent antisera indicate that it contains antibodies only for one kind of antigen while
polyvalent serum contains antibodies against many antigens/antigenic determinants.
Generally, agglutination is done on the slide. Suspension of bacterial colony is made in
saline and drop of it is mixed with proportional amount of antisera on slide and
agglutination is seen.
Uses:
To identify particular strain of an organism involved in a particular epidemic i.e. To
know the serotype of vibrio cholera, Salmonella and Shigella.
5. Typing: Typing is done to differentiate different organisms in separate groups.
Typing can be based on the following criteria.
(I): Similar biochemical reactions – Biotyping
i.e.E. coli and Edwardsiella which show the same IMVIC reactions are placed in one
group.
(II): According to antigenic similarities - Serotyping
i.e. Neisseria meningitides is divided into 3 serogroups – A, B & C
(III):According to the susceptibility of bacteria to bacteriophage – Phage typing.
(IV):According to the inhibition of bacteria to bacteriocin – Bacteriocin typing.

Bacteriocins are antibiotic like substances produced by bacteria and they inhibit the
growth of different strains of that particular bacterial group form which they are
75
 produced.
i.e. E. coli produces colicin.
Pseudomonas produces pyocin
Cornybacteriumproduces Diphthericin

6. Pathogenicity Tests:
Guinea pigs, rabbits and mice are mostly used for animal inoculation tests, which are of
great value in anthrax, tuberculosis, diphtheria, plague, gas gangrene, and tetanus. The
animals may be inoculated by subcutaneous, intramuscular, intravenous, intraperitoneal
or intracerebral routes. Oral route or nasal spray may also be used.

76
 Oxidase Test Slide Agglutination

WORK:
1. Demonstrations:

77
 ● Instruments: Incubator, Nichrome wire loop & straight wire
● Anaerobiosis Instruments: Candle jar, Gaspak, McIntosh and Fildes anaerobic jar,
RCMM
● Culture techniques
● Various colonial morphology on culture plates.
● Growth characteristic in liquid media. Turbidity, deposits & pellicle formation.
● Biochemical tests. Catalase, coagulase, IMViC tests, sugar utilization tests.
2. Questions.
A. Enumerate various inoculation techniques.
B. Enumerate different methods of anaerobiosis
C. Enumerate various inoculation techniques.
D. Write Sugar reaction of E.coli& Klebsiella.
E. Write IMViC reaction of E.coli& Klebsiella.

78
 PRACTICAL NO-11
LABORATORY DIAGNOSIS OF BACTERIAL INFECTIONS- 3
ANTIBIOTIC SENSITIVITY TESTS

ANTIBIOTIC OR DRUG SENSITIVITY TESTS:

The isolated organism is subjected to drug sensitivity test in vitro for selecting
appropriate antibacterial drug. Antimicrobial drug sensitivity tests are useful in treatment
of infections. It can be done by one of the two principle methods.

Types:
 Disc diffusion method
Kirby – Bauer
Stoke’s method
 Minimum inhibitory concentration (MIC)
1. Broth dilution method
2. Agar dilution method
3. E-test
4. Automated methods: Vitek (Biomurex), Phoenix (B.D.)
 E-Test method
a). Disc diffusion method: Kirby – Bauer
Principle:
On a solid media, bacterium to be tested is inoculated by lawn culture method. A disc
containing particular antibiotic is placed on the inoculated medium, the antibiotic is allowed to
diffuse in surrounding medium and so the gradient is established; the concentration of
antibiotic is highest near the site of application of disc and decreases with distance. The
sensitivity of this inoculated bacterium is determined by measuring the diameter of zone where
bacterial growth is inhibited surrounding the antibiotic disc. If the zone of inhibition is more
than standardized value, it indicates that the bacterium is sensitive to this particular antibiotic.

Material required:
1. Culture media:
- Mueller - Hinton agar: For testing majority of non-fastidious bacteria. Or
- Blood agar or chocolate agar (Prepared with MHA and 5 % sheep blood): For testing
fastidious bacteria like S.pneumoniae, N. gonorrhoeae, H.influenzae
2. Materials for inoculation of bacterium:
- The pure growth of bacteria to be tested (obtained on solid media).
- Sterile swab,
- Peptone water / Normal saline,
- 0.5 Mc farland standard (a turbidity standard - suspension with equivalent turbidity are
having approximately 1.5 X108 bacteria / ml)
79
 3. Antibiotics:
- The antibiotics to be tested should depend upon
1. Type of organism
2. Type of infection
3. Availability of antibiotics for therapy in that area.
- The specific concentrations of antibiotics are individually impregnated in separate discs of 6
mmdiameter. These antibiotic containing discs are also available commercially, which can
be preserved forlonger period.
4. Equipments:
- Burner / spirit lamp
- Incubator
- Scale
Procedure:
 The pure growth of bacteria isolated from clinical specimen is processed for antibiotic
sensitivity.
 Preparation of bacterial suspension: 5 to 6 isolated colonies are picked up with sterile
loop or swab and inoculated in peptone water or normal saline and mix well for
preparation of bacterial suspension. The turbidity of bacterial suspension is adjusted with
0.5 Mc farland standard.
 A sterile swab is moistened with this bacterial suspension and then cultured on Muller
Hinton agar by lawn culture method.
 Within 15 minutes after inoculation, the selected antibiotic discs are applied over the
plate (not more than 6 discs should be applied over a plate). The plates are incubated in
inverted position at 37°C.
 After overnight incubation, the plate is observed for bacterial growth - there should be
confluent lawn of growth along with uniform circular zone of inhibition.
 Zone of inhibition around the individual antibiotics are measured and compared with
zone interpretative standards (as described by CLSI or EUCAST). The results are
interpretated as susceptible / resistant / intermediate.

Examples of some drugs (Disc concentrations and interpretation)

Zone of inhibition in mm.
Drug Concentration
Sensitive Intermediate Resistance
Ampicillin 10 mcg ≥ 30 21.29 ≤ 20
Chloramphenicol 30 mcg ≥ 18 13.17 ≤ 12
Colistin 10 mcg ≥ 11 09.10 ≤ 08
Erythromycin 15 mcg ≥ 18 14.17 ≤ 13
Kanamycin 30 mcg ≥ 18 14.17 ≤ 13
Penicillin 10 mcg ≥ 30 21.29 ≤ 20
Streptomycin 10 mcg ≥ 17 13.16 ≤ 12
Sulphonamide 30 mcg ≥ 20 15.19 ≤ 14
Tetracycline 30 mcg ≥ 20 15.19 ≤ 14

Clinical Interpretation of results:

Susceptible:
The isolate will be inhibited by usually achievable concentration of antibiotic when it is
used in recommended therapeutic dose.
Resistant:
The isolate will not be inhibited by usually achievable concentration of antibiotic when it
is used in recommended therapeutic dose.

Intermediate:
At the achievable concentration of antibiotic when it is used in recommended therapeutic
80
 dose, the response rate of isolate may be lower than that of susceptible one. This category
implies clinical efficacy in certain condition
i. In body sites where the drugs are physiologically concentrated i.e. quinolones in urine
ii. When higher than normal dose of drug can be used.

Primary sensitivity tests: Urine or fluid specimen containing bacteria is directly

inoculated on a solid medium and medicated disc are placed on the surface of the plate and
incubated.

b). Disc diffusion method: Stoke’s method:

The outer side of the plate is inoculated with a standard organism and the test organism in the
middle of the plate and the zone of inhibition around the disc is compared.
Result: The zone inhibition of isolate is compared with zone inhibition standard bacterial
strains and sensitivity of the particular drug is determined.*Sensitive / *Moderate sensitive /
*Resistant

c). MIC by broth dilution method:

It is a laborious procedure but useful in assessment of therapeutic dose.
Method: Serial dilutions of the drug in broth(well of microtiter plate or test tube) are
inoculated with the bacterium under test and incubated.
Observation: Lowest concentration of drug inhibiting bacterial growth represents minimum
inhibitory concentration (MIC), bacteriostatic effect. Minimum bactericidal concentration
(MBC) is then determined by subculture from the tubes of MIC test into solid media.
Bacterial growth on solid media indicates presence of surviving bacteria. The lowest
concentration of the drug that kills all the bacteria is found out from the appropriate tube,
which will not show any growth on subculture.

81
d). MIC by E-test strip:
It uses a rectangular paper of filter paper of filter paper strip unique to each antibiotic that has
the dot of dried antibiotic on the underside. This is placed on an agar plate already swabbed
with the patient’s isolate & read after incubation. It produced a µg/ml value that correlates
fairly well with the minimal inhibitory concentration.

E-Strip :Here MIC = 0.126 ug/ml

82
 WORK:
1. Demonstrations:
● Antibiotic sensitivity test. Disc diffusion test.
2. Draw the labelled diagram of Antibiotic sensitivity testby Disc diffusion test
3. Questions.
A. Enumerate various methods of antibiotic sensitivity testing.
B. What is drug resistance?

Antibiotic Sensitivity Test:Disc diffusion

Methods for detection of bacterial property or mechanism which is responsible for its
resistance to a particular antibiotic or group of antibiotics

For Staphylococci:
1. Beta lactamase (penicillinase) production:
Detection methods:Acidometric method, Iodometric method, Chromogenic cephalosporins
(nitrocephin)
Interpretation:Staphylococcus sp. producing beta lactamase which de-activate the penicillins.
These bacteria are resistant to majority of the penicillin group of antibiotics (i.e. Penicillin G,
ampicillin, amoxycillin, piperacillin etc.). In this case only methicillin group (i.e. cloxacillin) or
penicillins plus beta lactamase inhibitors combinations (e.g. Ampicillin with sulbactum or
Amoxycillin with clavulinic acid) can be effective.
2. Methicillin Resistant Staphylococcus (MRSA / MRS)
Detection methods:If the staphylococcus is found resistant to methicillin (nowadays oxacillin
or cefoxitin discs), it is considered Methicillin Resistant Staphylococcus.
Interpretation:Methicillin Resistant Staphylococcus is considered resistant to all the beta
lactam group of antibiotics including Penicillins, Cephalosporins and carbapenems. Here drug
of choice for treatment is Vancomycin.
For gram negative bacilli
1. Extended Spectrum Beta Lactamase (ESBL) production:
Detection methods:By disc diffusion method if a bacterium is found sensitive to
ceftazidime+clavulinic acid disc; and its zone of inhibition is significantly more than that of
ceftazidime alone as per standard guidelines, the bacteria is considered ESBL producer.
Interpretation:The ESBL producing gram negative bacilli are considered resistant to all the
penicillins, cephalosporins and monobactum. Here drug of choice for treatment is carbapenems.
2. Carbapenemase Production:
Detection method:Comparison of Imipenem &imipenem+EDTA sensitivity.
Interpretation:Such bacteria are resistant to all the beta lactam antibiotics& carbapenems.
Here drug of choice for treatment is Aztreonem.
83
Antibiotic stewardship programs (ASPs):
A hospital based programs dedicated to improve antibiotic use, which can both optimize the
treatment of infections and reduce adverse events associated with antibiotic use. These programs
help clinicians to improve the quality of patient care and patient safety through increased infection
cure rates, reduced treatment failures, and increased frequency of correct prescribing for therapy
and prophylaxis. They also significantly reduce antibiotic resistance. In recognition of the urgent
need to improve antibiotic use in hospitals and the benefits of antibiotic stewardship programs, in
2014 CDC recommended that all acute care hospitals should implement Antibiotic Stewardship
Programs.
Interventions for ASPs:
1. Hospital level intervention:
Each hospital should implement the policies for optimal use of antibiotics:
This should include
- Documents regarding indications, dose and duration of commonly prescribed antibiotics
- Recommendations of antibiotic options in common infectious diseases like UTI, Pneumonia,
septicemia, Enteric fever, SSTI etc. This recommendation should be on the bases of national
guidelines and local antibiotic sensitivity pattern.
-These policies should be followed by all the prescribing doctors.
2. Clinician level:
- Clinician must follow the policy for prescribing antibiotics
- Review of antibiotic usage:
The clinician must review the antibiotic selection 48 hours after the antibiotic initiated with
answer to following questions (Clinician must review with the backup of laboratory reports)
1. Does this patient have an infection in which antibiotic will be effective?
2. If really antibiotic is needed then patient is receiving a right antibiotic with proper dose and
route of administration?
3. Can more targeted antibiotic be used - whether it should be escalated or de-escalated?
Escalation: Switch over the antibiotic therapy from lower spectrum to higher spectrum
molecule
De escalation: Switch over the antibiotic therapy from higher spectrum to lower spectrum
and more specific molecule)
4. How long the patient should receive the antibiotic?

Exercise:
1. Interpretation of culture and sensitivity report
2. To review the antibiotic therapy in a given case
Case: A 24 year old female suffering from Urinary tract infection was given tab. Cefixime
since 2 days.
Investigations:
- USG abdomen revealed pyelonephritis
- Urine culture report revealed Escherichia coli with more than 1 lac colony count per
ml of urine; Bacteria produced ESBL.
- After 48 hours of treatment, patient does not respond well to therapy
Questions:
Q.1 Whether Cefixime should be continued or not?
Q.2 Any other from beta lactam group of antibiotic can be selected?

84
 PRACTICAL NO-12
LABORATORY DIAGNOSIS OF FUNGAL INFECTIONS

The comprehensive diagnostic approach to fungal infections can be divided under two broad
headings:
A. Clinical diagnosis
B. Laboratory diagnosis

A. CLINICAL DIAGNOSIS:
The clinical criteria may give presumptive diagnosis of fungal infections.
Clinical classification of fungal infections (mycoses) & their causative agents
Mycoses Definition Most common causative fungi
Superficial Fungal infections that involve Dermatophytes
mycoses hair, skin or nails without Candida
direct invasion of the deeper Malassezia
tissue. Piedraia
Subcutaneous Fungal infections that are Fonsecaea
mycoses confined to the subcutaneous (Chromoblastomycosis)
tissues without dissemination Sporothrix (Sporotrichosis)
to the distant sites. Madurella(Mycetoma)
Cladosporium
(Phaeohyphomycosis)
Deep/invasive Fungal infections primarily Histoplasma
mycoses involving the lungs but may Blastomyces
become widely disseminated Coccidioides
& involve any organ system. Paracoccidiodes
Opportunistic Fungal infections that occur Aspergillus
mycoses primarily in patients who are Zygomycetes (Mucor,Rhizopus)
immunocompromised. Candida
Cryptococcus

B. LABORATORY DIAGNOSIS:
I. SPECIMEN COLLECTION:
The site as well as nature of specimens is most important to proceed for final diagnosis.
a) Superficial mycoses:
Prior to collection of specimen, clean the affected area with 70% alcohol. (To remove any
tracesof skin products or medications).Collect skin scales, crusts, pieces of nail or hairs in
sterile petridish or paper envelope
• Skin scales: Dermatophytic lesions usually spread outward in concentric fashion with
healing in central region. Therefore, material should be collected by scraping outward from
edges of lesions with scalpel blade held at an angle of 90O to skin surface. If multiple lesions
are present, then choose the most recent for scrapping as old loose scale is not satisfactory.
• Hairs: The specimen from the scalp are best obtained by scrapping with blunt scalpel so that
they include hair stubs, contents of plugged follicles &scales. The base of the infected hairs
should be collected by clipping or plucking the hairs.
The ringworm of scalp, especially caused by anthropophilic species infection may produce
fluorescence of infected hairs & may be detected by wood’s lamp examination.
• Nail pieces: The nail clippings/scrapings should be taken from discolored, dystrophic or
brittle parts of nails. It should be collected from proximal part of nails because fungus in
distal part of nails is often non-viable. (fungus may be visible on microscopy but fail to
grow in culture)
85
b) Subcutaneous mycoses:
Scraping, crusts, aspirated pus, granules& tissue biopsies are to be collected.
Mycetoma:
When the mycetoma is caused by a fungus, the swelling is called a Eumycetoma, and when
caused by bacteria it is called an Actinomycetoma. The color, size, consistency &
microscopic appearance of granules are used to diagnose & differentiate mycetoma. Black
granules indicate Eumycetoma, White to yellow granules Actinomycetoma&pale coloured
granules are produced in both type of mycetoma.In mycetoma (A clinical condition
characterized by 3S = swelling, sinus, sulfur granules), the discharge from sinuses is
aspirated after cleaning the surface. Along with the discharge, granules must be collected.
When it is difficult to aspirate the pus, a sterile gauze piece is applied over the sinus then
pressure is applied over it to squeeze out some discharge. A biopsy of the lesion may also be
taken.
c) Systemic/Invasive mycoses:
In patients with invasive mycoses specimens may be taken from as many as potential sites as
possible. The sources include tissue biopsy, pus, feces, urine, sputum, body fluids, blood, &
scrapings & swabs from edge of lesions.
II. TRANSPORTATION OF SPECIMENS:
Specimens are placed in sterile containers & transported to the laboratory immediately.
Specimens that are not processed immediately should be held at room temperature.
III. Microscopic methods:
Fungal elements can be detected in the clinical specimens by direct microscopic examination
of material from the lesion.
 Potassium hydroxide (KOH) preparation :
Keratinized tissue specimens such as skin scrapings and plucked hair samples are treated
with 10% KOH which digests the keratin material so that the fungal hyphae will be
clearly seen under the microscope. Heat the slide gently over the flame and leave it aside
for 5-10 minutes before examination.
• About 10% is the usual concentration of KOH used.
• About 20-40% KOH is needed for the specimens such as nail that otherwise takes
longer time to dissolve.
• Biopsy specimens as they take longer time to dissolve are usually dissolved in 10%
KOH in a test tube and examined after overnight incubation. Glycerol (10%) can be
added to prevent drying. DMSO (dimethyl sulfoxide) can be added to help in tissue
digestion.
 Gram stain :
It is useful in identifying the yeasts (e.g.Cryptococcus) and yeast like fungi (e.g.
Candida). They appear as gram-positive budding yeast cells.
 India ink and Nigrosin (Negative) stains :
They are used as negative stains for demonstration of capsule of Cryptococcus
neoformans.
 Calcofluor white stain:
It is more sensitive than other stains; binds to cellulose and chitin of fungal cell wall and
fluoresce under UV light.
 Histopathological stains:
They are useful for demonstrating fungal elements from biopsy tissues. This is useful for
detecting invasive fungal infection.
• Periodic acid Schiff (PAS) stain: It is the recommended stain for detecting fungi.
PAS positive fungi appear magenta/deep pink, whereas the nuclei stain blue.

86
 • Gomori methenamine silver (GMS) stain: It is used as an alternative to PAS for detecting
fungi. It stains both live and dead fungi, as compared to PAS which stains only the live fungi.
GMS stains the polysaccharide component of the cell wall. Fungi appear black whereas the
background tissue takes pale green colour.
• Mucicarmine stain: It is used for staining the carminophilic cell wall of Cryptococcus and
Rhinosporidium.
• Masson Fontana stain: It is used for pigmented (or pheoid) fungi.
• Hematoxylin and Eosin (H and E) stain.
 Lactophenol cotton blue (LPCB) :
It is used to study the microscopic appearance of the fungal isolates grown in culture. It
contains:
• Phenol acts as disinfectant.
• Lactic acid preserves the morphology of fungi.
• Glycerol prevents drying.
• Cotton blue stains the fungal elements blue.
Observation- Fungi are stained blue. Fungal elementslike hyphae (septate, aseptate),conidia
etc. can be well appreciated.
IV. Culture: Fungal culture is frequently performed for isolation and correct identification of the
fungi.
 Culture Media
• Sabouraud's dextrose agar (SDA): It is the most commonly used medium in diagnostic
mycology. It contains peptone (1%), dextrose (4%) and has a pH of 5.6. This may not support
some pathogenic fungi.
• Neutral SDA (Emmons' modification): It differs from original SDA in having neopeptone
(1%) and dextrose (2%) and pH of 7.2.
• Corn meal agar and rice starch agar: They are the nutritionally deficient media used for
stimulation of chlamydospore production.
• Brain heart infusion (BHI) agar and blood agar: They are the enriched media, used for
growing fastidious fungi like Cryptococcus and Histoplasma.
• Niger seed agar and bird seed agar: They are used for the selective growth of Cryptococcus.
• CHROM agar Candida medium: It is used as selective as well as differential medium for
speciation of Candida.
• Czapek-Dox medium: It is used as a selective growth medium, used for growing saprophytic
fungi like Aspergillus, Candida, Penicillium and Paecilomyces.
 Culture Condition
• Temperature: Most of the fungi grow well at 25-30°C except the dimorphic fungi that grow
at both 25°C and 37°C.
• BOD incubators (biological oxygen demand): It is a special incubator used in diagnostic
mycology, which is capable of maintaining low temperature.
• Incubation time: Culture plates should be incubated for 2-3 weeks. Cultures are routinely
incubated in parallel at room temperature (22°C) for weeks and at 37°C for days.
• Antibiotics such as cycloheximide (actidione), chloramphenicol and gentamicin can be added
to the culture media to inhibit bacterial growth.
 Culture Identification
The correct identification of the fungus is based on the macroscopic appearance of the colonies
grown on culture and microscopic appearance (LPCB mount of colonies).
a). Macroscopic Appearance of the Colony
• Rate of growth :
 Rapid growth (<5 days): It is seen in saprophytes, yeasts and agents of opportunistic mycoses.
 Slow growth (1-4 weeks): It is observed in dermatophytes, agents of subcutaneous and
systemic mycoses.
87
 • Colour and morphology of colony on the obverse
• Pigmentation: It can be seen on the reverse side of the culture media.
• Texture: It refers to how the colony would have felt if allowed to touch. It may be of
various types such as — glabrous (waxy/leathery), velvety, yeast like, cottony or
granular/powdery.
• Colony topography: Colony surface may be rugose (radial grooves), folded,
verrucose or cerebriform (brain-like).
b). Microscopic Appearance of Fungi
• Teased mount: A bit of fungal colony is teased out from the culture tube and the
LPCB mount is made on a slide and viewed under microscope. Identification is based
on the following:
 Nature of hyphae (such as septate or aseptate, hyaline or phaeoid, narrow or wide) and
 Type of sporulation (conidia or sporangia).
• Slide culture: Though this is a tedious procedure, it gives the most accurate in situ
microscopic appearance of the fungal colony. A sterile slide is placed on a bent glass
rod in a sterile petri dish. Two square agar blocks measuring around 1 cm2 (smaller
than the coverslip) are placed on the slide. Bits of fungal colony are inoculated onto
the margins (at the centre) of the agar block. Then the coverslip is placed on the agar
block and the petri dish is incubated at 25°C. After sufficient growth occurs, LPCB
mounts are made both from the coverslip and the underneath slide.
• Cellophane tape mount: The impressions are taken by placing the cellophane tape on
the colonies present on the surface of SDA plate, and then LPCB mount is made from
the cellophane tape. This is easy to perform than slide culture and in-situ fungal
morphology is also maintained.
c). Other Methods of Identification
• For Candida: Germ tube test and carbohydrate assimilation tests.
• For dermatophytes: Hair perforation test, dermatophyte test medium.
• Cryptococcus: Urease test can be done.

V. Immunological Methods
These tests are available to detect the antibody or antigen from serum and/or other body
fluids.
• Antibody detection can be done by ELISA, immunodiffusion test, agglutination test and
complementfixation test (CFT). Because fungi are poor antigens, the efficacy of serology
varies with different fungal agents. Can be used for chronic and invasive fungal diseases.
• Antigen detection:
• Cryptococci :latex agglutination and Immunochromatographic strip from test for CSF
and serum
VI. Tests for Metabolites:
• Aspergillus: detection of Aspergillus specific galactomannan in serum.
Tests to demonstrate Delayed Hypersensitivity
Skin tests are available for demonstrating delayed type of hypersensitivity for pathogens
like Histoplasma, Blastomyces, Coccidioides, Paracoccidioides, Dermatophyte,
Sporothrix and Candida.

VII. Molecular Methods

Polymerase chain reaction (PCR) and MALDI-TOF from culture as well as from the
specimens.

WORK:
1.Demonstrations:
88
 ● Gram stain smear – Yeast cell and Filamentous forms of fungus.
● Lactophenol cotton blue stains: Filamentous forms of fungus
● Culture on SDA : candida and Aspergillus.
2.Draw figures of various demonstrations.
3.Questions:
a). Morphological classification of fungi.
b). Enumerate various culture media used for diagnosis of fungal infections.
c). Describe pathogenicity of candida species.
d). Which are opportunistic fungi?

89
 Yeast
Hyphae

Candidaspp. Aspergillus spp.

On SDA On SDA

90
 PRACTICAL NO-13
LABORATORY DIAGNOSIS OF PARASITIC INFECTIONS(Stool Microscopy)

The following are the main ways in which parasitic infections are diagnosed in the laboratory.
Microscopic examination: The majority of intestinal, urinary, and blood parasites can be
detected microscopically in unstained or stained preparations, either directly or following
concentration techniques.
Culture techniques: Only a minority of parasitic infection is diagnosed routinely by culture
techniques.
Immunodiagnosis: With the development of reagents, which are both more sensitive and
specific, immunodiagnosis techniques are becoming increasingly used in diagnosis and in studies
involving the epidemiology and control of parasitic diseases.

1. DIRECT EXAMINATION OF FECAL SPECIMENS:

Collection:
Freshly collected stool specimen is always recommended for parasitic examination as it can
also reveal motile forms of parasites, if they are present in the specimen. Stool should be
collected in suitable, clean, wide mouth , Leak proof container. Normally passed stool is
recommended. However, samples obtained after purgatives or enema may be collected to
recover parasites in certain conditions. Sample should be transported within one hour to the
laboratory for observing motility of parasites.
Gross examination:
- On gross inspection of sample, adult worm may be seen in stool samples, e.g., round worm,
thread worm,segments of tape worms etc.
- Presence of mucus and blood will indicate dysentery, which is a common presentation of
acute intestinal amoebiasis.
- Foul smelling white stool indicates steatorrhoea, which is a common presentation of
giardiasis.
- Mention: Color, Consistency, (i.e. whether it is formed, semiformed, unformed, or fluid),
- Mention whether it contains blood, mucus or pus.

Microscopic examination:
A. Normal saline & Iodine wet mount preparation:
Place a drop of fresh physiological saline on one end of a slide and a drop of Lugol‘ iodine
(5%) on the other end. A small amount of feces is mixed by a stick to form a uniform
smooth suspension. Cover slip is placed on the mount and examined under low power
(10X), followed by high power (40X) objective.
Saline Mount:
Advantages:
● In a saline preparation motile parasites such as trophozoites, flagellates, larvae, and
ciliates can be identified.
● Cysts can also be detected but they are much more easily seen in iodine preparation
because it stain nuclei and glycogen mass. of trophozoites and larvae can be
demonstrated.
● Helminth eggs can be readily identified. Bile staining property can be appreciated-
Bile stained eggs appear brown and non bile stained eggs appear colorless.
Iodine Mount
Advantages:Nuclear details of cysts are better visualized; helps in species identification.
Disadvantages:Iodine immobilizes and kills parasites, hence motility of the trophozoites,
flagellates, larvae, and ciliates Bile staining property cannot be appreciated.

Other structures:
91
 • Presence of pus with or without blood suggests dysentery.
• Charcot - Leyden crystals may be present in stool sample; which are generally seen in
parasitic infections & are particularly suggestive of amoebic dysentery.
Normal structures found in feces.
Care must be taken not to report as parasites those structures, which can be
normally found in feces such as muscle fibers, vegetable fibers, starch cells, pollen
grains, fatty acid crystals, soaps, spores, yeasts, and hairs.
Concentration techniques for fecal parasites:
Wet mount preparation can easily reveal ova and cysts of parasites if they are abundant in
the sample. If the sample contains fewer parasites, they may not be detected by direct
microscopic examination. In such condition, they can be detected by using various
concentration techniques prior to microscopic examination
1. Sedimentation techniques: Formol-ether sedimentation technique:
Procedure:
• 1 to 2 grams of stool is mixed with 10 to 15 ml of saline thoroughly.
• The mixture is filtered through double layered gauze with the help of funnel. This will
remove large particles.
• The filtrate is now centrifuged at 2000 rpm for 5 minutes.
• The supernatant is discarded and the sediment is mixed with 7 ml of formal saline. It is
allowed to stand for 5 to 10 minutes.
• 3 ml of ether is added to the above suspension and vigorously mixed on vortex mixer.
• The suspension is now centrifuged at 2000 rpm for 2 minutes. The tube is now
allowed to stand for a minute.
• Then, four distinct layers will be visible in the tube. The upper two layers are carefully
discarded.The deposit at the bottom layer is resuspended in the 3rd layer of formal
saline.
• A drop of suspension is placed on a slide and examined for the presence of parasitic
ova / cysts.
2. Floatation techniques:
a. Saturated salt solution floatation technique:
Procedure:
- 1 to 2 grams of stool sample is mixed with 3 - 4 ml of saturated salt solution in a test
tube.
- After mixing thoroughly, more amount of saturated salt solution is added up to the
brim of the test tube.
- A glass cover slip is placed on the brim of the above test tube, in such a way that the
supernatant of the suspension remains in touch with the cover slip. It is allowed to
stand like this for 30 minutes.
- After 30 minutes, the ova which tend to float in saturated salt solution will get
concentrated in the supernatant at the air interface and hence, will get stuck to the
cover slip.
- The cover slip is now picked up and placed on a clean glass slide for microscopic
examination.
b. Zinc sulphate floatation technique:
- 1 to 2 grams of stool is thoroughly mixed with 8 to 10 ml of distilled water.
- This suspension is filtered through a double layered gauze piece. The filtrates are
again mixed with distilled water and centrifuged at 2000 rpm for 10 minutes
- The deposit is now mixed with saturated zinc sulphate solution and allowed to stand
for 30minutes.
- After 30 minutes, fluid from supernatant is carefully aspirated with a dropper and
placed on a glass slide for microscopic examination.

Note:- Unfertilized eggs of round worm cannot be concentrated by this technique

92
 - The recommended technique for hospital laboratories is the formal ether technique.
It is rapid and gives good concentration of parasitic cysts, eggs, and larvae, in fresh or
preserved feces.

B. Special staining methods for fixed smear:

These staining methods help in identification of ova and cysts of the parasites. These are
identified due to their distinct staining properties. Permanent slides can also be prepared &
preserved for further reference and academic purposes.
1. Iron haematoxylin:
This is used for differentiation among various amoebic cysts. This is done on the basis of
specific & peculiar shapes of their nuclei.
2. Modified Z N stain:
The oocysts of Cryptosporidium and Isospora are weakly acid fast. A smear is prepared
from the stool suspension on a slide & is stained with hot or cold ZN staining method.
Here, 1% H2SO4is used instead of 20% as the decolorizing agent.
3. Trichrome stain:
This staining method can be used for the demonstration of various protozoa. It is especially
useful for the detection of very small oocysts of Microsporidia.

C. Other methods:
Duodenal contents:
Examination of duodenal contents is useful for the detection of Giardia lamblia, when
repeated examination of faeces fails to demonstrate this parasite in the faeces. It is
collected by duodenal intubation.
Entero Test
It is a frequently used test to collect duodenal contents for microscopic examination and
demonstration of the parasites. It uses a gelatin capsule attached to a thread containing a
weight. One end of the thread is attached to the outer aspects of patient’s cheek, and then,
the capsule is swallowed. Capsule gets dissolved in stomach releasing the thread which is
carried to duodenum by peristalsis and due to weight attached to the thread. The thread
gets unfolded and takes up duodenal samples. Four hours later, the thread is withdrawn and
shaken in saline to release trophozoites which can be detected microscopically by wet
mount or permanent stained smear. This procedure eliminates the need for duodenal
intubation.
Anal swab:
Cellophane tape preparation
The principle of this method is that the eggs are deposited by female Enterobius
vermicularis on the surface of the skin surrounding the anus during night. These eggs stick
to the sticking surface of cellophane tape, which can be visualised under the microscope.
The slide is examined microscopically under a low-power objective (10×) of the
microscope with low illumination. The specimen is best collected in the morning before
bathing and going to the toilet.

Table: The intestinal parasites found in stool specimen

93
 Sr. Parasite Form of parasite found in stool
No.
Protozoa:
1 Entamoebahistolytica Trophozoites, cysts
2 Giardia intestinalis Trophozoites, cysts
3 Balantidium coli Trophozoites, cysts
Helminths (worms):
1 Ascaris lumbricoides (Round worm) Adult worm, ova (eggs)
2 Ancylostoma duodenale, Necator americanus Ova (eggs)
(Hook worm)
3 Trichuris trichiura (Whip worm) Ova (eggs)
4 Enterobius vermicularis (Thread worm / pin Adult worm, ova (eggs)
worm)
5 Taenia solium, Taenia saginata (tape worms) Segments of adult worm, ova(eggs)
6 Hymenolepsis nana (dwarf tape worm) Ova(eggs)
7 Diphyllobothrium latum Segments of adult worm, ova(eggs)
8 Schistosoma japonicum & Schistosoma Ova (eggs)
mansoni
9 Clonorchis sinensis Ova (eggs)
10 Fasciola hepatica Ova (eggs)
11 Fasciolopsisbuski Adult worm, ova (eggs)
Opportunistic parasites causing infection in immuno-compromised patients:
1 Cryptosporidium parvum Oocysts
2 Isospora belli Oocysts
3 Cyclospora cayetanensis Oocysts
4 Microsporidia Oocysts
5 Blastocystis hominis Cysts
6 Strongyloidesstercoralis Larva
Non-pathogenic parasites commonly found indiarrhoeal stools:
1 Trichomonas hominis Trophozoites
2 Entamoeba coli Trophozoites, cysts

IMPORTANT FEATURES OF COMMONLY SEEN FECAL PARASITES

PROTOZOA:
MorphologicalF
Parasites Microscopic Features
orms
*Average size is about 25*20 µm.
Trophozoite *Active amoeboid movementin fresh warm specimens.
*Contains ingested red cells.
E. histolytica
*Round, measuring 10-15 µm.
Cyst *Contains 1, 2, or 4 nuclei.
*Chromatoid bars can be seen in immature cysts.
*Pear-shaped, usually measuring 10-12*6 µm.
Flagellate *Upper end has a concavity with a sucking disc.
*There are 8 flagella.
Giardia
lamblia *Oval in shape, measuring about 10*6 µm.
*Contains the remains of axonemes and parabasal bodies.
Cyst
*Thread-like remains of flagellate may also be seen.
*4 nuclei.

94
 *Large, measuring 50-200*40-70 µm.
Ciliate *Rapid revolving motility.
*Beating cilia can be seen, especially around the cytostome.
B. coli *Large, measuring 50-60 µm.
*Thick-walled.
Cyst
*Cilia may be seen in younger cysts.
*Macronucleus visible.
Isospora *Oval, measuring about 32*16 µm.
Oocysts
belli *Usually contain a central undivided mass of protoplasm.

CESTODES:
Morphological
Parasites Microscopic Features
Forms
*Appears white and opaque and measures about 20 mm long by 6
Segment mm wide when freshly passed.
T.saginata *Uterus has a central stem which has more than 13 main side
branches on each side.
*Appears gray-blue and translucent and measures about 13 mm
Segments long by 8 mm wide when freshly passed.
Taenia
T.solium *Uterus has a central stem which has up to 13 main side branches
on each side.
*It is round to oval, measuring 33-43 µm.
Egg
*A thick, brown, radially striated wall surrounds the
T.saginata
embryo.
T.solium
*Hooklets (3 pairs) are present in the embryo.
*It is colorless, oval or round, measuring 30-45 µm.
*Hooklets are present in the embryo.
H. nana Egg
*At the each end of egg thread-like structures called polar filaments
are usually visible.
*It is pale yellow and oval in shape, measuring about 70*45 µm.
*It has an operculum.
*Contains a mass of granulated yolk cells surrounding an
D. latum Egg
undeveloped ovum.
*A small projection is sometimes visible at the non- operculated
end of the egg.

TREMATDES:
Morphological
Parasites Microscopic Features
Forms
*It is pale yellow-brown, large and oval, measuring
about 140*85 µm.
F. buski
Egg *Contains an unsegmented ovum surrounded by many yolk
F. hepatica
cells.
*Has a small operculum.
*It is pale yellow-brown, large, and oval, measuring about
150*60 µm.
*Has a characteristic side spine.
S. mansoni Egg *Contain a fully developed miracidium.
*A viable egg shows flickering of the excretory flame cells.
*A non-viable egg is dark-colored and shows no structural
detail or flame cell movement.

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NEMATODES:
Morphological
Parasites Microscopic Features
Forms
*It is yellow-brown and the shell is covered by an uneven
albuminous coat.
Fertilized egg *Oval or round and measures 60*40 µm.
*Contains a central granular mass which is the
A. lumbricoides unsegmented fertilized ovum.
*It is darker in color and has a more granular albuminous
Unfertilized covering.
egg *More elongated, measuring about 90*45 µm.
*Contains a central mass of large refractile granules.
*It is colorless and has a clear shell.
*Oval in shape and usually flattened on one side. It measures
E. vermicularis Egg
about 55*30 µm.
*Contains a larva.
*It is actively motile.
*It is large, measuring 200-300*15 µm and is unsheathed.
S. stercoralis Larva *Shows a typical rhabditiform bulbed esophagus.
*It can be distinguished from a hookworm larva
by its shorter buccal cavity.
*It is yellow-brown and measures about 50*25 µm.
*Has a characteristic barrel shape with a colorless
T. trichiura Egg protruding Mucoid plug at each end.
*Contains a central granular mass which is unsegmented
ovum.
*It is colorless with a thin shell which appears as a black
line around the ovum.
*Oval in shape, measuring about 65*40 µm.
A. duodenale
Egg *Contains an ovum, which usually appears segmented. If
N. americanus
specimen is more than 12 hours old, a larva may be seen
inside the egg. If the feces is more than 24 hours old than
larva may hatch and seen free in the feces.

Work:
Demonstrations: (1) Microscopic observation – Protozoal cyst, Helminth egg.
(2) Helminth adult specimen.
Exercise: (1) Examination of stool for Protozoal cyst, Helminth egg
(2) Draw the diagram of various trophozoite & cyst of protozoan
(3) Draw the diagram of various helminth eggs.

Trophozocte ofE. histolytica Trophyozocte ofE. Coli

96
 Cyst. OfE. histolytica Cyst ofE. Coli

Trophozocte ofGiardia Cyst of Giardia

Egg of Ascaris lumbricoides (Fertilized) Egg of Ascaris lumbricoides (Unfertilized)

97
Decoricated egg of A. Lumbricodes Egg of Hookworm

Egg of Trichuris trichiura

Egg of Enterobius
i l i

Egg. of H. nana Egg of Taenia sp.

98
 PRACTICAL NO-14
LABORATORY DIAGNOSIS OF VIRAL INFECTIONS

Specimen collection and transport:

Viral shedding is usually greatest during the early stages of infection, so the best specimens are
those collected as early as possible. Specimens should be collected as aseptically as possible.
Aspirated secretions are often preferable, but swabs are easier to use for collection. Tissue
samples must be kept moist. Viral transport medium can be used. It is optimal to process viral
specimens for culture immediately. However, if specimens cannot be processed immediately
after collection, they should be stored at 40 C. Specimens should not be frozen unless a
significant delay (greater than 4 days) in processing occurs. In that case, specimens should be
frozen and held in a –700 C freezer.
Appropriate Specimens:
A commonsense approach in selecting specimens for isolation is to collect the specimens from
the infected site. However, if systemic, congenital, or generalized disease is involved, then
specimens from multiple sites, including blood and CSF, as well as from the portals of entry or
exit are appropriate.
A. Microscopic methods:
1. Demonstration of Inclusion bodies:
Definition: Structure with distinct size, shape, location & staining properties seen under light
microscope within virus infected cells are called inclusion bodies.
Methods to demonstrate inclusion bodies:
1. The inclusion bodies are large enough to be visualized by routine microscope. The
inclusion bodies can be stained with Giemsa stain, eosin methylene blue stain and H & E
stain.
2. The inclusion bodies can also be stained with fluorescence dyes & visualized under
fluorescent microscope.
Examples of inclusion bodies:
Intracytoplasmic inclusion bodies: Negri bodies: Rabies, Molluscum bodies: Molluscum
contagiosum
Intra-nuclear bodies:Herpes virus, Yellow fever virus, Adeno virus, Polio virus
2. Florescent microscope:
This method can detect viral particles / viral antigens / inclusion bodies in the specimen
Principle: Specimen is smeared on the slide and fixed. A fluorescent dyes tagged on antibodies
which are specific against viral antigens is used to stain the smear. The stained smear is
examined under fluorescent microscope.
Examples:
Anti rabies antibodies tagged with auramin dye are used to detect rabies viral particle /
inclusion bodies in the smear from brain specimen or impression smear from cornea.
3. Electron microscope:
This type of microscope can demonstrate as small as 20 nm sized viral particles. However this
method cannot be employed for routine diagnostic laboratory and its use is limited up to
research level.
Clinical application:
Rarely it can be applied for detection of rota virus or hepatitis A virus from stool specimen.
B. Molecular techniques for detection of viral genome
1. Polymerase chain reaction
- PCR& its modification like Real Time PCR: detects virus specific genetic sequence-template
Can be used for diagnosis of HIV virus, Hepatitis B virus, Hepatitis C virus, Rota virus,
Dengue virus, Chikunguniya, SARS-Cov-2 virus and many other viruses.
Viral quantity in the specimen can be detected by this method: e.g. HIV, HBV, HCV viral
load.
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 2. Other molecular techniques
- Gene probe
C. Immunological techniques
1. Detection of viral antigens
Various serological techniques used for detection of antigens:
ELISA, immunocromatography (ICT), Reverse passive haemagglutination (RPHA)
These technologies are useful for diagnosis of various viral infections:
- Hepatitis B surface antigen in Hepatitis B viral infection
- NS1 antigen in Dengue viral infection
- p24 antigen detection in HIV infection
2. Detection of viral specific antibodies
Detection of virus specific IgG or IgM antibodies in patient's serum:
- Enzyme Linked Immuno-sorbent Assay (ELISA)
- Immunochromatography
- Passive agglutination
- Immuno-fluroscence
These technologies are useful for diagnosis of various viral infections:
- Human Immuno deficiency Virus
- Arboviruses i.e. Dengue, Chikunguniya, Japanese encephalitis, Crimean -
congohaemorrhagic fever
- Hepatitis viruses: Hepatitis A, Hepatitis C, Hepatitis D & Hepatitis E, Antibodies against
hepatitis B surface antigens and core antigens
- Herpes viruses: i.e. Herpes, Cytomegalo virus
- Measles, Mumps, Rubella

D. Virus culture:
Specimen collection & transport:
Appropriate specimen which most likely contains etiological viral agent should be collected
and transported to the laboratory without delay.
In case of delay in transportation, the specimen must be transported in viral transport media
(A buffered solution containing antibiotics for bacterial growth inhibition and protein for
maintenance of viability of viruses.)
Viruses are obligate intracellular organisms. So, they need cells for their growth.
There are 3 different methods for virus culture
1. Animal inoculation:
Laboratory animals used for study are:
Mice (infant mice are preferred), Guinea pig, Rabbit etc.
The route of administrations:
Intra-cerebral, subcutaneous, intra-peritoneal, intra-nasal etc.
The growth of virus in inoculated animals may be evident by:
Death, disease or development of visible lesion
Uses:
- Diagnosis of viral disease (this method is not used nowadays).
- To study pathogenesis, immune response and epidemiology of agents
2. Egg inoculation:
The specimen can be inoculated at four different sites in embryonated egg depend upon type of
viruses
i. Chorioallantoic membrane (CAM): e.g. Variola or vaccinia viruses form pocks on
CAM
ii. Allantoic cavity: Used to produce large population of viruses particularly for vaccine
production.e.g. Influenza viruses, paramyxoviruses, rabies viruses
iii. Amniotic sac: Used for isolation of influenza viruses.
iv. Yolk sac: For isolation of some viruses including arboviruses; rickettsiae, chlamydiae.

100
3. Tissue culture / cell culture:
Tissue culture: Specimen may be inoculated in tissue or a small piece of organ maintained
in laboratory to give good yield of viruses. However this method is rarely used.
Cell culture:
Principle: The cells are dissociated from the tissue or organ in the laboratory with the help of
proteolytic enzymes like trypsin. These cells are washed and suspended in the medium
(Minimum Essential Medium) which supports viability and growth of cells. The suspended
cells are then dispensed in bottles, tubes or petri dishes. The cells adhere to glass surface of
these containers. On further incubation, they will grow, multiply and form a sheet of cells
on the surface of container. Specimen is inoculated over it; after proper incubation, viruses
present in specimen infect the cells and produce their growth effect (Cytopathic effect).
This is evidence for presence of virus in the inoculated specimen.
Minimum Essential Medium (MEM)
This medium is composed of essential amino acids, vitamins, salts, glucose and buffering
system. Phenol red is added as an indicator.
Development of cell line:
- The cells suspended in MEM.This is supplemented with 5% fetal calf serum.
- Antibiotic is added for prevention of bacterial growth.
- This suspension is dispensed in bottle, tube or petri dishes & incubated at 37°C with 5 %
carbon dioxide.
- Cells will adhere to the glass surface & divide to form a monolayer sheet over entire glass
surface which is covered with the medium.
Examples of cell culture:
A. Primary cells culture: These are formed by normal cells of the body; they have limited
growth potentials; they cannot be maintained by serial passage. e.g. Monkey kidney cell
culture
B. Diploid cell strain: These cells can be maintained for maximum up to 50 serial passages.
e.g. human fibroblast.
C. Continuous cell lines: These usually developed from cancer cells, which are capable of
continuous serial passage for indefinite times e.g. HeLa, HEp-2, Vero cell lines.
Detection of virus growth in cell culture:
Specimen inoculated in cell culture is incubated to allow the growth of virus present in it.
The viral growth in cells can be evident by,
1. Cytopathic effect: Virus can change the morphology of cells in which they grow. These
are known as cytopathic effects (CPE). e.g. Enterovirus will lead to degeneration of cells
& make them refractile and crenated. CPE in cells can be detected by inverted
microscope.
2. Metabolic inhibition: The metabolism of infected cells will be inhibited by intracellular
growth of virus. This can be detected by pH indicator present in cell culture medium
(MEM). The virus infected cells would not produce acid.
3. Hemadsorption: The washed erythrocytes will be adsorbed over virus infected cells. e.g.
in case of influenza and parainfluenza viruses.
4. Immuno-fluorescence: Viral antigens will be expressed over infected cells. The
fluorescence conjugated antiserum against these viral antigens will bind to infected cells
and stain them. These fluorescence stained cells can be demonstrated by fluorescent
microscope.

WORK:
1. Demonstrations:
● Serological tests – HIV, HBsAg.
● Microtitre plate.
2. Questions.
A. Describe important points for the storage of specimens for the viral diagnosis.
B. Describe usefulness of molecular methods in diagnosis of viral infections.
C. Classify DNA and RNA viruses.
101
102
 PRACTICAL NO-15
HOSPITAL WASTE MANAGEMENT

MEDICAL WASTE:
Medical waste can be classified as,
1. Non-infectious waste: (80%)
It constitutes a major portion of the entire hospital waste generated. It does not contain infection-
spreading microbes, as it is not in contact with any body fluids.
Non-infectious waste is broadly classified into:
a. General office waste: Comprising wrapping paper. Office paper, cartons, packaging
material including plastic sheets, newspaper and bouquets etc.
b. Kitchen waste: Includes leftover food, swill, pearls and dirty water generated from the
hospital kitchen. Kitchen waste is further divided into two categories:
i. Biodegradable kitchen waste: Which includes peels of fruit and vegetable skins, left
over food, tea dregs and other natural kitchen waste.
ii. Non-biodegradable waste: This waste includes wrappings, foil, plastic, paper and
other material, which is essentially man made.

2. Infectious waste: (20%)

a. Used sharps and items that could cause a cut or puncture.
b. Pathological waste including tissues, organs, blood and body fluids.
c. Syringes, IV tubing, blood bags and other items contaminated with blood and body fluids.
d. Items such as plaster casts etc. that may be defined as infectious waste only when
contaminated with blood or pus. These are non-infectious waste, when they have had no
contact with body fluids.
WASTE SEGREGATION:
Segregation is the key to any waste management scheme. Through segregation, different
ategories of wastes are sorted and placed in different containers or bags. Segregation should be
carried at the point of generation to keep general waste from becoming infectious.
Segregation comprises of separation of different streams based on the types of treatment and
disposal practice. The waste of each category specified will be disposed off into separate
receptacles. The best way of segregation is color-coded system, where each receptacle has its
own color.
 Techniques available for treatment of infectious waste :
1.Chemical Treatment :Chemical such as formaldehyde (6.8%), glutaraldehyde (2%),
hydrogen peroxide and chlorine in various forms (Most commonly as hypochloride solution
1%) are commonly used for the treatment of waste.
2. Autoclave :
Uses : Autoclaving is used for treatment of
1) Microbiological and pathological waste
2) Blood and blood products
3) Body fluids
4) Sharps and plastic waste

Advantages.
1) Economical
2) Easy to operate
3) Makes waste completely noninfectious
4) Disinfects waste without hazardous emissions

103
 3. Hydroclaving :
1) The hydroclave is an expansion of the autoclave technology
2) There is indirect heating without any contact with the waste by introducing steam into the
outer jacket while the waste is kept inside another chamber and turned mechanically with
the help of a series of large rotating rods which spin continuously rupturing the waste bags
and ensuring complete exposure to heat.
Use :Can be used for treatment of pathological and microbiological waste, sharps animal
waste, etc.

Advantages :
1) Hydrolyse the organic components of the waste
2) No pretreatment of waste is required
3) Complete fragmentation and dehydration is achieved
4) Reliable for the treatment of pathological and highly infectious waste
5) Reliable in volume and weight reduces the transportation and land filling costs
6) Environmentally friendly with no harmful emissions or discharges
7) Economical and simple for operation and maintenance
8) Sterilization and higher temperature and pressure but faster heat penetration than
autoclave.
9) Water equivalent to sterile so disposed of sewer
Disadvantage :No disadvantages are known.

4. Incineration :
Uses :This method is used for treatment of
1) Microbiological and pathological waste.
2) Soiled dressings
3) Anatomical waste
4) Cytotoxic waste
Advantages :
1) Ensures complete disposal of waste
2) Reduces volume of waste
Disadvantages :
1) Generates highly toxic gases such as dioxans& furans.
2) Not recommended for sharps and plastic waste.
3) High cost
4) Expensive to maintain and operate

COLOR CODING AND CONTAINERS FOR TREATMENT AND DISPOSAL

OFBIOMEDICAL WASTES

Type of Bag Treatment

Category Type of Waste or Container and Disposal
to be used options
(a) Human AnatomicalWaste
(b)Animal AnatomicalWaste
c) Soiled Waste
d) Expired or DiscardedMedicines Yellow
Incineration or Plasma
(e) Chemical Waste coloured non-
Yellow Pyrolysis or deep
(f) Chemical LiquidWaste chlorinated
burial
(g) Discarded linen, mattresses, beddings plastic bags
contaminated with blood or body fluid.
h) Microbiology, Biotechnology and other
clinical laboratory waste
104
 Type of Bag
Type of
Category or Container Treatment and Disposal options
Waste
to be used
Red Autoclaving or microwaving/
Contaminate colourednon hydroclaving followed by shredding
Red d Waste chlorinated or mutilation or Combination of
(Recyclable) sterilization and
Plastic bags or
containers shredding.
(1) Autoclaving or
(2) Dry Heat Sterilization followed
by shredding or mutilation or
Puncture
encapsulation in metal container
White Waste sharps proof, Leak
or cement concrete;
(Transluce including proof, tamper
(3) Combination of shredding cum
nt) Metals proof
autoclaving; and sent for final
containers
disposal to iron foundries or
sanitary landfill or designated
concrete waste sharp pit.
(1) Disinfection (by soaking the
washed glass waste after cleaning
(a) Glassware: Cardboard
with detergent and Sodium
boxes with
Blue b) Metallic Body Hypochlorite treatment) or
blue colored
Implants (2) Autoclaving / microwaving /
marking
hydroclaving and then sent for
recycling

BIOMEDICAL WASTE
Sr.No. Waste Example
1 Human anatomical waste Human tissues, organs, body parts
Experimental animal carcasses, body parts, organs,
2 Animal anatomical waste
tissues
Items contaminated with blood, body fluids like
3 Soiled waste dressings, plaster casts, cotton swabs, discarded blood
bags.
Antibiotics, cytotoxic drugs including all items
Expired or Discarded
4 contaminated with cytotoxic drugs along with glass or
Medicines
plastic ampoules, vials etc.
Chemicals used in production of biological and
5 Chemical waste
used or discarded disinfectants.
Silver X-ray film developing liquid, discarded
6 Chemical liquid waste Formalin, infected secretions, aspirate body fluids,
liquid from laboratories and floor washings.
Blood bags, Laboratory cultures, stocks or specimens
Microbiology,
of microorganisms, live or attenuated vaccines, human
7 Biotechnology and other
and animal cell cultures used in research, devices used
clinical laboratory waste
for cultures.

105
 Wastes generated from disposable items such as tubing,
Contaminated bottles, intravenous tubes and sets, catheters, urine
8 waste bags, syringes (without needles and fixed needle
(Recyclable) syringes) and vacutainers with their needles cut and
gloves.
Needles, syringes with fixed needles, needles from
Waste sharps including needle tip cutter or burner, scalpels, blades, or any other
9
Metals contaminated sharp objects that may cause puncture
and cuts.
Broken or discarded and contaminated glass including
10 Glassware
medicine vials.

WORK:
1. Demonstrations: Various equipments used for hospital waste disposal
● Various equipments used for hospital waste disposal.
● Color coded bag used of waste segregation and disposal.
● Chemical used for treatment of BMW.
● Bio hazard symbol
2. Questions.
A. What is BMW ?
B. How many types of medical waste?
C. Describe various color codes, type of waste to be collected in each color coded
bag/containers.
D. How will you dispose syringe & needle after use.
3. Exercise: Tick (√) mark appropriate container for corresponding BMW

Cardboard boxes
Yellow bag/ Red bag/ White, Puncture
BMW Items: with blue colored
container container proof container
marking
Swab

PPE kit plastic

Used needle

Used syringe

106
 PRACTICAL NO-16
IMMUNOLOGY AND SEROLOGY

Antigen:
Antigen is a substance which when introduced into the tissues of an animal by any route
provokes an immune response after a latent period which can be demonstrable by, (i) production
of antibody in blood and other body fluids which reacts with antigen, (ii) specific cell mediated
immunity, (iii) increased reactivity known as hypersensitivity.
Antibody:
Antibody is the substance produced in response to introduction of antigen with which it reacts
specifically in some observable way.
Antigen-Antibody reactions:
Antigen and antibody by definition combine with each other specifically and in an observable
manner. These reactions can be used for the detection and quantitation of either antigens or
antibodies. Antigen-antibody reactions in vitro are known as serological reactions.
Sensitivity: Sensitivity is the ability of a test to detect the minimum amount of antigen or
antibody present in the serum (chance of false positivity)
Specificity: Sensitivity is the ability of a test to detect the true antigen or antibody present in the
serum (chance of false negativity)

Role of serological tests in infectious diseases:

1. For diagnosis of infectious diseases:
Certain infectious diseases are very difficult to diagnose by microscopic examination or culture
methods. For example,
a.Viral infections: These agents are too small to be demonstrated under routine laboratory
microscopes and also very difficult to culture. Hence, a diagnostic void is created which is
filled up by serology.
b. Occult infections: The infecting agent is present at such a site in the body which cannot be
accessed for sample collection; or the local site of infection remains unidentified. In such cases,
indirect evidences in the form of serological reactions come in handy.
c. All the infections where the infecting agent cannot be recovered from the clinical samples; and
we need laboratory evidence of infection, especially in cases with highly suspicious clinical
background, wherein to stamp upon a disease, a diagnostic laboratory report is needed.
For diagnosis of such diseases, we may take indirect evidence of infections,
- Microbial infection will induce antibody production. Such antibodies are present in patient's
serum. To detect these antibodies, patient's serum is allowed to react with laboratory reagent
containing commercially (in vitro) prepared antigens of the microorganism belonging to the
same species.
- Similarly, microbial antigen present in patient's serum may be detected with the use of its
homologous antibodies present in the laboratory reagent, prepared commercially.
2. Detection of a particular antibody titre in patient's serum
Titer: Antibody titer of serum is the highest dilution of the serum which shows observable
reaction when reacted in vitro with its specific antigen.

SEROLOGICAL REACTIONS:
1. Precipitation:
When a soluble antigen combines with its specific antibody in the presence of electrolytes, at
optimum temperature and pH, it forms an insoluble precipitate, this reaction is called
precipitation.When precipitates remain suspended as floccules, the reaction is known as
flocculation.

107
 A. Slide precipitation / flocculation test
VDRL (Venereal Disease Research Laboratory) test used for diagnosis of syphilis.
The antigen formerly used in the VDRL test was a colloidal suspension of tissue
cardiolipin, which in the presence of cholesterol and lecithin, reacted with cardiolipin
autoantibodies to form floccular type agglutination. Today chemically synthesized
cardiolipin is used as the sole antigen.
The VDRL test uses serum that has been heat treated for 30 minutes at 560 C. The serum
must be allowed to cool to room temperature before being tested. The test is usually
performed as a slide technique. A measured drop of diluted antigen suspension is added to
a measured volume of serum on a slide. After rotating the slide for 4 minutes, the
preparation is examined microscopically for clumping of antigen particles.
Positive sera are reported as:
 Reactive‖ if the clumps are of medium or large size, or
 Weakly reactive‖ if clumps are small.
 All reactive or weakly reactive sera require diluting to estimate the antibody titer.
RPR Test:
The antigen used in the RPR card test is similar to that used in the VDRL test. The antigen
particles, however, are either carbon containing or dyed to enable the reaction to be read
macroscopically on a card. No heating of the patient‘s sera is required and plasma or serum
can be used for the RPR card test. A reactive serum shows definite clumping of the antigen
particles or slight roughness. A reactive serum requires serial diluting to estimate the
antibody titer.
B. Tube precipitation / flocculation test
For quantification and standardization of toxins and toxoids.
C. Gel Precipitation test (Immunodiffusion)s
e.g. Elek’s gel precipitation test. This test is used to detect the toxigenicity of diphtheria
bacilli.
D. Immuno-elecrophoresis
E. Counter immuno electrophoresis
2. Agglutination test:
When a particulate (insoluble) antigen reacts with its homologous antibody in a suitable
environmental condition in terms of temperature, pH & electrolytes, it will form large, clumped
aggregates visible to the naked eye. This reaction is known as agglutination.
A. Slide agglutination
A drop of suspension containing insoluble antigen (kit reagent) is allowed to react with the
patient's serum on a slide. If the serum contains antibodies against that specific antigen, it
will form clumps (agglutination). These clumps may be visible to the naked eye or can be
demonstrated by a magnifying lens.
This test may also be used to detect antigen; wherein a suspension of antigen is allowed to
react with antisera containing known antibodies.
Examples of commonly used slide agglutination tests:
a. Blood grouping and cross matching
b. For antigenic typing of bacteria and identification of unknown bacterial cultures:e.g.
i. Lancefield typing of Streptococcus pyogenes
ii.The isolated bacteria can be identified by characterization of their antigenic structures
with the help of specific antisera. For example if an isolated colony is suspected to be
that of Salmonella typhi or S. paratyphi, we need to use three different antisera,
containing antibodies against "H" antigen of S.typhi, S.paratyphi A and S.paratyphi B,
respectively.

108
 Test procedure: A drop of saline is placed on three different slides. The isolated bacterial
colony is picked up with a sterile wire loop and emulsified uniformly in the saline. This
procedure is done on each slide. One drop each of the three antisera is mixed with this
bacterial suspension on respective individual slides and mixed by rotation. The isolated
bacterium is identified by formation of agglutination with its homologous antisera.
B. Tube agglutination:
A serial dilution of the patient’s serum is mixed with the antigen in the tubes. Agglutination
will appear if the concentration of antibody is optimum in the mixture. The highest dilution
which shows agglutination, will be the titer of the antibody in the patient’s serum.
Examples of tube agglutination tests:
i. Widal test - For diagnosis of typhoid fever.
Widal test this test is done to diagnose typhoid fever caused by. Salmonella typhi, S.
paratyphi A and S.paratyphi B. It is an agglutination reaction by which antibody is
estimated for salmonella antigen.
Salmonella has two major antigens against which antibodies appear in the blood.
1.Somatic antigen ‘ O’ Ag.
2.Flagellar antigen ‘H’ Ag.
‘O’ Ag is mosaic and is shared by Salmonella typhi, S.paratyphi A, S. paratyphi B, while H
Ag differs in these three species.
So for perfomingwidal reaction ‘O’ Ag, ‘H’ Ag of Salmonella typhi. S. paratyphi A and
S.paratyphi B are used.
Time for perfomingwidaltest :
As the antibodies appear in optimum amount in second weak of Salmonella Infection so that
test should be perfomed in second week of illness.
Specimen required :Serum
Procedure:
- Serial dilution of patient's serum (1:40 to 1: 320) is prepared in four two different sets of
tubes so asfinal dilution of serum in each set tubes after adding respective Salmonella Ag
should be 1: 40 in
1 tube, 1:80 in 2nd tube, 1:160 in 3th tube and 1:320 in 4th tube.
st

- Incubated at 37⁰ C overnight in waterbath.

- Conical Dreyer's tube is used for H antigen. Round bottomed Felix tube is used for O
antigen
Result :
1. Agglutination in ‘H’ antigen tube are cottoy wooly, loose and fluffy.
2. Agglutination in ‘O’ antigen tube are chalky, compact and powdery.
3. The Widal test is reported by giving the titer for both O and H antibodies. The antibody
titer is takenas the highest dilution of serum in which agglutination occurs.
Interpretation of the Widal test:
1. The agglutination titer will depend on the stage of the disease. Agglutinins usually
appear by the end of the first week. The titer increases steadily till the third or the fourth
week, after which it declines gradually.
2. Demonstration of a rise in titer of antibodies, by testing two or more serum sample is
more meaningful than a single test.
3. The result of a single test should be interpreted with caution. It is difficult to lay down
levels of significance though it is generally stated that titers of 1/100 or more for O
agglutinins and 1/200 or more for H agglutinins are significant.
4. Agglutinins may be present on account of prior disease, unapparent infection or
immunization. H agglutinins persist longer than O agglutinins. Serum from an
individual immunized with TAB vaccine will generally have antibodies to S. typhi, S.

109
 paratyphi A and B, while in case of infection antibodies will be seen only against the
infecting species.
5. Persons who have had prior infection of immunization may develop an anamnestic
response during an unrelated fever. The anamnestic response shows only a transient
rise, while in enteric fever the rise is sustained.
6. Treatment with chloramphenicol causes poor agglutinin response.
ii. Weil-Felix reaction -For diagnosis of various Rickettsial diseases (typhus fever)
- This test is based on the sharing of common antigens by Rickettsiae and Proteus bacilli
& hence, sera from the patients of rickettsial diseases will react with the surface
antigens of the Proteus spp. Test kits (commercially available) have three types of
surface antigens of Proteus bacilli: OX 2, OX19 & OX K.
- Patient's serum is allowed to react with each of these three antigens separately, in three
different test tubes.
- Sera from the patients suffering from typhus fever and spotted fever will agglutinate
OX 2 & OX 19antigens; while sera from the patients having scrub typhus will agglutinate
OX K antigens.
iii. Paul-Bunnell test: - For diagnosis of infectious mononucleosis
Patient's serum is allowed to react with the sheep RBCs in a test tube. The infectious
mononucleosis patient's serum contains cold agglutinins of IgM type, which agglutinate
sheep RBCs. Hence, if the patient suffers from infectious mononucleosis, agglutination
will be visible.
iv. For diagnosis of Brucellosis:
Tube agglutination method is used to detect anti Brucella antibodies and its titer in
patient's serum.
(Widal test, Weil-Felix reaction and Brucella reaction are collectively known as triple
Agglutination test.)

C. Passive agglutination test:

When a soluble antigen is allowed to react with its specific antibody precipitation occurs. If
this soluble antigen is coated over a carrier particle, and then it is allowed to react with its
specific antibody, agglutination will occur, because the carrier molecule will render the
soluble antigen insoluble. Thus, by coating soluble antigens over carrier particles, the
precipitation reaction can be converted into agglutination reaction. This is known as passive
or indirect agglutination test.
Benefits of passive agglutination over precipitation:
- More convenient to perform.
- More sensitive for detection of the antibodies.
- Better visibility of the results of the antigen-antibody reactions.
Reversed passive agglutination:
Instead of antigen, if the soluble antibody (reversed) is coated (adsorbed) over a carrier
particle, and the antigen-antibody reaction allowed to occur, it is known as reversed
passive agglutination. It is used for detection of antigens.
Commonly used carrier particles
Latex particles, carbon particles, RBCs, etc.
Examples of passive agglutination
i. Latex agglutination:
a). Anti-streptolysin O (ASO): Patient's serum is mixed with a reagent containing
Streptolysin O antigen coated over latex particles. Occurrence of agglutinations suggests
that the patient's serum contains ASO antibodies; which in turn indicates that the patient
has been infected with Group B beta haemolyticStreptococci. These antibodies cross
react with human heart valve. So in suspected case of rheumatic heart disease, ASO is
used as a diagnostic marker.

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 b. C- Reactive Protein (CRP):It is an acute phase reactant. Its level rises in various
inflammatory conditions.

c. Rhematoid (RA) factor:It is an anti gamma globulin antibody. Its level rises in
autoimmune disorders i.e. Rhematoid arthritis.

ii. Haem-agglutination
a. Treponema PallidiumHaem-agglutination (TPHA) test: The test reagent contains
treponemal antigens which are coated over RBCs. This is used for serological
diagnosis of syphilis.
b. Rose-Waaler test: Used for detection of RA factor.
iii. Co agglutination:
- Cell walls of certain Staphylococcus aureus strains have an affinity to the Fc portion of
antibodies.These strains are used as carrier particles for coating antibodies on them.
- Co-agglutination techniques are used for detection of various capsular antigens of
capsulated bacteria from CSF, in suspected cases of pyogenic meningitis. e.g.
Streptococcus pneumoniae, Neisseriameningitidis, Haemophilus influenzae etc.

3. Complement Fixation Test (CFT)

This test consists of two systems/ steps /principles
1. Test system
System: Reagent 1 = antigen + free complement.
Step : Patient's serum is allowed to react with the test reagent. (Test system)
Principle: When an antigen-antibody reaction occurs, the so formed complex fixes any
complement in the system.
Interpretation:
If antibodies are present in the patient's serum, they will bind to the antigen and the antigen
antibody complex so formed will further consume(fix) the free complement in the system.
This mixture will now contain no more free complement.
If patient's serum does not contain antibodies, there will not be any Ag-Ab reaction and
the complement will remain free in the mixture(system).
2. Indicator system
System: Reagent 2 = Sheep RBCs coated with anti sheep RBC antibodies. (Indicator
system)
Step: Mixture of step 1 + Reagent 2.
Principle: Free complement in a system lyses RBCs.

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 Interpretation:
Positive(No haemolysis): If the patient's serum contains antibodies, it will consume
complement during first step & complement will not be available during second step. In
absence of complement, there will not be lysis of the indicator system.
Negative (hemolysis of sheep RBCs): If the patient's serum does not contain antibodies, the
complement would remain free after first step. During second step, the free complement will
lyse the indicator system.

4. Enzyme - Linked Immuno Sorbent Assay (ELISA)

ELISA test is used to detect either antigen or antibody in serum or any body fluid.
Principle:
A commercially available ELISA kit contains
i. A solid phase to which antigens or antibodies are already bound. The most commonly
used solid phase device is microtiter plate having 96 wells.
ii. A reagent enzyme conjugated over antibody.
Test procedure: Enzyme conjugated reagent is mixed with the specimen in the well of
microtiter plate.
The antigen-antibody reaction is detected by addition of chromogenic substrate, with which the
conjugated enzyme will react. The intensity of colour produced will be in direct proportion
to the concentration of antigen or antibody in the specimen. In the microtiter plate, the
intensity of colour change can also be detected by automation.

Types of ELISA:
1. Indirect ELISA:
- The microtiter well is already coated with the specific antigen to which antibody in the
patient serum is to be detected.
- The specimen (patient’s serum) is added in the well.
- The antibody, if present in the specimen will react with the coated antigen in the well.
- Any free antibodies are washed off with wash buffer available in the ELISA kit.
- The presence of antibodies bound to solid phase antigen is detected by addition of anti-
gammaglobulin antibodies conjugated with an enzyme. These will get attached to bound
antibodies.
- Any free labeled antibodies are again washed away.
- A chromogenic substrate is added to the well.
- The colour production is measured by specialized spectrophotometer (ELISA reader).
- Formula: Ag + Ab + AAb.E + S = colour

Interpretation:
If the specimen contains antibody: The colour of substrate will change. Its intensity will be
in direct proportion to the concentration of antibody in the specimen.
If the specimen does not contain antibody: No colour change occurs in the microtiter plate
at the end of the test.
Uses: For detection of antibodies in cases of infection with
- Human Immunodeficiency Virus, Hepatitis A virus, Hepatitis C virus, Hepatitis E virus,
TORCH etc.

2. Competitive ELISA:
- The microtiter plate is coated with the specific antigen.
-The ELISA kit contains conjugated antibodies which are idiotypically similar to the
antibodies to be detected.
- This reagent is mixed with patient's serum and poured in the well. The sample antibodies
and conjugated antibodies (present in reagent), both will compete for binding to the solid
phase antigen. More the antibodies in the specimen, the conjugated antibodies will find less
opportunities for binding to the solid phase antigens.
- After proper incubation, the remaining antibodies are washed away from the well.
- The concentration of conjugated antibodies will be detected by addition of the substrate.
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 -Increased intensity of the colour produced suggests that the conjugated antibodies got more
chance to bind with the coated antigens due to less concentration of antibodies in the
specimen. So, intensity of colour change will be inversely proportional to the antibody
concentration in the specimen.
Use: For detection of antibodies e.g Anti Hepatitis D antibodies

3. Sandwich ELISA:
- The microtiter plate is coated with specific antibodies.
- The test sample is added to the well. If the sample contains that specific antigen, it will react
with the coated antibodies and get fixed.
- The remaining unbound contents of the system are washed off.
- Another antibody, homologous to the antigen, which is conjugated with an enzyme is added
in the reaction well.
- These conjugated antibodies will get attached to the fixed antigen in the well.
- In the next step, substrate is added.
- The intensity of colour production will be in direct proportion to the concentration of antigen
in the sample.
- Formula: Ab + Ag + Ab.E + S = colour
Use: For detection of antigens e.g.
- Hepatitis B surface antigen detection
- Malarial antigen (HRP2, pLDH) detection
- HIV viral p24 antigen detection
4. Membrane based ELISA:
In these methods, a specific antigen is immobilized at a specific place over a nitrocellulose
membrane in the form of a dot or line. This membrane is fixed over an absorbent membrane,
which helps to soak all the free unbound material through the membrane during the test
procedure.
- Patient's serum is pipetted over the membrane in the sample well provided in the test device.
- The specific antibody to be detected, if present in serum, will bind with its specific antigen
coated over the membrane.
- The rest of the unbound material will be soaked off by the absorbent material.
- The antibody on the membrane is detected by addition of anti Ig antibody conjugated with
colloidal gold.
- This conjugate binds to the antigen antibody complex and forms red dot or band, which is
suggestive of a positive reaction.
- Formula: Ag + Ab + AAb.CG = red dot
Advantage of the test:
Rapid, easy to interpret and it does not require sophisticated equipments.
Use: For detection of various antibodies in patient serum e.g.
- Anti HIV antibody
- Anti HCV antibody

5. Immuno-chromatography:
This is nowadays most popularly used immunological method for detection of various antigens
and antibodies from the patient serum.
Principle:
For detection of antibody:
The antigen (against which antibody is to be detected) is immobilized in a form of a line (test
line) over a nitrocellulose test strip.
The lower half of the strip contains an absorbent pad in which colloidal gold conjugated to
similar Ag is loosely impregnated.
The sample is added to the absorbent pad (or absorbent pad is immersed in sample).
Sample moves forwards by capillary action towards the test line.
If the sample contains the specific Ab, it will get attached to the colloidal conjugated Ag and
flow forwards within the test strip. This Ab will then crosslink with the antigen present over the

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 test line and give a red band there.
Test strip also contains an internal control - anti gamma globulin antibody which is coated
(control line) above the test line. Formation of control band suggests validity of the test.
Formula: Ag + Ab + Ag.CG = colour
Uses: Detection of Anti HAV antibodies, Detection of Anti HEV antibodies.
For detection of antigen:
Monoclonal Ab specific against the Ag to be detected is coated over the test line.
In the absorbent pad, colloidal gold conjugated with a similar monoclonal antibody is
loosely impregnated.
The Ag present in the sample will bind to the conjugated Ab and move forward and then
crosslink with the Ab coated over the test line and form the band.
Formula: Ab + Ag + Ab.CG = colour
Use: Detection of HBsAg, Malarial antigens (pLDH& HRP2)
Both antigen and antibody detection system can be incorporated in a single device:
e.g. For diagnosis of Dengue infection: Anti Dengue IgM antibody, Anti Dengue IgG
antibody and NS1 antigen of Dengue virus- all these three parameters can be detected in a
single device.

6. Immuno fluorescence test:

Principle:
Fluorescent dyes when exposed to UV light, will absorb it but emit the visible light (wavelength of
absorbed and emitted light is different). This emitted light can be visualized by fluorescent
microscope. Rhodamine and fluorescein isothiocyanate are the most commonly used fluorescent
dyes. These dyes can be conjugated with antigen or antibody to detect their homologous
counterparts.
Use:
- For diagnosis of Syphilis
- For detection of antibodies against EB virus, influenza and para-influenza viruses
- For detection of rabies virus from corneal smear / smear prepared from post mortem brain
specimen.
7. Neutralization test:
Used for detection of virus (viral neutralization test); and bacterial exotoxins (toxin
neutralization test).
8. Radio-immunoassay (RIA):
A radioactive agent (radioisotope) is used to detect antigen antibody reaction. Radioactivity is
measured with automated reader. This test is used for qualitative as well as quantitative analyses
for the antigen in the serum or body fluids.
9. Western blotting:
It is used to identify the microbial antigen in the patient‘s serum. Proteins (antigen) in the patient‘s
serum are separated by a process called electrophoresis. The proteins (antigens) are then
transferred to a filter by blotting. Next, antibodies tagged with a dye are washed over the filter. If
the specific antigen is present in the serum, the antibodies will combine with it and will be visible
as a colored band on the filter. Western blotting is used to diagnose HIV infection.

114
 Western blot: HIV Antibody

WORK:
1. Demonstrations:
● Serological tests. Precipitation & Agglutination
● Immunochromatography Tests
● Immunoassays: ELISA.
2. Label the figure of Antibody Capture ELISA & Antigen Capture ELISA
3. Interpret the tube widal result given in figure.
4. Questions.
B. Define Antibody and Immunogloobulin?
C. Enumerate types of Antigen – Antibody reaction.
D. Write three differences between V.D.R..L. and R.P.R.
E. What is the diagnostic titers of ASO.?
F. Enumerate the different types of ELISA.

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 LABEL COMPONENT OF ELISA

Antibody Capture ELISA Antigen Capture ELISA

Interpret the tube widal result given in figure

116
 Gastrointestinal tract Infections
PRACTICAL NO-17
Laboratory Diagnosis Of Diarrhea And Dysentery

Following are the different terminologies used to describe the diarrheal diseases.
1.Gastroenteritis: It is defined as the inflammation of mucosa of the gastrointestinal tract
(stomach - "gastro" and intestine - "entero") often leading to diarrhea, vomiting and abdominal
discomfort.
2. Diarrhea: It is defined as a condition in which loose or liquid stool pass for three or more times
a day. It is the most common presentation of gastroenteritis. Infective diarrhea is often considered
synonymous to gastroenteritis.
3. Dysentery: It is a condition associated with frequent passage of stool containing mucus, blood
or pus (exudates). It is characteristically associated with abdominal cramps, tenesmus and fever.
4. Food poisoning: It is a condition of acute gastroenteritis originating due to consumption of
food containing large number of bacteria or their products like toxins; which after entry in
gastrointestinal tract, without needing further multiplication or toxin production immediately and
with very short incubation period leads to pathogenesis and clinical manifestations.
5. Colitis: Inflammatory condition of large bowel (colon) is called colitis. Many organisms may
cause only colitis or enterocolitis leading to watery diarrhea or dysentery.
Patients of infectious diarrhea have two kinds of presentations:
1. Watery diarrhea
2. Dysentery
Causative agents of watery diarrhea
A. Bacteria:
Vibrio cholerae (rice watery diarrhea)
Entero-Pathogenic Escherichia coli (paediatric diarrhea)
Entero-Toxigenic Escherichia coli (traveler's diarrhea/Montezuma’s revenge/ Delhi belly)
Campylobacter spp.
Salmonella sp. (S.typhimurium, S.enteritidisetc)
Clostridium difficile (antibiotic associated colitis)
B. Viruses:
Rotavirus (most common cause of paediatric diarrhea)
Norwalk virus
Calici virus
Astrovirus
Adenovirus
C. Parasites
Giardia intestinalis (usually leads to fatty diarrhea)
Cryptosporidium parvum (Paediatric diarrhea or diarrhea in immuno compromised patients)
Isospora belli (diarrhea in immuno compromised patients)
Helminths: Hymenolepsis nana, Trichuris trichiura, Strongyloidesstercoralis, Ascaris
lumbricoides, Ancyclostoma duodenale etc.
D. Fungus:Candida albicans
E. Bacteria associated with food poisoning
Bacterial food poisoning is of two types
1. Toxic type: Bacterial toxins lead to gastroenteritis
Staphylococcus aureus (Common with milk and milk products)
Bacillus cereus (Common with fried rice)
Clostridium perfringens
Clostridium botulinum (Common with canned food)
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 2. Infective type: Direct bacteria invade mucosa and cause gastroenteritis
Vibrio parahaemolyticus
Campylobacter jejuni
Yersinia enterocolitica
Salmonella typhimurium

Causative agents of dysentery:

According to the aetiological agents, dysentery is divided in two types
A. Bacillary dysentery: caused by bacteria
Shigella spp.
Entero-Invasive Escherichia coli
Entero-haemorrhagicEscherichia coli
Vibrio parahaemolyticus,
Campylobacter jejuni
B. Amoebic dysentery: Caused by Entamoeba histolytica

Laboratory diagnosis
A. Stool analysis:
Sample collection:
Stool is the most informative sample to evaluate aetio-pathogenesis of diarrheal diseases. In
paediatric patients or critically debilitated patients with severe watery diarrhea, if it is not possible
to collect sample, rectal swab may be taken. Intestinal aspiration with help of endoscope may be
taken to diagnose certain parasitic infection (e.g. hook worm present in small intestine,
E.histolytica present in the ulcers of large intestine).
Sample transport:
Sample as it is should be immediately transported to laboratory.
If there is delay in transport, according to suspected bacterial pathogens. It should be transported
infollowing transport media,
1. In suspected cases of Cholera:
Venkatraman - Ramakrishnan (VR) medium
Cary-Blair medium
Alkaline peptone water (if sample is transported within a day)
2. In suspected cases of infection by enterobacteriacae members: Glycerol buffered saline
3. In suspected viral infections: The sample should be refrigerated if not processed within
2 hours. Rectal swabs may be stored & transported in Stuart’s medium.
Sample processing:
1. Gross examination:Direct appearance may help to evaluate infective aetiology
- Rice watery stool is most commonly seen in Vibrio cholerae
- Presence of gross blood or pus is indicative of dysentery
- Foul smell suggestive of fatty diarrhea is a common presentation of giardiasis
- Helminths like round worm, hook worm, thread worm, tape worm, whip worm may be
grossly visible in the stool.
2. Microscopic examination:
Stool should be examined microscopically for following findings
1. Pus cells: Presence of pus cells is suggestive of invasive infection with marked inflammation
2. RBCs: Presence of RBCs is suggestive of dysentery
3.Parasites: On microscopic examination, following parasites may be seen in stool,
Cysts or/and trophozoites of Entamoeba histolytica, Giardia intestinalis.
Oocysts of Cryptosporidium parvum, Isospora belli.
Ova of H. nana, hook worm, round worm, whip worm, tape worm and thread worm.
Larvae of S.stercoralis.

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 4. Fungus: Yeast cells - Candida may be demonstrated. Its significance should be evaluated
with otherclinical background; generally if pseudohyphae are seen along with budding yeast
cells, it is consideredsignificant.
5. Bacteria: A large number of bacteria are normally present in stool. It is not possible to
differentiate normal bacteria from pathogenic bacteria by microscopic examination.
However, presence of Vibriocholerae may be indicated by demonstration of its specific
darting type of motility with hanging droppreparation.
6. Other structures: i.e. Charcot-Leyden (CL) crystals are present in amoebic dysentery.

3. Culture examination:
Stool specimen normally contains large number of commensals. These bacteria may affect
selective isolation of pathogenic bacteria. Escherichia coli, a common cause of diarrhea is also
a common commensal of normal stool. So, for culture examination of stool, we need different
sets of culture media containing enrichment and selective media along with simple and
differential media according to suspected aetiological agents.
Culture media used:
1. Simple media: Nutrient agar
2. Differential media: Mac conkey agar
3. Enrichment media: Selenite F broth or tetrathionate broth: For Salmonella and Shigella
Alkaline peptone water: For Vibrio cholerae
4. Selective media:TCBS: For Vibrio cholerae
Wilson-Blair medium: For Salmonella
DCA or XLD agar: For Shigella and Salmonella
Isolated bacteria are identified and processed for antibiotic sensitivity testing. Typing of
bacteria may be needed for epidemiological purpose.
4. Antigen Detection:
E. coli O157:H7- Enzyme linked Immuno Assays (EIAs).
Rotavirus- Enzyme linked Immuno Assays & Latex Agglutination Tests (LATs).
C. difficile- Toxin A and/ toxin B detection.

B. Analysis of other specimens:

Rectal swab:If it is not possible to collect stool sample, rectal swab may be taken and
processed for culture examination.
Intestinal biopsy: May be taken by endoscopy which may show pathogenic organism on HPE
e.g. E.histolytica present in ulcer of large intestine, H.pylori present in gastric biopsy.
Vomited material: Particularly useful in case of food poisoning for detection of bacterial
toxins
Food sample: Should be taken to find out agent responsible for food poisoning.

Escherichia coli:
Most numerous facultative anaerobes in the human gut comprising approximately 109/g of feces.
Characteristics: Gram-negative rod; motile; with or without capsule; non-fastidious, facultative
anaerobes; bile tolerant.
Transmission: Normal habitat is gut of man and animals; may colonize lower end of urethra and
vagina. Spread is by contact and ingestion (fecal-oral route); may be food-associated; may be
endogenous. Possesses O (somatic), H (flagellar), K (capsular) and F (fimbrial) antigens, which
can be used to characterize strains by serotyping.

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 Diseases:
1. Urinary tract infection
2. Diarrhea
3. Neonatal meningitis
4. Septicemia
5. Wound infection
Laboratory Diagnosis:
Sample Collection:
1. Feces for culture and toxin detection in case of diarrhea
2. Urine for microscopy and culture in case of UTI
3. Pus for culture in case of Wound infection
4. C.S.F. for microscopy and culture in case of neonatal meningitis
5. Blood for culture in septicemia
Methods for Identification of Organism:
1. Microscopy: E. coli is Gram negative, motile rod.
2. Culture: Nutrient agar, MacConkey agar
MacConkey agar: large, spreading.
3. Biochemical Reactions:
IMViC: I & MR: positive, Vi & C: Negative
TSI: A/A with gas, H2S: Negative
Peptone water sugar: Glucose, Sucrose, Lactose & Mannitol fermented with Acid & gas
Urease & PPA: Negative
Antibiotic Sensitivity Test:

WORK:
1. Demonstrations:
● Gram stain smear – Gram-negative bacilli.
● Cultural characters – MacConkey‘s agar.
● Biochemical reactions: IMViCtest, TSI agar, Urease & PPA, Sugar set.
2. Draw figures of E. coli colony on MacConkey’s agar
3. Questions.
A. Describe salient characteristics of enterobacteriaceae.
B. Enumerate the various diarrheagenic types of E. coli.

E. coli (MacConkey’s agar)

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GENUS SHIGELLA:
Contains four species of importance to man as causes of bacillary dysentery:
Shigella dysentery, Shigella boydii, Shigella flexneri and Shigella sonnei.
Characteristics: Gram-negative rods. Non-motile. Non-capsulated.
Transmission: Human pathogens spread by fecal-oral route, especially in crowded conditions.
Small infective dose.
Diseases: Bacillary dysentery, very rarely invasive
Laboratory Identification: Non-fastidious, bile-tolerant, Lactose non-fermenters.
LABORATORY DIAGNOSIS:
Sample Collection: Feces for culture
Transport medium:Buffered glycerol saline
Methods for Identification of Organism:
1. Microscopy: Shigella is Gram negative, non-motile & non capsulated rod.
2. Culture: Nutrient agar, MacConkey agar, D.C.A (Deoxycholate Citrate Agar) & S.S.
(salmonella- shigella) agar
● MacConkey agar: Colonies are colorless except shigella sonnei which is late lactose
fermenter.
● D.C.A agar and S.S. agar (Selective Medium): Colonies are colorless.
3. Biochemical Reactions:
IMViC: OnlyMR: positive
TSI: A/Alk without gas, H2S: Negative
Peptone water sugar: Glucose & Mannitol fermented with Acid only
Sh. Sonnei late Lactose fermenter
Sh. Dysentery does not ferment Mannitol
Urease & PPA: Negative

Identification is confirmed by polyvalent and monovalent sera.

Antibiotic sensitivity test

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 WORK:
1. Demonstrations:
● Gram stain smear – Gram-negative bacilli.
● Cultural characters – MacConkey‘s agar.
● Biochemical reactions: IMViC test, TSI agar, Urease & PPA, Sugar set.
2. Draw figures of Shigella colony on MacConkey’s agar
3. Questions.
A. Enumerate the different species of Shigella.
B. Important differences between Amoebic & Bacillary dysentery.

Shigella (MacConkey’s agar)

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GENUS VIBRIO:
Most important species, V. cholerae, causes cholera. V. parahaemolyticus also causes diarrheal
disease.
Characteristics: Curved Gram-negative rods, highly motile by means of single polar flagellum.
Many species salt tolerant; some salt requiring.
Transmission: V. cholerae is a human pathogen; no animal reservoir. El Tor biotype survives
better in the environment than classical V. cholerae. Infection is acquired from contaminated
water or food. V. parahaemolyticus infection acquired from consumption of contaminated fish
and seafood.
Diseases: Cholera caused by V. cholerae. V. parahaemolyticus causes diarrheal disease.
Other species may cause wound infections.
LABORATORY DIAGNOSIS:
Sample Collection: Stool or Rectal swab if stool sample is difficult to obtain e.g. children
Transportation of sample: Alkaline peptone water, Cary-Blair medium
Methods for Identification of Organism:
1. Microscopy:
● Gram`Stain:Vibrio are Gram negative, curved, motile bacilli.
● Hanging Drop Method: Darting type of motility seen.
2. Culture: Nutrient agar, MacConkey agar, Thiosulphate Citrate Bile salt Sucrose (TCBS)
● Nutrient agar: 1 to 2 mm translucent, convex colonies.
● MacConkey agar: Colonies are colorless.
● T.C.B.S. medium (Selective Medium): Colonies are yellow due to fermentation of
sucrose.
● Blood agar:hemodigetion
3. Biochemical Reactions: Important which help in diagnosis of Cholera
IMViC: I &C:positive, MR: Negative, Vi: Variable
TSI: A/A without gas, H2S: Negative
Peptone water sugar: Glucose, Sucrose & Mannitol fermented with Acid only
Urease & PPA: Negative
Oxidase test: Positive
Cholera red reaction: Positive
String test: Positive
V. cholerae is classified into two biotypes Classical and El Tor, based on certain
biochemical properties.
4. Typing by Antisera:Confirmation of V. cholerae is done by agglutination reaction with O1
andO139 antiserum.V. cholerae is subclassified in to Inaba, Ogawa and Hikojima serotypes
usingInaba and Ogawa antisera.

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 WORK:
1. Demonstrations:
● Gram stain smear – Gram-negative bacilli.
● Motility test: V. cholerae.
● Cultural characters: TCBS agar.
● Biochemical reactions: IMViC test, TSI agar, Urease & PPA, Sugar set, Oxidase test
2. Draw figures of V. cholerae colony on TCBS agar
3. Questions.
A. What are the important differences between classical and El Tor vibrio?
B. Which species of vibrio causes food poisoning?

V. cholerae (TCBS agar)

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 Gastrointestinal tract Infections
PRACTICAL NO-18
LABORATORY DIAGNOSIS OF ENTERIC FEVER

SALMONELLA
Salmonella and Shigella are not normal inhabitants of the human gut unlike other members of
the Enterobacteriaceae. Both genera are responsible for diarrheal diseases, which may be severe;
Salmonella typhi is also invasive and gives rise to systemic infection.
Salmonella Taxonomy:
Kauffman-White classification recognizes each serologically distinct salmonella as a species.
These are then arranged in groups. Further serologic distinctions recognized as subtypes.
Kauffman-White classification:
Somatic Flagella (H) antigen
Group Name
antigen Phase-I Phase-II
A S. paratyphi A 1,2,12 a -
B S. paratyphi B 1,3,5,12 b 1,2
C1 S. typhimurium 1,4,5,12 i 1,2
S. paratyphi C 6, 7, Vi c 1,5
D S. typhi 9, 12, Vi d -
S. enteritidis 1,9,12 g, m -

Characteristics: Gram-negative, motile rods. Capable of aerobic and anaerobic respiration.

Transmission: Widespread in animals; encountered in food chain. Acquired by ingestion of
contaminated food or person to person via fecal-oral route. S. typhi human pathogen only.
Spread via fecal-oral route, usually via contaminated water or food. Carriers are important source
of organisms
Diseases:
1. S. Typhi: Typhoid fever (enteric fever), Osteomyelitis, Abscesses & meningitis
2. S. paratyphi A: Paratyphoid fever (enteric fever)
3. S. paratyphi B: Paratyphoid fever (enteric fever)
4. S. paratyphi C: Septicaemia
5. S. typhimurium: Food poisoning

LABORATORY DIAGNOSIS:
Sample collection:
According to the site of infection and clinical features: Blood, Feces & Urine.
In special situation: Bone marrow & Duodenal aspiration to detect carrier
1. Blood: It should be collected during rising temperatures. 5 to 10 ml of blood is collected in
50-100 ml of bile broth or Brain-heart infusion broth. Ratio between blood and broth
should be 1:5 . After overnight incubation at 37ºC if turbidity is seen then the broth is
subcultured on solid media.
2. Feces: In the third week. In 80% of patients, organisms can be detected Selenite F and
Tetrathionate broth are used as enrichment media. Subculture is done on Wilson-Blair
medium.
3. Urine: Chances of isolation are high in the fourth week of infection.

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Methods for Identification of Organism:
A. Direct methods:
1. Microscopy: Salmonella are Gram negative, motile, non- capsulated & non-sporing rod
2. Culture: Nutrient agar, MacConkey agar, Wilson and Blair medium
● Nutrient agar: Colonies are large, circular, low convex & smooth.
● MacConkey agar: Colonies are colorless and non-lactose fermenter (NLF)
● Wilson and Blair medium (Selective Medium): Black colony except Paratyphi A
● XLD Agar: Pink colony with black center
3. Biochemical Reactions:
IMViC: I &Vi: Negative, MR: Positive, C: Negative (except Paratyphi B)
TSI: A/Alk with gas (except S.typhi), H2S: Positive (except Paratyphi A)
Peptone water sugar: Glucose & Mannitol fermented with Acid & Gas (S.typhi does
notproduce gas)
Urease & PPA: Negative
Antibiotic sensitivity test
B. Indirect Methods:
1. Antibody Detection:
• SerumWidal test: Positive in 2nd week of infection.
• Typhi dot (EIA): Positive in 1st week of infection.
• Enterocheck-WB (Immunochromatography): Positive in 1st week of infection
2. Antigen Detection:
• Performed during first week from blood & urine.
• Demonstrated by Staphylococcal coagglutination test.
C. For Carrier Detection:
1. Bile culture
2. Vi antibody titer: > 5 is significant.

WORK:
1. Demonstrations:
● Gram stain smear – Gram-negative bacilli.
● Cultural characters: MacConkey ‘s agar.
● Biochemical reactions: IMViC test, TSI agar, Urease & PPA, Sugar set
● Widal test
2. Draw figures of Salmonella colony on MacConkey’s agaragar
3.Questions. (A) What is clot culture?(B) What is an anamnestic reaction in Widal test ?

Salmonella (MacConkey’s agar)

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 HEPATOBILIARY SYSTEM INFECTIONS
PRACTICAL-19
LABORATORY DIAGNOSIS OF VIRAL HEPATITIS

Viral Hepatitis:It is caused by:

• Infectious hepatitis: Hepatitis A & E viruses
• Serum hepatitis : Hepatitis B, C & D viruses

HEPATITIS A VIRUS:
Characteristic:
• 27 nm RNA virus
• Spread by the fecal-oral route
• Incubation period 2-6 weeks
• Affecting mainly children
• No chronic infection or carrier state

Viral markers of HAV:

Laboratory diagnosis:
1. Direct detection – Electron Microscope (specimen-Faeces)
2. HAV Ag detection by ELISA (specimen-: Blood / Stool)
3. HAV Ab – IgM by ELISA (It remains in blood for 2-6 months)
4. Liver function tests

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HEPATITIS B VIRUS:
Characteristic:
• 42 nm DNA virus
• 22 nm spherical form (HBsAg)
• 22 x 200 nm tubular form (HBsAg)
• 27 nm nucleocapsid (HBcAg, HBeAg, partially double stranded DNA)
• Spread by parenteral, sexual & perinatal route
• Incubation period 1-6 weeks
• Affecting mainly adult.
• Chronic infection or carrier state present

Clinical outcome of HBV:

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Hepatitis B Viral Markers

HbsAg: First marker detected before elevation of transaminases & onset of clinical illness.
Remains in circulation through the icteric or symptomatic course of the disease.
Anti Hbs: Protective antibody.
HbcAg: Not demonstrable in circulation, it can be detected in hepatocyte by IF.
Anti Hbc: Appears week or 2 week after the appearance of the HbsAg. Earliest antibody
marker to be seen in the blood.
HBeAg: Suggests active intrahepatic viral replication.
AntiHBe: Important in chronic infection & carriers.

Laboratory diagnosis:
1. Direct detection
• IF: detection of HBcAg (specimen-Hepatocyte)
• PCR: HBV DNA (specimen-Blood)
• PCR: Viral DNA polymerase (specimen-Blood)
2. HBsAg detection by ELISA, EIA, Immuno-chromatography (specimen-: Blood)
3. HBeAg detection by ELISA (specimen-: Blood)
4. Anti HBsAg Ab by ELISA (specimen-: Blood) (Protective antibody)
5. Anti HBcAg IgM Ab by ELISA (specimen-: Blood) (Suggests acute hepatitis B)
6. Liver function tests

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Interpretation of markers in various stages of hepatitis B infection

Symptoms
Anti-HBe
Anti-HBc
Anti-HBs

enzymes
HBeAg
HBsAg

Liver

DNA
Interpretation

+ – – – – Absent Normal + Early acute hepatitis (incubating)

+ – IgM ++ – Present Elevated ++ Acute hepatitis B, high infectivity

+ – IgG ++ – Present Elevated ++ Chronic hepatitis B, high infectivity

Chronic hepatitis B, low infectivity
+ – IgG – + Present Elevated +
(Chronic inactive hepatitis)
Immunotolerant chronic HBV
+ – IgG + – Absent Normal ++ infection (previously called as super
carriers)
Chronic inactive HBV infection
+ – IgG – +/– Absent Normal +
(previously called as simple carriers)
– + IgG – +/– Absent Normal – Recovery

– + – – – Absent Normal – Post-vaccination

IgG
– – – – Absent Normal + Occult hepatitis B infection
(+/–)

Work:
Demonstration:
● Serological tests of hepatitis viruses

Questions:
A.Enumerate different hepatitis viruses producing hepatitis & their mode of transmission.
B. Write about the interpretation of common serological markers of HBV.
C. How will you prevent hepatitis B viral infection?
D. What is the protective titre of Anti HBsAg Ab.

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 Cardiovascular system & Blood stream infections
PRACTICAL-20
Laboratory Diagnosis Infective Endocarditis And Rheumatic Fever

Bloodstream Infections:
Blood is normally sterile, Bloodstream infections refer to the presence of microorganisms in
blood, which constitute one of the most serious situations among infectious diseases, as they are a
threat to every organ of the body
● Bacteremia - presence of bacteria in blood without any multiplication.
● Septicemia - bacteria circulate and actively multiply in the bloodstream and may produce
their products (e.g. toxins) that cause harm to the host.
● Presence of viruses, parasites and fungi in blood are described as 'viremia', 'parasitemia' and
'fungemia' respectively.

Etiological agents of Bloodstream infections

Bacteria Viruses
Primary blood stream infections HIV
Salmonella Typhi Dengue
Brucella Chikungunya
Leptospira Ebola
Borrelia Epstein Barr virus
HACEK group Parasites
Secondary blood stream infections Plasmodium
Staphylococcus Babesia
Beta hemolytic streptococcus Leishmania
Enterococcus Trypanosoma
E.coli Toxoplasma
Klebsiella Wuchereriabancrofti
Enterobacter Brugiamalayi
Acinetobacter Fungus
Pseudomonas Candida
Histoplasma
Blastomyces
Coccidiodes

A. Clinical conditions and agents causing bacteraemia

1. Systemic infections:
- Bacteraemia is common in several infections like,
a. Endocarditis: e.g.Viridans group of Streptococci, Enterococcus faecalis, Staphylococcus
aureus, Staphylococcus epidermidis, HACEK group (Haemophilus sp., Actinobacillus
actinomycetemcomitans, Cardiobacterium hominis, Eikenellacorrodens and Kingella sp.)
b. Osteomyelitis: Staphylococcus aureus
c. Pneumonia: Streptococcus pneumoniae, Haemophilus influenzae etc.
d. Meningitis: Neisseria meningitidis, Streptococcus pneumoniae, Haemophilusinfluenzae
etc.
- Bacteraemia is also common with certain systemic infectious diseases like typhoid,
brucellosis
- Bacteraemia is occasionally associated with some infections like urinary tract infection and
gastroenteritis e.g.Escherichia coli.

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2. During surgery especially which involve mucous membranes: In such conditions, normal
flora of mucous lining may get a chance to enter through the breach in mucous membrane into the
blood stream.
a. G I surgery: Escherichia coli, Klebsiella pneumoniae or other enterobacteriacae
members,Enterococcus faecalis
b. Dental surgery: Viridans group of Streptococci
3. Due to intra-vascular catheter, prosthesis or any foreign body entering arteries or veins
In-situ catheter associated infections form a critical bulk of blood stream infections;
e.g.Staphylococcus epidermidis
4. Neutropenic patients: Neutrophils form the first line of defense against bacterial entry in the
blood stream. So the patients with neutropaenia are prone to develop
bacteriaemia.Candidaemia is also frequently seen in debilitated and neutropenic patients.

B. Clinical conditions and agents causing Septicaemia

a). Neonatal septicaemia: Group B beta haemolyticStreptococci, E.coli, Klebsiella sp.,
Enterobacter sp.,Listeria, Candida sp. etc.
b). Septicaemia in Intensive Care Units:Pseudomonasaeruginosa, Klebsiella pneumoniae,
Acinetobacterbaumanii, Candida sp. etc.

Laboratory methods for diagnosis of Blood stream infections:

There are two modes to evaluate suspected cases of blood stream infections
1. Culture methods:
Two kinds of samples are recommended for culture examination
a. To isolate the invading organism from blood sample - Blood culture
b. To isolate the organism from its source in body - By culture of sample from the local
site suspected to be the source of infection.
2. Serological methods:
a. Serological tests to evaluate septicaemia: C-reactive protein& Procalcitonin
b. Serological tests to identify particular aetiological agent causing bacteraemia and
scepticaemia
- Typhoid fever: Widal test, COAG(co-agglutination) test for detection of Salmonella
Antigen
- Brucellosis: Tube agglutination test

Blood culture
Purpose:
To isolate the bacteria present in blood.
Indications:
1.For diagnosis of diseases which are frequently associated with bacteraemia and
identification of aetiological agents causing those diseases.
e.g. Infective endocarditis, Meningitis,
Pneumonia, Osteomyelitis,
Typhoid, Brucellosis
2. For evaluation of bacteraemic conditions in patients having high risk of bacteraemia.
e.g. - Patient who has underwent GI surgery.
- Patients with Central venous line, prosthesis etc.
- Patients who are febrile and neutropenic.
3. For evaluation of any infection associated with sepsis and may lead to septicaemia.
4. Pyrexia of unknown origin (PUO).

Blood culture media:

Following media are commonly used for culture of blood specimen
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 1. Glucose broth
2. Taurocholate broth
3. Brain-heart-infusion broth
4. Trypticase soya broth
5. Hartley's Broth
6. Thioglycollate broth
The quantity of culture media in bottle is kept less (20-25 ml) for pediatric patients and more
(40-50 ml) for adult patients.
Supplements in culture media:
Several substances may be added to blood culture bottle to improve isolation of bacteria from
blood samplese.g. -
1. Resin or charcoal particles: These neutralize the substances that may be present in
blood andinterfere with bacterial growth (i.e. antibiotics, antibodies, complements etc.)
2. SPS (Sodium Polyanethol sulphonate) or liquoid: It prevents formation of blood clots
and also helps in neutralization of bactericidal substances present in blood.

Sample collection:
Guidelines should be followed for blood sample collection.
 Sample should be collected prior to the commencement of antibiotic therapy because
antibiotics may hamper bacterial growth. If the patient is already on antibiotic therapy,
blood should be collected just prior to the next dose of antibiotic.
 In a febrile patient, bacteremia is more likely to be present at the time of rising fever. So,
sample collection at the time of rising fever, i.e., fever spike; will have higher isolation
rates.
 Collection site should be thoroughly disinfected with alcohol and betadine prior to the
collection of blood to prevent contamination of blood with skin commensals. The
disinfectants should be allowed to dry off from the skin before venipuncture is
performed.
 The amount of blood collected in blood culture bottle should be optimum. For paediatric
patients, 2 to 5 ml and for adults, 5 to 10 ml of blood should be collected in the respective
bottles. (ratio of 1:10)
 Bacterial shedding is intermittent in subacute bacterial endocarditis, so three samples
should be collected at 1 hours interval or two samples collected at 12 hours interval.
 Blood culture bottle inoculated with patient's blood should immediately be transported to
the laboratory. If transport is delayed, then it should be kept at room temperature (never
be refrigerated).
Blood culture methods:
1. Conventional blood culture method: Culture bottles are made of glass & are transparent.
Bacterial growth is evident by development of turbidity, Hemolysis of Rbcs, Gas bubble
or formation of surface pellicle in the medium. Examine daily for upto 7 days (slow
growers like Brucella upto 14 days). If there are signs of bacterial growth, subculture the
broth on solid media and examine a Gram stained smear for bacteria and then do standard
follow up for identification of organism.
2. Automated blood culture methods: After inoculation of culture bottles with blood sample,
they are incubated in specialized instruments which have automated systems for bacterial
growth detection. e.g.
a. BACTEC: Bacterial growth is detected by fluorometric method
b. BacT Alert: Bacterial growth is detected by colorimetric method

Sensitivity of blood culture

 Blood culture will be positive if the concentration of bacteria is 1 to 10 live bacilli per ml
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 of blood.
 In children, bacterial concentration is relatively high. So blood culture positivity is higher
in paediatric patients.
 Due to low bacteraemia in adults, isolation rates are lower. But it can be increased up to
some extent by taking multiple blood cultures (2 or 3) from multiple sites.

Modifications of blood culture:

1. Clot culture: This a double diagnostic method used for detecting Salmonella in cases of
typhoid fever, in which approximately 5 ml of patient’s blood is collected with proper
asepsis. It is allowed to clot. The serum thus separated is used for performing widal test
and for detection of Salmonella antigens; the clot that remains is cultured. For this, the
clot is broken with the help of a sterile glass rod & inoculated into the blood culture
medium along with streptokinase, which helps breaking the clot, with the release of the
bacteria that may be enmeshed within the clot.
2. Castaneda method: This is a blood culture method in a biphasic culture medium,
specifically used for the diagnosis of brucellosis. A single blood culture bottle contains
both solid as well as liquid medium.

Solid
Liquid medium
medium

Blood is inoculated in the bottle & allowed to incubate. The liquid medium is allowed to run

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 on the slant of the solid medium by keeping the bottle in a tilted position & then the bottle is
re-incubated in upright position. The bacteria that may be present in the liquid medium get
cultured on the solid medium, bypassing the need for subculture (which need to open the
bottle) & hence, reducing chances of contamination.

Infective Endocarditis
Infective Endocarditis refers to microbial invasion of the heart valves or mural endocardium-
characteristically results in formation of bulky friable vegetation, composed of mass of platelets,
fibrin, microcolonies of organisms, and scanty inflammatory cells.
It is classified as acute and subacute endocarditis.

Acute endocarditis Subacute endocarditis

Evolution is rapid Evolution is slow
Involves normal cardiac valve Involves previously damaged heart (scarred
or deformed valve)
Implicated organism is of high virulence, Implicated organism is of low virulence,
e.g. S. aureus e.g.viridans streptococci
Causes substantial morbidity and Follows a gradually progressive course of
mortality even with appropriate weeks to months; most patients recover after
antibiotic therapy and/or surgery antibiotic therapy
Less common type, accounts for 10–20% More common type, accounts for 50–60% of
of all cases all cases

Agents of Infective Endocarditis

Staphylococcus aureus Fastidious gram negative coccobacilli
(HACEK group)
Coagulase negative staphylococcus Enterobacteriaceae
Streptococci Pseudomonas spp
Enterococci Candida species
Pneumococci Diphtheroids

Laboratory Diagnosis
Blood Culture
● Isolation of the causative microorganism from blood cultures is critical for diagnosis,
AST, and planning of treatment and should be collected before antibiotic therapy.
● Two blood culture sets should be collected at an interval of 12hr between 1 and 2
st nd

set.
● Alternatively – Three blood culture sets can be collected over one hour interval.

*Blood culture set - ‘pair of bottles’; collected from different venipuncture sites.

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 Laboratory diagnosis of Rheumatic fever
RF (Rheumatics fever) and RHD (Rheumatic heart disease) are nonsuppurative complications of
Group A streptococcal pharyngitis (sore throat) due to a delayed immune response typically
affecting children of school-going age with a peak prevalence in the 5–14 years age group, and
causing significant cardiac valve damage due to repeated episodes of ARF( Acute rheumatic
fever).
Pathogenesis of Rheumatic Fever
Autoimmune theory: Streptococcal antigens show molecular mimicry with human antigens,
hence antibodies produced against previous streptococcal infection (mostly sore throat) cross react
with human tissue like heart, brain, joint, skin to produce lesions. Frequent episodes of Rheumatic
fever can lead to cardiac lesions like inflammatory myocardial lesions, degeneration of heart
valves which is known as RHD.
Clinical manifestations:
Symptoms of rheumatic fever vary and typically begin 1 to 6 weeks after a bout of sore throat
caused by group A streptococcus. Most common symptoms of RF maybe fever, swollen and
painful joints, subcutaneous nodules, skin rashes, uncontrolled movements of arms, legs, or facial
muscles etc. and those of RHD may be breathlessness, chest pain, swelling which may be
accompanied by auscultatory murmurs.
Diagnosis
For diagnosis of RF, Jones criteria can be used which are in the form of major and minor criteria
and supporting evidence of previous streptococcal infection.
Laboratory diagnosis
Laboratory tests like ASO titre as well as acute phase proteins detection like CRP, Procalcitonin,
serum ferritin etc. play an important role in confirming a diagnosis and in the follow-up of
rheumatic diseases.
1. ASO titre (Anti Streptolysin-O)
Antistreptolysin O (ASO) titre is a rapid latex agglutination test for the qualitative and semi-
quantitative determination of antibodies against streptolysin O, a haemolytic exotoxin produced
by group A Streptococcus bacterium.
Measurement techniques
• Slide agglutination test (Qualitative and semi-quantitative)
• Immunoturbidometry
• Nephalometry
Procedure: (Slide agglutination)
• ASO latex reagent is a stabilized buffered suspension of polystyrene latex particles that
have been coated with Streptolysin O.
• Patient’s serum is added to this reagent following kit manufacturer’s guidelines and
examined for agglutination.
Interpretation:
• Most test have a detection limit of 200 IU/ml, i.e. positive agglutination indicates ASO
level more than 200 IU/ml. Titre should be determine in case of positive reaction.
• Rising titre is more significant in diagnosis of Acute Rheumatic Fever hence repeat test is
recommended.
• Antibiotics may give false negative results by inhibiting streptococcal antibody response,
while increased Beta-lipoprotein levels, liver disease, and tuberculosis may give false
positive results.

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 2. CRP (C Reactive Protein) detection test
CRP is an acute phase reactant, a protein made by the liver and released into the blood within a
few hours after tissue injury, the start of an infection, or other cause of inflammation.CRP was
so named because it was first identified as a substance in the serum of patients with acute
inflammation that reacted with the antibody against the somatic capsular polysaccharide (C-
polysaccharide)of pneumococcus.
Measurement techniques
• Slide agglutination test (Qualitative and semi-quantitative)
• Immunoturbidometry
• Nephalometry
• ELISA (Enzyme Linked Immunosorbent Assay)
• CLIA (Chemiluminescent immunoassay)
Indications of CRP measurement test:
• Bacterial infections including:
 Meningitis and encephalitis
 Infective endocarditis
 Septicaemia
 RHD
 Pneumonia
• Viral infections.
• Rheumatoid arthritis.
• Inflammatory bowel disease (Crohn’s disease, Ulcerative colitis).
• Pelvic inflammatory diseases. (PID)
A high-sensitivity CRP (hs-CRP)
Test measures low levels of CRP using laser Nephalometry. The test gives results in 25
minutes with a sensitivity as low as 0.04 mg/L.
The risk of developing cardiovascular disease is quantified as follows:
• low: hs-CRP level under 1.0 mg/L
• average: between 1.0 and 3.0 mg/L
• high: above 3.0 mg/L
Reference range of CRP and clinical interpretation
• Normal range: 0.3mg/dL- 0.8 mg/dL ( 3mg/L – 8 mg/L )
• Mild elevation up to 10 mg/L can be seen in obesity, pregnancy, depression, diabetes,
common cold, gingivitis, sedentary lifestyle, cigarette smoking, etc.
• False negative CRP can be found in patients with liver failure of clinical conditions
having compromised liver functions.
• Hepatic synthesis of CRP starts 6 to 8 hours after onset and peak concentrations are
reached between 36 to 50 hours after infection has started.
• The half-life of CRP is 19 hours making it come to normal range rapidly on treating the
infection or inflammation, hence it has a significant value as a prognostic marker.
Proinflammatory cytokines (e.g., IL-1, IL-6, and tumour necrosis factor α-[TNF-α]) appear
within one hour and Procalcitonin (PCT) after 5 hours of the start of bacterial infection.

Work:
1. Demonstration:
● Blood culture bottle & blood culture techniques.
● ASO test, CRP test.
2. Questions:
A. Enumerate different blood culture medias.
B. Which blood culture medium is preferred in conventional blood culture technique.
C. Blood culture technique in case of sub acute bacterial endocarditis.
D. What is the normal level of CRP in serum.
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138
 Cardiovascular system & Blood stream infections
PRACTICAL-21
Laboratory Diagnosis Of Malaria And Filaria

Examination of Blood:
Examination of blood smear stained with Giemsa stain is the most common method of detecting
Plasmodium spp., Babesia spp., Trypanosoma spp., and some species of Microfilariae. Although
motile organisms such as Trypanosoma spp. And Microfilaria can be detected on a wet
preparation of a fresh blood specimen under low and high power magnification, identification is
made on the basis of characteristics seen on a permanently stained smear.

COMMON PARASITES FOUND IN BLOOD

Specimen Parasite Form
Plasmodium Trophozoite, schizont, gametocyte
Trypanosoma Trypomastigote
Babesia Trophozoite & Merozoite
Leishmania Amastigote
Blood
W. bancrofti Microfilaria
B. malayi Microfilaria
L. loa Microfilaria
Mansonella Microfilaria

Peripheral blood smear.

Collection of blood:
Capillary blood can be collected by finger pricking. Venous blood may be collected in tube
containing anticoagulant like EDTA which should be examined as soon as possible.
Blood should be collected as soon as possible in suspected case of malaria especially before
administrating anti malarial drugs.
Nocturnal periodicity: Microfilariae are usually present in greatest numbers in the peripheral
blood during night hours, which correspond to the peak biting times of their insect vectors hence
blood should be collected during night hours.
Methods of examination of blood:
o Direct wet mount examination.
o Examination of blood smears after permanent staining.
o Examination of buffy coat region. (quantitative buffy coat)
o Concentration of blood.
Direct wet mount examination.
Place a drop of blood, collected by finger prick, on a glass slide and cover it with coverslip.
Examine under low power objective lens of microscope.
• For detection of microfilariae and trypanosomes by their motility.
Permanent staining.
Thick and thin, two types of smears can be prepared with their own merits and demerits for the
purpose of permanent staining. Such stained smears are to be examined under oil immersion field.

Methods for preparation of peripheral blood smear:

Thick smear:
- A drop of blood in middle of the slide.
- Using a pipette or stick, the blood is evenly distributed over the area of about 15 X 15 mm
on the slide.
- It should be possible to just see (but not read) the news print on the paper through the
smear.
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 - Label the slide legibly.
- Smear is allowed to dry. It need not be fixed.
- Stain by Romanowsky’s stains.
- A thick film is best for detection of parasites, because organisms are concentrated in
relatively small areas.
- At least 100 oil immersion fields should be examined before a negative result
is reported.
Thin smear:
- A small drop of blood is placed near one end of slide.
- A dry grease free glass slide (or a smooth edged slide spreader) is kept at an angle over
theblood drop, which in turn will spread along the entire edge of spreader that is in
contact withblood on slide.
- The spreader is now rapidly moved over the surface of slide to make a thin blood film.
- A good thin smear should have a smooth tail and be free of vertical lines and holes.
- It should be possible to read the newsprint on a paper through the smear.
- The smear is fixed with absolute methanol or absolute ethanol for 1 minute and then
allowed to dry.
- Stain by Romanowsky’s stains.
- Species identification should be made from a thin film, because the characteristics of the
parasite and the RBC can be seen.

Thick smear Thin smear

RBCs are lysed. RBCs are fixed, mostly in a single layer
Larger volume of blood can be Less volume can be examined.
examined.
Blood elements are more concentrated, Good for species identification and
hence species identification of differentiation of Plasmodium.
Plasmodium not done.
Good screening test. More time consuming, not appropriate for
screening.

140
 Note: Thick and thin smear may be prepared on the same slide with due care.

Stains:
• Romanowsky’s stains include Giemsa, Leishman’s, Wright’s, Field’s and Jaswantsingh and
Bhattacharya’s stains.
• These stains contain combination of methylene blue (basic) which stains the nucleus and
eosin (acidic) which stains the cytoplasm.
• In general parasite stains bluish and the chromatin red to purple red
Giemsa stain-
• 1:10 dilution of working solution is prepared from Giemsa stain inphosphate buffer.
• Fixed thin smear or dried unfixed thick smear is immersed in the working solution in
couplin jar for 20 to 30 minutes.
• Slide is washed with phosphate buffer or tap water and examined.
Fielde’s stain-
Fill up two couplin jars with Field stain A and Field stain B separately.
- Dip the smear in Field stain B for 5-6 seconds and wash in running water.
- Dip the smear in Field stain A for 20-30 seconds and wash in running water.
- Wipe the back of the slide clean.
- Air dry the smear and examine under microscope.
Leishman’s stain-
-Stain contains Leishman’s powder in absolute ethalnol. It is kept under sunlight or in
incubatorfor 1 hour for 3 days in a glass stoppered brown bottle for maturation.
-The smear is poured with about 10 drops of stain for 2 minutes followed by adding up of
doublethe volume of distilled water and mixed gently by rocking the slide.
-After 15 minutes, the slide is washed with buffered distilled water and air dried for
examinationunder the microscope.
Jaswant-Singh-Bhattacharya stain (JSB stain)-
-Standard method used by the laboratories under the National vector borne disease control
program in India. (NVBDCP)
-Stain has two solutions, solution 1 and solution 2.
-Fixed and air-dried smear is immersed in solution 1 for 30 seconds and then washed with
water.
-Slide is immersed in solution 2 for 1 second and washed with water.
-Slide is immersed again in solution 1 for 30 seconds. Slide is washed again as above till
thesmear gives pink background, then air dried to be examined under microscope.

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Methods for diagnosing Malaria:
1. Peripheral blood smear examination: Gold standard
Thick smear - more sensitive, species can not be identified.
Thin smear - speciation can be done based on the following features:
P. vivax - amoeboid ring form and schizont
P. falciparum - ring forms (multiple ring form, accole form, headphone shaped ring
forms), banana shaped gametocyte
2. Quantitative buffy coat (QBC):Not commonly used as it is replaced by simple &rapid
malarial antigen detection card
3. Antigen detection card based on immune-chromatography (RDTs):
• Detect pan malarial Ag (pLDH, aldolase), falciparum specific Ag (HRP-II)
• Most of the kits are designed to detect a combination of two antigens, one is P.falciparum
specific (HRP-II) and the other is pan malarial antigen (pLDH).
Advantages of RDTs
1. Simple and rapid.
2. More than 90% sensitivity if >100 parasites/µl.
Disadvantages of RDTs
1. Gametocytes cannot be detected.
2. False positive bands appear in Rheumatoid arthritis factor positive cases.
3. Ineffective in less than 40 parasites/µl for HRP2 detection and in less than 100
parasites/µl for pLDH detection.
4. Culture: RPMI 1640 medium (not commonly used)
5. Molecular diagnosis: PCR targeting 18S rDNA (not commonly used)

Methods for diagnosing Filariasis:

Methods to diagnose filariasis.
1. Detection of microfilariae by microscopic examination.
 Demonstration of microfilariae in smear prepared from peripheral blood (Thin / Thick
film / Concentration method) collected at correct time (10 p.m to 4 a.m, peak 2 a.m.)
 Demonstration of microfilariae in smear prepared from peripheral blood (Thin / Thick
film / Concentration method) collected during day time after hetrazine provocative test(
hetrazine 2mg/kg body weight), blood collected after 15-20 minutes.
 Detection of microfilariae in urine, hydrocele fluid or other body fluids.
2. Detection of Adult worm: lymph node biopsy or calcified worm in X-Ray.
3. Detection of filarial antigens in serum: ELISA or IC strip/card.
4. Detection of antibodies
5. Molecular method: DNA probe, PCR.
6. Indirect evidence: Blood examination – Eosinophilia.

Work:
Demonstration:
• Morphological forms of Plasmodium vivax and Plasmodium falciparum.
• Microfilaria.
Questions:
A. Enumerate stains used for peripheral smear for malarial parasites
B. Enlist different morphological forms of P. vivax and P. falciparum as seen in
peripheral blood smear.
C. Draw diagrams of different morphological forms of P. vivax and P. falciparum
D. Draw diagram of microfilaria of W. bancrofti.
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 vivax in peripheral blood smear

(P. vivax) (P. vivax) (P. vivax)

Ring form Schizont form Gametocyte form

P. falciparum in peripheral blood smear

(Pl. falciparum) (Pl. falciparum)

Ring form Gametocyte

Microfilaria of
143 W. bancrofti
144
 Cardiovascular system & Blood stream infections
PRACTICAL-22
Laboratory Diagnosis of HIV infection

Kinetics of immune response:

• Viremia- Viral entry into the body leads to a transient period of high-level viremia and p24
antigenemia.
• Window period- 3 to 12 weeks. Virus remains in the blood but antibody does not appear in
the blood.
• Later on Humoral response is evidenced by formation of antibodies against different
antigens.

Pre-requisite for HIV testing (3Cs):

• Consent in written format should be taken before the test is done.
• Confidentiality of a positive test result is must.
• Counselling (Pre-test and Post-test Counselling) should be provided to motivate the
individual.

Laboratory tests for diagnosis of HIV infection:

A) Screening tests (Antibody detection): (ERS) (ELISA/ Rapid/ Simple test)
Specimen: blood in plain tube
1. ELISA
Advantages:
• Highly sensitive
• Adaptable to large number of samples and cost effective.
Disadvantages:
• Require special equipments like ELISA reader and washer
• Require training of laboratory technician. Takes 2-4 hours
2. Rapid/ Simple test
Advantage:
• Easy to perform and provide quick results (takes < 30 minutes).
• Do not require special equipments.
Disadvantage:
• False positive results
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 Most commonly used rapid tests in India:
a. Dot blot assays (Immunoconcentration) (vertical flow)
Principle: This is a type of solid phase immunoassay where HIV antigens are immobilized
on a porous membrane. Specimen & reagent pass through the membrane & are absorbed
into the underlying absorbent pad. As the specimen passes through the membrane, HIV
antibodies if present, bind to the immobilized antigens. The conjugate binds to the Fc
portion of the HIV antibodies and produces a distinct coloured dot against a white
background.

b. Immunochromatography (lateral flow)

c. Dip stick/ comb tests

B). Supplemental test (Antibody detection) test:

Western blot:

146
 • Principle: It detects individual antibodies in serum separately against various antigenic
fragments of HIV such as, Antibody to gag gene products, pol gene products and env gene
products. The antigen antibody complexes appear as distinct bands on nitrocellulose strip.
• Sample taken: Blood in plain vacutte
• WHO criteria i.e. presence of at least two envelope bands (out of gp120, gp160 or gp41)
with or without gag or pol bands
• CDC criteria-presence of any two out of p24, gp 120, gp160, gp41 bands

C). Confirmatory tests

1. p24 antigen detection
2. HIV RNA detection
• The ‘gold standard’ method for confirmation of HIV diagnosis.
• Real time RT-PCR- for estimating viral load
• Sample taken: blood in EDTA (plasma)
Uses:
• For confirmation of diagnosis of HIV/AIDS (can detect even few copies of viral RNA)
• Diagnosis of HIV during window period (earlier than all available methods :12 days post
exposure)
• Viral load monitoring- monitoring the response to antiretroviral therapy.
3. HIV DNA detection:
• Early Infant Diagnosis (<18 months age)
• Sample – Blood drawn from heel and taken on filter paper and dried - DBS (dried blood
spots)

D). Non–specific Immunological Methods:

● Low CD4 T cell count
● Altered CD4:CD8 T cell ratio

NACO STRATEGY FOR HIV DIAGNOSIS

• NACO (National AIDS Control Organization, India) has formulated a strategic plan for HIV
diagnosis.

147
148
PROGNOSIS / MONITORING OF HIV-monitoring the response to antiretroviral therapy
• CD4 T cell count – - by flow cytometry method
• HIV RNA load– most consistent and best tool at present

149
 Work:
Demonstration:
1.Rapid kits
2. ELISA Plate
Questions:
a. What is NACO ?.
b. Define window period? What is duration of window period for HIV infection?
How can we diagnose HIV infection during window period?
c. What is ICTC?
d. What is DAPCU ?
e What is full form of PPTCT ?

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 RESPIRATORY TRACT INFECTIONS
PRACTICAL-23.
Laboratory diagnosis of Respiratory tract infections

Anatomy: Part of respiratory tract above the glottis or vocal cord is called upper respiratory tract
& Below glottis or vocal cord is called lower respiratory tract

Upper Respiratory tract Infections:

Types of upper respiratory tract infection
1. Local inflammation: i.e. Sore throat (Tonsilitis or Pharyngitis), Rhinitis, Epiglottitis,
Sinusitis, Otitis media etc.
2. Abscess formation: Faucial or Tonsillar
3. Ulcer formation: mouth, throat etc.
4. Diphtheria
5. Pertussis
6. Quinsy (Infection of retropharyngeal space)

1. Common etiological agents of Upper respiratory tract infections:

Bacteria Virus
Streptococcus pneumoniae Rhinovirus
Streptococcus pyogenes coronavirus
Haemophilus influenzae Influenza viruses
Corynebacterium diphtheriae Parainfluenza virus
Borrelia vincentii Adenoviruses
Fusiform bacteria Fugal
Candida albicans

151
2. Laboratory diagnosis of Upper respiratory tract infection
Specimen collection:
• Nasal / throat swab: two throat swab samples should be collected from affected part, one for
direct examination and one for culture.
• Part of membrane & swab from fragile margin of membrane
• Nasopharyngeal swab or aspirate for viral diagnosis
Processing the specimen
A. Microscopy
1). Gram stain:
Bacteria can be identified based on their morphology and shape.
Interpretation: Bacteria demonstrated in Gram stain examination should be carefully
interpreted i.e. presence of gram positive cocci in chains suggestive of Streptococcus
species which may be commensals (streptococcus viridians) or pathogenic (streptococcus
pyogenes).
2). Albert stain:
To demonstrate Corynebacterium diphtheriae
Interpretation: presence of bluish-black metachromatic granules either at one end or both
ends and cuneiform arrangement (Chinese letter pattern arrangement) of bacilli
suggestive of C.diphtheriae
3). Immunofluorescence microscopy:
To demonstrate presence of antigens of viruses causing URTI
B. Culture:
For bacteriological culture specimen should be inoculated onto following culture media
• Blood agar
• Chocolate agar
• MacConkey’s agar
• Loeffler’s serum slope and potassium tellurite blood agar (for isolation of
Corynebacterium diphtheria in suspected cases of Diphtheria).
• Sabouraud dextrose agar (SDA) is used if fungal infection is suspected.

Interpretation:
- Bacterial growth can be identified by colony morphology / pigment production /
haemolysis on blood agar & / or colour of colony on Mac Conkey agar.
- On Loeffler’s serum slope media C.diphtheriaeproduces small, circular glistening,
white with yellowish tinged colonies within 6-8 hours while on potassium tellurite
blood agar C.diphtheriae produces black colonies due to reduction of tellurite to
tellurium after 48 hours. Tellurite blood agar commonly helps in isolation of
C.diphtheriae from carrier.
- On SDA: Candida species produces smooth, creamy, white, pasty colonies.
Aspergillus / mucor produce growth of mycelium.

C. Molecular test
- Polymerase chain reaction method is used for diagnosis of various virus infections i.e.
Swine flu / bird flu / SARS-Cov-2
- Multiplex PCR assay are available where multiple primers targeting the genes specific
for each of the suspected agents of URTI are used.

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Laboratory Diagnosis of Diphtheria:
Sample collection:
1. Throat and nasopharyngeal swab
2. A portion of pseudomembrane
3. skin swab from for diagnosis of cutaneous diphtheria
Methods for identification of organism:
Direct methods:
1. Staining
Gram staining: Gram-positive rods are seen arranged as Chinese letters or
palisade (commensal corynebacteria)
Albert staining: C. diphtheriae stains green and beaded due to the presence of
dark staining granules in rods. These granules, known as volutin granules or
metachromatic granules, are energy storing inorganic poly-phosphate units
Methylene blue staining: C. diphtheriae stains unevenly with dark blue staining granules
in rods
2. Culture:Loffler‘s serum medium, Potassium tellurite blood agar
3. Biochemical reactions:
Sugar fermentation test: ferment Glucose and maltose with acid production
4. Animal inoculation:
Intracutaneous injection in to Guinea pig leads to death of the animal.
Indirect methods:
Toxigenicity testing of C. diphtheriae by Elek‘s gel precipitation test and Schick test
Diphtheria is a notifiable disease. So any positive laboratory result should be inform to
higher authority.

Lower Respiratory tract Infections:

Types of lower respiratory tract infection
1. Bronchitis:
• Acute Bronchitis:It is a self limited inflammation of the bronchial tree due to infections.
• Chronic bronchitis: It is defined as a cough with expectoration for at least 3 months/year
during a period of 2 consecutive years.
2. Bronchiolitis: Inflammation of bronchioles
3. Lung abscess
4. Pleuritis
5. Alveolitis
6. Bronchiectasis
7. Tuberculosis
8. Pneumonia:
(1) Community acquired—patients acquire infection in the community,
(2) Hospital acquired—patients acquire infection in the healthcare facility.

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Common etiological agents of lower respiratory tract infections:

Infectious Agent Group Name of Pathogen

Gram positive bacteria Streptococcus pyogenes

Streptococcus pneumoniae
Staphylococcus aureus
Bacillus anthracis (Anthrax)
Nocardia sp.
Gram negative bacteria Haemophilus influenzae
Pseudomonas aeruginosa
Klebsiella spp.
Other enterobacteriacae
Moraxella catarrhalis
Acinetobacter sp.
Yersinia pestis (Plague)
Miscellaneous bacteria Mycobacterium tuberculosis(Tuberculosis)
Mycoplasma pneumoniae (Atypical pneumonia)
Legionella pneumophila
Fungi Candida sp.
Aspergillus sp.
Cryptococcus neoformans
Histoplasma capsulatum
Blastomyces dermatitidis
Pneumocystis jirovecii
Parasites Paragonimuswestermanii
Entamoeba histolytica
Echinococcus granulosus
Viruses Orthomyxoviruses (Influenzae)
Paramyxoviruses (parainfluenzae, respiratory syncitial virus
etc.)
Adenoviruses
Herpesviruses (i.e. CMV)
Enteroviruses
Coronaviruses (SARS, MERS, SARS CoV2)

Common etiological agents of Pneumonia:

Community acquired Hospital acquired
Bacteria Bacteria
Streptococcus pneumoniae Pseudomonas
Haemophilus influenzae Acinetobacter
Mycoplasma pneumoniae Escherichia coli
Chlamydophila pneumoniae Klebsiella pneumoniae
Virus Methicillin resistant Staphylococcus aureus
Influenza
Corona
Respiratory syncytial virus
Parainfluenza
Adenovirus

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In addition to these, certain infectious disease may involve upper and lower respiratory tract
simultaneously e.g. croup (laryngo-tracheo-bronchitis)
Normal flora of mouth and throat
Oral cavity and throat have plenty of normal microbial flora which are commonly isolated from
sputum sample. Few of them can also cause infections. Isolation of such organisms should be
evaluated with clinical background of the patient. These are Staphylococcus spp., Streptococcus
spp, Micrococci, Lactobacilli, Diphtheroids, Neisseria, Moraxella spp., Fusobacterium etc.
Laboratory diagnosis of Respiratory tract infection
Specimen collection:
1) Sputum:
Sputum is a good sample majorly for URTI. It may be collected for LRTI too, but is not an
ideal sample. Still, it is the most commonly collected sample for bacteriological analysis.
2) Swab:
Swab also is a common sampling method For URTI. In case of LRTI, bacteriological infectious
agents can’t be generally identified by swabs. However, swabs are a major specimen when it
comes to viral LRTI. Generally, nasal swabs, throat swabs & nasopharyngeal swabs are taken
in decreasing order of preference when a viral etiology is suspected for either URTI or LRTI.
3) Aspiration:
a.Tracheal or endotracheal aspiration:
This specimen is usually taken to diagnose LRTI, particularly in patients with
endotracheal intubation or with tracheostomy. The specimen is aspirated with a special
device known as a mucus extractor. One end of the device is inserted through the
endotracheal tube or tracheostomy site, the other end opens in a sterile container.
Sample is collected by creating negative pressure in the container with the help of
suction machine (which is attached to the container by another tube.)
b.Broncho alveolar lavage (BAL):
This is a specimen collected at the time of bronchoscopy. Lavage is taken after saline
wash directly from infection site (bronchi, bronchioles). This is the most informative
sample for lower RTI as it completely bypasses the oral cavity & throat and hence, does
not contain the commensals. If intracellular bacteria are present in more than 25 % of
inflammatory cells; they serve as indicators of pneumonia.
c.USG/CT guided percutaneous needle aspiration:
If the lesion is present at a distal site of the lungs, it is very difficult to extract specimen
even with bronchoscope or bronchial wash. In such cases, sample is taken by
4) Blood culture: Done in cases of bacteremia associated with pneumonia; e.g.,
S. pneumoniae.
5)Lung biopsy: This is an option in life threatening, difficult to diagnose infections like
Herpes simplex pneumonia or Pneumocystis pneumonia.

Transport of specimen:
 Sample should be immediately transported to the laboratory.
 Special transport media are recommended in certain cases if transportation of the swab
specimen is expected to be delayed.
e.g. 1. Streptococcal infection: Pike's medium
2. Viral infection: Special viral transport media.

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Gross examination of sample:
Sputum samples may be purulent, muco-purulent, bloody or salivary. In pyogenic infections
sputum will be purulent or muco-purulent. In case of tuberculosis blood may be present in
sputum. Salivary samples are unsatisfactory for microbiological evaluation of RTI

Microscopic examination:
Sputum/aspiration/lavage: Smear prepared from the specimen should be examined by
a. Gram stain: To demonstrate and identify bacteria by their morphological study;
i.e. Gram negative or Gram positive; cocci or bacilli.
b. Z N stain: To detect Acid Fast Bacilli.
c. KOH wet mount preparation: To detect presence of fungi
d. Giemsa stain: For cytological examination.
e. Special fungal stains may be needed in suspected cases of fungal infections e.g. GMS
(GomoriMethnamine Silver) stain for Pneumocystis jirovecii.
These preliminary examinations help clinicians to start or alter the empirical therapy according
to the group of organisms involved; e.g., if KOH mount shows fungal hyphae in BAL,
appropriate antifungals can be started immediately.
Culture examination:
Culture media:Sample should be cultured on the following media
1. Simple media: Nutrient agar
2. Differential media: MacConkey agar
3. Enriched media: Blood agar, Chocolate agar
4. Selective media: e.g. For Tuberculosis: L J medium.
5. For suspected fungal etiology: SDA
Incubation of culture plates:
1. For bacterial cultures:
One set of inoculated plates should be incubated at 37° C temperature, aerobically & the
second set of plates should be incubated at 37° C temperature, in presence of 5-10% CO2
(i.e.candle jar / CO2 incubator)
2. For fungal cultures:
One set of inoculated SDAs should be incubated at 37° C temperature, and the other set
should be incubated at 25° C temperature; so that any dimorphic fungi (e.g.Histoplasma
capsulatum) may also be diagnosed. These cultures have to be followed for at least a week.

Identification and Antimicrobial sensitivity testing:As per standard procedure::

Serology (Antibody detection tests): Helps in diagnosis of atypical pneumonia
Antigen detection tests:
Molecular tests: Real time PCR, Biofire Filmarray

Work:
Demonstration:
1. C. diphtheriae-Gram stain & Alberts stain
2. Streptococcus pneumoniae- Gram stain.
3. Blodd& Chocolate agar: Hemolysis.
4. Loffler‘s serum medium, Potassium tellurite blood agar
5. Biochemical reaction: Glucose and maltose fermentation test to differentiate C.
diphtheria from diphtheroid.
6. Eleck’s gel test
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 7. Klebsiella pneumoniae- Gram stain, colony characteristics, identification test.
8. Viral transport medium.

Questions:
A. Describe the Lancefield classification of streptococci.
B. Describe various types of hemolysis on blood agar.
C. Write in brief about the morphology of C. diphtheriae.
D. What are the coryneform and diphtheroid bacteria?

Gram stain: Albert stain:

C. diphtheriae C. diphtheriae

Gram stain: Gram stain

Streptococci S. pneumoniae

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 K. pneumonia on Β hemolytic colony
MacConkey’s agar on Blood agar

α hemolytic colony Colony on

on Chocolate agar Pottasiumtellurie

Colony on Elek` gel

Loffler's Serum precipitation test

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 RESPIRATORY TRACT INFECTIONS
PRACTICAL-24.
Laboratory Diagnosis of Tuberculosis

Mycobacteria:
Mycobacteria are widespread both in the environment and in animals. The major human
pathogens are M. tuberculosis and M. leprae, but awareness of the importance of other species is
increasing with their recognition as pathogens in AIDS patients.
Characteristics: Aerobic rods with a Gram-positive cell wall structure, but stain with difficulty
because of the long-chain fatty acids (mycolic acids) in the cell wall. Acid-fastness can be
demonstrated by resistance to decolorization by mineral acid and alcohol (Ziehl-Neelsen stain).
Mycobacteria grow more slowly than many other bacteria of medical importance, but the genus
can be divided into: rapid growers (form visible colonies within seven days); slow growers (form
visible colonies only after 14 days‘ incubation). M. leprae does not grow in culture media.
Transmission: Droplet spread aided by ability of organism to survive in the environment (M.
tuberculosis, M. leprae). Milk borne spread of M. tuberculosis to humans from infected cattle has
been important in the past. Social and environmental factors and genetic predisposition all have a
role. Leprosy requires close and prolonged contact for spread.
Diseases: M. tuberculosis causes tuberculosis in humans and animals. M. leprae is restricted to
human and causes leprosy. Mycobacteria other than tuberculosis (MOTT) are associated with a
range of conditions, usually in immunocompromised hosts. The avium-intracellular complex has
important association with AIDS patients.

Mycobacterium tuberculosis:
Morphology: M. tuberclosis is a straight or slightly curved rod, occuring singly, in pairs or in
small clumps. M. bovis is straighter, stouter and shorter. M. tuberclosis stains beaded while M.
bovis stains more uniformly. The organisms are acid alcohol fast.
Cultural characters: Mycobacteria are aerobes. They grow on Lowenstein–Jensen (L-J)
medium. Growth is slow. Growth may appear as on 10-14 the day or late and requires incubating
for about 6 weeks. M. tuberclosis shows Rough dry, wrinkled irregular tough in consistency
(hard and difficult to emulsify) and buff-color (begin with creamy white and then develop buff
color). M. bovis grows as flat, white, smooth, moist colonies which emulsify easily.
Biochemical Reactions: Only M. tuberclosis gives niacin test positive.
Pathogenicity: It causes tuberculosis. In primary tuberclosis lungs, intestines, tonsils and skin
are affected. In secondary tuberculosis any organ may be involved more commonly meninges,
larynx, bones, joints, spleen, urinary tract are affected.

LABORATORY DIAGNOSIS OF TUBERCULOSIS:

Sample collection
A. Pulmonary TB:
a. Sputum: Collected in disposable wide mouth container. Early morning sputum samples
collected for three consecutive days are ideal. For direct microscopy, according to RNTCP
guidelines, two sputum samples are sufficient. First is spot sample & second is the early
morning sample. Steam inhalation or expectorants may enhance productivity of sputum.
b. Gastric aspirate: Children cannot collect sputum properly and usually swallow majority of
expectorated sputum. This material can be recovered from stomach by gastric aspiration or
lavage.
c. Broncho alveolar lavage (BAL): It is the best sample, particularly when the sputum is
expectorated minimally.
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 d. Lung aspirate: Trans-thoracic, CT guided aspiration directly from the site of lesion in lung
would be the most informative sample; especially when sputum and BAL specimens seem
to be unsatisfactory.

B. In EPTB: (Extra pulmonary TB) Specimens vary depending on the site involved
a. Urine: In cases of sterile pyuria, a high suspicion of renal tuberculosis should be
considered.
Early morning urine samples collected for 3 consecutive days are ideal. Every day samples
are centrifuged and deposits are processed further.
b. Body fluids:
- CSF in cases of meningitis.
- Pleural fluid for diagnosis of pulmonary tuberculosis.
- Ascitic fluid for diagnosis of abdominal tuberculosis.
- Synovial fluid in cases of arthritis.
All the above samples can be processed for demonstration of Acid Fast Bacilli, culture, NAT,
ADA measurement and cytological examination for evidence of tuberculosis infection.
c. Pus: In case of cold abscess, pus can be taken aseptically & examined for demonstration of
bacilli, culture examination and NAT.
d. Lymphnode: In case of lymphadenopathy, lymphnode biopsy or FNAC should be taken for
histopathological and cytological examination. This can also be processed for demonstration
of Acid Fast Bacilli, culture and NAT.

Digestion, decontamination and concentration of specimen

Purpose:
• Preparation of samples for microscopic and culture examination.
• For concentration of bacilli to have higher positivity rate.
• For homogenization of sample to process further.
• For decontamination of samples, this kills all the organisms except Mycobacteria.
• Applicable to Sputum / Broncho alveolar lavage / Gastric aspirate
Methods:
• Digest sputum by equal part of 4% NAOH at 370 C with frequent shaking. It is then
centrifuged at 300 r.p.m. for 30 minutes and discarrd supernatant. The sediment
neutralised with 8% HCL and used for smear, culture.
• Cetylpyradinium chloride (CPC) + NaCl
CPC is considered the best decontaminating agent. It does not affect the viability of
Mycobacteria even after prolonged incubation. Particularly, when the sample is to be
collected from a remote place, sputum should be collected in CPC containing container
and then transported to Laboratory. The sample will be decontaminated during transit
time and Mycobacteria will be preserved. The mixture of CPC with sample is then
centrifuged and deposit is used for microscopic and culture examination.
• N-acetyl L - cysteine (NALC) + NaOH
The concentration of NaOH is kept 2%, which can decontaminate samples without
affecting the viability of Mycobacteria. The added NALC will act as a mucolytic agent
and homogenize sample thoroughly.
Microscopic Examination: For demonstration of Acid Fast Bacilli
1. Z N stain: Easiest and quickest test. Sensitivity: 10,000-100,000 bacilli / ml
• Routine Z N stain: Involving the heating of the smear as a mordant.
• Cold stain (Kinyoun stain) : A modified Z N stain that does not involve the heating
process, but has increased concentration of phenol in carbolfuschin, increased duration of
stay of the primary stain on the smear & a wetting agent; tergitol, to suffice the need of

160
 the mordant instead of heat. This method prevents alteration in morphology of the
Mycobacteria.
2. Auramine-phenol: Can increase sensitivity by 2.5 times, needs fluorescent microscope.
Culture examination: Sensitivity is very high, as low as 10 bacilli / ml of specimen can be
detected.
Methods:
1. Solid culture media: L. J. medium
- The processed specimen is cultured on L.J. medium slants.
- The bacterial growth (colonies) will appear after 20 - 40 days of incubation.
- The isolated Mycobacteria are processed further for biochemical reactions, to
differentiate M. tuberculosis (MTB) from Mycobacterium other than tuberculosis
(MOTT).
The following tests will be positive for MTB
1. Niacin test
2. Nitrate reduction test
3. Catalase test.
2. Liquid culture media: Middle brook medium, Dubo's medium
3. Automated culture methods:Processed sample is inoculated in the specified Middle
brook medium. The Mycobacterial growth in the medium will release carbon dioxide. This
can be identified by automated methods based on various principles. Growth can be
detected as early as within 11-14 days of incubation. Can be used for growth detection &
antitubercular sensitivity testing.
• BACTEC MGIT: will detect bacterial growth and carbon dioxide released by fluoro-
metric method.
• BacT Alert: will detect bacterial growth and carbon dioxide released by colorimetric
method.
Culture identification: Manual biochemical tests,MALDI-TOF
Antibiotic sensitivity:
1. On Solid media (Conventional method):
• 1 % Proportional method: Commonly used.
Mycobacterial isolate is inoculated simultaneously in 1. Control LJ medium (not
containing any anti tuberculous drug), 2. L J Medium slants containing a critical
concentration of individual anti-TB drug to be tested for sensitivity. They are incubated
aerobically at 37° C and observed at regular intervals till growth appears on control slant.
At the same time, if test slant shows bacterial colonies more than 1% of colonies on control
slant, the bacteria is interpretated as being resistant to that particular anti-TB drug which is
present on the test slant.
• Resistance ratio method
• Absolute concentration method
2. In Liquid medium (Automated method)

Nucleic acid technologies (NAT):

1. Detection of M.Tb in specimen: Sensitivity 1-10 bacilli / ml. The bacterial specific DNA /
RNA sequence is detected in specimen.
Technologies available are
• CBNAAT (Cartridge based nucleic acid amplification test)/GeneXpert:
Turnaround time is 2 hours
• Polymerase chain reaction:
• Gene probe

161
 2. Mycobacterial species identification:
• Line Probe Assay: Turnaround time is 2-3 days
3. Anti-TB sensitivity:
• Gene-Xpert (detects resistance to Rifampicin)
• Line Probe Assay (detects resistance to first and second line AKTs)

CBNAAT (Cartridge based nucleic acid amplification test)/GeneXpert:

For identification and detection of resistance to rifampicin. Turnaround time is 2
hours.

Line probe assay:For identification and detection of resistance to 1st and 2nd line
antitubercular drugs. Turnaround time is 2-3 days.

Modalities providing indirect evidence of TB infection:

1. Mantoux test (Tuberculin Test):Tests indicate infection; and not necessarily disease
The reaction to the intradermally injected tuberculin is a classical example of a
delayed (cellular) hypersensitivity reaction.
Procedure:
A standard dose of five tuberculin units (TU) (0.1ml) is injected intradermally on the volar
aspect of forearm (left side is recommended uniformly). Skin induration measured be read
between 48 and 72 h after administration. The basis of reading is the presence or absence
of induration, which may be determined by inspection and by palpation. For
standardization, the diameter of induration should be measured transversely to the long
axis of the forearm and recorded in millimeters.
Interpretation:
- Reaction of more than 15 mm in size is considered positive
- No reaction or reaction size < 5 mm is considered negative
- 5 to 15 mm of reaction size should be interpretated by correlation with other
clinical and epidemiological parameters
2. Interferon gamma (INF-γ) - assay – IGRA: Tests indicate infection; and not necessarily
disease It is an in-vitro assay to test cell mediated hypersensitivity against tuberculin
antigen.
Procedure:Patients’ blood is incubated with tuberculin antigen in the laboratory; If patient
will have prior exposure to TB bacteria, the sensitized T cells on exposure to tuberculin
antigen, will release INF-γ. This released INF-γ can be measured by ELISA test.
3. Adenosine Deaminase (ADA) assay:
- ADA is an enzyme produced by lymphocytes in the presence of intracellular tubercle
bacilli.
- In case of effusion, ADA should be analyzed in fluid. ADA can be measured by
automated and semi-automated methods
- The cutoff value for ADAPleural fluid, abdominal fluid, Synovial fluid: 50 IU / ml.
CSF: 10 IU / ml
- Increased ADA level (above cutoff value) in fluid with higher lymphocyte count is most
likely associated with tuberculosis.

WORK:
1. Demonstrations:
● ZN stain smear – M. tuberculosis and M. leprae.
● Culture media – L.J. medium and Dorset egg medium.
● BCG vaccine, Tuberculin syringe& Armadillo.
162
2. Draw figures of various demonstrations.
3. Questions.
A. What are the microscopic differantiative features of M. tuberculosis and M. bovis ?
B. Classify atypical mycobacteria. How will you differentiate from M. tuberculosis ?.

ZN stain: ZN stain:
M. tuberculosis M. Leprae

Colony Dorset egg

Tuberculin syringe
on L. J . medium medium

163
164
 MUSCULOSKELETAL& SOFT TISSUE INECTIONS
PRACTICAL-25.
Laboratory diagnosis of Musculoskeletal & Soft Tissue infections

Various types of infections and their etiology:

Type of infection Description Aetiological agents
Bacteria: Staphylococcus aureus,
Infection and inflammation of Pseudomonas aeruginosa
1. Folliculitis
hair follicle Fungus: Candida albicans, T.rubrum,
Malassezia
2. Furuncles Skin infection with painful
Staphylococcus aureus
(Boil) swelling and collection of pus.
Individual boils clustered
together leading to large
3. Carbuncles Staphylococcus aureus
infected mass filled with fluid,
pus and dead tissue
Diffuse inflammation of
connective tissue with Staphylococcus aureus,
4. Cellulitis
involvement of dermal and Streptococcus pyogenes
subcutaneous layers of skin
Highly infectious bacterial skin
Streptococcus pyogenes
5. Impetigo infection commonly seen in
Staphylococcus aureus
pre-school children
Acute infection of skin and
superficial lymphatics. Lesions
6. Erysipelas Streptococcus pyogenes
are typically raised and
demarcated.
Also known as flesh eating
disease. It is a rapidly Streptococcus pyogenes, Staphylococcus
7. Necrotizing
progressing infection of skin aureus, Clostridium perfringens (may lead
infections
and subcutaneous tissue which to gas gangrene). Majority times it is a
(Necrotisingfascitis)
can spread across the fascial polymicrobial infection.
plane.
Infection at the site of surgery Staphylococcus aureus, Staphylococcus
8. Surgical site
which occurs within 30 days epidermidis, Pseudomonas aeruginosa,
infections
after operation. members of enterobacteriaecae family, etc.
Staphylococcus aureus is most common.
Collection of pus due to
9. Pustule / Abscess M.tuberculosis is associated with cold
inflammatory processes
abscess.
Only myositis in absence of other
surrounding site involvement is more
commonly associated with autoimmune
10. Myositis and Infection of muscle with or
disorders rather than directly due to
pyomyositis without pus collection.
infection. Pyomyositis is usually caused by
Staphylococcus aureus

11. Infection B. anthracis (cutaneous anthrax),

following animal Bartonella henslae (Cat scratch
contacts disease),E.rhusiopathiae (Erysipeloid)
12. Infection after Mixed aerobic and anaerobic bacteria,
animal bite Rabies virus

165
13. Infection after
Mixed aerobic and anaerobic bacteria
human bite
Chronic subcutaneous infection Eumycetoma: Caused by fungi
characteristically associated (e.g.Madurellamycetomatis)
14. Mycetoma
with multiple sinuses which Actinomycetoma: Caused by bacteria
discharge typical grains (e.g.Nocardia sp.)
Loss of epidermal & part of
15. Cutaneous ulcers B. anthracis, M. marinum,
dermal tissues.
Initially with Gram positive bacteria
Generally associated with
16. Burn wounds followed by Pseudomonas aeruginosa
bacteremia
&members of Enterobacteriaceae family.
17. Systemic a) Petechiae in meningococcaemia.
infections with skin b) Cutaneous ulcers & bullae in V.
manifestations vulnificus bacteremia.
Systemic disease primarily
18. Leprosy involving skin, peripheral Mycobacterium leprae
nerves and nasal mucosa.
Flat, non-palpable
discoloration of skin (<5 Dermatophytes
19. Macule
cm size). If size exceeds 5 Viral rashes (e.g. enterovirus, measles)
cm, is called as patch
Elevated lesions usually <5 • Virus:
20. Papule mm in size that can be felt or Molluscum contagiosum,
palpated
Warts (Human Papilloma
Multiple papules my virus)
become confluent to form
21. Plaque
plaque which are palpable • Parasite
lesions >5 mm scabies (Sarcoptesscabiei)
• Bacteria
Staphylococcus aureus,
22. Nodule Firm lesions >5 cm size Mycobacterium marinum
• Fungi
sporothrix
Fluid-filled lesions with a Herpes simplex virus,
23. Vesicle
diameter less than 0.5 cm varicella-zoster virus
• Bacteria
Clostridium
Fluid-filled lesions with a
24. Bulla Staphylococcus aureus
diameter more than 0.5 cm
• Virus
Herpes simplex virus
Dermatophytes
25. Scale Excess dead epidermal layer
Streptococcus pyogenes

Sample collection:
- Pus
- Aspirated fluid
- Swab
- Wound exudates
- Tissue biopsy

166
Diagnosis of specific etiological agent:

Staphylococcus aureus:
1. Gram staining: Gram positive cocci arranged in grape like clusters.
2. Culture:Culture media:
Blood agar: circular,smooth,beta hemolytic colonies
Nutrient agar: circular, smooth, convex opaque,golden yellow pigmented (localized
pigment production)
MacConkey agar: small, pinpoint pink colonies.
3. Biochemical reactions/Enzyme detection: Coagulase test, Catalase test, Mannitol
fermentation, Phosphatase production, DNase production.
Test S. aureus S. epidermidis S. saprophyticus
Coagulase production + - -
Golden yellow colony + - -
Acid from Mannitol + - -
Phosphatase + - -
Β Hemolysis on Blood agar + - -
Novobiocin sensitivity S S R

a). Coagulase Test:

Principle: These enzymes are of two types, soluble or bound. Both of them finally convert
fibrinogen into fibrin. There are two methods for this test.
Slide Method for Bound Coagulase:It detects bound coagulase which coverts
fibrinogen directly to fibrin without requiring a coagulase reacting factor. Clumping of
bacteria occurs within 10 seconds when suspension of organisms is treated with a drop of
undiluted human or rabbit plasma.
Test tube Method for Free Coagulase:it detects free coagulase which converts
fibrinogen to fibrin by activating a coagulase reacting factor present in the plasma.0.5 ml,
1:10 diluted citrated human or rabbit plasma is added to equal volume of bacterial broths.
Observe for coagulum at 4, 6 hours up to overnight.
b). Catalase Test: It is used to differentiate staphylococci (catalase positive) from
streptococci (catalase negative).

c). Phosphatase test:Culture grown on phenolphthalein diphosphate incorporated

medium is exposed to ammonia vapour. This gives pink colour to colony due to the
presence of free phenolphthalein. Most of pathogenic staphylococci produce phosphatase.

d). Mannitol fermentation: S. aureus ferment mannitol and produce acid

Streptococcus pyogenes:
Direct methods:
1. Gram staining: Gram-positive cocci arranged chain.
2. Culture:
Blood agar: β hemolysis and Bacitracin sensitive
Glucose broth: for blood culture
3. Biochemical Reactions:
1.Catalase negative.
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 2.Bacitracin sensitivity: Bacitracin (0.04 unit/disc) sensitive. Any zone around the disc is
considered sensitive.
Indirect methods:
1. A.S.O. titer (Antistreptolysin O titer): It is important in the investigation of post
streptococcal diseases. Titer more than 200 IU/ml is significant
2. Estimation of DNase B antibody: used for diagnosis of post streptococcal Acute
glomerulonephritis and Rheumatic fever. Titer more than 300 IU/ml is significant.

Pseudomonas aeruginosa :
1. Microscopy: Gram-negative motile rod.
2. Culture:Nutrient agar, MacConkey agar & Cetrimide agar (Selective Media)
Nutrient agar: Bluish green colonies due to pigment production by bacteria.
Types of pigment produced: Diffusible pigment
• Green blue (Pyocyanin),
• Yellow-green pigment (Pyoverdin),
• Red (Pyorubin)
• Black (Pyomelanin).
MacConkey agar: Colonies are colorless and lactose non-fermenter (LNF).
3. Biochemical Reactions:
• Oxidase test: Positive
• Citrate utilization test: usually positive
• Peptone water sugar: G S L M: No acid, No gas.
• TSI agar slant: Red /Red (no reaction)

WORK:
Demonstrations:
Staphylococcus:
Gram stain smear – Staphylococcus
Nutrient agar: Pigmented colony.
Blood agar: β hemolytic colony.
Biochemical Test: Catalase, Mannitol fermentation, and coagulase test

Streptococci:
Gram stain smear – Streptococci
Blood agar: Colony with hemolysis – Alpha, Beta and Gamma
Bacitracin sensitivity
Hemolysis – Alpha, Beta, Gamma on blood agar

Pseudomonas:
Gram stain smear – GNB
Peptone water/Nutrient broth: Pellicle
Nutrient agar : Pigmented colonies. MacConkey Agar: Pale colony
Biochemical tests – Oxidase and Peptone water sugar fermentation

Questions:
A. What is MRSA?
B. Describe various types of hemolysis on blood agar.
C. What is the importance of pseudomonas?
D. Enumerate the pigment produced by pseudomonas?
E. Name the selective media used for pseudomonas

168
169
Gram positive cocci
in chain
Pseudomonas
(Nutrient agar)

170
 Pseudomonas
(MacConkey agar)

171
 Musculoskeletal & Soft Tissue infections
PRACTICAL-26.
Laboratory Diagnosis of Anaerobic infections

Clostridium :Clostridia are anaerobic Gram positive spore bearing bacilli. Their natural
habitat is soil, some of them are found in large intestine of man and animals. A few of them
produce powerful exotoxins. They cause (1) Tetanus (cl. Tetani) (2) Gas gangrene (Usually a
mixed infection caused by Cl. perfringens, Cl. Oedematiens, Cl. Septicum). (3) Botulism (caused
by ingestion of contaminated food containg preformed toxin of Cl. botulinum)

Gas Gangrene:It is a rapidly spreading, edematous myonecrosis, occurring in association with

severely crushed wounds contaminated with pathogenic clostridia, particularly with Clostridium
perfringens.
Laboratory diagnosis of Gas gangrene:
Based on clinical diagnosis of gas gangrene, treatment should be started as early as possible,
laboratory diagnosis has the role only for confirmation of clinical diagnosis and species
identification.

Sample collection:
1.Necrotic tissue
2. Muscle fragments
3. Exudates from deeper parts of the wound.
Microscopy:
Thick boxcar shaped capsulated Gram positive bacilli without spore. Scanty pus cells is
characteristic feature.
Culture:
Grows in anaerobic condition.
Grows best at 37⁰ C but may grow at 45⁰ C
RCMM:Saccharolytic activity, meat particle is turned pink.
Blood agar: Target hemolysis (narrow zone of theta toxin & wide zone of Alfa toxin.
Biochemical reaction:
Predominately saccharolytic activity: meat particle is turned pink.
Mild proteolytic activity: So does not digest meat particle but liquefy gelatin.
Litmus milk: Stormy fermentation.
Toxin detection:
Nagler’s reaction:Cl. perfringens grown on 20% human serum or egg yolk agar with Cl.
perfringens antitoxin spreaded on one half of plate. Colonies in half without antitoxin will be
surrounded by zone of opacity.

Tetanus:It is an acute disease, manifested by skeletal muscle spasm and autonomic nervous
system, caused by Clostridium tetani.

Laboratory diagnosis of tetanus

Based on clinical diagnosis of tetanus, treatment should be started as early as possible, laboratory
diagnosis provides supportive evidence for confirmation.

Sample collection:
Excised tissue bits from depth of wounds.

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Microscopy:
Gram positive bacilli slender with spherical terminal spore (drumstick appearance).
Culture:
Grows in anaerobic condition
Can grow in simple media.
RCMM: Meat particle not digested or fermented.Mild proteolytic activity: So does not
digest meat particle but liquefy gelatin
Blood agar: Swarming growth.
Toxigenicity test: In animal to demonstrate tetanospasmin.
Culture filtrate is inoculated in tail root of test mouse. Control animal received tetanus antitoxin
serve as a control. Symptoms develops in test animal in 12-24 hours beginning with stiffness of
tail.

WORK:
Demonstrations:
Gram stain smear – Gram-positive bacilli.
Anaerobic media
Draw the figure of :C. perfringens &C.tetani in Gram`s stain
Questions:
1. Enumerate the toxins of C. perfringens
2. What is Nagler’s reaction?
3. What is gas gangrene?
4. What is the mechanism of tetanus toxin?

C. perfringens C. tetani
(Gram stain) (Gram stain)

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 Central nervous system infections
PRACTICAL-27.
Laboratory Diagnosis of Central Nervous System Infections

Central nervous system infections comprises of following clinical diseases:

Meningitis: It is an inflammation of the leptomeninges surrounding the brain and spinal cord,
with involvement of the subarachnoid space.
Encephalitis: It is an acute inflammation of the brain parenchyma by invasion of infectious
agents, most often the viruses.
Encephalopathy: It is a general term used for any diffuse diseases of the brain that alters the brain
function or structure.
Types of meningitis with etiological agents

Type of meningitis Characteristic Etiological agents

Acute pyogenic Acute inflammation of Newborn:

meningitis meninges Group B Streptococci
CSF = Large no. of pus cells E. coli
(Polymorphs) Klebsiella pneumoniae
Pseudomonas aeruginosa
Listeria monocytogenes
Children:
Haemophilus influenzae type b
Neisseria meningitidis
Streptococcus pneumoniae
Adults:
Neisseria meningitidis
Streptococcus pneumoniae
Listeria monocytogenes
Hospital acquired:
Pseudomonas aeruginosa
Staphylococcus sp.

Tuberculous Secondary to tuberculosis Mycobacterium tuberculosis

meningitis lesion

Aseptic meningitis CSF = Negative bacterial Herpes simplex viruses

culture Japanese encephalitis virus
Increased lymphocytes and Enterovirus
pleocytosis Cryptococcus neoformans
Toxoplasma gondii
Free living amoeba
Treponema pallidum
Leptospira

174
Laboratory Diagnosis of bacterial meningitis:
Sample collection:
1. Cerebrospinal fluid
2. Blood for culture in septicaemia
Transportation of sample: Transport sample as early as possible. Never refrigerate the
sample. It should be kept at 35º-37ºc.
Processing of the sample:
1. C.S.F. sample is collected in three sterile test tubes- one each for cell count, biochemical
analysis and bacteriological examination
2. The smears are prepared directly from the sample if it is turbid. if slight cloudy, centrifuge
the sample and sediment is used for preparation of smears and culture

1. Microscopic examination
a. Gram stain: to identify pyogenic bacteria
b. Z N stain: to detect M.tuberculosis
c. Negative stain (India ink or nigrosin): to detect Cryptococcus neoformans
d. Wet film: to detect parasites like Naegleria fowlerii, Acanthamoeba, Toxoplasma gondii

Appearance in CSF Gram stain Suggestive of

Gram-positive cocci in pair, capsulated, flame or lanceolate- Streptococcus
shaped pneumoniae
Gram-negative diplococci, capsulated, with adjacent sides
Neisseria meningitidis
flattened (lens or half-moon shaped).
Pleomorphic Gram-negative coccobacilli, capsulated Haemophilus influenzae
Escherichia coli or others
Gram-negative bacilli, arranged singly
GNB
Gram-positive cocci in short chain Streptococcus agalactiae
Gram-positive short bacilli Listeria monocytogenes

2. Culture examination
CSF:
a. set of culture plates including blood agar, chocolate agar and MacConkey agar are
inoculated directly from sample and incubated at 37° C in candle jar (or CO2
incubator).
b. CSF is centrifuged with precautions to avoid contamination and the deposit is
inoculated in a similar set of culture plates.
c. 1-2 ml of CSF is inoculated in a liquid medium ( i.e. Glucose/BHI broth ) or in
automated culture system bottle. This will allow growth of even minute number of
bacteria if present. When the growth is indicated or observed in liquid medium, it is
subcultured in similar set of plates to isolate them. The isolated bacteria are processed
for their identification and antibiotic sensitivity testing.
Blood: Blood culture can be collected in conventional blood culture bottles – Glucose/BHI
broth or preferably in automated blood cultures (e.g.BacT/ALERT)
The culture plates are observed for growth after 24-48 hours of incubation. The
isolated bacteria are processed for their identification and antibiotic sensitivity
testing.

175
3. Detection of antigens or antibodies: This is generally done from the supernatant of
the centrifuged CSF sample.
Capsular polysaccharide of aetiological agent may be detected by latex agglutination.
The following capsulated organisms may be detected by this method,
- N.meningitidis (Meningococci),
- Haemophilus influenzae,
- Streptococcus pneumoniae (pneumococci)
- Cryptococcus neoformans

4. Detection of antibody:
Antibodies are demonstrable in the following infections, which can be detected by ELISA or
immunofluorescence
- Herpes simplex virus
- Japanese encephalitis virus
- T. pallidum (neurosyphilis)

CSF chararacteristic in Meningitis:

Characteristics Pyogenic meningitis Tuberculous meningitis Viral meningitis

CSF pressure Highly elevated Moderately elevated Slightly

(mm of water) (>180) elevated/normal
Total leukocyte 100–10,000 10–500 25–500
count (per mm3)
Predominant cell Neutrophils Lymphocytes Lymphocytes
type
Glucose (mg%) <40 mg/dL 20–40 mg/dL Normal
(decreased to absent) (slightly decreased)
Total proteins >45 mg/dL 100–500 mg/dL 20–80 mg/dL
(mg%) (usually >250; (moderate to markedly (normal or slightly
markedly increased) increased) elevated)

Streptococcus pneumoniae
⮚ Culture: It produces α-hemolytic colonies on blood agar, described as
draughtsman-shaped or carrom coin appearance
⮚ Biochemical identification: It shows bile soluble, ferments inulin and sensitive to
optochin

Neisseria meningitidis
⮚ It produces non- hemolytic colonies on blood agar, which on smear shows gram-
negative diplococci
⮚ Biochemical identification: Meningococci are catalase and oxidase positive. They
ferment glucose and maltose but not sucrose
⮚ Serogrouping: Slide agglutination serogrouping (SASG) test is done to identify the
serogroups of meningococci isolates by using appropriate antisera

176
 Haemophilus influenzae
⮚ Culture: Blood agar with S. aureus streak line shows satellitism.
⮚ Biochemical identification: Disk test for X and V factor requirement shows growth
surrounding combined XV disk

Streptococcus agalactiae
⮚ Culture: It produces β-hemolytic colonies on blood agar, which on smear shows
gram-positive cocci in short chain
⮚ Biochemical identification: It shows CAMP test positive and resistance to
bacitracin
⮚ Serogrouping with group specific antisera shows Lancefield group B

Gram-negative bacilli meningitis

⮚ Escherichia coli and Klebsiella produce lactose-fermenting colonies on
MacConkey agar; identified by standard process.

WORK:
Demonstrations:
Gram stain smear: N. meningitidis
Crptococcal growth on SDA.
Questions:
1. Enumerate bacteria producing meningitis in neonate.
2. How to differentiate morphologically Candida from Cryptococcus.

N. meningitidis
(Gram stain) Cryptococcus
(India ink)

Cryptococci on
SDA

177
 Genitourinary Infections
PRACTICAL-28.
Laboratory diagnosis of sexually transmitted infections

Types of STI with etiological agents:

Sexually transmitted Disease Etiological agents
infections
Syphilis Treponema pallidum
Chancroid Haemophilusducreyi
Genito-ulcerative
Genital herpes Herpes simplex viruses
disease
Lymphogranuloma venereum Chlamydia trachomatis
Donovanosis Klebsiella granulomatis
Gonococcal urethritis Neisseria gonorrhoeae
Chlamydia trachomatis (D-K)
Ureaplasmaurealyticum
Urethritis Mycoplasma genitalium
Non-gonococcal urethritis
Herpes simplex viruses
Candida albicans
Trichomonas vaginalis
Genital warts Condyloma acuminatum Human papilloma virus
Bacterial vaginosis
Trichomonas vaginalis
Vulvovaginitis
Candida sp. and non albicans
spp
Other Pelvic inflammatory disease Neisseria gonorrhoeae
(PID) Chlamydia trachomatis (D-K)
HIV
Systemic
Hepatitis B Virus
infections
Hepatitis C Virus

Genital ulcer:
Disease Genital ulcer Lymphadenopathy
Syphilis Painless, indurated single Painless, moderate swelling (no bubo)
Herpes Multiple, painful Absence or moderate swelling (no bubo)
Chancroid Painful, soft single or multiple Painful, soft, marked swelling leads to bubo
LGV Painless Painful and soft
Absent (pseudo bubo may be present due to
Donovanosis Painless beefy red ulcer
subcutaneous swelling)

Laboratory Diagnosis
 Specimen Collection
• Discharge from the infected area - vaginal or urethral discharge in a sterile container
• Sterile swabs (if the discharge scanty): Charcoal impregnated swabs for Gonococcal
infection
• Fluid from the vesicles: genital herpes
• Blood in plain tube: serological test e.g. syphilis

178
 Transport:
• Immediately: Most of organisms are delicate, not viable for long outside body
• Transport medium: Modified Stuart’s medium
 Microscopy:
• Wet mount examination: vaginal discharge
- Trichomoniasis: Pus cells along with motile trophozoites
- Candidiasis: budding yeast cells along with pseudo hyphae
• Gram-stained smear:
• Bacterial vaginosis—clue cells (vaginal epithelial cells studded with gram variable
pleomorphic coccobacilli) :Gardnerella vaginalis
• Gonorrhea—intracellular kidney-shaped gram-negative diplococci
• Candidiasis—gram-positive budding yeast cells along with pseudo hyphae
• Giemsa stain:
• Klebsiella granulomatis - Donovan’s bodies (macrophages filled with bipolar stained
bacilli)
• Chlamydia trachomatis - inclusion bodies
• Dark field microscopy and silver impregnation (Fontana’s method) –
• syphilis - spirally coiled bacilli
 Culture: Specimens are inoculated onto the appropriate culture media/cell line
• Thayer-Martin medium—for N. gonorrhoeae
• Chocolate agar added with isovitalex and vancomycin— for H. ducreyi
• McCoy cell line—for Chlamydia trachomatis
• Sabouraud’s dextrose agar (SDA)—for Candida species
• Vero cells, monkey kidney cell line - herpes simplex virus.
 Serology:
• VDRL or RPR test, TPHA, TPI -syphilis
• CFT-chlamydia infection
 Molecular Test
• Multiplex PCR and real-time PCR: For simultaneous detection of pathogens such as:
C.trachomatis, Gonococcus, T. pallidum and H. ducreyi and HSV.

NEISSERIA GONORRHOEAE:
Characteristics:
Non-motile, NON CAPSULATED, Gram-negative diplococci with fastidious growth
requirements& capnophilic.
Diseases:
• Gonorrhea
• Pelvic inflammatory disease
• Salpingitis in females
• Ophthalmia neonatorum in infants
Transmission:
• Human pathogens; no animal reservoir.
• This may be carried in genital tract, nasopharynx and anus.
• Spread by sexual or intimate contact.
LABORATORY DIAGNOSIS:
Sample collection:
1.Urethral discharge collected by calcium alginate swab
2.Cervical swab from non-puerperal women
Transportation of sample: Samples are transported in Amies transport medium.

179
 Direct methods:
1.Gram staining: Gram-negative diplococci with concave adjacent sides (kidney shape),
seen intracellularly in polymorphonuclear cells.
2. Culture: Culture media: Chocolate agar, Modified Thayer martin medium Grow well at
37˚ C in moist atmosphere containing 5-10% Co2
3. Biochemical reactions:
• Oxidase test positive
• Sugar fermentation test: Glucose: Acid-No gas. Maltose: No reaction
4. Antibiotic sensitivity test

Indirect methods: Direct fluorescent antibody test and Slide agglutination test

GENUS TREPONEMA
Regularly coiled spirochetes with a longer wavelength than Leptospira. Several species and
subspecies are important human pathogens; others are members of the normal flora, especially in
the mouth. T. pallidum and its subspecies pertenue and T. carateum are most important species.
Characteristics:
Individual cells too small to visualize by direct light microscopy;
Can be seen with dark ground illumination, or after silver impregnation or immunofluorescent
staining. Cells are actively motile by means of flagella contained within periplasmic sheath
(Endoflagella).
Transmission:
Very susceptible to heat and drying, so successful transmission depends upon very close contact.
T. pallidum is spread by close sexual contact and may also be vertically transmitted in utero.
Yaws and pinta spread by direct contact from infected skin lesions. No animal reservoir.
Diseases:
• T. pallidum: Syphilis.
• T. pallidum: Pertenue
• T. carateum: Non-sexually transmitted treponematoses, yaws and pinta.
Identification:
T. pallidum and closely related species cannot be grown in artificial media; diagnosis of
infection depends upon microscopic examination of fluid from primary lesions (Dark field
microscopy and silver impregnation-Fontana’s method) and on serology (VDRL or RPR test,
TPHA, TPI).

Work:
Demonstration:
Gram stained slide of Gonococci
Serological test for syphilis (RPR)
Draw the diagram of: N. gonorrhoeae (Gram stain), T. palladium (Fontana stain)
Questions:
1. Describe in brief the morphology of Treponema.
2. Enumerate different clinical stages of syphilis.
3. Enlist standard test for syphilis.
4. What is BFP in RPR test?
5.Difference between VDRL and RPR.

180
 N.
T. palladium
gonorrhoeae(Gra
(Fontana stain)

181
 Genitourinary Infections
PRACTICAL-29.
Laboratory Diagnosis of Urinary Tract Infections (UTI)

UTI: Disease caused by microbial invasion of the urinary tract that extends from the renal cortex
of the kidney to the urethral meatus.
Classification:lower UTI and upper UTI - depending upon the anatomical sites involved
Lower UTI Upper UTI
Sites involved Urethra and bladder Kidney and ureter
Symptoms Local manifestation: Local and systemic manifestation
dysuria, urgency, (fever, abdominal pain, vomiting)
frequency
Route of spread Ascending route Both ascending (common) and
descending
Occurrence More common Less common

Definition
• Bacteriuria-Presence of bacteria in the urine
• Pyuria-Presence of pus cells/WBCs in the urine
• Significant bacteriuria: Normal urine is sterile; it may get contaminated during voiding with
normal urethral flora. A count of >105 CFU count/ml of mid-stream urine is considered to be
significant in most cases (Kass concept)
• Asymptomatic bacteriuria: Without symptoms, Common in pregnancy (5-7%)
• Lower UTI:
 Urethritis – Dysuria(sensation or pain during micturition), frequent urination, urgency
 Cystitis–Dysuria, frequent urination, Suprapubic pain, tenderness and occasionally
hematuria.
• Upper UTI
 Acute Pyelonephritis-flanks pain, renal tendernessBurning, hematuria, fever, chills and
vomiting(Systemic manifestation)+ symptoms of lower UTI present
Etiology of UTI
• E. Coli is commonest bacteria (70%) causing UTI without catheter and urological
abnormalities or calculus
• Other bacteria like Klebsiella, Proteus mirabilis, Enterobacter, Enterococcus spp.,
Staphylococcus aureus, Pseudomonas aeruginosa, Acenatobacter, Staphylococcus
saprophyticus- (in sexually active female), M.tuberculosis, fungus- Candida and parasite-
Trichomonas vaginalis, Schistosoma haematobium may be responsible for UTI
Note:
 Bacteriuria without Pyuria:Enteric fever, Bacterial endocarditic, diabetes
 Pyuria without bacteriuria: Renal tuberculosis, Gonococcal urethritis, Chlamydia
trachomatis infection, Ureoplsma, Leptospirosis, Trichomonas, patient treated with
antimicrobials, urological abnormality
Laboratory diagnosis:
Specimen collection: Urine should be collected in wide mouth screw capped sterile container
• Clean Catch Midstream early morning Urine:
 Female: Clean area with soap and water hold labia apart and begin voiding in commode.
After several ml have passed collect mid-stream urine
 Male: clean glans penis with soap and water, retract foreskin. After several ml have
passed collect mid-stream urine
182
 • Suprapubic aspirate:
 In neonates and small children when urine is difficult to collect
 Needle aspiration above symphysis pubis through abdomen into bladder.
• In catheterised patients:
 Urine aspirated from catheter tube after clamping distally and disinfecting through
syringe and needle
 Never collect from urine bag
Transportation
• Label and transport as early as possible to laboratory
• If not possible: if delay > 2 hours, refrigerate the urine at 4-6°C or add boric acid
preservative (0.1gm/10 ml urine)
Microscopic Examination:
a). Wet preparation:
• Pus cells:> 10 pus cells/HPF correlate with significant infection
• RBCs: Stone, Acute glomerulonephritis
• Crystals-Calcium oxalate: stone
• Epithelial cells- if in large no -inflammation or vaginal contamination of urine
• Yeast cells- Candida
• Motile trophozoites: Trichomonas vaginalis
• Egg: Schistosoma haematobium.
b). Gram stain: In gram stain presence of at least one bacterium in oil immersion field
(examining 20 fields) in uncentrifuged urine correlate with significant bacteriuria
(>105 /ml)
Culture:
Media used for isolation:
• Nutrient agar: Used for colony count & demonstration of pigment production by
bacteria
• MacConkey agar: Differentiate lactose fermenter from non-lactose fermenter.
• CLED agar (cysteine lactose electrolyte deficient agar)
 Allow to grow Gram positive &Gram negative bacteria.
 Swarming of Proteus is inhibited.
Method for isolation:
1. Semi-quantitative Urine Culture by Standard Loop: Known volume of uncentrifuged
mid streamed urine is streaked by calibrated wire loop on surface of solid medium. Plate
is incubated at 37⁰C degree for 24 hours . Colony developed is counted and expressed
as colony forming unit per ml of urine (CFU/ml of urine).If colony count >105CFU/ml
then it is considered Significant bacteriuria.
2. Pour plate method:Culture & colony count by Pour plate method (not commonly
used).
Identification: bacteria isolated on culture plates are identified by Gram stain, motility and
biochemical reactions
Antibiotics Sensitivity Test: By disc diffusion method
 Routinely following antibiotics for UTI are used
• Quinolones (Norfloxacin, Ciprofloxacin, Levofloxacin),
• Nitrofurantoin,
• Ampicillin/Sulbactam, Amoxicillin/clavulanic acid,
• Cotrimoxazole,
• cefuroxime,
• Aminoglycosides (Amikacin, Gentamicin)
• Novobiocin: to identify Staphylococcus saprophyticus.

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 Note:
• For Proteus spp - Do not use Nitrofurantoin and tetracycline
• Chloramphenicol must not be tested / given for UTI

Demonstration of Significant bacteriuria: Semi Quantitative Method: Standard loop

technique :

• Un-centrifuged urine(0.01 ml)with loop of 4 mm diameter is streaked on Nutrient agar plate.

• Plates incubated at 37⁰C degree for 24 hours.

• Colony counts (CFU/ml of urine) = number of colonies developed on Nutrient agar X 100.
• If colony count >105CFU/ml then it is considered Significant bacteriuria

Culture smear
Organisms Culture and motility Biochemical reactions
testing
Mac: Lactose Gram stain: GNB Oxidase: -
fermenting Motility: Motile IMViC: + + - -
Escherichia coli colony GSLM: AG AGAGAGAG
TSI: A/A Gas+, H2S: -
U- PPA-
Mac: Lactose Gram stain: GNB Oxidase: -
fermenting Motility: Non IMViC: - - + +
Klebsiella Spp. colony motile GSLM: AG AGAGAGAG
TSI: A/A Gas+, H2S: -
U+ PPA-
Mac: Non Gram stain: GNB Oxidase: -
Lactose Motility: Motile TSI: A/Alk Gas+/-, H2S: +
Proteus species fermenting U+ PPA+
colony
BA: Swarming

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Pseudomonas Spp. Mac: Non Gram stain: GNB Oxidase: +
Lactose Motility: Motile GSLM: - - - -
fermenting TSI: Nil/Alk Gas-, H2S: -
colony U- PPA-
NA: Diffusible
Bluish-Green
pigment
Staphylococcus NA: Golden Gram stain: GPC Catalase: +
aureus yellow colony in cluster Coagulase: +
BA: β Mannitol fermentation: +
hemolytic
Staphylococcus NA: White Gram stain: GPC Catalase: +
saprophyticus colony in cluster Coagulase: -
BA: Non Mannitol fermentation: -
hemolytic Novobiocin: Resistant
Enterococcus Spp. NA: Pin point Gram stain: GPC Catalase: -
colony in chain or pair Bile aesculin: +
Mac: Lactose (spectacle shaped)
fermenting
BA: Non
hemolytic

Work
Demonstration:
Standard loop
E. coli, Klebsiella colony on MacConkey agar
Proteus colony on MacConkey& Nutrient agar (swarming)
Pseodomonas colony on Nutrient agar (Pigment production)
Enterococci - Gram stain, colony on MacConkey & Blood agar.
Draw the diagram: of Semi-quantitative technique of Urine Culture by Standard Loop
Questions:
A.. Instruction to be given to patient for proper collection of urine sample.
B Transportation of urine sample for culture
C. Define significant bacteriuria.

Semi-quantitative Urine Culture by Standard Loop

Miscellaneous
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 PRACTICAL-30.
Microbiology of Water, Air and Food

A. Water
Purpose of drinking water testing
• For detection of fecal contamination of drinking water supply
• For assessing the quality of water purification system
• For investigation of outbreak of waterborne diseases
Water borne pathogens

Microorganisms Water borne pathogens

Bacteria ● Vibrio cholerae
● Salmonella Typhi, Salmonella Paratyphi
● Shigella sp.
● Yersinia enterocolitica
● Campylobacter jejuni
● Diarrheagenic E coli

Viruses ● Hepatitis A virus

● Hepatitis E virus
● Poliovirus
● Rotavirus

Protozoa ● Entamoeba histolytica

● Giardia lamblia
● Balantidium coli
● Cryptosporidium
● Isospora
Helminths ● Ascaris lumbricoides
● Enterobius vermicularis
● Trichuris trichiura

Indicator organisms
Coliforms: Most reliable indicator of potable water quality
Fecal thermotolerant E. coli: Best indicator(confirmation) of recent fecal contamination of
water
Fecal Streptococci: Confirm remote fecal contamination
Clostridium perfringens :Remote fecal contamination
Pseudomonas aeruginosa: Less reliable indicator, useful in hospital settings
Bacteriophages: Indicate fecal pollution and viral pollution.

Specimen collection:
Sample should be collected in sterilized screw cap bottles. At least 150 ml of water should be
collected.

Water collection from tap:

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 Open the tap fully and allow the water to flow for at least 2 minutes before collection.
Open the screw cap bottle and collect the water sample directly from the free flow of tap
Fill at least 2/3rd portion of the bottle
Close the screw cap tightly, label the location of collection and send it to the Microbiology
laboratory

Methods of water analysis:

Presumptive coliform count (Multiple tube method)

Differential coliform count (Eijkman test)

Fecal streptococci detection

Clostridium perfringens detection

Membrane filtration method

Examination of specific water borne pathogens

Presumptive coliform count (Multiple tube method):

• Also called as MPN count (Most probable number), a statistical method used to estimate the
viable numbers of coliform bacteria in sample of water.
• Used for checking quality of potable water

Standard table for MPN count of coliform bacteria

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188
 Result:

Quality of water Presumptive coliform E.coli count per 100 ml

count per 100 ml of water of water
Excellent 0 0
Satisfactory 1-3 0
Suspicious 4-10 0
Un satisfacory >10 0.1 or more

B. Air
Purpose of microbial examination of air
Air is very important vehicle for transmission of pathogen. Human is the important source of
spreading bacteria in the environment during coughing and sneezing.
The bacterial content in the air depends on increased density of human population.
Important in Critical areas where the safe working depends on the air’s bacterial count
being kept at very low level.
Critical areas:
• Operation theaters (OT)
• ICUs

Methods:
• Settle plate method: Microorganisms settle on to the surface of the plate via gravity.
Blood agar plate exposed to OT air for half hour, lid closed and incubated overnight at
37⁰ for bacterial colony formation. Colony count should be <12.
• Slit sampler method: Quantitative culture by air sampling device

C. Food
Purpose of food examination:
• For the investigation of outbreak of foodborne diseases.
• For the identification of organisms causing food poisoning

Methods:
Food material is homogenized in sterile solution like Ringer’s solution and cultured onto
appropriate medium. Vomitus and stool can be collected for microbiological examination.

Work:
Demonstrations
Multiple tube method for water culture
Methods of air sampling
Questions:
1. Enlist the various water borne diseases
2. Mention indicator organisms for fecal contamination of water
3. Enlist methods used for bacteriological examination of water
4. Enlist various organisms causing food poisoning and its common food sources.

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 Miscellaneous
PRACTICAL-31.
Confidentiality In Laboratory Results

Patient confidentiality:
Confidentiality is the right of an individual to have personal, identifiable medical
information kept private. It means that personal and medical information given to a health care
provider will not be disclosed to others unless the individual has given specific permission for
such release. Confidentiality respects patient privacy and autonomy.
Ethically, confidentiality is derived from the principles of autonomy (the patient determines
who shall know his or her medical history) and fidelity (the fiduciary relationship of the patient
and physician requires trust and confidence). Confidentiality allows the physicians to obtain all
the information necessary to make a complete diagnosis and motivate the patient to participate in
therapy.
The patient has the right to confidentiality. The physician should not reveal confidential
communications or information, without the consent of the patient, unless provided for by law,
for the need to protect the welfare of the individual, or for public interest.The confidentiality of
physician-patient encounters is a basic medical ethic, reflected in the Hippocratic Oath.

What information is confidential?

All identifiable patient information whether written, computerised, visual or audio recorded
or simply held in the memory of health professionals, is subject to the duty of confidentiality.
Confidentiality of Laboratory results must be maintained at all times.

Give medical information to patient first, Not the family/friends

The doctor must convey medical information to the patient first. Without direct instruction
from the patient, the family/friends should never receive the patient's confidential medical
information. It is patient’s decision whether he/she wants her family/friends to know about
his/her medical condition. There is a rare exception in the case of a patient with a psychiatric
illness whom to inform of the medical illness may induce a suicidal attempt.

Release of information to family, friends or colleagues

Confidentiality also includes keeping a patient's medical information private even from his
friends and family unless the patient expressly consents that it is okay to release the information
to them. The patient may have a good relationship with his family and friends but this is
absolutely no excuse to assume that the patient wants his/her medical information passed on to
them. It may seem rude and unreasonable but you must tell the patient's family members/friends
that you must ask your patient for permission before you release patient’s medical information.
The doctor must keep the medical information private from a patient's co-workers as well.

Release of information to other doctors

Information transfer between doctors involved in the care of patient is common. However,
the medical information about the patient can only be transferred if the patient has signed a
consent form requesting the transfer of information. The patient must sign the consent form, not
the health care providing doctor. The referred doctor must demand for the signed consent form
for release of information by the patient from the referring doctor.

Release of information to the governmental organizations and courts

If the doctor receives a court order from authorized law personnel, that constitutes a search
warrant then the doctor must furnish the information that he/she requests. If the investigator does
not have a search warrant, then the doctor must refuse him access to the patient’s files. The
doctor is not under any obligation to provide any information related to patient to third parties
unless it is at the request of the patient.
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 Exceptions to confidentiality:
The principle of confidentiality is never absolute and has always been subject to limits in the
interest of society, public welfare, and the rights of other individuals. The patient’s right to
confidentiality is less important than another person’s right to safety. In such cases the
confidentiality can be broken in order to protect others. For example, a mentally ill patient tells
the psychiatrist that he/she intents to harm someone. In such case, the psychiatrist must inform
the law enforcement and the potential victim. Other case where it is lawful to break the
confidentiality includes partner notification for sexually transmitted diseases such as syphilis and
HIV infections.

Exceptions to patient confidentiality include the following:

• Suicidal/homicidal patients
• Abuse (children, elderly, and/or prisoners)
• Duty to protect- State-specific laws that sometimes allow physician to inform or somehow
protect potential Victim from harm.
• Epileptic patients and other impaired automobile drivers.
• Reportable Diseases (eg, STDs, hepatitis, food poisoning); physicians may have a duty to warn
public officials, who will then notify people at risk. Dangerous communicable diseases, such as
TB or Ebola, may require involuntary treatment.

Medical records
The doctor/health care facility physically owns the medical record, but the information
within it is the property of the patient. The patient has an absolute right to free access to the
information it contains without providing any reason to the doctor/health care facility. The
information within medical records is covered by the same rules of confidentiality. You cannot
release the information within medical records without patient’s consent to anyone. No one
except those involved directly with patient care has a right to access the information within
patient’s medical records. Doctors/health care facility cannot hold medical records as “hostage"
to compel a patient to pay medical bills. The need of information to take care of patient is more
important than the doctor’s right to payment.

How to maintain confidentiality?

Doctors often violate ethics not because they mean to, but because they are careless. As a
matter of fact, they sometimes act with good intentions.
• Handle medical records as confidential documents.
• Do not leave patient information and laboratory results unattended on printers, desks etc.
• Protect information on Computer screens by screen saver / time out functionalities.
• Should a person call requesting results and there is a question about the person’s identity, the
requestor is asked for his/her name and phone number where they can be called back.
• Check that fax numbers are correct before sending confidential information and laboratory
results.
• Patient information should never be discussed with friends or relatives in a social setting.
• Do not discuss with family or friends patients details and if asked inform them that you are
not permitted to disclose any information. This includes patient names.
• Do not discuss patient information with the media.
• Every health care organization should have a policy that defines confidentiality and delineates
who is responsible for maintaining it. A good policy will state that every person who works for
the organization is responsible for ensuring patient confidentiality and for reporting policy
violations. It also will state that managers are responsible for implementing and enforcing the
policy as it pertains to their areas. Information about patients should be accessed or discussed
only on a need-to-know basis, according to job duties. To protect against lawsuits, organizations
should present the policy to new employees in orientation, and have all employees sign a
statement that they are aware of the policy.
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