BACTERIOLOGY: REVIEW NOTES

Gram negative cocci (aerobes): Branhamella, Neisseria

BACTERIOLOGY Gram negative cocci (anaerobes): Veilonella
Gram positive bacilli (aerobic): Bacillus, Corynebacterium, Erysipelothrix,
Lactobacillus, Listeria, Mycobacterium, Nocardia
Bacteria are single cell prokaryotic microorganisms. Gram positive bacilli (anaerobic): Actinomyces, Clostridium, Cutibacterium
(previously Propionebacterium)
They divide by binary fission. Gram negative bacilli (aerobic): Acinetobacter, Aeromonas, Alcaligines,
Bordetella, Brucella, Enterobacteriaacea, Francisella, Legionella,
Pasteurella, Pseudomonas, Vibrio
TAXONOMY Gram negative bacilli (anaerobic): Fusobacterium, Bacteroides

Prokaryotes – w/out true nucleus BASIC STRUCTURES

A. Eubacteria (true bacteria)
B. Cyanobacteria (blue-green algae) Cell wall (murein layer) – defines the shape of the
C. Archaebacteria (survive in extreme environments bacteria, some components are responsible for
e.g. hotsprings, volcanic vents) pathogenicity (eg. M protein – S. pyogenes, A protein
– S. aureus and mycolic acid - Mycobacteria). Main
BACTERIAL CELL – comprises mostly with 70% constituent is PEPTIDOGLYCAN.
water, 30% carbohydrates, proteins, lipids, and
enzymes etc. GRAM STAINING
*size 0.5-1 micron

Methods employed in the identification of bacteria in

the laboratory
1. Morphology and Staining of bacteria (microscopic)
2. Culture
3. Biochemical reactions – enzymes within the
bacterial cell
4. Serological – antigen and antibody reactions
5. Animal inoculation

GENERAL RULES FOR BACTERIA

1. All cocci are gram (+) except Neisseria, Branhamella
(Moraxella) (both aerobic gram (-)) and Veilonella
(anaerobic gram (-))
2. All bacilli are gram (-) except Bacillus (aerobic) and
Clostridium (anaerobic) (ONLY SPORE FORMING), GRAM POSITIVE GRAM NEGATIVE
Mycobacterium, Corynebacterium, Listeria, Teichoic acid (Mg++) Outer membrane (bilayer
Erysipelothrix (gram (+) bacilli H2S +), Lactobacillus, sheet of LPS) with porins
Kurthia, Rothia, Peptococcus, Peptostreptococcus,
and LPS
Cutibacterium (previously Propionebacterium - gram
(+) anaerobic) and fungus like bacteria: Nocardia Thick peptidoglycan peptidoglycan
(aerobic) and Actinomyces (anearobic)
3. Spirals or Spirochetes (BLT: Borrelia, Leptospira Mucin or MPLS layer Periplasmic space
and Treponema) are hard to stain -> gram (-).
4. NO cell wall: Mycoplasma and Ureaplasma -> gram Cytoplasmic membrane Cytoplasmic space
(-)
5. Acid fast organisms stain as gram ghost (gram (+)) Stains violet Stains pink
e.g. Mycobacteria other AFB include Nocardia,
Rhodococcus and Legionella micdadei.

Gram positive cocci (aerobes): Micrococcus, Staphylococcus,

Streptococcus
Gram positive cocci (anaerobes): Peptococcus, Peptostreptococcus,
Sarcina

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 BACTERIOLOGY: REVIEW NOTES

Gram positive Gram negative Special stains – these methods demonstrate the
PS: Crystal violet different bacterial structure.
Mordant : Grams’ Iodine A. Cell wall – Djar method
Decolorizer: 95% alcohol colorless B. Capsule – Tyler, Anthony, Grin, Welch, Hiss
CS: Safranin C. Spore – Acetic acid, Dorner’s, Wirtz, Schaeffer and
*do not dip, over wash
Fulton
*Teichoic acid - magnesium RNA – iodine – CV = gram positive
*increased lipids which are soluble to alcohol = gram negative D. Flagella – Gray, Leifson, Caesares-Gil, Loeffler’s,
Van Ermenger, Fisher and Cohn
ACID FAST STAINING E. Metachromatic granules – Albert, Neisser,
Ljubensky, LAMB
Ziehl- Kinyoun AFB Non-AFB
Neelsen (Cold) INSIDE THE CELL WALL
(HOT
method) Cytoplasmic Membrane - site of energy production
PS Carbol fuchsin
Mordant Heat Tergitol
among prokaryotes, selectively permeable
Decolori Acid alcohol (3% HCl in colorless
zer (95% ethanol) Mesosomes – point of attachment of chromosomes
Counter Methylene Malachite ZN KN
stain blue green Inclusions
*Principle: Acid fast bacteria are hard to stain due to mycolic acid
but once stained these bacteria are hard to decolorize.
1.Muchs granules – lipids, Mycobacterium tuberculosis
*mycolic acid or hydroxymethoxy acid = AFB, 2x3 size of smear 2. Babe Ernst Granules/Metachromatic granules/Vo-
with coiling using coconut midribs lutin granules polyphosphates, C.diptheriae (stains:
*wash only with Distilled water – contamination with Mycobacterium Burkes modification of gram stain and LAMB –
gordonae (tap water bacillus) Loefflers Alkaline Methylene Blue)
*start timer when steam is visible, filter stain – Carbol fuchsin with
precaution (carcinogenic) 3. Bipolar bodies – Yersinia pestis,Wayson stain
4. Ribosomes
Methods to facilitate staining:
1. Steaming – temporarily remove the mycolic acid Endospores – (endo – within, inside vs spores of plants
from the cell wall to allow the dye to enter the cell and - external) resting cells, highly resistant to dessication,
stain the organism. heat and chemical agents, composed of: Calcium
2. Increase the concentration of the phenol and dye dipicolinate or Dipicolinic acid.
3. Prolong the contact of the stain with the organism.
4. Addition of wetting agent like Tergitol. OUTSIDE THE CELL WALL

Two modifications of Ziehl-Neelsen stain Capsule – prevents phagocytosis, antigenic

1. Pappenheim stain – differentiate Mycobacterium demonstration thru serotyping: QUELLUNG – swelling
tuberculosis from Mycobacterium smegmatis. enhanced by media containing serum or milk, animal
RGT: Carbol fuchsin, Rosolic acid, Acid alcohol then tissue or fluid. Stains: Hiss, Nigrosine and India Ink
Methylene blue
Pili – hair-like extension of the cell, USES: bacterial
Result: MTB M. smegmatis conjugation, adherence to surfaces

2. Baumgarten stain (diluted Alcohol Fuchsin) – differ- Flagella – for locomotion

rentiate Mycobacterium tuberculosis from
Mycobacterium leprae.
RGT: Carbol fuchsin, 95% Ethyl alcohol, Nitric acid
then Methylene blue

Result: MTB M. leprae

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 BACTERIOLOGY: REVIEW NOTES

Methods of determining motility of bacteria in the lab

1. HDP
2. Use of SIM (Sulfide Indole Motility)
3. Staining the flagella
4. Serological test (e.g. Widal)
5. Swarming test
6. Fluorescence test

2. Fixed state- bacteria are dried and fixed on the slide

and stained - form, arrangement and staining reaction
can be observed.
STEPS:
Bacterial smear preparation – 1st step
*best demonstrated at 25°C, 37°C inhibits flagella of Listeria and
Yersinia Heat fixation or using alcohol
* hanging drop or wet mount, used SIM (Sulfide Indole Motility) – Purpose: For adherence of the bacteria into the slide
slant, streak and stab technique so it will not be washed off during staining procedure.
*stains : Leifson, Gray and Fisher & Cohn
STAINING
Glycocalyx (slime layer) Purpose: Bacterial cells are made visible so that one
can appreciate their appearance better. To
MORPHOLOGICAL STUDY OF BACTERIA differentiate one bacteria from another bacteria based
1. Living state – necessary for bacteria that are not on their staining reactions and helps in the
readily stained or easily cultivated -bacteria are identification and some of its special structures.
unstained, bacteria appear colorless -morphology is *Stains – salts composed of a (+) and (-) ion, one of
less distorted -shape/form and motility can be which is colored. (*basic dye – color is in the cation & acidic dye –
observed. color is in the anion)
Two methods:
1. Wet method – shape of bacteria is observe Types:
2. HDP or Hanging Drop Preparation – shape and 1. Simple
motility of bacteria are observed. 2. Differential
3. Special
Two types of motility 4. Indirect or negative (e.g. India ink or Nigrosine)
1. True motility – bacteria are moving in one direction,
movement is directional. VIRULENCE FACTORS
2. Brownian movement/False movement/Molecular 1. Adherence factors – pili or fimbriae
movement/On-the-spot vibratory movement - bacteria
are just vibrating in liquid medium through which they 2. Antiphagocytic factors – capsule
are suspended. The movement of bacteria is random
and they move only on the bombardment of water 3. Enzymes – e.g. hyaluronidase*Spreading factor/ Duran
molecule. Reynal factor, coagulase and fibrinolysin or kinase

Factors affecting the results of HDP

1. Using old culture which is already crowded with inert 4. Toxins – endotoxin or exotoxin (e.g. TSST, PVL,
living and dead bacteria tetanospasmin, BoTox)
2. Production of acid and other toxic products in old
culture destroying bacteria/killing bacteria.
*ideal culture: 18-24 hrs.old

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 BACTERIOLOGY: REVIEW NOTES

Exotoxin Endotoxin 3. Dark field microscopy

Source both gram (+) mostly Gram 4. Fluorescent microscopy
and gram (-) (-) 5. Electron microscopy – TEM/ SEM
Release metabolic released
mechanism products upon lysis of BACTERIAL GROWTH FACTORS
released by living cells 1. NUTRIENTS
cells a. Carbon source –synthesis of cellular component
Composition peptides or lipids I. Lithotroph/ Autotroph – inorganic substance
proteins II. Heterotroph/Organotroph – organic substance
Heat stability heat labile heat stable b. Nitrogen source – for proteins (DNA or RNA)
except for Staph c. Minerals – e.g. sulfur, Mg++, Ca++, Fe++
toxin (food d. Salts – halophilic – salt loving (eg. S.aureus)
poisoning) e. Water f. Others – others X and V factors –
Immunologic O - converted to X - easily Haemophilus, protein – Mycobacterium tuberculosis
toxoid converted to
toxoid 2. OXYGEN AND CARBON DIOXIDE AVAILABILITY
Pharmacologic Cytotoxin – kills generalize *superoxide dismutase
host cell, to non- a. Obligate/strict aerobes -ambient air, which 21%
Enterotoxin – specific oxygen and small amount of Carbon dioxide (0.003%)
damage the cell (fever, (e.g.Pseudomonas, Bordetella, Brucella, Francisella)
of the GI tract, septic shock b. Obligate /strict anaerobes -without oxygen, 5-10%
Neurotoxin – or DIC) Carbon dioxide, 80-90% Nitrogen (e.g. Bacteroides
interfere nerve and Clostridium)
impulse c. Facultative anaerobes – aerobes that can survive
transmission without oxygen (e.g. Staphlococcus and
Toxicity highly toxic low dose Streptococcus)
Lethal dose small dose larger dose d. Aerotolerant anaerobes - anaerobes that can grow
Diseases Tetanus, UTI, in the presence of oxygen
Botulism Typhoid e. Microaerophilic – reduced oxygen only appx. 5-10%
fever and increased Carbon dioxide (8-10%)(e.g.
*Limulus lysis test – to detect endotoxin in body fluids and surgical Campylobacter).
instruments f. Capnophilic -increased concentration of carbon
RGT: blood of horse shoe crab (Limulus polyphemus)
Principle: in the prescence of endotoxin, amoebocytes (WBC will release
dioxide (5-10%) and appx 15% oxygen (e.g. Neiserria,
lysate (proteins) + result: clumping HACEK – Haemophilus, Actinobacillus, Cardiobacter,
Eikenella, and Kingella)

3. TEMPERATURE
MICROSCOPY AND TYPES a. Psychropillic/Cryophilic – bacteria that grow on
1. Bright-field microscopy <10°C (e.g. Listeria monocytogenes, Yersinia
2. Inverted/ Phase Contrast microscopy enterocolitica, Escherichia coli)

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 BACTERIOLOGY: REVIEW NOTES

b. Mesophilic – bacteria that frow on 20- 40°C, best at c. Inoculate MHA *multiple interrupted
37°C most pathogen grow on this temperature. d. Apply antibiotic disc *3-5 mins, not longer than 15mins before
c. Thermophilic – bacteria that grow on 50-55°C (e.g. inversion

Thermophilus aquaticus – source of Taq polymerase e. Incubate for 16-18 hrs.

enzyme). f. Measure zone of inhibition
g. Refer to chart and interpret (Sensitive/Interme-
4. PH – optimum pH is neutral to slightly alkaline diate/Resistant)
a. Acidophile – L.acidophilus , doderlein bacilli pH 3.0 4. E-test – strip test with varying concentration of
b. Alkaliphile - pH 8.0 – 10.0 Vibrio cholera (APW- antibiotic along its length (elliptical)
Alkaline Peptone Water) Advance: MIC can be reported when growth inhibition
intersects.
GROWTH CYCLE 5. Automated – Vitek®, Mini-API® and BacT/Alert®
Principle: OD, turbidimetry, colorimetry
*MIC (Minimum Inhibitory Concentration) – lowest concentration of
antibiotic with no growth.
*MBC (Minimum Bactericidal Concentration) - lowest concentration
of antibiotic that kills 99.9% of bacteria.

ANTIMICROBIAL AGENTS TARGET SITES

1. CELL WALL SYNTHESIS
a. Betalactams: penicillin, cephalosporins, carbapenems,
monobatams
b. Glycopeptides: vancomycin, teicoplanin
c. Cylcoserine
d. Bacitracin
2. PROTEIN SYNTHESIS
a. Aminoglycosides: gentamicin, tobramycin, kanamycin
1. Lag/ Adjustment phase – little or no multiplication b. Tetracyclines
2. Log/ Exponential phase – organisms grow at c. Chloramphenicol
maximum rate, antibiotics are most effective d. Erythromycin
e. Fusidic acid
3. Plateau phase - living and dead are equal, toxins 3. NUCLEIC ACID SYNTHESIS
begin to accumulate, nutrients deplete. a. Sulfonamides
4. Decline/ Death phase – living < dead organisms b. Trimetoprim
c. Quinolones
SUSCEPTIBILITY TESTING d. Rifampicin
1. Tube or Microplate - dilution, MIC and MBC 4. CELL MEMBRANCE FUNCTION
a. Polymixin: Colistin
2. Agar Dilution – agar + appropriate media *Bacteriostatic - inhibit bacterial growth but some survive
3. Disc Diffusion – Kirby –bauer, Mueller-Hinton agar – *Bactericidal - no bacterial cell survive
4mm, pH 7.2-7.4 *Germicidal/Disinfectant – agent capable of killing microorganisms
a. Prepare inoculum by picking 4-5 colonies that produce infection
b. Incubate at TSB for 24 hrs then standardized to *Antiseptic – applied topically to living cells
*Aseptic technique – methods to keep all unwanted
0.5 Mcfarland standard (99.5 ml 1% H2SO4+ 0.5ml microorganisms (contaminants) out of the field of work or
BaCl) appx. 1.5 x 108 CFU/ml observation.

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 BACTERIOLOGY: REVIEW NOTES

STERILIZATION –complete removal or destruction of II. Selective

all form of microorganisms. ANTIBIOTICS – are substances produced by some
I. Physical Method organisms or it may refer to a similar substance
Moist Heat produced by chemical synthesis which in low
a. Autoclave – (Charles Chamberland, BI: G. concentration inhibits the growth of organisms.
stearothermophilus) 121°C at 15 lbs psi for 15 mins,  Steptomycin – Streptomyces griseus
steam under pressure  Penicillin – Penicillum notatum
b. Fractional/ Intermittent/ Discontinuous  Bacitracin – Bacillus licheniformis
i. Tyndallization 100°C for 30mins, 3 cons days (1st:  Chloramphenicol – Streptomyces
vegetative cells, 2nd: spores & 3rd: remaining spores) venezuelae.
ii. Inspissation 75-80°C for 2 hrs, 3 cons days
Dry Heat– action: oxidation Essential properties of chemotherapeutic agents:
a. Oven – (BI: B. subtilis var. niger), 160-180°C 1-2 hrs 1. Show selective toxicity
b. Direct heat/ flame burner - heating the loop or 2. Bactericidal than bacteriostatic.
needle 3. Not show resistance to susceptible microorganisms
c. Incineration- 870-980°C 4. Not produce adverse side effects
5. Not be allergenic
Filtration - for antibiotics, Liquid: cellulose acetate or 6. Remain active even in the presence of body fluids
cellulose nitrate with vacuum and Air: HEPA, ULPA and exudates.
Ionizing radiation – (BI: B.pumilis), short wave , high
energy gamma rays Factors affecting disinfecting potency
II. Chemical Method 1. concentration of agent
1. Ethylene Oxide (ETO) - gas chamber (BI: 2. pH
B.subtillis var. globigii) 3. contact time
2. Formaldehyde vapor and vapor phase H2O2 4. temperature
– sterilization of HEPA filters 5. nature of organism
3. Gluteraldehyde – sterilization of
bronchoscopes
4. Peracetic acid

DISINFECTION – destruction of pathogenic organism.

I. Physical
a. Pasteurization
1. Batch – (Low Temperature Holding) 63°C for 30
mins
2. Flash –(High Temperature Holding) 72°C for 15
mins
b. Boiling - 100°C
II. Non-ionizing Radiation – long wavelength low
energy UV light (e.g. mercury lamps)

C. Chemicals
I. Non-selective
a. Alcohol – 70% alcohol
b. Halogen – iodine or chlorine
c. Quaternary Ammonium Compounds (QUATS)
d. Phenol
e. Heavy metals – mercury, copper and silver
*phenol coefficient – expression of the bactericidal power of a particular
substance as compare to pure phenol
*standard organisms: S.aureus( +); S.typhi (-)

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 BACTERIOLOGY: REVIEW NOTES

GENETIC EXCHANGE MECHANISM OF BACTERIA

PATRICK R. DE VERA,RMT, MSMT 7

 BACTERIOLOGY: REVIEW NOTES

BIOSAFETY LEVEL and BSC CLASSIFICATION

Biosafety is the application of safety precautions that reduce a laboratorian’s risk of exposure to a potentially infectious
microbe and limit contamination of the work environment and, ultimately the community.

Biosafety Level (BSLs)

 There are four (4) biosafety levels. Each level has a specific controls for containment of microbes and
biologic agents.
 The primary risks that determine levels of containment are infectivity, severity of disease, transmissibility,
and the nature of the work conducted.
 Origin of the microbe, or the agent in question, and the route of exposure are also important.
Biosafety Levels

Centers for Disease Control and Prevention Classification of Biological Agents

Biosafety Cabinet (BSC) is the primary means of containment developed for working safety with infectious
microorganisms. Class II-BSCs are the most commonly used in laboratories.

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 BACTERIOLOGY: REVIEW NOTES

Biosafety Cabinet

LABORATORY SPECIMEN COLLECTION AND 3 tubes (#1 CC, #2 Micro, #3 Hema if #4 Micro) are
HANDLING usually collected if only 1 tube (Micro- Hema-CC- other
General guidelines test).
1. Specimen should be collected from the actual site of *bacterial meningitis guide
infection. S.agalactiae : neonatal meningitis (infants)
2. Specimen should be collected before the initiation of H.influenzae : 1-5 years old
antibiotic therapy. *ARD – Antimicrobial Removing Device – resin containing S.pneumoniae: 29 years old
medium that inactivates most antimicrobial by non-selectively adsorbing them to the surface
L.monocytogenes: infants, elders, immunocompro-
of the resin medium. Used for patients that received antibiotics. It can remove at least 25
mised
antimicrobials.

3. Timing of collection should be considered. Respiratory tract specimen – S.pyogenes most

4. Quantity of specimen. common pathogen, Viridans Streptococci is the most
5. Specimen should be collected under sterile, abundant normal flora
antiseptic conditions. Upper Respiratory Tract
6. Use of proper method of collection. nasal swab – moist the swab on the nares.
7. Specimen should be plated immediately. nasopharyngeal swab – flexible swab inserted through
8. Transport media could be use (e.g. Transgrow, Cary the nose and into the posterior nasopharynx. Rotate for
and Blaire, Stuart, Amies). 5 secs (e.g. H. influenzae, N. meningitidis and B.
pertussis)
Blood – used to evaluate bacteremia and septicemia, throat swab- swab in the posterior pharynx, tonsils and
collected 30-40 mins before the fever spikes. Blood inflamed areas.
culture bottles contain anti-coagulant (0.025% w/v Lower Respiratory Tract
Sodium Polyanethol Sulfonate – SPS: prevents sputum – early morning specimen, 3 consecutive days,
coagulation, neutralize the bactericidal effect of human expectorated (deep cough) (mainly of Mycobacterium
serum and prevents phagocytosis) tuberculosis)
*1 set (2 bottles): 1 aerobic, 1 anaerobic, incubated for 7 days
*2-3 sets from different puncture sites, one hour interval within 24
Gram stain then check for contamination of SECs (>25
hrs PMNs less than 10 SECs per LPF).
*20-30ml of blood per set for adults, 5-10ml of blood per set for
pedia Gastro Intestinal tract specimen – used for
*disinfect skin prior to extraction by applying 70% alcohol then determining enteric pathogens. It could either be stool
iodine then another alcohol
*no changing of needle required (pea size), rectal swab or gastric secretions.
* Signs of growth include: hemolysis, formation of bubbles, turbidity Specimen: three (3) specimens, one per day for 3
(PELLICLE FORMATION), and appearance of small colonies in days. * should not be contaminated with toilet water or urine Incubate for 2
broth or surface of RBCs days.
*bacteremia*septicemia
Urinary tract specimen – mid stream clean catch
CSF –collected through lumbar puncture, used to
urine, early morning specimen. Other specimen may
evaluate meningitis (organisms are piliated or with
include catheterized urine or suprapubic urine. Colony
capsule).

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 BACTERIOLOGY: REVIEW NOTES

count of >105 CFU/ml bacteria indicates infection. SPECIMEN HANDLING

Incubate for 2 days. *Refrigerate at 4°C if there is any delay in processing
Colony count is done using an inoculating loop -> BAP *Specimen that need immediate plating are a.)
a. 1ul (0.01ml) – multiply by 100 specimen containing gonococci and b.) specimen
b. 10ul (0.001ml) – multiply by 1000 containing Bordatella pertussis.
*length of refrigeration for 2-3 hrs w/o appreciable loss
Formula: of pathogen (urogenital swab, throat swab, rectal
CFU/ml = # of colonies x calibrated loop used swab, stool, sputum and swabs from wounds)
*Use of swabs: X – Cotton/ O - Dacron or Calcium
Interpretation: alginate
Significant bacteriuria =>105 CFU/ml *for 24 hrs. – urine
Doubtful bacteriuria = 103 to 105 CFU/ml *specimens that must be processed at once (CSF and
Possible contaminant = < 103 CFU/ml Gastric Aspirate)

*atleast 2 bacteria/HPO – correlates with the presence SPECIMEN TRANSPORT

of significant bacteria 1. Stuart – gonococci, contains anaerobic salt solution
(Thioglycolic acid, 0.5 agar and MB)
Genital tract specimen – used to determine the cause 2. Cary and Blair – V.cholera- 22 days, S.typhi – 49
of vaginitis, urethritis and cervicitis as well as child birth days, and Yersinia pestis– 75 days (Thioglycolate,
infections. phosphate, NaCl in 0.5 agar pH 8.3)
*often used to determine STDs (e.g. N.gonorrhea) 3. Amies – Vibrio sp., contains charcoal – adsorb
*Transport medium such as JEMBEC or Gono-Pak are bactericidal/static subs. With pH of 7.3
used 4. Glycerol saline – enteric fever bacilli (Salmonella),
*mostly fastidious organisms require CAP. for stool samples.

Wound and abscess – for impetigo, erysipelas and SPECIMEN STORAGE

cellulitis, also infections for burn patients, post-surgical 1. CSF: room temperature
wound infections and human /animal bite infections. 2. Urine, stool swab, sputum, foreign devices such as
Preferred specimen for wound and abscess culture is catheter: 4°C refrigerator
needle and syringe aspirate. 3. Serum for serology: -20°C freezer (1 week)
*needle and syringe enhances the recovery of 4. Tissues or specimen for long term storage: -70°C
anaerobes
*Proper aseptic technique must be applied. Care must METHODS OF OBTAINING A PURE CULTURE
be taken during specimen collection to prevent *ATCC – American Type of Culture Collection
contamination with normal skin flora. 1. Streak method
2. Pour-plate method
Eye and Ear discharge (normally sterile) - Specimen 3. Use of a selective medium
of choice: purulent material from the surface of the 4. Animal inoculation
lower conjunctival sac (eye) and swab or scrapping
(ear) DIFFERENT TECHNIQUES OF INOCULATION
1. Liquid culture media
Anaerobic Specimen Collection -best obtained by 2. Solid culture media – tube method
needle or syringe biopsy (e.g. Transtracheal aspirate- a. Butt (stab halfway)
thoracentesis, Urine thru suprapubic bladder catheter, b. Butt and slant (streak then stab)
cystostomy and nephrostomy, percutaneous aspiration 3. Plated Culture media
from bladder) a. Radial – center then away
b. Overlap – for sensitivity testing
c. Multiple Quadrant – divided by 4
d. Interrupted – streak then reverse 180° then streak

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 BACTERIOLOGY: REVIEW NOTES

e. Multiple interrupted – 1-4 streaks, most commonly C. According on how the medium is dispensed or
used, with spaces *incubate and refrigerate – distributes
inverted position to prevent contamination of water 1. Plated: weigh-dissolve - autoclave - dispense then
droplets wait to harden
2. Tubed: weigh-dissolve – dispense – sterilize - *if
Types of colonies based on serologic characteristics: solid wait to harden (slant, butt and slant and butt)
1. Mucoid – glistening and water like in appearance. D. According to Use
Characteristics of bacteria producing capsule. 1. Simple/Basal/Supportive/General Isolation/ pur-
2. Smooth – appears homogenous and uniform without pose – support the growth of most non-fastidious
appearing as liquid or mucoid. Characteristic of freshly bacteria. Used in preparation of enriched medium.
isolated wild type colony of enteric bacteria. Maintains stock culture of control strains of bacteria.
3. Rough –appear to be granulated and rough. Non- Used in subculturing pathogens in another culture
pathogenic bacteria produces rough colonies except media. (e.g. TSA, NA, TSB)
Mycobacterium tuberculosis in LJ media or 7H10 2. Enrichment –enhance the growth of an organism
Middlebrook agar. (e.g. Selenite broth & Tetrathionate broth – Salmonella
& Shigella, Alkaline Peptone water - Vibrio)
CULTURE MEDIA – artificial soil for microorganisms 3. Enriched – contains nutritive supplements needed
containing nutritional requirements and environmental for the growth of a particular organism (supplements
requirements for microbial growth. used: blood, serum, extra peptone, vitamins) (e.g.
Agar– polysaccharide extracts of seaweeds which is BAP, CAP). Contain nutrient supplement for fastidious
commonly used as base medium from seaweeds (red bacteria (e.g. BAP – 5% defibrinized sheep/horse/rab-
algae – Gelidium amansii or Gelidium sesquipedale bit blood *add defibrinized blood when temperature drops to 60°C).
and Gracilaria sp. in the Philippines it is locally known *X do not use human blood – it contains citrate -
as “gulaman”), polymer made up of subunits of the inhibits beta hemolytic Streptococci and dextrose –
sugar galactose. alters the hemolytic pattern
*Haemophilus – blood type O horse blood to
Classification of culture media: demonstrate the beta hemolytic species H. haemo-
A. According to consistency or physical state lyticus and H. parahemolyticus) and CAP *add defibrinized
1. Liquid – contains no agar, hardening or solidifying blood when temperature drops to 80°C – S.pneumoniae,

substances (e.g. broth or milk, BHI or TSB) Haemophilus and Neisseria.

2. Semi-solid – contains 0.5-1% agar (e.g. SIM- Sulfide 4. Differential – distinguish organism growing together
Indole Motility *specimens must be submitted to by differences in cultural characteristics (e.g. EMB,
lab for atleast 30 mins) MacConkey, BAP). Provides distinct colonial
3. Solid – contains 2-3% agar appearances of microorganism to aid in their
a.) Plated (e.g. BAP, CAP, EMB, SSA) identification. Enzymes involved include Lactose
b.)Tubed (e.g. TSA, SCA) permease and β – galactosidase.
*liquefiable – 2-3% agar, plated media or tubed
media (e.g. BAP, CAP) Enterobacteriaceae
*non-liquefiable – e.g. chopped meat – for
anaerobes, rice grain – for fungi RAPID LACTOSE FERMENTER
B. According to composition (Lactose permease and β – galactosidase)
1. Synthetic/defined – commercially prepared media. Escherichia
Exact composition is known. Klebsiella
2. Non-synthetic – exact composition is not known. Enterobacter
Contains atleast one component that is not chemically
defined (not pure, no chemical formula) (e.g. blood, LATE LACTOSE FERMENTER
serum, plant/animal/yeast extract). (β – galactosidase)
3. Tissue – made up of living cells/ not grown on a cell Serratia Hafnia Yersinia
free medium (e.g. McCoy cells – Chlamydia, W138 – Salmonella arizonae Citrobacter
normal human skin, HeLa cells – cervical CA cells) Shigella sonnei

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 BACTERIOLOGY: REVIEW NOTES

NON-LACTOSE FERMENTER Inhibitor: Sodium thiosulfate and sodium citrate –

(No enzyme) alkaline pH to inhibit gram-positive organism and
Proteus coliforms
Providencia Bile salts – inhibit the growth of gram-positive
Morganella organisms
Salmonella pH: Neutral Red or Brilliant Green
Shigella H2S indicator: Ferric Citrate
Edwardsiella Salmonella: colorless colonies with black center
Erwinia Shigella: colorless colonies without black center

Selective/ Differential media for gram negative enteric 5. Selective – promotes growth of desirable organism
bacilli but at the same time inhibit the growth of other
organism (e.g. SSA, BSA, PEA). Substances added to
Eosin-Methylene Blue Agar make it selective:
CHO: Lactose a) Inhibit gram (+): Gentian violet, Sodium
Fermenter: pink-purple colonies desoxycholate, other bile salts
Non-fermenter: colorless colonies b) Inhibit gram (-): Sodium azide, Potassium Tellurite
Characteristic colonies on EMB c) Prevents swarming: Alcohol, Chloral hydrate
Escherichia.coli: pink to purple colonies with Greenish
Metallic Sheen For Mycobacterium tuberculosis
Klebsiella: pink MUCOID colonies that tend to sling Medium: Lowenstein – Jensen Slants
Enterobacter : pink colonies with dark center (“fish eye Inhibitor: Malachite Green
colonies”)
For Corynebacterium diptheriae
MacConkey agar Medium: Mueller Tellurite
CHO: Lactose Inhibitor: Potassium Tellurite
Inhibitor: Crystal violet and Bile salt – against gram
positive organisms For Neisseria gonorrhea
pH indicator: Neutral Red Medium: MHA enriched Chocolate agar plate +
Fermenter: pink colonies antibiotics (depending on the modification)
Non-fermenter: colorless colonies
THAYER MARTIN
Hektoen Enteric Agar  Vancomycin - inhibits gram (+) bacteria
CHO: Lactose, Sucrose and Salicin  Colistin – inhibits gram (-) bacteria except
Inhibitor: Bile salt – against gram-positive organisms N.gonorrhea
pH indicator: Bromthymol blue  Nystatin – inhibits fungi
Hydrogen Sulfide (H2S) indicator: Ferric Ammonium MODIFIED THAYER MARTIN
Citrate  Vancomycin - inhibits gram (+) bacteria
Fermenter H2S positive: yellow colonies with black  Colistin – inhibits gram (-) bacteria except
center N.gonorrhea
Fermenter H2S negative: yellow colonies w/out black  Nystatin – inhibits fungi
center  + Trimethoprim Lactate – prevents the
Non-fermenter H2S positive: green colonies with black swarming of Proteus sp.
center MARTIN LEWIS
Non-fermenter H2S negative: green colonies w/out  Vancomycin - inhibits gram (+) bacteria
black center  Colistin – inhibits gram (-) bacteria except
Salmonella-Shigella Agar N.gonorrhea
Selective and Differential for Salmonella and Shigella  Anisomycin – inhibits fungi
CHO: Lactose  + Trimethoprim Lactate – prevents the
swarming of Proteus sp.

PATRICK R. DE VERA,RMT, MSMT 12

 BACTERIOLOGY: REVIEW NOTES

NEW YORK CITY AGAR pH indicator: Phenol Red

 Vancomycin - inhibits gram (+) bacteria H2S indicator: Ferrous Ammonium Sulfate
 Colistin – inhibits gram (-) bacteria except Sulfur source: Sodium Thiosulfate
N.gonorrhea
 + Trimethoprim Lactate – prevents the Lysine Iron Agar
swarming of Proteus sp. CHO: Glucose (slant - lysine deamination, butt – lysine
 + Ampothericin B – inhibits fungi decarboxylation)
Amino Acid: Lysine + small amount of protein
Selective/ Differential for Staphylococcus sp. pH indicator: Bromcresol Purple
Medium: Mannitol Salt Agar H2S indicator: Ferric Ammonium Citrate
Inhibitor: 7.5 – 10.0% NaCl
CHO: Mannitol Simmon Citrate Agar
pH indicator: Phenol Red Carbon source: Citrate
Fermenter: pH indicator: Bromthymol blue
S.aureus – yellow colonies
Non-fermenter: 9. Antimicrobial Susceptibility Testing
S.epidermidis – pink colonies, Novobiocin sensitive Medium: Mueller Hinton agar/broth
S.saprophyticus – pink colonies, Novobiocin resistant Agar depth: 4mm
pH: 7.2-7.4
Selective/ Differential for Vibrio sp. size of plate: 24mm from disc center to center.
Medium: Thiosulfate Citrate Bile Salt Sucrose
CHO: Sucrose Steps when performing AST
pH indicator: Bromthymol blue 1. Pick 4-5 colonies into TSB. Incubate at 37°C
Fermenter: V.cholerae & V.alginolyticus - yellow for 2-5 hrs.
colonies 2. Compare turbidity with 0.5 McFarland
Non-fermenter: V.parahemolyticus – green colonies standard (99.5ml of 1% Sulfuric Acid + 0.5ml
BaCl) approx.. 1.5 x 108 CFU/ml.
For gram positive bacteria 3. Inoculate to MHA plates (multiple-overlap
Inhibitor: PEA (phenylethyl alcohol) inhibits gram- technique then rim).
negative bacteria 4. Apply antibiotic discs 3-5 mins but not longer
than 15 mins before inversion.
6. Special or Specific – support the growth of specific 5. Invert the plates and incubate at 37°C for
organisms (Petragnani medium – M. tuberculosis, 16-18 hrs.
Thayer Martin – N.gonorrhea, McBride medium – L. 6. Measure the zone of inhibition using a
monocytogenes, Dieudonne’s – V.cholera). Vernier caliper (naked eye, underside of the
*Overheating culture media may result to increased plate).
acidity, production of precipitate and decomposition of 7. Interpret susceptibility from the standard
carbohydrates. chart zone (reported as S – sensitive, R-
resistant and I - intermediate).
7. Transport media – semi-solid, contains charcoal to
absorb fatty acids which are toxic to some bacteria. Possible sources of Errors
(e.g. Stuart’s, Amies, Cary and Blair, Transgrow- 1. Use of mixed culture
N.gonorrhea). 2. Inoculum too light – false sensitive
3. Inoculum too heavy - false resistance
8. Biochemical test medium – enzymes produced by 4. Too much moisture on agar – false
bacterium. resistance due to overgrowth
5. Very dry agar surface – false sensitive due
Triple Sugar Iron Agar to poor growth
CHO: Slant: 1% Lactose (10), 1% Sucrose (10) 6. Improper storage of disc *Betalactams such as
Butt: 0.1% Glucose Penicillin and Methicillin are 1st to degrade.

PATRICK R. DE VERA,RMT, MSMT 13

 BACTERIOLOGY: REVIEW NOTES

7. Indicators of improper storage TYPES:

8. Reading and clerical errors a.) Slide method: 1 drop 3% H2O2 + loopful of organism
9. Deterioration of turbidity standard or control
strains b.) Direct (culture media) method: 1 drop 3% H2O2 +
*Increased cations (Mg++ and Ca++) can cause an increased colony
resistance of P.aeruginosa to aminoglycosides (X protein
synthesis) and susceptibility to sulfonamides (X nucleic acid RESULT:
synthesis), *two (2) concentric rings around the disc: remedy (+) effervescence or bubble formation *Staphylococci,
- measure the diameter of the outer zone).
Bacillus and Micrococci
controls:(+) Staphylococcus epidermidis
BIOLOGICAL ACTIVITIES OF MICROORGANISMS
(-) Streptococcus pyogenes
A. Enzyme production – coagulase, catalase etc.
STAPHYLOCOCCI
Coagulase test – best criterion for the pathogenicity of
B. Production of hemolysin – BAP (5ml of blood/100ml
Staphylococcus aureus.
agar base)
a. α (alpha) – incomplete or partial hemolysis
PRINCIPLE: S.aureus elaborates COAGULASE =
greenish zone
coagulation or clotting of plasma
b. β (beta) - complete hemolysis/ clear zone
c. γ (gamma) – no hemolysis
RGT: EDTA rabbit plasma * plasma is used because
of the presence of fibrinogen that will react with the
C. Production of pigment in bacteria
organism to form clump or coagulation.
a. Serratia marcescens – red (prodigiosin)
b. Chromobacterium violaceum – violet/purple
TYPES:
c. Pseudomonas aeruginosa – blue (pyocyanin ) -
a.) Slide method- demonstrates BOUND coagulase. It
green (pyoverdin)
serves as a rapid screening test. 1 drop NSS + 1
d. Pseudomonas fluorescens –yellow- green
loopful organism + 1 drop plasma – Mix for 1 min
(pyoverdin)
e. Staphylococcus aureus – yellow-golden
b.) Tube method – demonstrates FREE coagulase. 0.3
f. Staphylococcus epidermidis - white
or 0.5ml of plasma + 2 loopfuls of organism - incubate
g. Actinomyces - silver
in water bath (37°C) for 30 mins
h. Micrococcus roseus - pink
RESULT: (+) clumping or coagulation
i. Prevotella melaninogenica - black
*control: Staphylococcus aureus
j. Sarcina aurentiaca – orange
Noted that:
False (+) prescence of citrate utilizing organisms such
BIOCHEMICAL REACTIONS – demonstrate the
as Pseudomonas and Enterococci. *If citrate is utilized
enzyme system within the microbial cells
by the organism Calcium is released which is essential
for the physiologic clotting of blood.
Test for gram (+) cocci
False (-) some strains of S.aureus will form clot within
Catalase test – is an enzyme produced by a wide
24 hrs. Some strains of S.aureus elaborate enzyme
variety of gram (+) and gram (-) organism.
Staphylokinase which is capable of removing clot
*Differentiate Stapylococcus from Streptococcus sp.
formation within 4 hrs.
*preliminary genus identification of gram (+) non spore
forming rods
Mannitol Fermentation test
PRINCIPLE: Catalase if present cleaves H2O2
PRINCIPLE: Staphylococcus aureus can ferment
(hydrogen peroxide) to O2 and H2 resulting to BUBBLE
MANNITOL
formation.
MEDIA: Mannitol Salt Agar (MSA) – with increase
*DO NOT perform on BAP
concentration of NaCl (7.5-10%)
*blood (Hgb) contains catalase = false negative result
pH indicator: Phenol Red
RESULT: (+) yellow zones around the colonies

PATRICK R. DE VERA,RMT, MSMT 14

 BACTERIOLOGY: REVIEW NOTES

STREPTOCOCCI TEST FOR GRAM NEGATIVE ORGANISMS

Bacitracin Disk test – useful presumptive test for
differentiating group A beta hemolytic Streptococci
(Streptococcus pyogenes) from other beta hemolytic
group

PRINCIPLE: Streptococcus pyogenes is susceptible to

low dose of Bacitracin

RGT: Taxo A- Bacitracin disc (0.04 unit of Bacitracin)

RESULT:
(+) zone of inhibition *Streptococcus pyogenes
(-) Group B (S.agalactiae) and Group C, Group D
(Enterococcus and Non Enterococcus)

Optochin Disk test – most widely used test for the

differentiation of alpha hemolytic Pneumococci from
other alpha hemolytic Streptococci.

PRINCIPLE: Streptococcus pneumoniae is sensitive to

Optochin disk (Taxo P)

RGT: Optochin Disk (Ethylhydrocuprein HCl)

RESULT:
(+) zone of inhibition *Streptococcus pneumonia
*S.pneumoniae is also (+) capsular swelling 1. CHO (carbohydrate) fermentation -anaerobic
(-) Viridans Streptococci (e.g. S.mutans, S. mitis, S. breakdown of simple sugars serve as main source of
sanguis, S. salivarius, S. constellatus and S. inter- energy of microorganisms.
medius)
2. Indole test
Test from gram (-) cocci PRINCIPLE: ability of bacteria to split indole and
Oxidase test – presumptive test to identify Neiserria alanine from tryptophan. Liberated Kovac’s or Ehrlich
and initially characterized gram (-) bacilli reagent giving it a deep RED color.
MEDIA: SIM, MIO (Motility Indole Ornithine), Indole
PRINCIPLE: Organisms produced cytochrome NO3 media and Rapid Spot test (filter paper strip w/
oxidase for electron transport chain. Presence of PDAB – (+) blue color)
enzyme is indicative of aerobic respiration.
Kovac’s rgt - PDAB (P-dimethyl aminobenzaldehyde)
RGT: 1 % Tetramethyl-para-phenylenediamine dissolve in amyl or isoamyl alcohol with concentrated
dihydrochloride or PADMA (Paraaminodimethyl Aniline HCl.
Monohydrochloride) RESULT: (+) 0.5ml + SIM = deep pink/red

RESULT: Ehrlich’s rgt - PDAB (P-dimethyl aminobenzaldehyde)

(+) dark purple *Neisseria and Pseudomonas *control: dissolve in butyl or absolute alcohol with concentrated
Pseudomonas aeruginosa HCl.
(-) absence of color *control: Escherichia coli, Serratia RESULT: (+) 0.5ml + SIM = pink/red ring below xylene
marcescens and Salmonella enterica layer *xylene – extracting agent

PATRICK R. DE VERA,RMT, MSMT 15

 BACTERIOLOGY: REVIEW NOTES

Results for SIM: *Composition : 1% lysine, glucose, Ferric Ammonium

Growth along the stab line = non – motile Citrate and Sodium Thiocyanate (detects H2S
Growth away from the stab line or surface = motile production)
Blackening of the medium = H2S production
(+) red/pink ring = Indole production (*control: RESULT:
Escherichia coli) SLANT
(-) no ring *control: Klebsiella pneumoniae, Serratia lysine deamination – (+) Red (-) Purple
marcescens, Salmonella enterica BUTT
lysine decarboxylation – (+) Purple
3. Methyl Red test fermentation of glucose – (+) Yellow
PRINCIPLE: Some organisms produce a large amount H2S production – (+) Black
of acids which is detected by the Methyl Red indicator (+) *control: Serratia marcescens
MEDIA: Clark and Lubbs Dextrose Broth (-) *control: Proteus mirabilis
RESULT:
(+) Red *control: Escherichia coli 7. Urease test
(-) Yellow *control: Klebsiella aerogenes PRINCIPLE: Some organisms can hydrolyze urea
rapidly releasing ammonia in the process.
4. Voges-Proskauer test MEDIA: Urea broth or Christensens Agar
PRINCIPLE/RGT/RXN: Dextrose is fermented; pH indicator: Phenol Red
organism will produce Acetoin or Acetylmethylcarbinol
RESULT:
Acetylmethylcarbninol + (KOH + Alphanapthol) (+) Cherry red *control: Proteus mirabilis
oxidation = Dimethylcarbinol + guanido = deep red (-) Orange *control: Escherichia coli

MEDIA: Clark and Lubbs Dextrose Broth 8. Decarboxylase/ Dihydrolase test

RESULT: PRINCIPLE: Some organisms decarboxylate or
(+) Red *control: Klebsiella aerogenes hydrolyze an amino acid to form an amine resulting to
(-) Yellow *control: Escherichia coli an alkaline pH range.
MEDIA: LOA (Lysine Ornithine Arginine) medium
5. Citrate test pH indicator: Bromcresol Purple and Phenol Red
PRINCIPLE: Some organism can utilize citrate as a
sole source of carbon releasing ammonia in the Amino acid Enzyme Product
process. Lysine Decarboxylase Cadaverine
MEDIA: Simmon Citrate Agar (SCA) Ornithine Decarboxylase Putrescine
pH indicator: Bromthymol Blue Arginine Decarboxylase Citrulline
RESULT:
(+) Prussian Blue *control: Klebsiella pneumoniae, RESULT:
Proteus mirabilis, Serratia marcescens (+) Red violet/ Purple *control: Serratia marcescens
(-) Green *control: Escherichia coli (-) Yellow *control: Proteus mirabilis

6. Lysine decarboxylase 9. Phenylalanine deaminase

PRINCIPLE: Some organisms can decarboxylase PRINCIPLE: Phenylalanine deaminase is an enzyme
amino acid Lysine converting it to amine Cadaverine that removes an amino group (NH3) from an amino acid
liberating O2. MEDIA: Phenylalanine Agar, overnight culture + 10%
MEDIA: Lysine Iron Agar (LIA) FeCl3
pH indicator: Bromcresol purple RESULT: (+) Green slant and fluid
*medium that detects H2S production, lysine
decarboxylation, phenylalaninedeamination and
glucose fermentation

PATRICK R. DE VERA,RMT, MSMT 16

 BACTERIOLOGY: REVIEW NOTES

10. ONPG (O-nitrophenyl-β-D-galactopyranoside) 14. Nitrite reduction

PRINCIPLE: Based on the utilization of lactose. Test PRINCIPLE: NO2 is reduced to nitrogen gas or other
for β – galactosidase that hydrolyze ONPG to compound (add zinc powder)
orthonitrophenol. True non-lactose fermenters do not RESULT: (+) colorless (-) Red
possess β- galactosidase.
MEDIA: TSIA or KIA MOLECULAR DIAGNOSIS
RESULT:
(+) Yellow *control: Escherichia coli 1. Polymerase Chain Reaction (PCR) – unknown
(-) appear clear or colorless*control: Proteus vulgaris sample ->extraction of DNA/ RNA ->amplicons (PCR
product)-> Identification
11. Malonate utilization  Conventional PCR – use of agarose gel as
PRINCIPLE: Determines if the organism can utilize migration medium. Uses a ladder (kB)
malonate as sole source of carbon examined under UV light
MEDIA: Malonate broth  Real time PCR/ Quantitative PCR/ qPCR –
RESULT: (+) Blue (-) Green or Yellow fluorescence indicates amplification of target
DNA/RNA (threshold) (e.g. GeneXpert®)
12. Gelatin liquefaction/hydrolysis test  Reverse Transcriptase – PCR (RT-PCR) – a
PRINCIPLE: Gelatinase producing organism can laboratory technique combining reverse
liquefy the gelatin media after incubation transcriptase of RNA to DNA and
RESULT: amplification of specific DNA targets.(e.g. )
(+) liquefaction*control: Proteus vulgaris  Multiplex PCR
(-) solid media*control: Klebsiella aerogenes  Nested PCR
2. LAMP (Loop mediated isothermal amplification) –
13. Nitrate reduction single tube technique for the amplification of DNA
PRINCIPLE: reduction of NO3 to NO2 by adding (e.g. GenProbe®).
Sulfanillic acid and α napthylamine. 3. MALDI-TOF (Matrix-Assisted Laser Desorption/Ioni-
RGT and RXN: Sulfanilic acid + NO3 -> diazonium salt zation-Time of Flight) – mass spectrometry, is an
(add zinc powder) ionization technique that uses a laser energy
RESULT: (+) Red; (-) no color absorbing matrix to create ions from large molecules
with minimal fragmentation.

REFERENCES:
Bailey & Scott’s Diagnostic Microbiolgy, Elsevier 13th edition, 2014.
Jawetz, Melnick, & Adelberg’s Medical Microbiology, McGraw-Hill 27th edition, 2014.
Graeter, Hertenstein, Accurso and Labiner, Medical Laboratory Science Examination Review, Elsevier, 2015.
Polansky, Valerie Dietz - Quick Review Cards for Medical Laboratory Science 2nd Edition, A. Davis, 2014.
“Biosafety and biosafety levels”. www.cdc.gov retrieved April 22, 2018.
“Introduction to Microbiology“. https://www.atcc.org retrieved April 22, 2018.

/prdvrmt2019

PATRICK R. DE VERA,RMT, MSMT 17

FAMILY MICROCOCCACEAE PATHOGENIC DETERMINANT OF STAPHYLOCOCCUS AUREUS
Genera: Micrococcus, Staphylococcus, Planococcus, Polysaccharide capsule Inhibits phagocytosis
Stomatococcus (Rothia) Slime layer and biofilm Adherence to inorganic surfaces;
ability to circumvent antibiotics (S.
Cultivation aureus, S. epidermidis)
BAP (5% Defibrinated Sheep blood) Peptidoglycan Resembles endotoxin of gram (-)
Activates complement and IL-1;
Broth: Thioglycollate, Dextrose, Brainheart infusion
serves as a chemotactic for PMNs
causing swelling and tissue
Human blood - not preferred, nonspecific inhibitor (citrate & damage
dextrose) Teichoic acid Species-specific; binds to
fibronectin
TEST TO DIFFERENTIATE STAPHYLOCOCCUS TO MICROCOCCUS Protein A Surface protein of S. aureus, binds
to Fc receptor of IgG &
TEST STAPHYLOCOCCUS MICROCOCCUS
complement
Aerobic growth Growth (facultative) Growth (obligate) - IgG1, IgG2 & IgG4
Anaerobic growth Growth (anaerobe) No growth(aerobe) immunoglobulin attaches to
Lysostaphin Susceptible Resistant Staphylococcal Protein A
Furazolidone Susceptible Resistant - IgG3 most efficient at
complement fixation but fails to
Bacitracin Resistant Susceptible
attach to Protein A
Modified Oxidase Negative (microdase) Positive Cytotoxins
Glucose Utilization Fermenter Oxidizer Alpha toxin - disrupts smooth
Growth on BAP Gram (+) cocci in Gram (+) cocci in muscles in blood vessels, toxic to
clusters “Grapelike tetrads or sarcinae, RBCs, WBCs, hepatocytes and
cocci “Clublike” platelets
Beta toxin - heat labile
Creamy white sphingomyelin which hydrolyses
pinhead colonies w/ phospholipid membranes causing
characteristic cell lysis
hemolytic pattern
Delta toxin - cytolytic to RBC,
Epidemiology of Staphylococci nonspecific membrane toxicity to
other mammalian cells, found in S.
aureus, S. epidermidis, S.
➢ Staphylococcus aureus haemolyticus
Colonizes anterior nares, nasopharynx, perianal area, skin, mucosa Gamma toxin – found in all S.
aureus strains, function with the
Mode of transmission: person to person, traumatic introduction, Panton-Valentin-Leukocidin
fomites, aerosols, abrasion (PVL) - toxic to WBCs initiating
deeper tissue infection
- predominant pathogen in adult joint infection
- common cause of food poisoning Leukocidin Type of neutrophils and
macrophages, inhibits
phagocytosis
➢ Staphylococcus epidermidis Enterotoxin Associated with
Skin flora, associated with bacterial endocarditis following insertion pseudomembranous colitis and
of prosthetic heart valves, blood culture contamination, hospital TSS
acquired UTI Exfoliative/Epidermolytic toxin Serine protease causing exfoliative
dermatitis or Scalded Skin
➢ Staphylococcus haemolyticus, Staphylococcus lugdunensis Syndrome
Normal flora of skin and mucous membranes Ritter disease (>90% of body is
involved)
Mode of transmission: person to person, implantation of medical Pemphigus neonatorum
devices (localized)
Toxic Shock Syndrome toxin-1 AKA: Enterotoxin F
(TSST-1) Induces IL-1 secretion causing
fever, vomiting,
Note: Exotoxins A & B desquamation/rash and
food poisoning hypotension – organ damage,
shock and death
Coagulase Forms fibrin clot
Staphylokinase Digest fibrin clot (fibrinolysin)
Hyaluronidase AKA: Duran-Reynal Factor/
Spreading factor
Digest connective tissue matrix
Lipase Digest oils, bacteria easily
colonizes skin eg. boils
Penicillinase Inactivates penicillin
Laboratory Diagnosis False positive:
1. Use of citrate anticoagulant
1. GRAM STAIN 2. Citrate utilizing organisms that release calcium causing clot
formation
Staphylococcus: gram (+) cocci in clusters
Micrococcus: gram (+) cocci in tetrads or sarcinae form Positive:
Staphylococcus aureus (human)
2. SHEEP BLOOD AGAR - enriched and differential at 35C Staphylococcus intermedius
increase CO2 or ambient air for 24 hours Staphylococcus schleiferi
Staphylococcus hyicus
S. aureus: pinhead creamy white colonies with smooth entire
margins, beta hemolytic VP, PYRase test
S. epidermidis: gray-white, extremely sticky (slime producing) Differentiates S. aureus from other coagulase positive
S. saprophyticus: large very glossy, butyrous, usually white but can Staphylococcus
be yellow/orange
VP test PYRase test
Alpha (greenish/brown) - partial lysis of red cells around the colony Staphylococcus + -
Beta (clear) - complete hemolysis aureus (human)
Gamma (no lysis) – non-hemolytic
Alpha Prime (small zone of Alpha surrounded by a zone of Beta Staphylococcus - +
hemolysis after refrigeration) - refrigerated sample
intermedius
3. PEA (Phenyl Ethyl Alcohol) & CNA (Columbia Nalidixic Acid) -
Staphylococcus + +
eliminates gram (-) contaminants; useful for STOOL specimen
schleiferi
4. MSA (Mannitol Salt Agar) - selective and differential
Staphylococcus - -
Inhibitor: 7.5-10% NaCl
hyicus
pH Indicator: Phenol red
CHO: mannitol

S. aureus: mannitol fermenter, yellow colonies 3. Modified Oxidase/Microdase test

S. epidermidis & S. saprophyticus: non-mannitol fermenter, red-pink Reagent: Tetramethyl p-phenylene diamine dihydrochloride in
colonies DMSO (Dimethyl Sulfoxide)
Positive: Micrococcus (Blue/Purple)
Negative: Staphylococcus (No color change)
5. CHROMAgar (Alain Rambach) - selective and differential
medium for MRSA
Contains Cefoxitin 4. Oxidation-Fermentation test
MRSA: Mauve-colored Medium: OF tube
Non-MRSA: White/Blue/Green CHO: Glucose
pH indicator: bromthymol blue
Acid (+): Yellow
Biochemical Identification
Alkaline (-): Blue
1. Catalase test - used to differentiate Staphylococcus from
Oxidizer: Micrococcus
Streptococcus
Fermentor: Staphylococcus
Principle: Staphylococcus uses catalase enzyme that can convert
hydrogen peroxide to oxygen and water. Common pH indicators used
Reagent: 3% hydrogen peroxide (H2O2) a. Bromcresol purple: purple to yellow
b. Andrade’s acid fuschin: yellow to pink
Positive: Staphylococcus (Effervescence or bubble formation) c. Phenol red: red to yellow
Negative: Streptococcus d. Bromthymol blue: green to yellow

2. Coagulase test - most important pathogenic determinant of 5. DNAse test - ability of organism to hydrolyze DNA
S. aureus. Medium: DNA
Methyl green: clear zone around colony
A. Slide Coagulase Toluidine blue: pink zone around colony
HCl precipitation: no precipitation after addition of 1N HCl
Detects cell-bound coagulase
Positive: S. aureus, Moraxella, Serratia (clearing of medium)
Reagent: Rabbit’s plasma (EDTA)
Negative: S. epidermidis & S. saprophyticus
Positive: Clumping
Negative: Confirmed with tube coagulase test 6. Novobiocin test - differentiates the coagulase negative
Staphylococci (CoNS) that are PYR positive, zone of 16 mm or
B. Tube Coagulase more in diameter indicates susceptible to novobiocin
Detects free coagulase
Reagent: 0.5 mL Rabbit’s plasma (Heparin) incubate at Sensitive: S. epidermidis
37c for 4 hours (check every 30 mins) If negative, Resistant: S. saprophyticus
incubate for another 20 hours, report negative if no fibrin
clot after 24 hours 7. Loeffler’s Serum Slant - enhances pigmentation of
Positive: Fibrin clot Staphylococcus

S. aureus: golden yellow colonies

S. citreus: lemon yellow colonies
S. albus formerly S. epidermidis: white porcelain colonies
8. MRSA GROUP B STREPTOCOCCUS
Drug of choice: vancomycin
Reedman syndrome ➢ Streptococcus Agalactiae

Associated with

Infant (transmission occurs vertically or during delivery, obstetric

complication)
Pneumonia
Neonatal meningitis

Adult
Endometritis
Wound infection

Medium: Todd Hewitt broth

Hemolysis: Beta

GROUP C and G STREPTOCOCCUS

➢ Streptococcus dysagalactiae
Large colony
Hemolysis: Beta
Resembles S. pyogenes infection
FAMILY STREPTOCOCCACEAE
➢ Streptococcus anginosus
GROUP A STREPTOCOCCUS Small colony
Hemolysis: Beta
➢ Streptococcus pyogenes Resembles S. pyogenes infection

Virulence factor GROUP D (ENTEROCOCCUS)

1. Protein M - bacterial protein binds that factor H PYR positive
2. Protein F (Fibronectin-binding protein) - mediates adherence to Bile-esculin positive
host epithelial cells Growth in 6.5% NaCl
3. Lipoteichoic acid - mediates attachment to mucosal cells Express Lancefield group D antigen
4. Hyaluronic acid capsule - prevents opsonized phagocytosis, Hemolysis: Alpha, Beta, Gamma
mask bacterial antigens
5. Streptodornase (DNAses) - degrade host DNA (DNAse) and Virulence factor
RNA Extracellular protein, serine protease and gelatinase - adhesion
6. Streptokinase (Fibrinolysin) - causes lysis of fibrin clots Cytolysins and resistance to antimicrobial agents
7. Hyaluronidase - solubilizes hyaluronic acid in connective tissue
8. Streptolysin O Associated with
- Subsurface hemolysin (oxygen labile) UTI (catheterization)
- Toxic to RBCs, WBCs, and platelets Bacteremia (hemodialysis, surgery)
- Induces antibody response - anti-streptolysin O, Nosocomial infection (multidrug resistant)
immunogenic Endocarditis (prosthetic heart valve)
9. Streptolysin S Pneumonia (ventilator)
- Surface hemolysin (oxygen stable)
- Lysis WBCs and non-immunogenic ➢ Enterococcus faecalis
10 Streptococcal pyrogenic exotoxins A, B, C (SPE) ➢ Enterococcus faecium
Exotoxin A - associated with Scarlet fever and Streptococcal shock-
like syndrome VIRIDANS STREPTOCOCCI (Group A, C, F, G, N)
Associated with Group D nonenterococcus
Bacterial Pharyngitis and tonsilitis Normal flora of the oral cavity, respiratory tract, and gastrointestinal
Pyodermal infections (Impetigo, Erysipelas, Cellulitis, Scarlet fever) (GI) tract mucosa
Necrotizing fasciitis (Suppurative fasciitis, Hospital gangrene,
Necrotizing erysipelas) ➢ Streptococcus bovis
Streptococcal Toxic Shock Syndrome Hemolysis: alpha, gamma
Post Streptococcal sequelae
Associated with
Complication Subacute bacterial endocarditis from patient with damage heart
Rheumatic fever (cross reactive antibodies against streptococcal valves
antigens and human heart tissue; fever and inflammation of heart Bacteremia, Septicemia
and blood vessels) Cavities (may enter the blood after dental procedures)
Acute Glomerulonephritis (Deposition of ab-ag complexes in
glomeruli) - ASO positive, dysmorphic RBC

Test for Scarlet fever

1. Dick’s test - susceptibility to Scarlet fever
Positive: Redness/ erythema
2. Schulz-Charlton test - neutralization/diagnostic tests
Positive: rashes fades/blanching phenomenon
STREPTOCOCCUS PNEUMONIAE BACTRACIN SXT NOTES
Capnophilic TAXO A
Gram positive diplococci (lancet or bullet shaped) A (S. S (>10 mm) R PYR +
Neufeld reaction (Quellung capsular swelling) pyogenes)

Virulence factor B (S. R R CAMP +,

Capsular polysaccharide agalactiae) Hippurate
Antigenic polysaccharides (80 serogroups) Hydrolysis +
Antiphagocytic C R S
D R R PYR +
Associated with Enterococcus Growth at
Pneumonia lobar and community acquired (rust colored sputum) (multidrug 45c and 10c
resistant)
Sinusitis Growth at
Otitis media 6.5% NaCl
Bacteremia D Non- R R Growth at
Meningitis Enterococcus 45c
Penicillin S
Colony morphology
1. Mucoid strains produce a large polysaccharide capsule. S. Pneumoniae Viridans
2. Umbilicated, depressed centers caused by autolytic enzymes Mouse virulence Death (+) Survive (-)
3. After 48 hours, colonies become nonviable. Inulin fermentation + -
Bile solubility + (soluble) - (insoluble)
Serology test Optochin S (>14 mm) R
Francis test - skin test for pneumococcal antibody
Taxo P
Laboratory identification
1. Gram stain: gram positive diplococci, lancet/bullet shape Chem name:
Ethylhydrocupreine
2. Growth on BAP: white, pinpoint + (capsular
Neufeld Quellung -
swelling)
3. Catalase
NUTRIOTIONALLY VARIANT STREPTOCOCCI
4. Bile esculin hydrolysis
Determine ability to grow in 40% bile and esculin hydrolysis
Positive result: esculetin reacts with ferric chloride to form black- ABIOTOPHIA & GRANULICATELLA
brown precipitate Streptococci that need cysteine or pyridoxal (vitamin B6)
Positive: Group D streptococcus (blackening) Satelliting-streptococci, thiol requiring streptococci
Negative: Viridans streptococcus
➢ Streptococcus adjacens
5. Bile solubility tests ➢ Streptococcus defectivus
Reagent: 2 % deoxycholate/sodium taurocholate
Positive: S. pneumoniae

TEST TO DIFFERENTIATE GROUP D ENTEROCOCCUS

FROM GROUP D NON-ENTEROCOCCUS
Bile esculin Growth in PYR
hydrolysis 6.5% NaCl
D + + +
Enterococcus
D non- + - -
enterococcus

6. PYRase
Test for pyrrolidonyl arylamidase
Substrate: L-pyrrolidonyl-B-napthylamide
Color developer: p-dimethylaminocinnamaldehyde
Positive: Red to Cherry Red (Group A Strep and Enterococcus)
Negative: No color change or orange color (Group D Non-
enterococcus)

7. CAMP reaction
Test for the synergistic hemolysis between group B. Streptococcus
and beta hemolytic S. aureus
Positive: enhance zone of hemolysis (Group B)
Negative: No zone of enhanced hemolysis in arrowhead pattern

8. Hippurate hydrolysis
Determines hydrolysis of sodium hippurate to benzoic acid
and glycine
Reagent: ninhydrin (glycine can be detected)
Positive: deep blue/purple (Group B strep and L. monocytogenes)

9. Bacitracin and sulfamethoxazole-trimethoprim susceptibility tests

GRAM-NEGATIVE COCCI 2. Utilization of CHO: CTA (Cystine Trypticase Agar)
Differentiation of Neisseria species through acid production from
NEISSERIA oxidation of carbohydrates
Gram-negative cocci (coffee bean/kidney shaped diplococci) Indicator: Phenol red
except N. elongata (rod)
Nonmotile, obligate aerobes, grow at 37c Glucose Maltose Sucrose Lactose
Preferred increased carbon dioxide M. - - - -
Catalase: positive except N. elongata catarrhalis
Oxidase: positive N. + - - -
gonorrheae
Capnophilic 5-10% CO2 M. + + - -
Candle Jar meningitidis
3-5% CO2 N. subflava + + +/- -
15% O2 N. + + - +
lactamica
Oxidase test - key identification test
Reagent: tetramethyl-p-phenylenediamine dihydrochloride ONPG
Positive: Dark purple in 10 secs positive
False positive: iron in the nichrome loop
➢ Neisseria meningitidis
PATHOGENIC Life threatening, acute, purulent meningitis
➢ Neisseria gonorrhoeae
Second leading cause of STD “Clap” Associated with
Male: urethritis Shock
Female: cervicitis (asymptomatic) Meningococcemia (meningococcal bacteremia)
Thrombocytopenia
Associated with Disseminated intravascular coagulation (DIC) - serious disorder in
Pharyngitis which the proteins that control blood clotting become overactive.
Disseminated infection (Fitz-Hugh Curtis syndrome) - pelvic Waterhouse Friderichsen syndrome - bleeding, hemorrhages in
inflammatory disease adrenal gland
• Bacteremia
• Gonococcal arthritis (joint fluid) Treatment
• Metastatic infections Ceftriaxone
Ophthalmia neonatorum - purulent conjunctivitis in newborn Rifampicin (≥8 hours close contact within 12 days)
• Crede’s prophylaxis (1% silver nitrate)
MORAXELLA
Transport medium Gram-negative diplococci
Jembec system Nonmotile, grow on blood agar
Citric acid and Sodium bicarbonate Catalase: positive
Oxidase: positive
Presumptive test DNase: positive
1. Oxidase (Taxo N) Carbohydrate utilization: negative, non-fermenter
Positive: Neisseria and Moraxella (purple)
Do not use nichrome wire (false positive) ➢ Moraxella Catarrhalis
Wagon wheel (wavelike periphery)
2. Superoxol test (modified catalase test) Hockey pock
Reagent: 30% H2O2
Positive: N. gonorrhoeae (vigorous bubbling) Associated with
Negative: N. meningitidis, N. lactamica (weak delayed bubbling) Sinusitis
Pneumonia (immunocompromised)
Definitive test Otitis media
1. Culture: Selective (Enriched CAP+antibiotics) Elderly: lower respiratory tract infection (rarely causes DIC such as
bacteremia or meningitis
Thayer Martin Vancomycin - inhibits gram positive
Colistin - inhibits gram negative, Specimen
except N. gonorrhea Male urethra
Nystatin - inhibits fungi Female: endocervix
Modified Thayer Martin Vancomycin + Colistin + Nystatin + Others: rectum, pharynx, joint fluid, urine (PCR)
Trimethoprim lactate - inhibits
swarming Proteus Immunologic assay
Martin Lewis Vancomycin + Colistin + Trimethoprim 1. Coagglutination method (N. gonorrhoeae, N. lactamica)
lactate + Anisomycin (fungi) 2. Fluorescent antibody testing – recognizes epitopes, principal
New York City Agar Vancomycin + Colistin + Trimethoprim outer membrane protein of N. gonorrhoeae, confirms diplococci
lactate + Nystatin (fungi) + lyse horse morphologic appearance
Also support the growth of
genital mycoplasma eg. blood
Mycoplasma hominis,
Mycoplasma urealyticum
GC LECT Lincomycin + Vancomycin + Colistin +
Trimethoprim lactate + Amphotericin
B
 Colonial Appearance and other characteristics on CAP RAPID LACTOSE FERMENTERS
N. gonorrheae Small, grayish white, convex, A/Ag H2S (-)
translucent, shiny colonies with either Lactose permease
smooth or irregular margins; may be Beta-galactosidase
up to five different colony types on
primary plates ESCHERICHIA COLI
N. meningitidis Medium, smooth, round, moist, gray IMViC ++-- TSI: A/Ag H2S (-)
to white; encapsulated strains are LOA ++-
mucoid; may be greenish cast in agar
underneath colonies UTI - 90%
N. elongata Gray, translucent, smooth, glistening; Sepsis
may have dry, claylike consistency Meningitis (also group B streptococcus) meningitis in infants
(BAP) Diarrheal disease
M. catarrhalis Large, nonpigmented or gray,
capsule, smooth; friable “hockey MUG test
puck” consistency; colony may be Enzyme: Beta D-glucuronidase
moved intact over surface of agar Positive (electric blue fluorescence): all except E. coli 0157:H7
(Hemolytic Uremic Syndrome)
GRAM-NEGATIVE BACILLI
Uropathogenic E. coli (UPEC)
ENTEROBACTERIACEAE “CPON NO GF FA” • Most common cause of UTI in humans
Gram-negative rods; facultative anaerobes; glucose fermenter Virulence factors
Motile (peritrichous flagella) except Klebsiella and Shigella Pili - adherence to epithelial cells
Catalase: positive except Shigella dysenteriae Cytolysins - inhibit immune effector cells
Oxidase: negative except Plesiomonas (new member) Aerobactin - chelate iron
Nitrate reducer (nitrosoreductase) except Erwinia and Pantoea
Enterotoxigenic E. coli (ETEC)
ALL ARE AEROGENIC (gas producer) EXCEPT SHIGELLA • Produces heat-labile and or heat-stable enterotoxins; genes of
(+/-) Salmonella, Proteus, Providencia both toxins reside on plasmid
Grow well on MacConkey agar (MAC), some as lactose fermenters • LTs are closely related in structure and function to cholera toxin;
(pink colonies) lactose non-fermenter (colorless) STs result in intestinal fluid secretion by stimulating guanylate
cyclase
ANTIGENIC STRUCTURE • “Montezuma’s revenge or turista”, “traveler’s and childhood
K antigen Capsular antigen diarrhea characterized by watery stool
(capsule) Heat-labile Medium: BAP
Some salmonella have capsular (K)
antigen, referred to as Vi (virulence) Enteroinvasive E. coli (EIEC)
O antigen Somatic antigen • Invades the intestinal epithelium causing shigella-like infection
(cell wall) Heat-stable • Dysentery (necrosis, ulceration, and inflammation of large
IgM antibody bowel); usually in young children living in areas of poor sanitation
H antigen Flagella • Stool with RBC, neutrophils, and mucus
(flagella) Denatured by heat or alcohol • Acid resistant (≥100,000 cfu/mL); explosive stool
Agglutinate with anti- H antibody mainly IgG
Enteropathogenic E. coli (EPEC)
Non motile at 37c • Non-invasive, produces no toxin
Shigella • Nosocomial, seen in newborn and infants
Klebsiella • Watery diarrhea with mucus but no blood
Yersinia
Enterohemorrhagic E. coli (EHEC)/Verotoxic E. coli (VTEC)
H2S Positive • Produce verotoxin - named for its cytotoxic effect on vero cells, a
Salmonella line of African green monkey kidney cells, a cytotoxin that
Proteus resembles Shiga toxin produced by S. dysenteriae
Arizona • HUS (E. coli O157:H7)- most severe manifestation of EHEC
Citrobacter - form of hemorrhagic colitis
Edwardsiella - transmitted by ingestion of ground beef or raw milk
Medium: MacConkey Sorbitol (colorless)
Laboratory Diagnosis
1. Culture Enteroaggregative E. coli (EAEC)
2. TSI • Probably involves binding by pili, ST-like, and hemolysin like
3. LIA toxins; actual pathogenic mechanism not known
4. Indole test • Watery diarrhea
5. MRVP

RAPID LACTOSE LATE LACTOSE NON LACTOSE

FERMENTER FERMENTER FERMENTER
Salmonella
Escherichia Citrobacter Shigella
Klebsiella Serratia Proteus
Enterobacter Hafnia Providencia
Salmonella arizonae Morganella
Shigella sonnei Edwardsiella
Yersinia enterocolitica Erwinia- plant
pathogen
KLEBSIELLA
IMViC --++ TSI A/Ag H2S (-) LATE LACTOSE FERMENTERS
LOA +-- LIA K/K A or K/A

Exhibit a mucoid growth, large polysaccharide capsule CITROBACTER

Tribe Salmonella (genera Salmonella, Citrobacter and Arizonae)
➢ Klebsiella pneumoniae Resembles Salmonella but are ONPG positive (LATE LF), LDC
“Friedlander’s bacillus” negative and KCN positive, growth
Encapsulated and mucoid colonies that tends to string Hydrolyzes urea slowly
Lactose fermenter
➢ Klebsiella ozaenae
Purulent sinus infection Associated with
UTI
➢ Klebsiella rhinoscleromatis
Granuloma of the nose and oropharynx Medium: Simmon citrate
Plasmid mediated ESBLs
➢ Citrobacter freundii
➢ Klebsiella oxytoca (only indole Extraintestinal pathogen
positive)IMViC: +-++ Isolated in diarrheal stool cultures

ENTEROBACTER Associated with (nosocomial infections)

IMViC --++ TSI A/Ag H2S (-) UTI
Pneumonia
Opportunistic infections: UTI, RT, and wound infections Intraabdominal abscesses
Endocarditis in IV abusers
➢ Enterobacter cloacae 80% produce hydrosulphide
Most predominant isolate 50% fail to ferment lactose

➢ Enterobacter sakazakii (Cronobacter sakazakii) ➢ Citrobacter diversus/koseri

Yellow pigment at 25c (room temp) Cause of nursery outbreaks of neonatal meningitis and brain
Medium: EMB (fisheye colonies) abscess

DECARBOXYLASE TEST NOTE:

LDC: Lysine Decarboxylase C. freundii H2S (+)
Product: Cadaverine C diversus & C. koseri H2S (-)
ODC: Ornithine Decarboxylase
Product: Putrescine C. Freundii Salmonella
ADH: Arginine Dihydrolase 70% hydrolyze urea Fail to hydrolyze urea
Fail to decarboxylate lysine Isolate decarboxylate lysine
LDC ODC ADH Growth on KCN No growth on KCN
K. pneumoniae + - -
K. oxytoca + - - SERRATIA
E. aerogenes + + - Common opportunistic pathogens in hospitalized patients
E. cloacae - + + (antimicrobial resistant)
P. agglomerans - - - DNAse, Lipase, Gelatinase: positive
Plesiomonas + + + ONPG: positive except S. fonticola

MOTILITY TEST Prodigiosin - red pigment (non-diffusible) at room temp

Differentiates Klebsiella (nonmotile) to Enterobacter (motile) ➢ S. marcescens, S. rubidaea, S. plymuthica

➢ Serratia marcescens
Most clinically significant

Associated with
Infection of urinary and respiratory tract
Bacterimic outbreaks
Septic arthritis (due to contamination of antiseptic solution)

➢ Serratia plymuthica
Osteomyelitis following motorcycle accident

➢ Serratia odoriera
Rancid potato-like odor

VP (+), Gas (+), TSI

Klebsiella
Enterobacter
Serratia
HAFNIA NON-LACTOSE FERMENTER
Gastrointestinal infections K/A
Lactose & Citrate test - differentiates Hafnia from Enterobacter
Hafnia alvei - Late lactose fermenter; Citrate (-) SALMONELLA
Enterobacter - Rapid lactose fermenter; Citrate (+) IMViC -+-+ TSI: K/A H2S (+)

YERSINIA Source of infection: water, milk and dairy products, shellfish (from
Nonmotile at 37c but motile at 22c contaminated water), dried or frozen eggs, meat and meat
except Y. pestis (non-motile both) products, household pets
Exotic pets (ciulla)
➢ Yersinia pestis
Plague (black death) - infection of wild rodents transmitted from one Associated with
rodent to another and occasionally by the bite of fleas Enteritis
Common vector: Xenopsylla cheopsis (rat flea) Enterocolitis/gastroenteritis (S. typhimurium) - most common
manifestation of Salmonella infection
3 types of plague Bacteremia (S. cholerasuis)
PNEUMONIC PLAGUE - highly infectious Typhoid fever - Pea soup stool
BUBONIC PLAGUE - buboes (swollen lymph nodes)
SEPTICEMIC PLAGUE - septic shock and death Cause of Enteric fever “Typhoid fever”
S. paratyphi A (serogroup A)
Inclusion: Bipolar bodies S. paratyphi B (serogroup B)
Stain: Wayson stain (safety pin appearance at 25c to 30c) S. cholerasuis (serogroup C) - bacteremia
Culture: Stalactite pattern (clumps of cells adhere to one side of S. typhi (serogroup D) - most important cause
tube)
Specimen collection (BUS)
Medium: Sheep Blood Agar (cauliflower after 48 hours) 1st week – blood
2nd week – urine, (STOOL ang isasagot pag given pareho)
➢ Yersinia enterocolitica 3rd week – serology, urine
Most common death of bacterial contamination of packed RBC
Gold standard: Bone marrow aspirate
Associated with Carrier: Bile acid/fluid
Enterocolitis
• Fever Medium: Bismuth Sulfite Agar - S typhi
• Diarrhea (black colonies with metallic sheen)
• Abdominal pain (resembles appendicitis in children)
Bacteremia Serological test
Widal test - classic tube dilution agglutination
Arthritis
Atleast 2 serum specimen, obtained at intervals of 7-10 days
Mesenteric lymphadenitis
Ab titer to O antigen ≥ 1:160 ACTIVE INFECTION
Ab titer to H antigen ≥ 1:160 PAST INFECTION
Medium: Cefsulodin Irgasan Novobiocin (CIN) bull’s eye
↑Ab to K (vi) Ag - carrier
colonies

Urease
Y. pestis -
Y. +
enterocolitica

SHEWANELLA
Resembles Salmonella H2S (+)

SUCROSE FERMENTATION
Shewanella is sucrose (+) while Salmonella sucrose (-)
SHIGELLA Resistant to:
IMViC -+-- TSI: K/A H2S (-) Cephalosporins
Aztreonam
Natural habitat is limited to the intestinal tracts of humans and other Penicillin
primates, where they produce Bacillary Dysentery
PANTOEA AGGLOMERANS
Group Catalase ONPG Mannitol Gained notoriety with a nationwide outbreak of septicemia resulting
S. dysenteriae A - - - form contaminated intravenous fluids
S. flexneri B + - +
S. boydii C + - + ➢ Pantoea agglomerans HG XII
S. sonnei D + +, + Produces a yellow pigment primarily a plant pathogen
LATE
LF ERWINIA AGGLOMERANS
Triple decarboxylase negative (lysine, ornithine, arginine)
➢ Shigella dysenteriae GROUP A
Catalase (-), ONPG (-), Mannitol (-) LABORATORY IDENTIFICATION

Associated with CULTURE

Seizures
HUS a. EOSIN METHYLENE BLUE (EMB) AGAR
Selective/differential for gram negative bacilli
PPM CHO: lactose and sucrose
Lactose non-fermenter
Motile Lactose fermenter: pink to purple colonies
MR (+) Non lactose fermenter: colorless
LDA (+)
Characteristic Colonies
PROTEUS E. coli (pink to purple colonies with green metallic sheen)
Produce infection only when they leave the intestinal tract Klebsiella (pink mucoid colonies)
Formation of Renal stones/calculi “staghorn calculi” - struvite (triple Enterobacter (pink colonies with dark center “Fish eye”)
phosphate or magnesium ammonium phosphate)
b. MacConkey Agar (MAC)
Associated with Selective/differential for gram negative bacilli
UTI Inhibitor: crystal violet, bile salts
Bacteremia CHO: lactose
Pneumonia pH indicator: neutral red
Nosocomial infections
Lactose fermenter: pink colonies
Source of antigen for the Will Felix reaction because they share Non lactose fermenter: colorless
polysaccharide for dx of rickettsial infection
Proteus vulgaris source of ox-2 ox-19 c. Hektoen Enteric (HEK)
Proteus mirabilis source of ox-k (Kingsbury) Selective/differential for gram negative bacilli
Inhibitor: bile salts
Indole CHO: lactose, sucrose, salicin
P. vulgaris + pH indicator: bromthymol blue
P. mirabilis - H2S indicator: ferric ammonium citrate

Lactose fermenter, H2S positive: orange to yellow w/ black center

PAD H2S
Lactose fermenter, H2S negative: orange to yellow
Proteus + +
Non lactose fermenter, H2S positive: blue green w/ black center
Serratia - - Non lactose fermenter, H2S negative: blue green

PROVIDENCIA d. Salmonella-Shigella Agar (SS)

Normal intestinal flora Selective/differential for Salmonella and Shigella
Multi drug resistant CHO: lactose
pH indicator: neutral red
Associated with H2S indicator: ferric ammonium citrate
UTI
Salmonella
Urease Non lactose fermenter, H2S positive: (colorless w/ black center)
P. rettgeri +
P. stuartii -/variable Shigella
Non lactose fermenter, H2S negative: (colorless)
EDWARDSIELLA
e. Xylose-lysine deoxycholate agar (XLD)
IMViC ++-- (similar to E. coli) but
E. coli (yellow colonies)
E. tarda is non-lactose fermenter, H2S (+)
Shigella (colorless - pink/red)
Isolated from cold and warm-blooded animals
Salmonella (red colonies w/ black center)
➢ Edwardsiella tarda
Most human species

H2S Lactose
P. tarda + NLF
E. coli - LF
Extended-Spectrum B-Lactamase (ESBLs)
VIBRIO ➢ Vibrio mimicus
Facultative anaerobe Gastroenteritis and ear infections associated w/ marine
Motility: Rapid darting, shooting star environment
Oxidase: Positive Non-halophilic
Halophilic except: V. cholerae and V. mimicus Sucrose (-)

Medium: APW and TCBS ➢ Vibrio vulnificus

Septicemia and wound infectious marine environment
1. APW (Enrichment) Halophilic
pH: 8.4 Sucrose (+)
Lactose (+)
2. TCBS (Selective and Differential) Predisposing factors: liver dysfunction, ↑ serum iron
pH: 8.6 - inhibits growth of other intestinal flora
CHO: Sucrose AEROMONAS
Indicator: Bromthymol blue
➢ Aeromonas hydrophilia
Composition: “water loving” most common isolate
1% Sodium chloride Gram negative straight rods
Bile salts - inhibits the growth of gram-positive organisms Medium:
SBA (Large, round, raised opaque, β hemolytic)
a. V. cholerae - SF, yellow colonies CIN (Lactose fermenter, pink centered)
b. V. parahaemolyticus - NSF, green colonies
PLESIOMONAS
➢ Vibrio cholerae
Characteristic: rice water stool ➢ Plesiomonas shigelloides
Curved or straight (comma-shaped) gram negative rods Straight gram-negative rods that occur singly, in pairs, in short
Polar/ peritrichous flagella chains and filamentous forms
Cholera toxin (choleragen) - increase CAMP Medium:
“Pfeipper’s phenomenon” - lysis of V. cholerae after injecting to an SBA
immunized host Bile Salt Agar (White to pink in inositol brilliant green)
LDC, ODC, ADH Positive “Positive Trio”
SEROGROUPS
Inaba (Philippines) CHROMOBACTERIUM VIOCELIUM
Ogawa (India)
Hikojima (Japan) Slightly gram-negative rods with rounded ends
Black or very Dark Purple colonies
Odor: Ammonium cyanide
3 MAJOR SUBGROUPS
Violacein: Purple pigment
V. cholerae O1 & V. cholerae O139 - epidemic cholera
Gold standard: Quorum sensing
V. cholerae non-O1 - strains that phenotypically resemble V.
Medium: MacConkey (Non lactose fermenter)
cholerae O1 but fail to agglutinate in 01 antisera

Vibriostatic test ✓ Sodium requirement and mannitol fermentation: key tests to

Reagent: 0/129 (150 ug) impregnated disks differentiate Aeromonas and Plesiomonas from Vibrio
Positive: susceptible
- Vibrio cholerae Vibrio Other Aeromonas Plesiomonas
- Plesiomonas shigellosis cholerae vibrio
Negative: resistant TCBS + + - -
- Aeromonas spp. String test + + - -
- Chromobacterium violaceum 0/129 (150 + - - +
ug) susp
String Test Broth w/o + - + +
Differentiate Vibrio from Aeromonas (lyse Vibrio cells but not those NaCl
Aeromonas) Broth w/ + + - -
Reagent: 0.5% Sodium deoxycholate 6.5% Na Cl
Positive: Vibrio Inositol + - - +
Negative: Aeromonas, Plesiomonas fermentation
Addt. Mannitol Mannitol LDC, ODC,
➢ Vibrio alginolyticus comments (+) (+) ADH
Blood, eye, and ear infections associated w/ marine environment
Halophilic
Sucrose (+)

➢ Vibrio parahaemolyticus
“Summer Diarrhea”
Gastroenteritis; contaminated seafood
Kanagawa phenomenon positive - heat stable hemolysin that
lyse human RBC in Wagatsuma agar
Halophilic
Sucrose (-)
CAMPYLOBACTER NON-FERMENTING GRAM NEGATIVE BACILLI
Gram negative rods that are associated with Gastritis & Diarrhea Grow in MacConkey as colorless colonies
S-shaped, seagull wings Most are oxidase positive; multi-drug resistant
Microaerophilic and capnophilic
Motile: Single polar flagellum (darting) Fail to acidify TSI agar (K/NC)
Optimum temp: 42c to 43c Fail to acidify O-F media, overlaid with mineral oil
Medium: Campys BAP, Skirrow’s
PSEUDOMONAS
HUMAN PATHOGEN Obligate and aerobe
➢ Campylobacter jejuni Motile and rod shaped
GUILLAIN BARRE SYNDROME Sweet or grape-like or corn-taco like odor
Hippurate Hydrolysis (+)
➢ Pseudomonas aeruginosa
ANIMAL PATHOGEN Grows well at 42c (Other Pseudomonas 37-42c or 35-42c)
➢ Campylobacter fetus Oxidase: Positive
Abortion in animals
No growth at 42c Medium:
SBA Beta hemolytic
HELICOBACTER Cetrimide agar serrated confluent growth
Associated w/ Peptic ulcer (rapid urease producer) Acetamide: positive
Microaerophilic and Capnophilic TSI: K/K or K/NC
Optimum temp: 35c to 37c
Medium: Campys BAP, Skirrow’s Pigments (Diffusible)
Motile: 4-6 polar flagella Pyocyanin - blue
Pyoverdin/Fluorescin - green
NONCULTURE METHOD
1. Gastric biopsy specimen Associated with:
2. Urea breath test UTI
3. Fecal antigen detection Nosocomial pneumonia
4. Microscopic examination of stained gastric tissue Hot tub whirlpool dermatitis (Spa)
5. DNA Amplification test (PCR) Septic Arthritis in IV drug abusers
Ecthyma gangrenosum - skin lesions
H. C. jejuni Mild otitis externa/media - Swimmer’s ear
pylori Septicemia in immunosuppressed patients and infants
Urease + Neg Severe wound infections in burn patients (blue green pus)
Nitrate Neg + Destructive eye infections (keratitis, corneal ulcers) - contact lens
Hippurate hydrolysis Neg + wearers
Chronic lung infection - Respiratory infections in patients w/ cystic
fibrosis
Note:
Hippurate Hydrolysis (+)
Note:
S. agalactiae
Grows well at 42c “CAP”
L. monocytogenes
Campylobacter
G. vaginalis
Aeruginosa
C. jejuni
Pseudomonas

Contaminated contact lens care solution “APA”

Acanthamoeba
Pseudomonas
Aeruginosa

➢ Burkholderia mallei (Pseudomonas mallei)

“Glanders disease”
Diseases of horses transmissible to man
Horses: pulmonary involvement
Human: fatal, begin as ulcer of skin and mucous membrane
followed by lymphangitis and sepsis

➢ Burkholderia pseudomallei (Pseudomonas pseudomallei)

“Vietnamese time bomb disease”
Melioidosis/Whitmore’s bacillus (glander like disease)
Gram stain: Bipolar
Medium: Ashdown (deep pink wrinkled colonies)
Odor: Earthy
➢ Burkholderia cepacia (Pseudomonas cepacia) CHRYSEOBACTERIUM (Flavobacterium) MENINGOSPETICUM
2nd most common cause of Cystic fibrosis Oxidase, DNAse, Gelatin hydrolysis, Indole (+)
Oxidase: Positive, no growth at 42c Nonmotile, MaC (-)
Motility: Lopotrichous Produces yellow pigment (flavin)
Medium:
OFPLB (Yellow) Associated with
MacConkey (Pink) Sepsis
Neonatal meningitis
Associated with:
Heart valve endocarditis HAEMOPHILUS, HACEK and OTHER
Plant: Onion bulb rot FASTIDIOUS GRAM-NEGATIVE BACILLI
Human: Foot rot
HAEMOPHILUS
➢ Burkholderia gladioli
Oxidase: Negative Non-motile, none-spore forming, facultative anaerobic
Ferment Carbohydrates (except H. ducreyi)
Preferred incubation: 35 to 37c
Environmental pathogen of plant
Oxidase and Catalase: positive
Associated w/ Cystic Fibrosis
Requires X and V factors
ACITENOBACTER
1. X factor/hematin/hemin
Gram-negative coccobacilli From degradation of hgb
Nonmotile, obligate aerobe Heat-stable
Drug resistant
2. V factor/NAD/Coenzyme I
TSI: K/K, Catalase (+) Oxidase (-) Produced by some bacteria (S. aureus) and yeast
Associated with UTI, wound, diarrhea Heat-labile
Medium: ➢ Haemophilus influenzae (Pfeiffer's bacillus)
MacConkey agar (Purple) Main cause of meningitis in children <5
EMB (Blue-grey, cornflower blue) Respiratory condition: EPIGLOTTITIS
Specimen: Nasopharyngeal swab
➢ Acitenobacter baumanni
Oxidizer, growth at 42c Virulence factor
2nd most common cause of nosocomial infections 1. Capsule - antiphagocytic and anticomplementary
a, b (most common), c, d, e, f
➢ Acitenobacter iwoffi 2. IgA protease - cleaves secretory IgA
Non-oxidizer, asaccharolytic 3. Outer membrane proteins and LPS

➢ Acitenobacter haemolyticus a. Encapsulated - systemic, and invasive infections

β hemolytic strain - Meningitis, epiglottitis, sepsis, pneumonia, arthritis
b. Non-encapsulated
Motility Oxidase Catalase LDC - Localized infections
Acitenobacter - - + - - May be carried asymptomatically (nasopharynx)
S. maltophilia + - + +
Pseudomonas + + + - Staph streak
Dew drop colonies around S. aureus “satellitism”
STENOTROPHOMONAS MALTOPHILIA
Old name: Xanthromonas ➢ Haemophilus aegyptius (Koch-weeks bacillus)
Oxidase: negative; DNAse: positive Resembles H. influenzae biotype III
Oxidizer of glucose; strong oxidizer of maltose PURULENT CONJUNCTIVITIS “pink eye” and BPF (Brazilian
Lavender green colony Purpuric Fever)
Odor: Ammonia-like Specimen: conjunctival swab
TSA: Yellow pigment
➢ Haemophilus ducreyi (smallest pathogenic bacteria)
Associated w/ Cystic Fibrosis and wound infection (farming Chancroid - soft chancre venereal disease
equipment) Incubation: 5% to 10% CO2 at 33c with high humidity, 7 days

ALKALIGENES FAECALIS Microscopic morphology

1. Small gram-negative coccobacilli to long filaments; may be
Oxidase, Catalase (+) encapsulated
Motility: Peritrichous
2. SCHOOL OF FISH, RAILROAD TRACKS, FINGERPRINTS from
Odor: Apple-like
genital lesions
OF (-/-), Assacharolytic
Specimen Processing and Isolation
Associated with a. Collected by pre-moistened swab, Stuart’s, or Amie’s
UTI b. Medium
Wound Chocolate agar
Diarrhea
Chocolate agar with bacitracin (H. influenzae)
Chocolate agar with 1% IsoVitaleX (H. ducreyi or H. aegyptius)
Oxidase Motility
Nairobi medium
Acitenobacter - - c. Incubation 5% to 10% CO2 at 35c to 37c, 24 to 72 hours
Alkaligenes faecalis + +
 Species Colony appearance KINGELLA
H. influenzae Translucent, smooth, and convex Short bacilli to coccobacilli with square ends in pairs/chains
Mousy or bleach-like odor in CHOC agar Fermenter (dysgonic); pits agar
Encapsulated strains are larger and mucoid
H. ducreyi Small flat smooth transparent to opaque Catalase: negative
Colonies can be pushed intact Oxidase: positive
Clumpy in saline
Associated with
Species Requirement for Bone infections
β
X V hemolysis D-ala Xylose
factor factor ➢ Kingella denitrificans
H. Influenzae + + - - + Non-hemolytic
H. parainfluenzae - + - + -
H. hemolyticus + + + - - ➢ Kingella kingae
H. - + + + - Beta hemolytic
parahaemolyticus
H. aegypticus + + - - - CAPNOCYTOPHAGA
H. aphrophilus - - - + -
Gram negative, fusiform shaped bacilli
H. - + - + -
paraphrophilus w/ one rounded end and one tapered and filamentous forms
H. ducreyi + - - - - Motility: Gliding

HACEK CALYMMATOBACTERIUM
Dysgonic (slower or poorer growing) Encapsulated, pleomorphic gram-negative bacillus usually
Normal biota of the oral cavity observed in vacuoles in large mononuclear cells
Fail to grow at MacConkey Donovan body: group of organisms seen within mononuclear cells
Fermenter (require serum) Closely related to Klebsiella

Associated with ➢ Klebsiella granulomatis

Subacute Bacterial Endocarditis *Calymatobacterium granulomatis (former name)
Periodontal diseases
Associated with
H. APROPHILUS DONOVASIS
Granuloma
“Foam loving” (need high concentration of CO2) Inguinale
With V factor dependent and independent strains
➢ Calymatobacterium inguinale
AGGREGATIBACTER ACTINOMYCETEMCOMITANS
Morse code bipolar, only catalase (+) PASTEURELLA
Glucose fermenter (require serum) Normal flora of cavity, gastrointestinal tract, respiratory tract of cats
& dogs
24 hours: for visible growth
After 48 hours: star shape with 4-6 points (100x light microscope) Mode of transmission
Wound infection (most common) of animal bite
CARDIOBACTERIUM HOMINIS
Pits agar; tend to form rosettes or long filaments Specimen
Normal flora Upper Respiratory Tract Sputum
Rare cause of Endocarditis Bronchial wash

EIKENELLA CORRODENS (Corroding bacterium) ➢ Pasteurella multocida

Fastidious, capnophilic rod; non-fermenter; biochemically inert Agent of Pasteurellosis (dog/cat bite/scratch) NOT DISEASE
Part of the gingival and bowel flora Agent of Shipping Fever in cattles (hemorrhagic septicemia)
Pitting of agar/corrodens
Odor: bleach-like

Catalase: negative
Oxidase: positive

Associated with
Human bite infection (clenched fist wounds)
BORDETELLA ➢ Brucella abortus (cattle)
Gram-negative bacilli “Bangs Bacillus”
Nonmotile except B. bronchiseptica Only capnophilic

Animal CO2 H2S Growth

➢ Bordetella pertussis
source requirement Thionine Basic
Agent of Whooping cough fuschin
Three Stages of pertussis (whooping cough) “CPC”
B. Cattle + + Inhibited G
Catarrhal - flulike symptoms, most contagious abortus
Paroxysmal - repetitive coughing episodes
B. Sheep, - - G G
Convalescent - recovery phase
melitensis goats
B. suis Pigs - - G Inhibited
Specimen: Nasopharyngeal swab
NP swab or NP aspirate collected with plastic shaft B. canis Dogs - - G Inhibited
Do not use aluminum or calcium alginate for PCR specimen
Use 2 minute safranin “O” or a 0.2% aqueous basic fuschin to Grade A milk (healthy cows)
enhance visibility <75,000 bacteria/mL raw
Not exceed 15,000/mL once pasteurized
Medium:
Bordet-Gengou medium (enriched potato blood-glycerol) Standard Plate Count
Colonies: MERCURY DROPLETS Estimate of the total number of viable aerobic bacteria present in
Regan-Lowe (Charcoal-cephalexin) - PREFERRED raw milk
Component:
- Horse blood FRANCISELLA
- Charcoal Faintly staining, gram-negative coccobacilli
- Antibiotics (Amphotericin, Cephalexin) Nonmotile, obligately aerobic

➢ Bordetella parapertussis ➢ Francisella tularencis

Pertussis-like syndrome, Mild form Disease of rodents (tularemia)
eg. rabbits transmissible to man
➢ Bordatella bronchiseptica
Inhabits respiratory tract of canines (kennel’s cough) Mode of transmission
Chronic respiratory tract in HUMANS Animal bite/scratch
Infected blood (common in hunters)
Urease Motility Nitrate Oxidase Vector: ticks
B. pertussis - - - +
B. + - - - Medium: Blood-Glucose Cystine Medium
parapertussis Requires cysteine, cystine, thiosulfate
B. + + + +
bronchiseptica LEGIONELLA
Gram-negative; poor staining 0.1 % fuschin not safranin
BRUCELLA
Small nonmotile, aerobic, gram-negative coccobacilli or short rods Source
Normal flora of urinary tract and gastrointestinal tract of sheep/goat 1. Natural eg. ponds creeks, wet soil
2. Artificial eg. air-condition towers, shower heads, plumbing
Mode of transmission systems
Ingestion of contaminated products: meats, milk
Farmers: direct animal contact Mode of transmission
Ingestion of contaminated products; meats milk
Associated with
Brucellosis Stain
Undulant fever Dieterie’s stain
Malta fever
Specimen
Specimen Sputum
Blood Bronchial wash
Bone marrow
Medium: Buffered Charcoal Yeast Extract (BCYE) brown
Medium: Castaneda bottle (biphasic medium) colonies
Agar & Broth
➢ Legionella pneumophila
Blood culture: 7 days reincubate if negative *Legionnaire’s disease
Slow grower (3-4 weeks reporting) *Pontiac fever

➢ Legionella micdadei
*Pittsburgh pneumonia

➢ Legionella bozemanni
*Wiga’s agent of pneumonia
STREPTOBACILLUS MONILIFORMS AEROBIC GRAM-POSITIVE BACILLI
Agent of Rat bite fever and Haverhill fever (ingestion of
contaminated food, unpasteurized milk, water) BACILLUS
Gram positive; aerobic
RAT BITE FEVER Round and central spore
direct contact w/ rat feces or saliva Catalase: positive
Motile except B. anthracis
HAVERHILL FEVER
Ingestion of contaminated food, such as unpasteurized milk or milk ➢ Bacillus anthracis
products and, less frequently, water “Bioterrorism agent”
Non-motile, non-hemolytic
Specimen Larges pathogenic bacilli and largest bacteria
Blood Causative agent of anthrax; most virulent
Serum
Ascites fluid Associated with
Cutaneous anthrax/malignant pustule/eschar - most common, least
Broth severe
Resembles “fluff balls” Woolsorter’s disease or pulmonary anthrax - inhalation of spores
Gastrointestinal anthrax - least common, most severe
Agar
Resembles “breadcrumbs” Medium
BAP “Medusa head/ rhizoid colonies”
SPIRILLUM MINOR/MINUS Bicarbonate Agar
Gram negative, helical, strictly aerobic organism From patient, encapsulated (D-glutamic acid capsule)
From culture, bamboo pole appearance/fishing rod arrangement
RAT BITE FEVER (SODOKU) MHA + Penicillin “String of pearls”
direct contact w/ rat feces or saliva
➢ Bacillus cereus
Associated with “Fried rice bacillus”
Arthritis (rarely) Motile, beta hemolytic
Swollen lymph nodes Large, feathery, spreading
Febrile episodes Food poisoning from rice, cereals vegetables and milk

ELIZABETHKINGIA MENINGOSEPTICA Toxins

Emetic (vomiting) after 1-4 hours of ingestion
Chryseobacterium meningosepticum (former name)
Diarrheic (diarrhea) after 18-24 hours
Flavobacterium meningosepticum (former name)
Specimen
Mode of transmission
Food (toxigenicity assay)
1. Contaminated medical device or solutions Vomitus
2. Birth canal to neonate
Stool
Medium: Blood Agar Plate (yellow colonies)
Associated with
POST TRAUMATIC EYE INFECTIONS
ACTINOBACILLUS
Endocarditis
Granulomatous disease to animals Bacteremia
Soft tissue infection in HUMAN following animal bites Infection of other sites - IV drug abusers or Immunocompromised
Morphology: Dots and dashes of Morse code patients

Tests B. anthracis B cereus

Catalase + +
Lecithinase + +
Motility Non-motile Motile
Hemolysis BAP Gamma (non- Beta
hemolytic)
ANAEROBIC GRAM-POSITIVE BACILLI “LBC” ➢ Clostridium tetani
Gram-variable straight rods with blunt ends
CLOSTRIDIUM Hemolysis: swarming β hemolytic
Gram positive; anaerobic Spore: Swollen round terminal “drumstick/tackhead
Oval sub-terminal spore except C. tetani (round and terminal) bacillus/lollipop/tennis racket (Mahon)”
Catalase: negative
Etiologic agent of tetanus
➢ Clostridium perfringens Devils green - results from entry of the organism or spores into a
*Bacillus aerogenes (former name) puncture wound
*Clostridium welchii (former name)
Virulence factor
Aerotolerant (superoxide dismutase) Tetanospasmin - a neurotoxin that causes a spastic type of paralysis
Boxcar, large square rods, with continuous muscle spasms, backward arching

Associated with Associated with

Food poisoning Tetanus - paralysis with continuous muscular spasms
Gas gangrene/myonecrosis Trismus (lock jaw)
Sulfhemoglobinemia Risus sarcodinicus (distorted grin)
Breathing difficulty
Virulence Factor
α toxin - type C food poisoning (enteritis necrotans) ➢ Clostridium botulinum
Enterotoxin “Flaccid paralysis”
Hemolysis: β hemolytic
Specimen Lipase: Positive
Food Spore: Swollen terminal “tennis racket” (Bailey’s)
Stool Oval, subterminal (Mahon)

Spores: usually absent Virulence factor

ETHANOL SHOCK SPORE TECHNIQUE Botulinum toxin - most potent exotoxin (neurotoxins associated w/
If spores are not present on Gram stain, the ethanol shock spore or flaccid paralysis)
heat shock spore test can separate Clostridium spp from the non- Strabismus “wandering eye”, “frown lines”
spore-forming bacilli.
Agent of
Double zone of hemolysis Wound botulism
Inner: Complete (beta zone, theta toxin) Food botulism “canned good” - pre-formed toxin
Outer: Incomplete (alpha zone, lecithinase) Infant botulism/floppy baby “honey” - organism multiply in GIT then
Lecithinase: Positive produced toxin

Nagler reaction: Lecithinase Precipitation Test Specimen

Principle: Neutralization Serum
Positive: Opaque zone on side w/o anti-toxin and no opacification Vomitus
on side w/ anti-toxin Stool (confirmatory)
Gastric aspirate (best specimen for infant)

Medium: EYA (iridescent sheen)

Confirmatory
Demonstration of toxin

➢ Clostridioides difficile
Important cause of antibiotic (clindamycin) associated
pseudomembranous colitis and diarrhea
Spores: oval, subterminal
Reverse CAMP Medium: Cycloserine-Cefoxitin-Fructose agar (CCFA)
S. agalacatiae instead of S. aureus Yellow ground glass colonies with chartreuse fluorescence
Positive: Bow-tie zone of hemolysis Odor: Horse barnyard

Virulence factor
Toxin A (enterotoxin)
Toxin B (cytotoxin)

Confirmatory
Toxin B (cytotoxin) in feces by tissue culture
Organism in feces (liquid or unformed)

Reverse camp positive

C. perfringens
C. jeikeium
A. haemolyticum
➢ Clostridium septicum Brown halo
Associated with malignancies (colorectal cancer) Useful in differentiating feature because only C. diphtheriae,
Neutropenic enterocolitis and myonecrosis C. ulcerans, and C. pseudotuberculosis produce a brown halo on
Resembles “Medusa head”, β hemolytic, smoothly swarming CTBA

Motility Lecithinase Lipase Lactose Glucose Organism Urease

Perfringens - + - + + C. diphtheria subs. gravis -
Botulinum + - + - + C. diphtheriae subs. mitis -
Tetani + - - - - C. diphtheriae subs. belfani -
Difficile + - - - + C. diphtheriae subs. -
intermedius
Swarming motility (Anaerobic BAP) C. ulcerans +
D. Tetani, C. septicum C. pseudotuberculosis +

Lipase positive Biotypes of Corynebacterium diphtheriae

C. botulinum 1. Gravis (most severe type)
C. novyi 1 to 2mm colonies on blood agar; largest colonial type
C. sporogenes Gray, non-hemolytic, starch/glycogen positive

CORYNEBACTERIUM DIPHTHERIAE 2. Mitis

“KLEBS LOFFLER BACILLUS” Medium “fried egg appearance” on blood agar (clear colonies w/
Agent of diphtheria white center
Pseudomembrane formation of the oropharynx leading to Black, beta hemolytic, starch/glycogen negative
respiratory obstructions “bleach-like odor” on tellurite medium
Characteristic: “bulls neck appearance”
3. Intermedius
Gram: positive Small colonies (0.5 mm) on blood agar
Catalase: positive Black colonies w/ gray borders on tellurite medium; non-hemolytic
Nonmotile
Nitrate reduction: positive 4. Belfanti
Urease: negative Most frequently recovered
Glucose/maltose: fermenter
Sucrose: not fermented Toxigenicity test
1. In vivo: Guinea pig inoculation test (+) death
*Catalase test - test to differentiate 2. In vitro: ELEK test, Definitive test (+) precipitin line
Corynebacterium diphtheriae - nonmotile Principle: Immunodiffusion test
Listeria monocytogenes - motile

“Babes Ernst granules/Metachromatic Volutin granules”

o Club shaped
o Barbed/dumbbell
o Palisade/picket pence, cigarette packets
o V or L forms
o Snapping formation
o Chinese character arrangement

Specimen
*Collected using a calcium alginate or dacron tip swab
Oropharyngeal/Throat swab Susceptibility test
Nasopharyngeal swab/Nasal swab Shicks test
WHO: collect 2 specimens (nasal and oropharyngeal swab) Positive: redness (erythema)

Stains
1. Loeffler’s alkaline methylene blue stain
2. Burkes modification of gram stain

Culture
1. LSS (Loeffler’s Serum Slant) - enhances pleiomorphism and
metachromatic granule formation
*Burke’s Modification of Gram stain - demonstration of
metachromatic granules
2. Pai’s coagulated egg - enhances pleiomorphism and
metachromatic granule formation
3. Modified Tinesdale afar - black with Tinsdale’s halo (brown halo)
4. Cystine Tellurite Blood Agar (CTBA) - Gun metal black colonies
*Potassium tellurite inhibits normal flora

Note: Staphylococcus and Streptococcus also produce gun metal

black on CTBA
DIPTHEROIDS (looks like Diphtheria) Motility Salicin
fermentation
➢ Corynebacterium jeikeium (formerly group JK) Listeria + +
Multi drug resistant. reverse CAMP Corynebacteria - -
Endocarditis, pneumonia, peritonitis

➢ Corynebacterium pseudodiphthericum CATALASE NEGATIVE GRAM-POSITIVE BACILLI “WELGA”

Normal flora: throat
ERYSIPELOTHRIX RHUSIOPATHIAE
➢ Corynebacterium xerosis Causative agent of Erysipeloid/Butcher’s cut/Diamond cut -
Normal flora: conjunctiva inflammatory swelling of hands and fingers (seal or whale finger)

➢ Corynebacterium acnes (P. acnes) Common in

Normal flora: skin Fish vendors
Butcher (butchers’ disease)
➢ Corynebacterium amycolatum
Most common isolated specie Gram: positive rod
Normal flora: Human conjunctiva, skin, nasopharynx Catalase: negative
Immunocompromised: endocarditis, septicemia, pneumonia, and Non-motile
neonatal sepsis Only gram-positive bacilli that is H2S (+)

➢ Corynebacterium minutissimum Medium:

Erythrasma - Superficial, pruritic skin infections SIM Gelatin stab (test tube brush-like or bottle brush-like growth)

LISTERIA MONOCYTOGENES ARCANOBACTERIUM HEAMOLYTICUM

Major source of infection is contaminated w/ food (cabbage, fruit,
dairy products, frozen meat, chopping board) Medium: EYA
Common cause of neonatal meningitis Lipase and Lecithinase: positive
Exhibit reverse CAMP reaction due to production of phospholipase D
Catalase: positive that inhibits β-lysin
H2S: negative
Hanging drop: tumbling motility

Associated with
Meningitis, pneumonia, abortion, stillbirth, endocarditis, conjunctivitis,
and urethritis
Perinatal human listeriosis (granulomatosis infanseptica)
*The only bacteria that can cross the placenta

Virulence factor
1. Listeriolysin O - allows it to escape the phagolysosome
2. Actin Rockets - allows it to “sling-shot” from one cell to another
LACTOBACILLUS
Medium: Non-pathogenic and has little clinical significance
Sulfide Indole Motility (SIM) umbrella like/inverted Christmas tree Anaerobic
McBride’s Medium
➢ Lactobacillus acidophilus
Virulence test Normal flora of mouth gastrointestinal tract, and vaginal canal
Anton test/Ocular test of Anton - organism is inoculated to the
conjunctival sac of rabbit VAGINA: Doderlein’s bacillus
Positive: Purulent Conjunctivitis GIT: Boas-Oppler Bacillus

Camp test w/ S. aureus Medium: Tomato Juice Agar

Positive block/rectangular hemolysis Yakult: L. casei Shirota strain
Vacuum-sealed sliced bacon

➢ Lactobacillus casei
“Shirota strain”

S. agalactiae Listeria
Cause meningitis in neonates
CAMP (+), Hippurate hydrolysis (+)
Gram (+)
Beta hemolytic
Catalase (-) Catalase (+)
Non-motile Tumbling motility
Bile Esculin hydrolysis (-) Bile Esculin hydrolysis (+)
Gram stain: COCCI Gram stain: BACILLI
GARDNERELLA VAGINALIS
Formerly Hemophilus vaginalis/Corynebacterium vaginalis ➢ Mycobacterium tuberculosis (Koch’s bacillus)
“Clue cells” in wet mount of vaginal fluid Obligate aerobe, gram positive or ghost/neutral
Slightly curved rod 0.2-0.6 micron in diameter & 1-4 micra in length
Medium: Require: CO2 for growth
Sheep Blood Agar Pinpoint; non-hemolytic
Human Blood Tween 80 β-hemolytic Virulence factor
*HBT is CNA with human blood Allows MTB survived intracellular survival
1. Cord factor (serpentine cords)
Presumptive test - prevents fusion of phagosome and lysosome
Whiff test - Fishy amine-like odor, after the addition of 1 drop of 10% - prevents recruitment of neutrophil
KOH to the vaginal washings - highly presumptive of MTB complex
2. Sulfatides, lipids (mycolic acids, phospholipids)
Vaginal discharge
Grayish, foul odor: G. vaginalis Resistant to drying
Greenish, foul odor, strawberry cervix: T. vaginalis 6-8 months: remains dried sputum when protected from sunlight
Cheesy, curdy discharge, fishy odor: Candidiasis (summer days) 8-10 days: droplets of air-dried sputum that may be infectious
20-30 hours: sputum, exposure to sun before they get killed
KURTHIA BESSONNII 2 hours: organisms from culture killed upon exposure to sunlight
Food in soil, opportunistic pathogen
Killing
Generally resistant to chemical disinfection; requires 24hr exposure
ROTHIA to 5% phenol (eg. lysol)
Normal flora of human mouth Easily killed by moistened heat, boiling for 10 minutes,
Rare cause of abscess of endocarditis pasteurization/autoclave

MYCOBACTERIUM TUBERCULOSIS and OTHER Langhans Giant cells

NONTUBERCULOSIS BACTERIA Formed by the fusion of epithelioid cells (macrophages), and
contain nuclei arranged in a horseshoe-shaped pattern in the cell
MYCOBACTERIUM periphery
Aerobic gram positive
Non-spore former except M. marinum Ghon Complexes
Nonmotile, very thin, slightly curved, or straight rods Combination of a peripheral lung injury and a calcified parahilar
node
Cell wall
N-glycolmuramic acid Treatment
↑Lipid content TBDOTS (Tuberculosis Directly Observe Treatment Strategy)
6 months
Mycobacterium tuberculosis Nontuberculous mycobacteria 2 months RIPE
complex (NTM) 4 months R,I
Non-cultivable
M. tuberculosis M. leprae M. lepromatosis Primary drugs for TB
M. bovis/BCG Slow-growing-nonchromogens Rifampicin - red orange urine
m. africanum M. avium complex Isoniazid - causes vitamin B6 deficiency (peripheral neuropathy)
M. caprae M. haemophilum Pyrazinamide - associated with uric acid crystals (gout, allopurinol)
M. canettii Slow-growing- Ethambutol - causes color blindness (red, green)
M. microti photochromogens Streptomycin - 1st (no longer use)
M. mungi M. kansasii M. marinum
M. orygis Slow-growing- Secondary drugs for TB
scotochromogens Ethionamide
M. gordonae M. xenopi Capreomycin
Rapid growing Ciprofloxacin
M. chelonae M. abscessus Ofloxacin
M. fortuitum M. smegmatis Kanamycin
Cycloserine
Rifabutin

CLASSIFICATION OF DRUG RESISTANT TB

Mono-resistant Resistant to any one TB
treatment drug
Poly-resistant Resistant to atleast 2 TB
drugs (but not both Isoniazid
and Rifampin)
Multidrug Resistant (MDRTB) Resistant to atleast Isoniazid
and Rifampin, the two best
first-line TB treatment drugs
Extensively Drug Resistant Resistant to Isoniazid +
(XDRTB) Fluoroquinolone + atleast 1 of
the 3 injectable second-line
drugs
Laboratory diagnosis
1. Skin test for TB Stains for MTB
Purified Protein Derivative (PPD) 1. Ziehl Nielsen (hot staining)
Type IV sensitivity reaction (delayed/cell mediated type) Mordant: steam/heat
• Heat killed ammonium sulfate 2. Kinyoun (cold staining) - preferred AFB in tissues
• Redness after 48 hours Mordant: 10% phenol/tergitol
3. Fite-Faraco’s - hematoxylin stain instead of Methylene blue
a) Mantoux - intradermal/intracutaneous (+) 5-10 mm duration as counter stain (eg. M. leprae)
b) Von pirquet - scratch test 4. Auramine-rhodamine stain (Truant’s)
c) Volmer - patch test piece of cloth to skin (+) yellow fluorescent organisms on black background
5. Spengler’s stain - color blind, (+) black
6. Pappenheim’s - differentiate MTB (red); M. Smegmatis (blue)
7. Baumgarten’s stain - differentiate MTB (blue); M. leprae (red)

CDC METHOD TO REPORT AFB

Number of AFB Seen Report
0 -
1-2/300 fields +/-, Repeated on second
slide
1-9/100 fields 1+
1-9/10 fields per OIF 2+
1-9/field 3+
>9/field 4+
NATIONAL TUBERCULOSIS ASSOCIATION METHOD
Number of Organism Seen Report
1 to 2 per slide Report number and
request for another
specimen
3 to 9 per slide Rare (1+)
10 or more per slide Few (2+)
1 or more per OIF Numerous (3+)
2. Chest X-ray (GHON complexes) NATIONAL STANDARD REPORTING SCALE (San Lazaro)
Primary infection: middle lobe infiltration 0 No AFB seen
Reactivation: upper lobe/apex infiltration in 300 visual fields
+n 1-9 AFB/100 visual fields
3. Sputum collection (2 spx in 1 day) 1+ 10-00 AFB/100 visual
1st: early morning fields
2nd: random 2+ 1-10 AFB/OIF in atleast
50 visuals
Gram stain - qualify specimen 3+ More than 10 AFB/OIF in
<10 SEC/LPF and >25 PMNs/LPF atleast 20 visual fields

Other Specimen METHODS FOR DECONTAMINATION AND DIGESTION OF

Sputum (2 specimens in 1 day) - DSSM BACTERIA
Gastric aspirate - infants 1. N-acetyl-Cysteine (NALC) and 4% NaOH
Secretion by bronchoscopy NALC: digesting agent (break disulfide bonds in mucus w/c trap
Blood MTB; mucolytic agent, liquefies mucus
CSF - Pellicle/Webb-like clot NaOH: decontaminating agent (remove normal flora and other
Urine contaminating organism)
P (3 P’s): Pleural, Peritoneal, Pericardial fluid - measure Adenosine 2. Trisodium phosphate and benzalkonium chloride
Deaminase (ADA) (Zephiran, QUATS) - decontaminating agent
✓ ≥ 40 units - tubercular effusion 3. Dithiothreitol and NaOH
4. Oxalic acid - harbor Pseudomonas and Proteus
4. DSSM: Direct Sputum Smear Microscopy
2x3 cm: Ideal size of the smear (thumb size) Media
DOTS: Directly Observed Treatment Strategy MTB culture (slow grower)
Dry prior to hear fixation to prevent aerosol Should be maintained for 2 months (60 days, 8 weeks)
Colonies: tan to buff (non-pigmented); cauliflower colonies
To remove adhering sputum on loop Rough/dry/warty granular resembling cauliflower
1. Dip in washed sand with 70% alcohol
2. Glass beads with 90-95% spirit Egg base medium
3. 5% cresol Need protein or eggs
Malachite green - inhibits normal flora/contaminants
Detection rate: 70%
Cure rate: 85% 1. Lowenstein-Jensen
300 OIO/fields examined before reporting as negative 2. Petragnani - heavily contaminated specimen (increase conc. of
malachite green)
3. American Thoracic Society Medium
4. Dorset Egg Medium
Agar base
1. Middle brook 7H10 and 7H11c RUNYOUN CLASSIFICATION OF MYCOBACTERIA OTHER
o Clear medium - examination of colonies THANTHE TUBERCLE BACILLI
2. Duboi’s Oleic Acid Albumin Medium
3. Mitchison’s medium Group I: Photochromogens (MASK)
Pigmented in light, non-pigmented in dark
Liquid media o M. marinum - swimming pool granuloma, NO3 (-)
1. Bactec, 12B, Septi-Chek, Middlebrook 7H9/7H12 o M. asiaticum
o M. simiae - niacin (+), NO3 (-)
Luciferase Reporter Mycobacteriophage (LRP) Assays o M. kansasii - “Yellow bacillus”, causes pneumonia, NO3 (+)
Can detect M. tuberculosis and characterize mycobacterial drug
susceptibility patterns within 24 to 48 hours in positive cultures Group II: Scotochromogens
(Luciferase - enzyme obtained from fireflies) Both pigmented in light and dark
o M. scrofulaceum - Tween 80 (-), Urease (+), causes
QuantiFERON-TB scrofula cervical lymphadenitis
Measures interferon gamma levels produced in whole blood in o M. szulgai - photochromogen at 25x
response to addition of specific tuberculosis antigens; relative o M. xenopi - nonphotochromogenic or scotochromogenic
specificity for Mycobacterium tuberculosis; not affected by o M. gordonae - “Tap Water Bacillus”, Tween 80 (+), Urease (-)
previous BCG vaccination o M. flavescens
Principle: ELISA o M. thermosistible
Specimen: Heparinized whole blood
Group III: Nonphotochromogens
RAPID CULTURE: Bactec Radiometric Culture Non-pigmented
Liquid broth in a bottle, with radioactive palmitate as a carbon o M. avium - “Lady Windermere Syndrome” (birds, chicken)
source. o M. avium-intracellulare complex - “Battey bacillus”
Mycobacteria grow and use the carbon, allowing early detection (1- ✓ CD4 count: <50 AIDS (stool)
2 weeks) even before colonies can be seen o M. ulcerans - “Inert bacillus”, Buruli ulcers, (-) biochemical test
o M. haemophilum - β hemolytic,
GeneXpert o M. terrae - “Radish bacillus”
PCR based (test to detect drug resistant tuberculosis) NAAT for o M. malmoens
detection of MTB from sputum specimens
Can also detect Rifampicin resistance (RpoB gene) Group IV: Rapid Growers (FPASCH)
o M. fortuitum - NO3 (+), Mac w/o crystal violet
➢ Mycobacterium bovis o M. phlei - “Hay bacillus”
TB in cattles (ingestion of milk) o M. abscessus - severe chronic pulmonary infection
Bacillus of Calmette and Guerin (BCG) for MTB - attenuated M. o M. smegmatis
bovis o M. chelonei - NO3 (-)

➢ Mycobacterium africanum Growth at 30c

Most common cause of TB in Africa (west and central) M. haemophilum
M. ulcerans
➢ Mycobacterium canetti M. marinum
Most common cause of TB in Africa (east)
➢ Mycobacterium leprae
OTHER MYCOBACTERIA “Hansen’s disease or leprosy”
Acid fast rod in nasal mucosa of patients with nodular variety of
➢ Mycobacterium gastri Hansen’s disease
J bacillus MOT: Person to person (inhalation or contact w/ infected skin)

➢ Mycobacterium genavensi Clinical Manifestation

Disseminated infections in AIDS, BACTEC (+) Leprosy (Hansen’s disease)
Lepromatous - Lepromine (-), CMI (-), many AFB, Multibacillary,
➢ Mycobacterium avium subsp. paratuberculosis “Leonine face”
Known to cause an inflammatory bowel disease Tuberculoid - Lepromine (+), CMI (+), few AFB, Paubacillary
(Johne’s disease) in cattle, sheep, and goats
Isolated from the bowel mucosa of patients with Chron’s disease, Laboratory diagnosis
chronic inflammatory bowel disease of humans Culture: foot pads of mice (cigar pockets)
Armadillo - susceptible to Hansen’s disease (experiments)
Lepra cells (macrophages w/ acid fast bacilli)

Skin test: Lepromine test

1. Fernandez (early reaction) 24-48 hours
2. Mitsuda (late reaction) 3-4 weeks
Specimen: Tissue juice - earlobe, nasal scrapings
Treatment: Sulfone Dapsone
Biochemical test for Mycobacteria BRANCHING/FUNGUS LIKE BACTERIA (gram-positive bacilli) “CAN”

1. Niacin Accumulation test ACTINOMYCES ISRAELII

Reagent: Cyanogen bromide Gram-positive, branching filamentous rods neither acid fast nor
Positive: yellow (M. tuberculosis) stained with fungus stain, anaerobic; catalase negative
Negative: (M. bovis)
Causes: Chronic Suppurative Granulomatous Disease
2. Nitrate Reduction Agent of: LUMPY JAW, Actinomycosis (sinus tracts fistulae, which
Filter paper strip erupts to the surface and drain pus. May contain “sulfur granules”
Positive: blue color (M. tuberculosis)
Sodium nitrate broth Colonies: Molar tooth
Positive: pink to red color (M. tuberculosis)
NOCARDIA
3. Heat Stable Catalase (68c)
Gram-positive, PARTIALLY ACID FAST, aerobic; catalase positive
Principle: Tween 80 + Mycobacteria + 30% hydrogen peroxide +
heat at 68c for 20 minutes
➢ Nocardia asteroides
(+) >45 mm height of gas bubbles
Most clinically relevant species; other species include N.
Reagent: 30% hydrogen peroxide
brasiliensis and N. otitidiscaviarum
Negative reaction: M. tuberculosis complex (M. tuberculosis, M.
Primary pulmonary infection resembling tuberculosis
bovis, M. africanum, M. ulcerans, M. leprae and M. microti)
Medium: Tap water Agar
4. TWEEN 80 HYDROLYSIS
Principle: Tween 80 converted to oleic acid by tween 80 lipase
Positive: red (M. kansasii - most useful)
Negative: no color change, amber (M. avium intracellulare)

5. Arylsulfatase
Reagent: Tripotassium phenolphthalein
Positive pink/red (M. fortuitum-chelonae)

6. Pyrazinamidase
Principle: Converted to pyrazinoic acid
Positive: MTB and M. marinum
Negative: M. bovis and M kansasii

7. T2H Susceptibility/Inhibition test

Positive: inhibition of growth (M. bovis)
Negative: growth/resistance (M. tuberculosis)

8. MPT64 Antigen
Positive: MTB

9. Iron uptake
Growth in 20% ferric citrate TROPHERYMA WHIPPLEI
Positive: M. fortuitum
Gram-positive actinomyces
Negative: M. chelonae
WHIPPLES DISEASE, found primarily in middle-aged men, PAS
10. Urease staining macrophages (mucopolysaccharide or glycoprotein) in
Growth in 5% NaCl every organ of the body
Positive: M. scrofulaceum, M gastri and M. bovis (most useful)
M. triviale - only slow grower that gives positive reaction
Negative: M. gordonae
SPIROCHETES: BORRELIA, TREPONEMA AND LEPTOSPIRA TREPONEMA
Spirochetes are motile without possession of flagella Can be visualized by dark-field microscopy
Tightly coiled (4-14 spiral)
Microscopes to visualize Spirochetes Motile with graceful flexuous movements in liquid (corkscrew
1. Dark field motility)
2. Fluorescent
3. Phase Contrast ➢ T. pallidum subs pallidum
Microaerophilic spirochete responsible for syphilis,
Stains Chronic systemic venereal disease with multiple clinical
Levaditi presentations.
Warthin starry “GREAT IMITATOR”
Fontana tribondeau “GREAT POX/EVIL POX”

BORRELIA Mode of Transmission

Can be visualized by bright-field microscopy 1. Sexual
Helically coiled bacteria transmitted through arthropod vectors 2. Parenteral - Killed by refrigerated citrated blood for 3 days
including lice and ticks; less tightly coiled (3-10 coils) 3. Late congenital syphilis may be bone pain, retinitis pigmentosa
Flexibly twisted organism resembling “stretched spiral” (a serious eye disease), Hutchinson's triad which is characterized
by pegshaped upper central incisors (teeth), and interstitial
➢ Borrelia recurrentis keratitis which consists of blurred vision, abnormal tearing, eye
Helically coiled “stretched spiral” pain and abnormal sensitivity to light

Epidemic: Louse-borne relapsing fever Stages of Syphilis

Vector: Pediculus humanus (Human Body Louse) 1. The primary stage:
High fever, muscle and bone pain, and confusion Hard chancre (painless and firm)
Specimen: Swab, aspirate
Endemic: Tickborne (Ornithodoros ticks) Test: Direct microscopy (Dark field or Fluorescent)
Positive: live Treponemes with corkscrew motility
➢ Borrelia burgdorferi
Agent of: Lyme disease 2. The secondary stage: systemic dissemination
Vector: Tick (ixodes/deer tick) Rash
Condylomata-lata, wartlike lesion in moist areas in the body
Three stages Specimen: Serum
Stage 1: Localized; appearance of lesion - ERYTHEMA Test: VDRL (qualitative + quantitative) confirm w/ FTA-ABS
CHRONICUM MIGRANS (bulls eye rashes)
Stage 2: Early dissemination through blood; affected areas may 3. Latent syphilis: Causes no symptoms
include bones, CNC, heart, and liver (+) sero tests
Stage 3: Late persistent; neurological abnormalities, arthritis, and
skin lesions 4. The late (tertiary) stage: Gummas
Neurosyphilis (Tabes dorsalis - degeneration of lower spinal cord)
Medium: Kellys medium Specimen: Serum and CSF
Barber Stoenner Kelly, at 33c for 6 weeks Test: VDRL (qualitative + quantitative) confirm w/ FTA-ABS
Positive: dead Treponemes
Serological test
1. Elisa - measures IgM (early), IgG (late) Congenital syphilis
2. Western blot Hutchinsonian Triad of Congenital syphilis
*OspC membrane protein - earliest response and development of - Notched teeth
IgM, flagellar antigens (flaA and flaB) or fibronectin binding protein - Keratitis
(BBK32) - Nerve deafness

➢ Borrelia hermsii/B parkeri Treatment

Agent of tick-borne relapsing fever 1. Heavy metals: Arsenic
Vector: Ornithodoros ticks (O. hermsii/O. parkeri) (Arsphenamine/Salvarsan 606)
Diagnosis: Wright/Giemsa blood/BM *Arsenic poisoning: Metallic taste/Garlic breath
Medium: Kellys medium 2. Penicillin G: (intravenous) able to cross the placenta

Jarisch-Herxheimer Reaction
Phenomenon wherein large quantities of toxin are released as the
bacterium dies during treatment
OTHER TREPONEMAL DISEASE
CHLAMYDIA AND RICKETTSIAE (obligate intracellular
➢ Treponema pertenue pathogen)
Yaws (chronic nonvenereal disease of skin and bones)
Transmission: traumatized skin comes in contact with an infected CHLAMYDIA
lesion Former name: Bedsonia
Obligate intracellular
➢ Treponema endemicum Infectious: ELEMENTARY BODIES (extracellular, direct fluorescent
Bejel (lesion of oral cavity, oral mucosa, skin, bones, and assay)
nasopharynx) Reproductive: RETICULATE BODIES (intracellular)
Transmission: mouth to mouth by utensils Diagnostic: INCLUSION BODIES (giemsa stain)
➢ Treponema carateum ➢ Chlamydia psittaci (Chlamydophila psittaci)
Pinta (ulcerative skin disease) Agent of Psittacosis/Ornithosis, a disease of birds, parrots
Transmission: traumatized skin comes in contact with an infected and cockatoos “Parrot fever”
person Inhalation of aerosols, bioterrorism agent
Resistant: Sulfonamide
➢ Treponema vincentii/denticola Produces non-glycogen “inclusion body” (giemsa) using Levinthal
VINCENT’S DISEASE - acute necrotizing ulcerative gingivitis, Cole Lillie stain
destructive lesion of gums
➢ Chlamydia pneumoniae (Chlamydophila pneumoniae)
LEPTOSPIRA TWAR strain: Taiwan Acute Respiratory Strain
Can be visualized by dark-field, phase contrast and IF microscopy Growth on human lines & Hep-2 cells
Tightly coiled, thin, flexible spirochetes with hooked ends Diagnosis: PCR, Immunofluorescence

➢ Leptospira biflexa Associated with

Nonpathogenic, found in water and soil Guillain Barre Syndrome
Mild respiratory tract infections
➢ Leptospira interrogans
“Weil’s disease” ➢ Chlamydia trachomatis
Cause of human and animal leptospirosis, zoonosis No. 1 cause of Non-Gonococcal Urethritis (STD)
Parasitic on vertebrates other than humans TRIC agent - trachoma, inclusion conjunctivitis (lead to blindness)
eg. rodents, cattle, dogs, cats, racoons, and bats Sensitive: Sulfonamide
Transport: 4c
Mode of Transmission
Direct contact w/ urine of animals who carry organism Subtypes Clinical Syndrome
A, B, Ba, C Endemic trachoma (multiple or
WEIL’S DISEASE: severe form of leptospirosis persistent infections that ultimately lead
Involves kidney, liver ad central nervous system to blindness)
D-K Urethritis, cervicitis, pelvic inflammatory
Principal Leptospiral Diseases (L. interrogans serovar) disease, epididymitis, infant pneumonia,
Icterohemorrhage: Weil’s disease and conjunctivitis
Canicola: Infectious jaundice
Autumnalis: Fort Bragg or Pretibial fever L1, L2, L3 Lymphogranuloma venereum
Grippotyphosa: Marsch fever
Hebdomadis: Seven day fever Laboratory Diagnosis
Mitis/Pomona: Swineherd disease Culture: McCoy cells (Tissue culture medium), Hep-2 HeLa, BGMK
Nonculture, non-amplified: DFA, EIA, OIA
Specimen Nonculture, amplified: Probe, PCR
1st week - Blood, CSF, Tissue (most sensitive; acute) Serology: CF, EIA, Micro I-F
2nd week - Urine (culture, chronic) Skin test: Frei’s test - delayed hypersensitivity (L1, L2, L3)
Medium: Specimen
Ellinghausen-McCullough-Johnson-Harris Culture (traditional diagnosis)
Fletcher’s animal serum + fatty acid (30c for 6-8 weeks) Male: urethral swab
Stuart’s Female: cervical swab
NAAT: Nucleic Acid Amplification test, PCR
Laboratory Diagnosis Urine from male and female (early morning first-voided urine)
1. Definitive test: Culture
2. Screening test: Macroscopic Agglutination test C. C. C.
serum + Ag (killed leptospira) = (+) agglutination trachomatis pneumoniae psittaci
3. Gold Standard: Microscopic Agglutination test Glycogen (+) (-) (-)
serum + Ag (live leptospira) = (+) agglutination under darkfield inclusion
EB Round Pear-shaped Round
morphology
Sulfa drug (+) (-) (-)
sensitivity
Natural host Humans Humans Birds
Human STD Pneumoniae Pneumonia
diseases Trachoma Pharyngitis FUO
LGV Bronchitis
RICKETTSIA MYCOPLASMA
Chick embryo (Rickettsia, Ehrlichia, Coxiella, Rochalimea) Smallest free-living organism, found in animal and plants
Gram negative obligate intracellular bacteria Lack of cell wall, pleomorphic in appearance
Infections spread through insect vectors such as lice, fleas, ticks Originally known as Pleuropneumonia like organisms (PPLOs) in
cattles
Signs of infection: fever, headache, characteristic rash that appears
on wrist and ankles ➢ Mycoplasma pneumoniae
Other manifestation: conjunctivitis, pharyngitis, mild respiratory Primary atypical pneumonia “Dry cough” or “Walking
distress pneumonia”
Eaton’s agent - community acquired pneumonia and
*All cannot survive outside animal host or insect vector except C. tracheobronchitis in children and young adults
burnetti Aerobic or with CO2, grows on BAP
*All MOT insect vector except C. burnetti (ticks/aerosols)
*All require tissue culture medium except R. quintana Medium: PPLO Agar, Edward’s Hayflick, SP4-Glucose
Diagnostic: inhibition of growth by specific antisera
Group Species Infection Transmission Confirmatory: Hemadsorption test - attachment of RBC on culture
Spotted fever R. rickettsii Rocky Mountain Ticks Serological: detection of cold agglutinins (Anti-I, IgM)
Spotted Fever
R. akari Rickettsial pox Mites
R. australis Australian/Queensland Ticks GENITAL MYCOPLASMA
Tick typhus Can colonize adult asymptomatically and are cause of
R. conorii Boutonneuse Fever Ticks
Mediterranean and
nongonococcal urethritis in males
Israeli Spotted Fever;
Indian Tick typhus, ➢ Mycoplasma hominis
Kenya Tick typhus
Typhus R. prowazekii Epidemic typhus Lice Agent of salpingitis (inflammation of fallopian tube) and postpartal
Sporadic typhus Flying squirrels fever in females
Brill-Zinsser disease Reactivation of Medium: A7/A8, SP4-arginine, NYCA
latent infection Large fried egg colonies
R. typhi Murine typhus Fleas
Scrub typhus R. tsutsugamushi Scrub typhus Mites, chiggers
O. tsutsugamushi ➢ M. urealyticum
Q Fever Coxiella burnetti Q fever Ticks, aerosol T strain, difficult to culture
Ehrlichiosis E. chaffeensis Human monocyte Ticks Requires sterol component
ehrlichiosis
E phagocytophila Human granulocyte Ticks Medium: Shepard’s A7/A8, SP4-urea, NYCA
(Anaplasma) ehrlichiosis Tiny fried egg colonies w/ dark brownish clumps
E. ewingii
Neorickettsia Sennetsu fever Ticks
sennetsu (former
Laboratory Diagnosis
E. sennetsu) PCR, Immunoblotting and Immunofluorescence
Rochalimeae R. quintana Trench fever Lice
BARTONELLA AND AFIPIA
Laboratory Test
BARTONELLA
Agglutination test Originally grouped with Rickettsia
Weil Felix test Short, gram-negative, rod-shaped, fastidious organisms
Principle: cross-reactions that occur between antibodies produced Oxidase negative
in acute rickettsial infections with antigens of OX (OX-19, OX-2, and Medium: blood enriched media
OX-K) strains of Proteus species
Organism Habitat(reservoir) MOT Clinical
Rickettsial infection OX-2 OX-19 0X-K manifestation
R. rickettsii RMSF + + - B quintana Uncertain; possibly Human body louse Trench fever
R. prowazekii Epidemic small rodents, Chronic
typhus humans bacteremia,
R. typhi Epidemic endocarditis,
typhus bacillary
R. tsutsugamushi Scrub typhus - - + angiomatosis,
chronic
R. akari Rickettsial pox lymphadenopathy,
- - - pericarditis
R. burnetti Q fever
R. quintana Trench fever B bacilliformis Uncertain, humans Sand flies Carrion’s disease,
Oroya fever,
Verruga Peruana
Serological test B henselae Domestic cats Domestic cat by bites Cat-scratch
and/or scratches, cat disease
Indirect Fluorescent Assay/Microimmunofluorescent assay fleas Bacteremia,
Gold standard for detecting rickettsial antibodies endocarditis,
bacillary
angiomatosis,
peliosis
hepatitis,
neuronitis

AFIPIA FELIS
Associated w/ cat-scratch disease (CSD), rare role in disease

Others
B. Henselae and B. clarridgeiae also cause cat-scratch disease
ANAEROBES OF CLINICAL IMPORTANCE GRAM-POSITIVE ANAEROBIC BACILLI

Enzymes in Oxygen PROPIONIBACTERIUM (CUTIBACTERIUM) ACNES

Catalase, Superoxide dismutase Anaerobic diphtheroid, skin flora
Catalase, Indole, Nitrate reduction (+)
Aerotolerant anaerobe Sulfur granules are only seen macroscopically (not through
Survived some oxygen exposure but performs metabolic processes microscope)
only in an anaerobic environment Aerotolerant
Cell wall composed of L-diaminopimelic acid and glycine
Obligate, strict anaerobe Blood culture contaminant/RBC contaminant
Strictly anaerobic requirement (0% O2), killed almost instantly in the Bacteria found in acne
presence of oxygen
BIFIDOBACTERIUM
Specimen Quality (Selection)
Diphtheroid
End of cells may be spatulated or bifurcated “dog bones”
1. Suitable Specimens for Anaerobic Culture
Tissue biopsy (necrotic tissues)
GRAM-NEGATIVE ANAEROBIC BACILLI (GIT FLORA)
Needle and syringe aspiration (serous exudates, abscess)
Stool and food in case of Clostridium perfringens
BACTEROIDES FRAGILIS
2. Unsuitable specimens for anaerobic culture Most abundant normal flora of the colon
Swabs Able to grow in 20% bile and can hydrolyze esculin
Voided or catheterized urine
Feces Laboratory diagnosis
Coughed (expectorated) sputum Capsule, Catalase: positive
Bronchial washing Medium: Bacteroides Bile Esculin agar (black)
Resistant: Kanamycin, Vancomycin, Colistin (multidrug resistant)
Reduced media
Anaerobic blood and Brucella blood agar PORPHYROMONAS ASSACCHAROLYTICA
PEA and CNA Blood agar Black pigment
Brick red fluorescence on UVL; No growth on KVLB
Anaerobic broth
Kanamycin-vancomycin-laked blood agar (KVLB) Resistant: Kanamycin, Colistin
Cycloserine Cefoxitin Fructose Agar (CCFA) Susceptible: Vancomycin
Bacteroides Bile Esculin Agar (BBE) Assacharolytic
Egg-yolk Agar (EYA)
Chopped meat broth, enrichment (enhance the growth of pathogens PREVOTELLA MELANINOGENICA
while inhibiting the growth of normal flora) Inhibited by 20% bile
✓ Extends the lag phase of contaminants, shortens the lag phase Brick red fluorescence on UVL; Growth on KVLB
of pathogens
Medium: KVLB agar (brown-black)
Thioglycollate broth Resistant: Kanamycin, Vancomycin
Anaerobes and aerobes Susceptible: Colistin
Nutritive factors: provides nutrition for the bacteria Saccharolytic
0.075% agar: minimizes convection currents → prevents
atmospheric oxygen FUSOBACTERIUM NUCLEATUM
Sodium thioglycollate: limits molecular oxygen within
the broth → low oxygen tension Spindle-shaped (fusiform) with pointed ends
Sodium chloride: osmotic equilibrium Breadcrumb, ground glass colonies
Resazurin: oxidation-reduction indicator Greening on exposure to air
Fluoresces chartreuse
Anaerobic incubation
Gas generator FUSOBACTERIUM NECROPHORUM
- 85-90% N2, 55% H2 and 5-10% CO2 Vincent’s angina, Lemierre’s disease
Catalyst Stereoscope and Chartreuse (+)
- Palladium pellets (speed-up the reduction of oxygen) Globular forms with swelling (bizarre-shaped)
Desiccant Medium: Egg yolk agar (lipase positive, iridescent sheen)
- Silica gel (blue → pink)
Redox indicator
- Methylene blue and resazurin
Chartreuse Fluorescence
Brick Red Fluorescence
Esculin hydrolysis

Iridescent sheen
Bile Resistance

Growth on KVLB

Specimen transport
C
K
V

Three kinds of anaerobic transport system

- Rubber-stoppered collection vial
- Oxygen-free transport tubes
- Anaerobic pouch
Contents of transport media B. Fragilis R R R + + + - - -
Reducing agent (thioglycolic acid, sodium thioglycolate, cystine) Porphyromonas S R R - - - + - -
Redox indicator (resazurin, ethylene blue) Prevotella R R V - - + + - -
Fusobacterium R S S - - ± - + +
Indicator With oxygen Without oxygen
Resazurin Pink Colorless
Methylene blue Blue Colorless
GRAM-POSITIVE ANAEROBIC COCCI FISH GRADING
Fluorescent Isothiocyanate (FITC)
PEPTOSTREPTOCOCCUS Positive: APPLE GREEN
Possess sweet fetid/foul smelling odor of the pus
1+ Faint
➢ Peptostreptococcus anaerobius 2+ Apple Green
Catalase: negative 3+ Bright
Indole: negative 4+Brilliant
SPS: sensitive
NUCLEIC ACID AMPLIFICATION TEST (NAAT)
➢ Peptostreptococcus niger (Staphylococcus like) Amplification used to increase the target microorganisms nucleic
Black to olive green colonies acid in a sample in a short amount of time.
SPS: resistant

GRAM-NEGATIVE COCCI POLYMERASE CHAIN REACTION

Simple in vitro chemical reaction that permits the synthesis of
VEILONELLA PARVULA essentially limitless quantities of targeted nucleic acid sequence
Normal inhabitant of the mouth, GIT, and genital tracts 1. Denaturation - separates double-stranded DNA; most PCR
Oxidase: positive protocols denature at 94c or 95c from 15 to 30 seconds
Nitrate reduction: positive 2. Annealing - anneals primers to target DNA; temperature usually
Indole: negative (orange) range from approximately 45c to 65c for about 30 seconds to 2
ATP production through lactose fermentation minutes
UV light: Fluorescence red 3. Extension - Taq DNA polymerase synthesize new strands of
DNA at 68c to 72c for 1 to 2 minutes
MOLECULAR DIAGNOSTIC TECHNIQUES IN MICROBIOLOGY
Magnesium chloride: function of polymerase
Specimen handling and certain organisms Buffer (pH 8.3): proper action of conditions (Trisaminomethane
1. Does not require the detection of viable organism hydrochloride and salt - potassium chloride)
2. The timing and collection devices used for molecular testing Storage: -20c
remain critical to the successful detection of nucleic acids
3. Plastic (nylon flocked) swabs are recommended for collection of REAL TIME PCR
bacteria, viruses, and Mycoplasmas from mucosal membranes PCR amplicons are assessed as they are accumulated at each
DO NOT USE calcium alginate swabs with aluminum shafts cycle
because they interfere with amplification of nucleic acids
4. White top tube (EDTA) for plasma RT-PCR (reverse transcriptase PCR)
DO NOT USE Heparinized plasma (inhibits polymerase enzyme)
Can be used to detect RNA viruses (commonly HIV)
5. First voided urine should be used for STIs
6. Transport iced or in the broth provided by manufacturer
OTHER AMPLIFICATION PROCEDURES
NUCLEIC ACID HYBRIDIZATION
NUCLEIC ACID SEQUENCE BASED AMPLIFICATION (NASBA)
Formation of hydrogen bonds between single strands of RNA or TRANSCRIPTION MEDIATED AMPLIFICATION (TMA)
DNA thar are complementary also called duplex formation
Used to detect a number of pathogens including viruses such as
HIV, HCV, varicella zoster virus, cytomegalovirus (CMV), rhinovirus,
Two components
enterovirus, measles, human papilloma virus, and bacteria such as
Target (template) - sequence that will be identified immobilize or
Mycobacterium tuberculosis, Neisseria gonorrhoeae, Chlamydia
suspended in solution
trachomatis, Mycoplasma pneumoniae and Campylobacter jejuni
Probe - single stranded RNA or DNA oligonucleotide labeled with a
reporter chemical, radionucleotide, or fluorescent particle
BRANCHED DNA DETECTION
Nonamplified typing techniques Quantification of RNA from hepatitis C virus and HIV and
Southern blotting quantification of DNA from hepatitis B virus
Plasmid profile analysis
Restriction enzyme analysis of chromosomal DNA ✓ HYBRID CAPTURE
Restriction Fragment Locus Polymorphism (RFLP) Chlamydia trachomatis, Neisseria gonorrhoeae, CMC, and HPV
Multilocus Enzyme Electrophoresis (MLEE)
✓ Pulsed Field Gel Electrophoresis (PFGE) - best for strain typing; CYLCING PROBE TECHNOLOGY
microbial typing gold standard mecA detection for methicillin resistance aureus

Solid support hybridization (Nonamplified typing techniques)

1. Southern blot - DNA

2. Northern blot - RNA
3 Sandwich hybridization - 2 probes are used
4. In situ hybridization - method of hybridization wherein DNA
or RNA transcript can be detected directly in tissue with labeled
probes
5. PNA FISH - novel fluorescent in situ hybridization (FISH)
technique that uses PNA probes to target species-specific
ribosomal RNA sequences (most common)
- can be used directly identify S. aureus and C. albicans
- differentiate Enterococcus faecalis from other Enterococci in
positive blood cultures
COMMON NAATs ON NON-BLOOD
SPECIMEN
 MYCOLOGY AND VIROLOGY

MYCOLOGY 2. Reproductive/ aerial – contains fruiting

bodies that produce the conidia or spores, for
reproduction.
Fungi are group of non motile eukaryotic (versus
bacteria (prokaryotic)) organisms that have definite Capsule – polysaccharide much larger than the
cell walls are devoid of chlorophyll, and reproductive bacterial capsule, virulence factor: antiphagocytic,
by means of spores (and conidia) or budding. mostly in yeast (e.g. Cryptococcus neoformans)
Hetero = “different”, trophic = “nourishment” Cytoplasm – contains the nucleus, nucleolus, nuclear
membrane, endoplasmic reticulum, mitochondria and
Mycosis (mycoses) vacuoles.
 Invasive treatment
 Immunosuppresive theraphy Hyphae – basic structural unit of fungi
 Immunocompromising infections (e.g. HIV) 1. Septate hyphae – with crosswalls/ divisions,
 Rise in common and uncommon mycoses present in all fungi EXCEPT in Zygomycetes
(organisms and tissue infected) 2. Aseptate hyphae (Coenocytic) - no
crosswalls/ divisions, present in Zygomycetes
BASIC STRUCTURES (Rhizopus, Absidia, Mucor)
Cell wall – antigenic,multilayered (90% polysaccha-
ride, chitin, 10% proteins and glycoproteins ), pro-
vides shape and rigidity to the cell (facilitate osmosis).

Cell membrane - bilayered phospholipids, contains

sterol (ergosterol and cholesterol), functions: protect
the cytoplasm, regulates intake of nutrients and
facilitates capsule and cell wall synthesis.

Mycelium – intertwining structure composed of tubular

filaments known as hyphae.
Spores – for reproduction (sexual or asexual).
1. Vegetative/ thallus – grows in a substrate and
absorbs nutrients.

Spores involved in Sexual Reproduction

1. Ascospores Contained in a saclike ascus (2-8 ascospores)
2. Zygospores Involve the fusion of two identical cells arising from the same hyphae
3. Oospores Involve the fusion of cells from two separate, non identical hyphae
4. Basidiospores Contained in a club-shaped basidium

Spores involved in Asexual Reproduction

1. Conidia Spores produced singly or multiply in ong chains by
- Arise from side of hyphae specialized vegetative hyphae known as
- Catenate conidia; chains conidiophores
- Echinulate; rough, spiny
2. Blastoconidia (blastospores) Develops as daughter cell buds off the mother cell and
is pinched off

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3. Chlamydoconidia (resting spores) Thick walled, resistant, resting spores produced by

rounding up and enlargement of terminal hyphal cells.
The spores germinate when favorable environmental
conditions occur
1. Terminal – end of hyphae
2. Intercalary - within hyphae
3. Sessile – side of hyphae
4. Arthroconidiaspores Involve the simple fragmentation of the mycelium.
Useful ID of C. immitis and G. candidum.
Appears “jointed”, rectangular/ barrel shaped spores
5. Sporangiospores Spores contained in a sporangia or sacs that are
produced terminally on sporangophore or aseptate
hyphae. Unique to Zygomycetes.

FIVE CATEGORIES OF FUNGAL INFECTIONS

A. Opportunistic fungi
 Immunocompromised patients
 Many different tissues
 Ubiquitous: environmental saprobes
 Monomorphs 2. Cryptococcus neoformans
 Same structural characteristics under all  Encapsulated yeast cell in bird and bat
conditions (e.g. Candida, Cryptococcus, droppings
Aspergillus, Zygomycetes)  Demonstration of the capsule by India Ink
stain
1. Candida albicans
 Gram stain: “Starburst pattern”
 Saprophytic in oral cavities, NF of the GI or
 Infection: Torulosis
vaginal tract, dimorphic
 Urease positive, inositol positive, nitrate
 Potential pathogen for immunosuppressed
positive
patients
 Cultured on:
 Infection: Candidiasis, Moniliasis (Oral –
SDA medium without cycloheximide
thrush; vaginal – vulvovaginitis)
Birdseed/ Nigerseed/ Staib’s – brown black
Onychomycosis (nails) Paronimycosis
colonies due to phenol oxidase (assimilates
(cuticle)
creatinine) *Guizotia abyssinica
Germ tubes are hyphae like extensions of young cells  Latex agglutination test for antigen in CSF
showing parallel sides, are non septate and will not 3. Aspergillus - *Czapek’s medium
constrict at their point of origin.
 Aspergillus fumigatus – fungus ball
Pseudohyphae look like germ tubes but are septate
Infection: Aspergilloma, allergy, otomycosis
and constricted at their point of origin.
Screening test: Germ tube test - serum + organism at (bread mold) “Farmer’s Lung disease”
35°C for 2-3 hrs  Aspergillus flavus –aflatoxin (toxicoses)
(+) C.albicans, C.dubliniensis (-) C.tropicalis *dried / processed nuts.
Confirmatory test: Chlamydospore Corn meal  Aspergillus niger – brown to black spore
C.albicans to corn meal agar – Incubate at RT for 48- 4. Pneumocystis jirovecii (previously P.carinii)
72 hrs. (+) Chlamydospores  Mistaken as a protozoan because it has a
trophozoite -> precyst -> cyst -> forms, no
ergosterol

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 #1 cause of pneumonia in AIDS (PCP)  Produce secondary metabolites that irritate

 #1 opportunistic infection in AIDS host defense which causes itching
 Specimen for diagnosis: BAL (Broncho-  Sometimes cutaneous and superficial
Alveolar Lavage) grouped (e.g. Trichophyton, Microsporum,
Epidermophyton)
5. Zygomycetes
 Causative agents: Rhizopus, Mucor or 1. Trichophyton species– skin, hair and nails
Absidia
 Infection: Zygomycosis or Mucormycosis Urease Pigment HPT
via inhalation of conidia T. rubrum (tear (-) red (-)
B. Superficial mycoses drop microconidia)
 Infections of outer. “dead layers”, person to T. mentagrophytes (+) (-) (+) v
(grape-like micro-
person contact, fomites conidia)
shape
 No host defense stimulation, pain or T.verrucosum “rat tail or string bean shaped macroconidia
discomfort “Clavate or pyriform” microconidia
*Thiamine and Inositol
 Usually treated because the infection is
T. tonsurans “Balloon shaped microconidia”
“unsightly”(e.g.Malassezia, Piedraia, *thiamine
Trichosporon, Exophialia) T. shoenleinii “Favic chandelier” hyphae

1. Malassezia furfur ***HPT - Hair Perforation Test or Hair Baiting Test

 Infection: Ptyriasis/ tinea versicolor – uneven

T. rubrum Urease negative
pigmentation of the skin
Macroconidia are pencil shaped
 “spaghetti and meatballs” (PAS), (+) SDA with Abundant wine-red water
olive oil. soluble pigment
2. Piedra Hortei
 Infection: black Piedra – brown-black crust
T. mentagrophytes Cigar shaped macroconidia
Scant-red pigment in some
outside hairshaft
strains
3. Trichosporon beigelii
 Infection: white Piedra – light brown nodules on T. tonsurans Macroconidia absent
beard Microconidia with flattened base
4. Exophiala werneckii “balloon forms” – aged
pleomorphic microconidia
 Infection: Tinea nigra - brownish spot, black
T. verrucosum Rat-tail macroconidia
macules
Microconidia are clavate
C. Dermatophytic/ Cutaneous mycoses
T. schoenleinii Conidia absent
 Deeper than the superficial it affects
Favic chandeliers and
keratinized tissue (skin, hair, nails), causes
chlamydospores
“tinea” latin word for ringworm.
T. violaceum Conidia absent
Swollen hyphae containing
Tinea capitis Scalp
cytoplasmic granules
Tinea barbae Beard area
Tinea corporis Middle part (arms, trunk or legs)
2. Microsporum species– skin and hair only
Tinea manus hands
Tinea cruris “jock itch”, groin area
Tinea unguium Nails (yellow discoloration) M. canis Large, multicelled, spindle
Tinea pedis “athlete’s foot” (zoophilic) shaped, rough macro-conidia
Terminal ends
 Still no living skin penetration

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(+) Wood’s lamp - green/yellow 3. Rhinosporidium seeberi (previously

fluorescence of ectothrix hairs Rhinocladiella aquaspersa)
(+) growth on rice grain medium  Infection: Rhinosporidiosis – polypoid
M. gypseum 3-9 celled, broadly spindle masses on the nose and the pharynx
(geophilic) shaped (spindle), rough walled  Acquired through swimming
macro-conidia 4. Loboa loboi
Oblong/ rounded terminal ends  Infection: Keloid like subcutaneous
Microconidia in single/ small nodules involving the extremities.
clusters 5. Exophiala jeanselmei, Phialophora
M. audouinii Conidia absent Wangiella dermatitidis
(anthrophilic) Apple-green fluorescence of
Cladosporum trichoides
ectothrix hair
(-) growth on rice grain medium  Infection: Phaeohyphomycosis – rare
infection caused by dematiaceous
3. Epidermophyton species – skin and nails only saprobes which invade organs of
immunosuppressed hosts.
E. floccosum Large multi-celled, club shaped, 6. Sporothrix schenckii
smooth walled macroconidia  “rose gardener’s disease”; dimorphic
“Dutch pants fuseaux” fungus
Microconidia not formed
 Infection: Sporotrichosis
 Mold phase: flowerette conidia
D. Subcutaneous mycoses  Yeast phase: cigar shape *Asteroid body
 Muscles, bone and connective tissues in tissue – central halo around
 Trauma, inoculation, thorns or scratch degenerating yeast; concentric radiating
 Usually remain localized (Chladosporium, eosinophilic material (Ag-Ab reaction).
Pseudallescheria, Phialophora, Exophiala, E. Systemic mycoses
Sporothrix)  “true”or “primary” pathogens that can
 Isolated from soil infect any tissue
 Specimen: Biopsy stained with PAS or H&E  Endemic to specific geographic areas.
(granules) Must travel through area to become
1. Fonsecaea pedrosoi– mixed sporulation infected (inhalation)
Phialophora verrucosa– vase like  Thermal dimorphs and yeasts (“two
Cladosporum carrionii – cauliflower like lesion bodies” based in temperature)(e.g.
 Infection: Chromoblastomycosis Blastomyces, Paracoccidiodes,Histo-
2. Pseudallescheria boydii, Madurella and plasma, Coccidiodes).
Leptosphaeria sp.
 Infection: Mycetoma or Madura foot–
granulomatous tumor of subcutaneous tissue
a. Eumycotic – true fungi; exophiala; most
common cause is Pseudoallescheria boydii
b. Actinomycotic – fungus like bacteria; do not
stain with fungal stains; actinomycetes and
nocardia

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ORGANISM/ DSE MOULD FORM (25°C) YEAST FORM (37°C)

Blastomyces dermatitidis Delicate, septate hyphae with round or pyriform Thick walled, large yeast cells with single
conidia born singly on conidiophores bud on a broad base
Infection: resembling lollipops
“North American Blastomycosis”
“Chicago disease”
“Gilchrist’s disease”
*Cottonseed Agar

Paracoccidiodes brasiliensis Small, septate, branched hpahe with intercalary Large, round to oval, thick walled yeast
and terminal chlamydoconidia cells with multiple buds which attach to
Infection: mother cell by narrow constrictions,
“South American Blasto- resembles a ship wheel, mariner’s wheel
mycosis” or a Mickey Mouse cap
“Lutz Splendore- Almeida
disease”

Histoplasma capsulatum Septate hyphae with round to pyriform Small, budding round to oval yeast cells,
*Leishmania – mistaken for microconidia on short branches or directly on intracellular to mononuclear cells
Histoplasma; differentiated by a hyphal stalk , thick walled knobby tuberculated possible with Giemsa or Wright’s stain
central nuclear body and failure macroconidia forms
to stain with fungal stains

Infection:
“Darling’s disease”
“Spelunkers’disease”
“Fungus Flu”
***isolated from droppings of
bats – “guano” and birds
(Starlings)

Coccidioides immitis Coarse, septate, branched hyphae that Large, round, thick walled spherules with
major biologic hazard to lab produce thick walled barrel-shaped rectangular endospores observed in tissue and
personnel arthroconidia that alternate with empty direct examination; not a true yeast
disjunctor cells
Infection:
“Coccidiomycosis”
“San Joaquin Valley fever”
“Desert fever”

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LABORATORY SAFETY  subculture sometimes necessary

 Standard precautions (temperature range, dimorphism and sexual
 No smoking, eating, drinking or applying and asexual developmental structures)
cosmetics  Culture is very important can be main
 Contact lenses (no removing or cleaning) identification
 No mouth pipetting  No or few biochemical (yeast are the
 Universal precautions exception)
 Class 2 or 3 biosafety hoods  Get rid of bacterial contamination (teasing
 Disinfectant: phenol based needles are used more than bacteriologic
 Biohazard containers loops O - electric incinerator
X - flame causes aerosols
SPECIMEN COLLECTION
 Important factors in isolating and identifying CULTURE MEDIA
a fungal pathogen  Incubated at 30°C/ room temp, culture held
 Correct type of specimen for 21 days
 Quality of specimen  Test tube for primary - less likely to become
 Rapid transport contaminated, less drying.
 Use of appropriate culture media  Petri dishes for subculture - Larger surface
 Processed within two hours for area growth.
SPECIMEN TRANSPORT  Use of inhibitory substances may be
 Sterile, leak-proof container required (e.g. Chloramphenicol, gentamicin,
 Dermatologic requires dry container cyclohexamide), May encounter some fungal
 No transport media, processed within a few inhibition
hours
 Specimen can be refrigerated at 4°C, only if Common Media for Isolation
processing is delayed 1. Saborauds Dextrose Agar (SDA) - most
 Blood and CSF: 30 to 37°C commonly, most fungi grow well
 Dermatologic: 15 to 30°C 2. Emmon’s modification: less glucose (e.g.
Blastomy-ces dermatitidis)
SPECIMEN PROCESSING: METHODS 3. Mycosel and mycobiotic - SDA +
1. Direct inoculation Chloramphenicol + Cycloheximide, selective recovery
 Adding several drops of specimen to media of dimorphs and dermatophytes
 For solid media, the specimen can be 4. Brain Heart Infusion (BHI) Agar - enriched to
streaked enhance recovery Cryptococcus neoformans of and
 Specimen types: Bronchial brush or wash, dimorphic transitions in Sporothrix and Paracocci-
aspirates, CSF, swabs, body fluids, hairs, diodes
scrappings  Plates or tubes
2. Concentration  Broth + penicillin for Zygomycetes
 Large volumes can be concentrated by  BHI + gentamicin + chloramphenicol,
centrifugation Cryptococcus neoformans from
 Specimen types: body fluids, CSF, urine contaminated specimen
3. Minced (homogenized) 4. Saboraud dextrose + BHI (SABHI)
 Some old specimens must be “destroyed” to  strengths of both
expose a buried pathogen to the media  enriched medium for Cryptococcus spp.
 Specimen typed: nails, tissues, biopsies Thermally dimorphic fungi, etc.
4. Culture of fungi 5. CHROMagar
 petri dishes or tubes (O2 requirements,  Selective and differential for presumptive
humidity) identification of genus Candida from primary
plates.

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 Morphology and colors of the yeast colonies  Need reduced light or phase-contrast
varies in species.  10% KOH added to specimen
 Candida albicans –light to medium green  Dissolves specimen quickly (fungi slowly)
 Candida tropicalis – light blue to metallic- 2. Calcoflour white
blue  Binds the cell wall and fluoresces blue-white
 Candida krusei – light rose with a whitish under UV light
border 3. Lactophenol cotton blue (Aman’s medium)
6. Inhibitory Mold Agar (IMA)  used to stain and preserve fungal elements
 Inorganic salts, chloramphenicol, gentamicin in culture isolates
 Inhibits bacteria 4. India ink
7. Dermatophyte test medium (DTM)  Used with CSF specimens
 Dermatophytes from heavily contaminated  Negative stain
specimens (pink-to-red color change)  Creates black background to visualize
 Commonly used in office practices capsular material (C. neoformans)
8. Czapek’s agar 5. Tissue examination: stains
 for Aspergillus sp.  Gram stain (Hucker’s modification) – for
9. Birdseed/ Nigerseed/ Staib’s medium yeast (gram positive)
 C. neoformans appear black colonies due to  Giemsa, Wright-Giemsa: (intracellular) to
phenol oxidase locate H. capsulatum in RE cells
10. Cottonseed medium  Hematoxylin and Eosin (H&E): Pink to
 for Blastomyces dermatitidis pinkish-blue
11. Rice medium  Meyer’s mucicarmine: C.neoformans – rose
 differentiates M. canis (+) from red
M. audouinii (-)  Gomori Methanamine Silver (GMS): black
 Papanicolau stain: pink to blue
Common Media for Subculture  Periodic Acid Schiff (PAS): red or purple
1. Potato Dextrose Agar (PDA)/ Potato Flake Agar  Acridine orange: green fluorescence - fungal
(PKA) elements/ Orange for epithelial cells
Incubation:
 Obligate filamentous: 25-37°C MACROSCOPIC EXAMINATION
 Dimorphic: 25°C and 37°C Growth conditions
 Yeast: 25°C or 37°C  Yeast: 2-3 days
 Aerobic, 3 to 4 weeks  Molds
2. Cornmeal Agar for yeast morphology – Rapid: Less than 5 days
recommended for promoting sporulation Intermediate: 6 to 10 days
 Candida to visualize chlamydoconidia Slow: more than 11 (sometimes 8 weeks)
 1% dextrose: used to differentiate T. rubrum Dimorphism
(red) from T. mentagrophytes  Pigment
 Front versus back of the plate
LABORATORY IDENTIFICATION  Texture
 Dictated the presence of aerial hyphae
DIRECT EXAMINATION o Glabrous: leathery or waxy
 Can identify yeast and filamentous forms o Velvety: Suede, plush
 Culture is used regardless o Yeast like: looks like
1. KOH preparation Staphylococcus
 Examine hair, nails, skin scrappings, fluids, o Cottony: Fluffy
exudates, and biopsy specimen o Granular: powdery
 Can see important fungal elements (hyphae, Topography
yeast)  Rugose: radial grooves, “folded”

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 Crareriform: central depression and raised 5. L-DOPA ferric citrate test

edge  for phenol oxidase of C. neoformans (+)
 Verrucous: Rough knobs black
 Cerebriform: Brainlike
Mold Identification
Examination of Molds
Three methods  Specimen source or infection
1. Tease/cut preparation: organism removed directly  Growth rate to reproduce structures
from culture plate, “teased” apart with teasing  Colony color front and back on plate
needles.  Microscopic morphology
2. Scotch tape preparation: Scotch tape pressed onto o Septate or aseptate hyphae
culture plate then transferred to microscope slide. o Conidiophore structure
3. Slide culture: organism is subcultured on a small o Microconidia/macroconidia
piece of agar then covered with a cover slip. Orga- o Other structures
nism grows onto the coverslip: remove and Advanced Techniques
examine. Best method. 1. Exoantigen test
 Rapid information of immnunoidentity
Examination of molds: Use one of the three methods  Extract soluble antigen from unknown
listed previously, followed by the addition of stain, isolate, concentrate
Lactophenol Cotton Blue (LPCB).  React with antiserum specific to known fungi
 Positive control necessary for definitive
Dermatophyte Identification identification
1. Hair perforation test  Test is read at 24 hrs
 5 to 10-mm sterile hair floated on sterile 1. A antigen – B. dermatitidis
water and yeast extract. Conidia or hyphae 2. 1, 2 & 3 antigen – P. brasiliensis
 inoculated onto water surface. Remove hair 3. H and M antigen – H. capsulatum
shafts and observe in LPCB weekly for 1 4. HS, HL and F antigen - C. immitis
month 2. DNA probe
*Trichophyton mentagrophytes: (+)  Rapid kits that use nucleic acid hybridization
**Trichophyton rubrum: (-) to identify fungi in culture.
2. Urease test  Highly specific to each fungus, because it is
 Tubes of urease agar are lightly inoculated based on DNA sequence.
for 5 days at room temperature  Needs to be performed on cultured
*Trichophyton mentagrophytes: (+) organisms – not for specimens
**Trichophyton rubrum: (-) or weak  Developed for Coccidiodes, Blastomyces,
3. Trichophyton Agars Histoplasma
 Originally numbers 1 to 4  Specialized clinical laboratories are using
 Most laboratories use only 1 and 4 DNA sequ-encing techniques to establish
Thiamine requirement: fungal infections.
 Trichophyton agar 1 (without thiamine) Yeast Identification
 Trichophyton agar 4 (with thiamine) MACROSCOPIC MORPHOLOGY
 10 to 14 days, observe for growth.  Colony color and texture
4. Rice grain growth  Color: white, tan, pink, salmon
 Sterile, nonfortified rice grain media, 10  Can have dematiaceous yeasts
days, observe for growth  texture: mucoid, butterlike, velvety, wrinkled
*Microsporum canis: (+) MICROSCOPIC MORPHOLOGY: wet preparation
**Microsporum audouinii: (-)  Hyphae
 Pseudohyphae
 Blastoconidia

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Cornmeal Tween 80 Agar Four main morphology types

 Encourages development of 1. Hyphae
chlamydospores 2. Pseudohyphae
 Relationships among hyphae, 3. Arthroconidia
pseudohyphae, and others 4. Chlamydioconidia or blastoconidia
 Clear media: can be observed under light a. Pseudohyphae or Blastoconidia only
microscope Candida krusei
 Specific organisms associated with specific Candida parapsilosis
morphology Candida keyfr
 Cornmeal agar morphology - used in Candida tropicalis
conjunction with carbohydrate usage b. Blastoconidia only
C.glabrata and C.neoformans
c. Arthroconidia - Trichosporon beigelii

_____________________________________________________________________________________________
 Assimilation – which can be used as a sole
PHYSIOLOGIC TESTS carbon source
1. Germ tube test Two Systems (Assimilation)
 Filamentous outgrowth of blastoconidia. a. API 20C: strip test
 Most basic and easiest to perform. b. Vitek: automated
 Requires the use of serum or plasma. 3. Urea hydrolysis
 Some commercially made broths (will last  Detected on simple urea agar
longer). Over-incubation and over-  Rapid and easy
inoculation are biggest problems.  Differentiate Cryptococcus from
 Other agents can form germ tubes Rhodococcus
 Not valid if not read after 2 hrs.  Positive: pink, negative: little or no change
 “true” germ tube: C. albicans 4. Temperature studies
 No constrictions are base, where the tube  Cryptococcus sp.: weak growth at 35°C and
attaches to the mother cell. no growth at 42°C
 constriction base indicates C. tropicalis  Candida sp.: several can grow well
 Other species have germ tubes exceeding 45°C
o C. stellatoidea (sucrose assimilation
is used to differentiate from C. Order of events
albicans) 1. Wet preparation
o C. dubliniensis (no growth at 4 hrs.) 2. Germ tube: germ tube negative and from
 Positive and negative controls are sterile site
necessary. 3. Cornmeal Agar morphology
2. Fermentation/Assimilation 4. Physiologic/Biochemical reaction
 Fermentation – carbohydrate is use in the 5. Temperature
absence of oxygen

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VIROLOGY
Viruses are the smallest infectious agents (ranging b. Papillomaviruses and Picornaviruses: the
from about 20 nm to 300 nm in diameter), contain only virion is identical with the nucleocapsid.
one kind of nucleic acid (RNA or DNA).
PRINCIPLES OF VIRUS STRUCTURE
Inert in the extracellular environment.
Electron microscopy
They replicate only in living cells (obligate intracellular  Use of heavy metal stains (e.g., potassium
parasites). phosphotungstate) to emphasize surface
structure. The heavy metal permeates the
BASIC STRUCTURES virus particle like a cloud and brings out the
surface structure of viruses by virtue of
“negative staining.”
 The typical level of resolution is 3–4 nm. (The
size of a DNA double helix is 2 nm.) However,
conventional methods of sample preparation
often cause distortions and changes in
particle morphology.

Cryoelectron microscopy

 Uses virus samples quick frozen in vitreous

ice; fine structural features are preserved, and
the use of negative stains is avoided.
Capsid - the protein shell, or coat, which encloses the  Three-dimensional structural information can
nucleic acid genome. be obtained by the use of computer image
processing procedures.
Capsomeres - morphologic units seen in the electron
microscope on the surface of icosahedral virus X-ray crystallography
particles. Capsomeres represent clusters of
polypeptides, but the morphologic units do not  Can provide atomic resolution information,
necessarily correspond to the chemically defined generally at a level of 0.2–0.3 nm.
structural units.  The specimen must be crystalline, and this
has only been achieved with small,
Envelope - a lipid-containing membrane that surrounds nonenveloped viruses.
some virus particles. It is acquired during viral
maturation by a budding process through a cellular Genetic economy requires that a viral structure be
membrane. Virus encoded glycoproteins are exposed made from many identical molecules of one or a few
on the surface of the envelope. These projections are proteins. Viral architecture can be grouped into three
called peplomers. types based on the arrangement of morphologic
subunits:
Virion - the complete virus particle. 1. Cubic symmetry (e.g., Adenoviruses) -
a. Herpesviruses and Orthomyxoviruses: this icosahedral pattern, the most efficient
includes the nucleocapsid plus a surrounding arrangement for subunits in a closed shell.
envelope. This structure, the virion, serves to Viral structures built from repeated
transfer the viral nucleic acid from one cell to identical protein subunits to form a nearly
another. spherical structure with rotational
symmetry.

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2. Helical symmetry (e.g., Orthomyxoviruses) - particle size. (preparations from tissue

protein subunits are bound in a periodic way extracts and in ultrathin sections of infected
to the viral nucleic acid, winding it into a cells).
helix. Viral morphology composed of a  Another method that can be used is
single type of capsomer stacked around sedimentation in the ultracentrifuge.
a central axis to form a helical structure.
3. Complex structures (e.g., poxviruses) - a CHEMICAL COMPOSITION OF VIRUSES
virus that possesses a capsid that is
1. Viral Protein: surface proteins, enzymes
neither truly helical nor icosahedral. This
2. Viral Nucleic Acid: either DNA or RNA that
morphology may have additional encodes the genetic information necessary
structures such as a protein tail or a for replication of the virus.
complex outer wall. 3. Viral Lipid Envelopes: lipid envelopes as
part of their structure.
MEASURING THE SIZE OF VIRUSES 4. Viral Glycoproteins: Viral envelopes contain
glycoproteins e.g. influenza virus membrane
 Viruses are filterable pathogenic agents. glycoproteins (hemagglutinin, neuramini-
 Direct observation in the electron microscope dase).
is the most widely used method for estimating

MULTIPLICATION CYCLE

ADSORPTION AND PENETRATION

UNCOATING

Early mRNA and protein SYNTHESIS

Viral genome replication (ECLIPSE STAGE)

Late mRNA an protein SYNTHESIS

ASSEMBLY

RELEASE (LYTIC OR LYSOGENIC)

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Proto-oncogene defines a normal gene that can become an oncogene due to mutations or increased expression.
These genes commonly code for proteins involved in the regulation of cell growth or differentiation.

Sense is a concept used to compare the polarity of nucleic acids.

 Positive sense – 5’ to 3’, Positive-sense RNA (5’ to 3’) signifies that a particular sequence can be directly
translated into protein.
 Negative sense – 3’to 5’, Negative-sense RNA (3’ to 5’) forms the complementary strand and must be
converted to positive-sense by an RNA polymerase prior to translation.

TYPES OF VIRUSES
DNA VIRUSES
Characteristics:
 All are D-S DNA except Parvovirus
 Replicate in the nucleus of the host cell except POXVIRUS (cytoplasm)
 Capsid: icosahedral or complex (nonconforming symmetry). All are ICOSAHEDRAL except
POXVIRUS (complex)
 All are ENVELOPED (ether sensitive) except PAPOVAVIRUS, ADENOVIRUS AND PARVOVIRUS

DNA VIRUSES

Double Stranded Single Stranded

Enveloped: Unenveloped:
Unenveloped:
HEPADNAVIRUS ADENOVIRUS
PARVOVIRUSES
HERPESVIRUS PAPILLOMAVIRUS
POXVIRUS POLYOMAVIRUS

FAMILY CHARACTERISTICS VIRUS DISEASE DIAGNOSIS/

COMMENTS
Adenoviridae dsDNA genome; Adenovirus *pharyngitis, CPE in cell lines
icosahedral capsid, *keratoconjunctivitis (swollen grapelike
unenveloped, 50 *pneumonia, clusters)
human serotypes *gastroenteritis in
children
Hepadnaviridae Party dsDNA genome, Hepatitis B virus *acute infection with ELISA/PCR
icosahedral, resolution (90%),
enveloped fulminant hepatitis Antigens
Dane particle virion most co infected with HbsAg – EXPO-
(David Dane) delta virus SURE*carrier
*chronic hepatitis state
persistence of HBsAg,

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 MYCOLOGY AND VIROLOGY

Hepatitis B surface followed by resolution,

antigen termed as asymptomatic carries HBeAg –
Australia antigen start INFECTIVITY
(Baruch Blumberg) (HIGH)
*serum hepatitis
Hepatitis D co- HBcAg
infection
Anti-bodies
*Anti-HBcAg –
IgM (Acute)
IgG (Chronic)
*window period
*Anti-HBeAg
*Anti-HBsAg -
Vaccination

Herpesviridae Herpes Simplex *primary infection - Tzanck smear –

Virus – 1 Gingivostomatitis, Giemsa stained,
*Latency infection - scraping from the
*latency site: pharyngitis, herpes base of the
Trigeminal ganglion labialis, keratitis, vesicle
encephalitis
Herpes Simplex *genital herpes Immunofluore-
Virus – 2 *neonatal herpes scent = (+)
*aseptic Meningitis multinucleated
*latency site: Sacral *cervical CA giant cells with
ganglion cowdry

Cell culture –
most diagnostic,
CPE occur in 1-5
days –
Identification by
IFT

Herpes Simplex *primary infection: Tzanck smear -

Virus – 3: Varicella Chicken pox multinucleated
– Zoster Virus (Varicella) giant cell w/
*Latency: Shingles cowdry type
*latency site: Dorsal (Zoster) inclusions (HSV,
root of ganglia *Reyes syndrome – HZV)
induced by aspirin
IFT- method of
choice

Lesions cultured
on A-549

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 MYCOLOGY AND VIROLOGY

Herpes Simplex *Infectious Atypical

Virus - 4 Epstein- mononucleosis lymphocytes,
Barr virus *Progressive Paul-Bunnell Test)
lymphoreticular
*infects B cells disease Heterophile
*Heterophile Abs *Oral hairy leuoko- antigen test:
plakia in HIV patients Monospot test
*Burkitt’s lymphoma
*Glandular fever or EBV specific Ab
Nasopharyngela CA test: EBVCA IgM,
EBNA

Hematology:
Downey cells

Herpes Simplex *Congenital disease of Blood (buffy coat)

Virus – 5 - the newborn, 40-day
Cytomegalovirus fever Body fluids, urine,
*mononucleosis like tissues, respiratory
“Salivary gland syndrome secretions cultured
virus” on HDF (Owl’s eye
inclusion) –
Giemsa/PAP

HSV-6 *Roseola (exanthem T -cells

subitum), 6th childhood
HSV-7 - X disease, malaise,
rash, fever, leukopenia
HSV-8 *Kaposi’s sarcoma Non-culturable
(AIDS- px) - purple
rashes on skin, cause
of cervical CA
Papillomaviridae Human Papilloma *Tropism for squa- Nonculturable
Virus mous epithelium –
“warts (verrucae)” – Pap smear for
HPV 6 and HPV 11 koilocytes

Disease: HPV vaccine

“Condylomata
acuminata - ano-
genital”
Cervical Squamous,
Vulvar or Penile CA –
HPV 16 and HPV 18

Polymaviridaea BK virus (B.K. Virus remains dormant

(Human initials - Px) in kidneys, reactivation
Papovavirus) in immunecompro-

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 MYCOLOGY AND VIROLOGY

mised patients causes

hemorrhagic cystitis

JC virus (John *Progressive

Cunningham - px) multifocal
leukoencephalopathy

Parvovirus ssDNA, icosahedral, Parvovirus B-19 *Erythema infectiosum Smallest DNA

no envelope, (5th disease) virus
parvovirus B-19 is the Slapped cheek rash PCR/viral DNA
only human parvovirus *Hydrops fetalis – from blood or
miscarriages, amniotic fluid
Poxvirus Largest and most Smallpox virus Smallpox is a CPE on culture or
complex of all viruses, generalized infection Pocks on
brick shaped with with pustular rash; chorioallantoic
nonconforming molluscum manifests membrane
symmetry referred to as benign modules of
as complex, dsDNA the skin.
*Smallpox (variola
major)
*Alastrim (variola
minor)
*Molluscum,
contagiosum – wart
like tumors, vaccinia
viruses
*Monkeypox and Orf
Iridoviridae dsDNA, icosahedral Iridovirus *African Swine fever
virus
Anelloviruses small (~30 nm in Torque Teno virus * No specific disease
diameter), icosahedral associations have
virions that lack an been proven. There is
envelope. The viral limited knowledge
genome is circular, about viral gene
single-stranded DNA, expression and
2.0–3.9 kb in size. replication.

RNA VIRUSES
Characteristics:
 All are S-S RNA except REOVIRUS. Replicates in the cytoplasm of the host cell.
 Generally HELICAL or ICOSAHEDRAL except the POSITIVE SENSE RNA VIRUS
 All are ENVELOPED except PICORNAVIRUS (smallest), CALICIVIRUS AND REOVIRUS
 All are NON-SEGMENTED except REOVIRUS, ORTHOMYXOVIRUS, BUNYAVIRUS and
ARENAVIRUS

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 MYCOLOGY AND VIROLOGY

RNA
VIRUSES
Single Single
stranded Double
Stranded stranded (non
(positive (negative
sense) enveloped)
sense)
Enveloped Enveloped Nonenveloped
Enveloped Nonenveloped
Icosahedral: Helical: Icosahedral:
Helical: Icosahedral:
FLAVIVIRIDAE CORONAVIRIDAE PICORNAVIRIDAE
ORTHOMYXOVIRIDAE REOVIRIDAE
TOGAVIRIDAE CALICIVIRIDAE
PARAMYXOVIRIDAE
RETROVIRIDAE FILOVIRIDAE
BUNYAVIRIDAE
ARENAVIRIDAE

FAMILY CHARACTERISTIC VIRUS DISEASE

Arenaviridae Enveloped, irregular Lymphocytic choriomenin- *LCM causes asymptomatic
capid, with 2 segmented gitis virus (LCM) to influenza-like to aseptic
ssRNA genome meningitis-type disease,
Lassa Fever virus
Arena – “sandy” (zootic -rats)
appearance
Machupo virus *Bolivian Hemorrhagic fever

Junin virus *Argentinian Hemorrahagic

fever
Sabia virus

*causes influenza like

disease to severe
hemorrhagic fever
Astroviridae Characteristic six point Astrovirus *Gastroenteritis among
star like structure young children/ diarrhea
outbreaks
Isolated among chicken,
ducks and turkeys
Bunyaviridae Segmented, ssRNA Arbovirus: *Encephalitis for arbo-
genome, spherical or California Encephalitis viruses, pneumonia or
pleomorphic capsid with Virus hemorrhagic fever for
envelope hantaviruses
La Crosse Virus

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 MYCOLOGY AND VIROLOGY

Non-arbovirus:
Sin Nombre Virus – “Four
Corners Virus” - rodents
Hantaan River Virus/
Korean Hemorrhagic
Fever – field mice (Korean
War)
Rift Valley Fever virus -
raw milk (cattles, goats
and buffaloes) – Rift
Valley, Kenya
Crimean Congo hemor-
rhagic Fever virus

Caliciviridae Nonenveloped, Norwalk – “Winter Nausea, vomiting and

icosahedral capsid Vomiting bug”: Norwalk, diarrhea, outbreaks of
surrounding ssRNA Ohio gastroenteritis in schools,
genome colleges, nursing homes,
cruise ships (Norwalk)
Hepatitis E virus *Hepatitis (water borne
hepatitis) similar to that
caused by heap A except for
extraordinarily high case
fatality rate among pregnant
women
Coronaviridae ssRNA, helical capsid with Coronavirus *Common cold, possibly
envelope Severe Acute Respiratory gastroenteritis, especially in
large halo or crown like Syndrome Corona Virus children
structure Novel Human Corona
Middle Eastern Viruses
Respiratory Syndrome 1. HCoV – 229E and HCoV –
Corona Virus OC43
2. NL63 – New Haven CoV
(2004)
3. Human Corona Virus
HKU1 (2005)
4. MERS – CoV (2012 and
2013)
5. MERS – CoV (2015 -
Korea)
Novel Corona Virus- 2019 6. nCoV-19 (2019 -Wuhan)

Filoviridae enveloped, long, fila- Ebola-Marburg viruses – *Severe hemorrhage and

mentous, and irregular Shepherd’s crook, “U” or liver necrosis
capsid forms with ssRNA “6” shaped virus (fruit
bats/bush meat/other pri-
mates)

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 MYCOLOGY AND VIROLOGY

Flaviviridae ssRNA, genome, sur- Arboviruses *acute hemorrhagic fever,

roundded by spherical and Dengue virus (Aedes “saddleback fever or
icosahedral capsid with aegypti) breakbone fever”
envelope
West Nile Encephalitis
virus (bird-mosquito-man)

Japanese B Encephalitis
virus (pig-mosquito-man)

St. Louis Encephalitis *Yellow fever

viruses (Culex)

Zika virus - mosquito *microencephaly

Hepatitis C virus/ NANB *Post Transfusion Hepatitis,
virus strong correlation between
chronic HCV infection and
Hepatitis G virus hepatocellular carcinoma

Orthmyxoviridae Segmented, ssRNA, Influenza A *Pandemic (Antigen shift

helical, with envelope with and drift) – humans, birds
spikes and pigs
AH1:N1 – Spanish flu (1918)
Hemeagglutinin (H) – 16 and Swine flu (2009)
Neuraminidase (N) - 9 AH2:N2 – Asian flu
AH3:N2 – Hongkong flu
Antigenic changes AH5:N1 – Avian flu,
1. Shift (genetic pandemic threat
reassortment) -
Pandemic Influenza B *Epidemic (Antigen drift) –
2. Drift (point humans and seals
mutation) -
epidemic Influenza C *humans, pigs and dogs

Flu Vaccine

Paramyxoviridae Single-stranded, helical, Measles virus Koplik’s spots in buccal

non-segmented, (Rubeola/ Morbilivirus) mucosa
enveloped Atypical measles occurs in
those with vaccine immunity
Hemeagglutinin (H) Subacute sclerosing
Neuraminidase (N) panencephalitis
Fusion (F) Mumps *Mumps/Parotitis (rare
cause of orchitis) – infertility
in men

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 MYCOLOGY AND VIROLOGY

Parainfluenza virus Adults: upper respiratory,

(common cause of rarely pneumonia
pneumonia) Children: *Croup, bronchio-
litis, pneumonia
Respiratory syncytial virus Infants:
(RSV) - common cause of bronchiolitis,pneumonia,
pneumonia croup
Children: upper respiratory
Henipavirus

Nipah virus (bats) *Encephalitis (pig to man)

Hendra virus *Respiratory disease horses

Metapneumovirus *2nd after RSV that causes

lower respiratory tract
infection among children
Picornaviridae Enteroviruses
(acid resistant)
Poliovirus *Poliomyelitis (Vaccines:
Inactivated: Jonas Salk; Live
attenuated/oral: Albert
Sabin)
Coxsackievirus A *Herpangina: Hand-foot-
mouth disease
Coxsackievirus B *Pleurodynia: pericarditis,
myocarditis (Devil’s Grip
disease)
Echovirus (Enteric Cyto- *Aseptic meningitis
pathic Human Orphan
virus)
Enterovirus 68-71 *Enterovirus 70: acute
hemorrhagic conjunctivitis
*Enteroviral meningitis
Hepatitis A virus *infectious hepatitis
(enterovirus type 72) *Hepatitis with short
incubation time, abrupt
onset, low morality, no
carrier state
Rhinovirus – 33°C *common cold/ Acute Viral
(acid sensitive) Nasopharyngitis
Reoviridae Segmented, dsRNA Rotavirus – wagon-wheel Gastroenteritis in infants
genome, icosahedral like) and children 6 months to 2
capsid, no envelope years (winter virus)
Orbivirus/ Colorado Tick Adults: asymptomatic
Fever Virus

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 MYCOLOGY AND VIROLOGY

Retroviridae ssRNA genome, icosa- HIV-1 Most diseases in humans is

hedral capsid with HIV-2 (African strain) caused by HIV 1; infected
envelope, reverse tran- Genus: lentivirus cells include CD4,
scriptase converts geno- Human T lymphotropic monocytes, asymptomatic
mic RNA into DNA viruses (HTLV-1 and 2) infection, acute flu-like
diseases, AIDS related
Major Proteins AIDS related Complex complex, AIDS, AIDS
associated malignancies
gag LymphoAdenopathy
capsid protein (CA) – Associated Virus T-cell leukemia and
(p24) lymphoma, and tropical
matrix protein (MA) – (p17) spastic paraparesis for
nucleoprotein (NC) – (p7) HTLV-1, no known disease
associated with HTLV-2
pol – (enzymes) (isolated on patients with
reverse transcriptase (p51) hairy cell leukemia)
integrase (p32) *screened in US bloodbanks
protease (p10) due to high incidence in drug
users
env
gp160 (gp140 and gp41)

Rhabdoviridae ssRNA genome, helical Rabies Lyssavirus (CNS *Rabies - latin “madness”
capsid with envelope, infection in humans and * FAT of rabies antigen (dog
bullet-shaped with spikes animals) brain), Seller’s stain of Negri
bodies

Western, Eastern, and *zoonotic (equine - horses)

Venezuela Equine Ence- *encephalitis
phalitis, Triple E or
sleeping sickness
Togaviridae
icosahedral Rubella virus * German measles (3-day
*reservoir: birds
rash), “blueberry muffin
baby”, Rubella – “little red”

Teratogenic virus (1st week

of pregnancy)

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 MYCOLOGY AND VIROLOGY

LABORATORY SAFETY 6. Use of biosafety hoods (*Biosafety Level 4

when working with high-risk agents such as
1. Aerosols: generated by homogenization of the filoviruses and rabies virus)
infected tissues, centrifugation, ultrasonic 7. Immunization if vaccine is available
vibration, or broken glassware.
2. Ingestion: from mouth pipetting, eating or SPECIMEN COLLECTION, TRANSPORT AND
smoking in the laboratory, or inadequate STORAGE
washing of hands  Early stage of infection before the height of
3. Skin penetration: from needle sticks, broken fever
glassware, hand contamination by leaking
 Sample the infected site
containers, handling of infected tissues, or
animal bites.  Tissues and swabs require a viral transport
4. Splashes: into the eyes or skin medium; aspirates do not require transport
medium
Good biosafety practices include the following:  DO NOT use CALCIUM ALGINATE swabs
(only dacron, rayon, or cotton swabs)
1. Training in and use of aseptic techniques  TRANSPORT: 4°C (1-2 days) - swabs
2. X of mouth pipetting  Stuart’s Medium
3. No eating, drinking, or smoking in the  Leibovitz-Emory
laboratory  Earl/Hanks Balanced Salt Solution
4. Use of PPE (e.g., coats, gloves, masks) not – Amino Acids, vitamins and
to be worn outside the laboratory bicarbonate, Penicillin-
5. Sterilization and decontamination. Streptomycin, Phenol Red
 STORAGE: -70°C (>3 days)

Disease/ manifestation Specimen Common Agents

Respiratory tract Croup, bronchiolitis, pneumonia Nasal aspirate, NP swab, Influenza, PIV, RSV, HSV,
throat swab, BAL Adenovirus,
Picornaviridae, Rotavirus
Gastrointestinal Gastroenteritis stool, rectal swabs Adenovirus 40& 41,
Enterovirus, Norwalk,
Rotavirus
Cutaneous Lesions and exanthems (rashes) vesicle aspirate, lesion HSV 1,2,3, Adenovirus,
swabs Measles, Rubella,
Enterovirus, Rotavirus
Ocular Conjunctivitis, keratitis conjunctival scrapings, HSV, Adenovirus,
corneal swabs/ scrapings Enterovirus
CNS Encephalitis, aseptic meningitis CSF, nasopahryngeal HSV, Arbovirus,
swabs Enterovirus, Mumps
Genital Urethritis, penile lesions vesicle aspirate or swab HSV
Congenital Lesions and exanthems throat swab, urine, serum CMV, HSV, Rubella
Disseminated multiple multiple sites Adenovirus, Enterovirus,
Rotavirus

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 MYCOLOGY AND VIROLOGY

CULTIVATION AND ASSAY OF VIRUSES 1. Development of cytopathic - refers to the

degenerative changes in association with viral
Cultivation of Viruses infection.
Viruses can be grown in cell cultures or in fertile eggs a. Rounding necrosis – Enterovirus
under strictly controlled conditions (e.g. embryonated b. Ballooning/ Giant cell – HSV
chicken eggs – Influenza, Tissue culture medium: c. Grape like cluster – Adenovirus
AGMK (vero cells)*African green monkey cells, A-549 *Adeno- d. Syncytium form – RSV/ Measles/
carcinoma human alveolar basal epithelial cell, WI-38*Winstar Institute Rubella
– aborted fetus, MRC-5*Medical Research Council cell strain 5 – e. Hemeadsorption – Influenza,
aborted Caucasian fetus – lung carcinoma cells). Parainfluenza, Measles and Mumps
Animals - primary isolation of certain viruses and for f. Refractile, round cell – Rhinovirus
studies (pathogenesis of viral diseases and of viral (33°C)
oncogenesis). g. No CPE – Influenza, Parainfluenza and
Mumps
 Serial passage – refers to the process of
growing the bacteria or virus in iterations 2. Appearance of a virus-encoded protein (e.g.
(repetitions). hemagglutinin - influenza virus). Specific antisera can
be used to detect the synthesis of viral proteins in
There are three basic types of cell cultures infected cells. Hemagglutination - refers to the
agglutination, or clumping, of red blood cells.
1. Primary cultures - dispersed cells (usually
with trypsin) from freshly removed host 3. Detection of virus-specific nucleic acid (PCR).
tissues. In general, they are unable to grow
for more than a few passages in culture (e.g. 4. Adsorption of erythrocytes to infected cells, called
Primary Monkey Kidney Cells). hemadsorption (e.g. hemagglutinin - parainfluenza,
2. Diploid cell lines/Secondary cultures - influenza) in cellular membranes
have undergone a change that allows their
limited culture (up to 50 passages) but that 5. Viral growth in an embryonated chick egg may result
retain their normal chromosome pattern (e.g. in:
Human Diploid Fibroblast).  Death of the embryo (e.g., encephalitis
3. Continuous cell lines are cultures capable viruses)
of more prolonged/indefinite growth that have  Production of pocks or plaques on the
been derived from diploid cell lines or from chorioallantoic membrane (e.g., herpes,
malignant tissues. They invariably have smallpox, vaccinia)
altered and irregular numbers of  Development of hemagglutinins in the
chromosomes. The type of cell culture used embryonic fluids or tissues (e.g., influenza)
for viral cultivation depends on the sensitivity
of the cells to a particular virus (Madin Darby INCLUSION BODY FORMATION
Canine Kidney, Hep-2 (Human Epithelial  Observed through a light microscope
Cells type 2 – Laryngeal CA), HeLa – (inverted microscope).
Henrietta Lacks – Cervical CA).  They become far larger than the individual
virus particle and often have an affinity for
A. Detection of Virus-Infected Cells Multiplication of a acid dyes (e.g., eosin).
virus can be monitored in a variety of ways:  The presence of inclusion bodies may be of
considerable diagnostic aid.

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 MYCOLOGY AND VIROLOGY

VIRUS INTRACYTOPLASMIC INTRANUCLEAR INTRANUCLEAR

EOSINOPHILIC BASOPHILIC EOSINOPHILIC
Rabies (fix with acetone) Negri bodies
Smallpox (Vaccinia, Variola) Guarnieri bodies
Smallpox (Variola) Paschen bodies
Fowlpox Bollinger bodies
Molluscum contagiosum Henderson-Patterson bodies
HSV Cowdry Type A
VZV Cowdry Type A
Yellow fever Torres bodies
Polio and Adenovirus Cowdry Type B
Adenovirus Cowdry Type B
CMV Owl’s Eye appearance
Rubeola Warthin-Finkeldey bodies

QUANTITATION OF VIRUSES PURIFICATION AND IDENTIFICATION OF VIRUSES

Purification
A. Physical Methods 1. Concentration by precipitation (e.g.
ammonium sulfate, ethanol, or polyethylene
 Quantitative nucleic acid-based assays - glycol or by ultrafiltration) or hemagglutination
Polymerase Chain Reaction (number of viral and elution (Orthomyxoviruses).
genome copies in a sample.)
 Serologic tests such as RadioImmuno- 2. Centrifugation
Assays (RIA), Enzyme-Linked Immuno- a. Rate-zonal centrifugation: a sample
Sorbent Assays (ELISA) and Hemagglu- of concentrated virus is layered onto
tination. a preformed linear density gradient
(sucrose or glycerol) band at a rate
B. Biologic Methods by the size and weight of the virus
particle.
 End point biologic assays (e.g. measure- b. High-speed centrifugation in density
ment of animal death, animal infection, or gradients (e.g. cesium chloride,
CPE). Titer - 50% infectious dose potassium tartrate, potassium
(ID50),reciprocal of the dilution of virus that citrate, or sucrose).
produces the effect in 50% of the cells or
animals inoculated. 3. Column chromatography: virus is bound to a
 Plaque assay - tissue culture. Monolayers of substance such as diethylaminoethyl or
host -> overlaid with medium containing agar phosphocellulose and then eluted by changes
or carboxymethylcellulose to prevent virus in pH or salt concentration.
spreading throughout the culture. -> After
several days, the cells initially infected have 4. Zone electrophoresis permits separation of
produced virus that spreads only to virus particles from contaminants on the basis
surrounding cells. Multiple cycles of of charge.
replication and cell killing produce a small
area of infection, or plaque. Identification of a Particle as a Virus
 Herpes and Vaccinia - form pocks when
inoculated onto the chorioallantoic mem- 1. The particle can be obtained only from infected cells
brane. or tissues.

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 MYCOLOGY AND VIROLOGY

2. Particles obtained from various sources are Stabilization of Viruses by Salts

identical regardless of the cellular origin in which the  Many viruses can be stabilized by salts in
virus is grown. concentrations of 1 mol/L (i.e., the viruses are
3. Particles contain nucleic acid (DNA or RNA), the not inactivated even by heating at 50°C for 1
sequence of which is not the same as the species of hour).
host cells from which the particles were obtained. o MgCl2, 1 mol/L, stabilizes
4. The degree of infective activity of the preparation Picornaviruses and Reoviruses.
varies directly with the number of particles present. o MgSO4, 1 mol/L, stabilizes
5. Destruction of the physical particle by chemical or Orthomyxoviruses and
physical means is associated with a loss of viral Paramyxoviruses.
activity. o Na2SO4, 1 mol/L, stabilizes
6. Certain properties of the particles and infectivity Herpesviruses.
must be shown to be identical (e.g., their sedimentation *The stability of viruses is important in the
behavior in the ultracentrifuge and their pH stability preparation of vaccines.
curves). pH
7. Antisera prepared against the infectious virus  Viruses are usually stable between pH values
should react with the characteristic particle and vice of 5.0 and 9.0.
versa. Direct observation of an unknown virus can be  Enteroviruses - resistant to acidic conditions.
accomplished by electron microscopic examination of  All viruses are destroyed by alkaline
aggregate formation in a mixture of antisera and crude conditions.
viral suspension.  Hemagglutination reactions, variations of less
8. The particles should be able to induce the than 1.0 pH unit may influence the result.
characteristic disease in vivo (if such experiments are Radiation
feasible).  Ultraviolet, x-ray, and high-energy particles
9. Passage of the particles in tissue culture should inactivate viruses.
result in the production of progeny with biologic and Ether Susceptibility
antigenic properties of the virus.  Enveloped Virus = sensitive to ether (ESE)
 Naked Virus = resistant to ether (NRE)
REACTION TO PHYSICAL AND CHEMICAL
*lipids in the viral envelope is soluble
AGENTS
to ether (fat solvent)
Detergents
Heat and Cold
 Nonionic detergents (e.g., Nonidet P40 and
 Icosahedral viruses: stable, losing little
Triton X-100) solubilize lipid constituents of
infectivity after several hours at 37°C.
viral membranes.
 Enveloped viruses are much more heat labile,
 Anionic detergents (e.g., sodium dodecyl
rapidly dropping in titer at 37°C.
sulfate) also solubilize viral envelopes; in
 Enveloped viruses tend to lose infectivity after addition, they disrupt capsids into separated
prolonged storage even at -90°C and are polypeptides.
particularly sensitive to repeated freezing and Formaldehyde
thawing.
 Formaldehyde destroys viral infectivity by
 Viral infectivity: X by heating at 50–60°C for reacting with nucleic acid.
30 minutes except (e.g., Hepatitis B virus, Photodynamic Inactivation
polyomaviruses).
 Vital dyes (e.g. toluidine blue, neutral red, and
 Viruses can be preserved by storage at proflavine). These dyes bind to the viral
subfreezing temperatures, and some may nucleic acid, and the virus then becomes
withstand lyophilization (4°C or even at room susceptible to inactivation by visible light.
temperature)
 Neutral red: commonly used to stain plaque
 Viruses that withstand lyophilization are more assays so that plaques are more readily seen.
heat resistant when heated in the dry state.

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 MYCOLOGY AND VIROLOGY

Antibiotics and Other Antibacterial Agents irradiation, or detergents (subunit vaccines) to

 Antibacterial antibiotics and sulfonamides: inactivate the vaccine virus.
X effect on viruses.
 Quaternary ammonium compounds PRIONS
(QUATS): in general, are not effective against
viruses.  Proteinacious infectious virions or
 Organic iodine compounds are also Transmissible spongiform encephalopathies
ineffective. Chlorine treatment -> stools (TSEs)
adequate to inactivate typhoid bacilli is  Neurodegenerative diseases that affect both
inadequate to destroy poliomyelitis virus human and animals
present in feces.  Target site: Brain – rapidly progressive and
 Alcohols: isopropanol and ethanol, are always fatal (Prions characteristics in the
relatively ineffective against certain viruses, cells are holes – creating a sponge like
especially picornaviruses. tissue lesion)

Common Methods of Inactivating Viruses for Human Prion Disease

Various Purposes  Creutzfeldt-Jakob disease (CJD)
 Gertsmann-Straussler-Scheinker Syndrome
 Sterilization: steam under pressure, dry heat,  Fatal familial insomnia
ethylene oxide, and γ-irradiation.  Kuru
 Surface disinfectants: sodium hypochlorite,
glutaraldehyde, formaldehyde, and peracetic Animal Prion Disease
acid.  Bovine Spongiform Encephalopathy (BSE)
 Antiseptics: skin disinfectants include  Chronic Wasting Disease (CWD)
chlorhexidine, 70% ethanol, and iodophores.  Scrapie
 Transmissible Mink Encephalopathy
 Vaccine: use of formaldehyde, β-
 Feline Spongiform Encephalopathy
propiolactone, psoralen + ultraviolet
 Ungulate Spongiform Encephalopathy

REFERENCES:
Bailey & Scott’s Diagnostic Microbiology, Elsevier 13th edition, 2014.
Jawetz, Melnick, & Adelberg’s Medical Microbiology, McGraw-Hill 27th edition, 2014.
Graeter, Hertenstein, Accurso and Labiner, Medical Laboratory Science Examination Review, Elsevier, 2015.
“ATCC® MYCOLOGY CULTURE guide tips and techniques for culturing yeasts and filamentous fungi“.
https://www.atcc.org retrieved April 22, 2018.
“ATCC® VIROLOGY GUIDE Tips and techniques for propagating virus in tissue culture and embryonated chicken
eggs”. https://www.atcc.org retrieved April 22, 2018.

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