Laboratory Diagnosis of Bacterial Infections

Laboratory diagnosis of bacterial infections is useful for the following purposes:

i) Identification: to identify the causative bacterial agent responsible for the disease
ii) Treatment: to provide accurate antimicrobial therapy
iii) Surveillance purpose: to assess the disease burden in the community by estimating the
prevalence and incidence of the infections
iv) Outbreak investigations: For investigation of outbreak in community or hospital
v) Post exposure prophylaxis: to start PEP in infectious disease (Eg- Anthrax, Plague)
vi) Initiation of appropriate infection control measure
Laboratory diagnosis of bacterial infections comprises of several steps as follows-
i. Specimen collection and Transport
ii. Direct detection from specimen
iii. Culture
iv. Secondary staining and identification by conventional and automation
v. Antimicrobial serology testing
vi. Serological test
vii. Molecular methods
viii. Typing methods

Specimen Collection and Transport

First step of laboratory diagnosis of an infectious disease is collection of appropriate specimen
correctly and its transport to laboratory.
Types of infections and specimens to be collected
Site of Infection Type of Infection Specimen Collected
Respiratory Tract Sinusitis Acute: Nasopharyngeal swab
Chronic: Sinus washing/
biopsy
URTI Nasopharyngeal swab/ Throat
swab
LRTI Sputum/ Endotracheal
aspirate/ Broncho-alveolar
lavage/Bronchial brushing/
Lung biopsy
Suspected Tuberculosis Sputum- Spot and Early
morning
Gastric aspirate for infant
Middle ear Acute/ Chronic suppurative Acute: Tympanic membrane
otitis media exudate
Chronic: Discharge from ear
Gastro intestinal tract Diarrheal disease Stool/ Rectal swab or rectal
mucus
Urinary tract Urinary tract infection Mid-stream clean catch urine/
Urine from catheterized
patients/ supra-pubic
aspiration of urine
Central nervous system Meningitis Cerebrospinal fluid
Cardiovascular system Blood stream infection/ Paired blood culture
sepsis/ infective endocarditis specimens
Sterile body area Infections from sterile body Sterile body fluid of that area
area (Pleural fluid/ synovial fluid/
Peritoneal fluid)
Skin and soft tissue SSTI Pus or exudates/ wound
swab/ aspirate from abscess/
tissue bits
Bones osteomyelitis Aspirate/ bone spicules
Genital tract Sexually transmitted disease Male: urethral discharge/
prostatic secretion
Female: urethral discharge/
high vaginal swab/ cervical
swab
Eye Conjunctivitis/ Conjunctival swab/ Corneal
keratitis/infections in aqueous scrapings/ Aqueous or
or vitreous humor vitreous fluid

Collection of respiratory tract samples

A) Throat swab: (two swabs always to be taken)
 A bright light from over the shoulder of the specimen collector should be focused into the
oral cavity so that the swab can be guided to the posterior pharynx.
 The patient is instructed to tilt the head back and breathe deeply.
 The tongue is gently depressed with a tongue blade to visualize the tonsillar fossa and
posterior pharynx.
 The swab is extended between the tonsillar pillars and behind the uvula.
 The patient is asked to phonate a long “ah” to lift the uvula and prevent gagging.
 The tonsillar areas and the posterior pharynx should be firmly rubbed with the swab.
 The swab should be placed immediately into a sterile tube or other suitable container for
transport to laboratory.
 Transport: Specimen immediately to be transported to the laboratory within 4 hours. Swab
for viral samples should be kept in viral transport media (VTM) and to be transported
immediately.
B) Nasopharyngeal swab:
 A bright light from over the shoulder of the specimen collector should be focused into the
oral cavity so that the swab can be guided to the posterior pharynx.
 The patient is instructed to tilt the head back and breathe deeply.
 With the thumb of one hand, gently elevate the tip of the nose.
 Moisten the tip of a small flexible nasopharyngeal wire swab with sterile water or saline
and gently insert it into one of the nares.
 Guide the swab backward and upward along the nasal septum until a distinct feel of
resistance indicates that the posterior pharynx has been reached.
 Leave the swab in contact with the posterior nasopharynx for 15 to 30 seconds if the patient
can tolerate the discomfort.
 Gently remove the swab.
 If while guiding the swab undue resistance is met, attempt the procedure through the
opposite nares.
 Transport: Specimen immediately to be transported to the laboratory within 4 hours. Swab
for viral samples should be kept in viral transport media (VTM) and to be transported
immediately.

C) Sputum:
1. Expectorated Sputum
 Food should not be taken 1-2 hours before expectoration
 Early morning sputum- containing pooled overnight secretions in which pathogenic
bacteria more likely to be concentrated
 Patient should brush the buccal mucosa, gum and tongue with a wet brush (not with any
paste) and gurgle with water before collection (no mouthwash or gurgle solution containing
anti-bacterial)
 Patient should take deep breath to produce bouts of cough
 To wait until he feels material coughed into his throat
 Sterile wide mouth jar with a tightly fit screw cap lid taken
 Patient is instructed to press the rim of the container under the lower lip to catch all of the
expectorated sputum
 To spit the deep coughed expectorated sputum directly into the container without spilling
over the rim
 To wipe off any spilled material with disinfectant moisture tissue (care not to enter the
container)
 Lid closed tightly
 Place of collection: Out-side the room, in the open air, making 6 ft distance from collector
or other person
 2. Induced sputum:
For those who cannot produce sputum,
i) Postural drainage
ii) Thoracic percussion To initiate strong cough
iii) With nebulized saline (0.85%)
For children, insane person
i) Gastric aspirate

D) Endotracheal aspirate:
The lower respiratory tract may be sampled by introducing a catheter through the larynx into the
trachea. If an endotracheal tube is in place or there is a tracheostomy, tracheal secretions is
aspirated through it. Endotracheal aspirate is treated same as sputum.
E) Broncho-alveolar lavage:
 Bronchoscope is introduced into the nose and advanced through nasopharyngeal passage
to trachea.
 Then it is introduced into area of interest of lung
 30-50 ml of physiologic saline is introduced through bronchoscope
 It is aspirated thereafter in a sterile tube.

F) Bronchial brushing:
 Bronchoscope is introduced into the nose and advanced through nasopharyngeal passage
to trachea.
 Then it is introduced into area of interest of lung
 Telescopic double catheter plugged at the distal end with polyethylene glycol to protect
endo-luminal brush is introduced through bronchoscope
 Once it is in correct area, inner cannula is pushed out to dislodge the plug
 Brush is then extended beyond the inner cannula and specimen collected by brushing
 Brush will contain 0.001-0.01 ml of secretion
 Brush is then withdrawn into inner cannula, which is withdrawn into outer cannula and
catheter is removed.

G) Fine Needle Aspiration and Lung Biopsy.

Fine needle aspiration is usually performed under radiologic guidance, particularly if a localized
lesion is present. This is an excellent means of obtaining material for cytology and culture; on-site
cytologic assessment is useful to guide culture. Trans-bronchial biopsy or open lung biopsy may
be necessary sometimes. This is invasive approach and is reserved for situations in which other
measures have failed. A small wedge is resected from the lung in which the disease process may
be studied histo-pathologically and microbiologically.
Transport: Specimen immediately to be transported to the laboratory within 4 hours. Swab for
viral samples should be kept in viral transport media (VTM) and to be transported immediately.

Collection of gastrointestinal tract samples

A) Stool:
Container: Wide mouth, clean, leak-proof, screw capped, detergent free container provided with
a small spoon.
Timing: Before starting antibiotic or anti-parasitic drugs.
Collection: Freshly passed stool to be collected directly into the container or with the help of
spoon (Not with wooden applicator stick). Stool should not be contaminated with urine or other
chemicals. Collection of stool from pans is not recommended.
For liquid stool, the specimen is best collected by introducing a lubricated catheter into the rectum
and letting the liquid stool flow directly into a screw capped container.
Rectal swabs may be taken for the following patients-
i) Newborn
ii) Severely debilitated patient
iii) Certain strain of Shigella
iv) Clostridium difficile associated diarrhoea
v) Recent gonococcal infection
For collection with rectal swab, Good quality cotton wool swab, absorbing 0.1-0.2 ml of fluid,
should be taken. The tip of the sterile swab beyond the anal sphincter should be passed, anywhere
from 1cm in infants to 4cm in larger adolescents. It should be rotated to sample the anal crypts for
30 seconds. Break the swab at the score line into the transport tube containing 1.0mL liquid media.
Precautions before collection for parasitic evaluation:
i) No consumption of much green leafy vegetables, egg, meat, fish or high protein
ii) Not any radiological examination with barium sulfate within one week
iii) To avoid certain medications: bismuth, mineral oil, anti-diarrheal or anti-parasitic,
antibiotics (Tetracycline, Metronidazole) within 10 days
For parasitic evaluation:
- Minimum 3 stools specimen necessary, 2 specimens from normal bowel movements, 1
preferably after purgative.
Transport:
Freshly passed stool to be transported to the laboratory immediately within 30 min – 1 hour. In
case of unavoidable delay, it may be kept at 40C or may be preserved for transportation in Cary-
Blair Media or Gram Negative Broth or Selenite F Broth or Alkaline peptone water (For Vibrio)
or Robertson’s Cooked meat Media with 10% NaCl (For Food poisoning).

Collection of Urinary tract samples

A) Urine:

a) For Male patient:

- Hands are cleaned properly
- Prepuce is retracted
- Glans penis is cleaned with wet cotton gauze
- The first portion of urine is allowed to be passed
- Next portion (midstream sample) is to be collected directly into a sterile wide mouthed leak
proof plastic container without spilling over the rim
- To wipe off any spilled material with disinfectant moisture tissue (care not to enter the
container)
- Lid is closed tightly.

b) For Female patient:

- Hands are cleaned properly
- Ano-genital toilet is to be done with careful cleaning with soap and clean water from front
to backside.
- Urine should be passed keeping the labia separated using fingers.
- The first portion of urine is allowed to be passed
- Next portion (midstream sample) is to be collected directly into a sterile wide mouthed leak
proof plastic container without spilling over the rim
- To wipe off any spilled material with disinfectant moisture tissue (care not to enter the
container)
- Lid is closed tightly.

c) For Catheterized patient:

- Hands are cleaned properly and gloved.
- A clamp is applied distally from the collection area and allowed the urine to accumulate.
- Part of rubber tube of Foley’s catheter (Collection area) is to be disinfected with alcohol
soaked sterile gauze.
- The cleaned area is to be punctured then with a sterile needle and syringe and urine is to
be aspirated directly. It may be collected directly from valve of three way Foley’s catheter.
- Urine must not be collected from the drainage bag.

d) Suprapubic aspiration:
- The suprapubic aspiration is ideal and avoids urethral contamination, but is invasive.
- The procedure is usually reserved for neonates and small children.
- It is to be performed when the bladder is full.
- The suprapubic skin overlaying the urinary bladder is disinfected and sterile drapes are put
in place.
- In the immediate site, where the aspiration is to be made, an anesthetic solution (1%
lignocaine) is injected subcutaneously.
- An 18-gauge short beveled spinal needle is extended into the urinary bladder and 10 ml of
urine is aspirated into the syringe.

e) Percutaneous nephrostomy (PCN) aspirate

- Percutaneous nephrostomy aspirate is urine collected directly from renal pelvis.
- If the sample is a PCN catheter sample, collection must be done as explained for
indwelling catheters and not from the drainage bag.

f) Cystoscopy specimens
- Cystoscopy specimen is urine collected from the bladder during cystoscopy.

g) Ileal conduit specimen

- Ileal conduit specimen is collected after cleaning stoma site.
- A fresh drain of urine is collected. It must not be collected from the urine drainage bag.

h) Intermittent catheter specimen

- A simple rubber catheter should be introduced into the urethra periodically to drain urine
from the bladder.
- It should be collected directly into a specimen container.

i) Broomhall method of urine collection:

- A non-invasive method of stimulating urine flow in a baby is by tapping just above the
pubis with two fingers at 1 hour after a feed: one tap/second is given for 1 min, an interval
of 1 min is allowed., then tapping is resumed.

j) Special mentions:
- For suspected Tuberculosis- Three consecutive early morning urine samples are to be
collected.
- For urethritis and prostatitis: Initial flow of urine, rather than mid-stream urine to be
collected.
Transport: Freshly voided urine is to be transported to the laboratory immediately within 1 hour.
In case of unavoidable delay >2 hours, it may be kept at 40C or may be preserved with 1.8% boric
acid in refrigerator up to 24 hours. In case of unavoidable delay >5 hours with untreated urine, it
should be discarded.

Collection of Blood:
A) Preparation of site:

- The site of venipuncture is to be selected first. If the patient is unusually dirty, wash the
intended site with
- soap and water prior to venipuncture.
- A tourniquet is to be applied, 3-4 inches above the intended site of venipuncture.
Alternatively, this can be done after cleaning.
- Examination gloves are put on.
- The area is to be vigorously cleansed with 70% isopropyl or ethyl alcohol to remove surface
dirt and oils.
- The venipuncture site is to be scrubbed gently but firmly with the cotton beginning in the
center and continuing in an outward direction circularly for an area of 4 to 5 inches in
diameter.
- It is to be allow to dry thereafter.
- The area is then to be swabbed or wiped in concentric circles of 2% w/v chlorhexidine with
70% isopropyl alcohol or 10% w/v povidone iodine/tincture of iodine, in a similar manner
as given earlier- beginning in the center and continuing in an outward direction circularly
for an area of 4 to 5 inches in diameter.
- It is allowed the povidone iodine to dry (2 minutes). For chlorhexidine gluconate (2%w/v)
or tincture Iodine (10%w/v), drying period is ~ 30 seconds.
- The area is not to touch after cleaning.
- The patient is to be instructed to clench and unclench the fist.
- Phlebotomy is to be performed thereafter using sterile needle and syringe.
- The tourniquet is to be released and the needle is to be withdrawn.
- Pressure is to be applied to the site of venipuncture and a bandage is to be placed over the
puncture site.
Skin preparation with either alcohol, alcoholic chlorhexidine (2% w/v), or tincture of iodine (10%
w/v) leads to lower blood culture contamination rates than does the use of povidone-iodine.

For pediatric patients

a) < 2months: Omit the iodine step, and clean two additional times with separate preparation
pads saturated with 70% isopropyl alcohol or ethyl alcohol
b) > 2 months: Chlorhexidine gluconate as a skin antiseptic is approved for use in pediatric
patients two months of age and older
 B) Preparation of the bottle:

- The septum or the foil (brown paper) of the blood culture bottle is to be removed and the
exposed parts of rubber stoppers or the cap on bottles or tubes is to be wiped with alcohol
or spirit.
- Blood is to be inoculated into the bottle or tube through washer and it is to be thoroughly
mixed to prevent contamination.
- It is to be closed or wrapped then again.
- The bottles are labelled properly with the patient’s name and the date and time of draw.
- Site of draw may be listed.
Note: In particular, please mention whether blood is collected from a central line or from
peripheral venipuncture.

 Recommended total volume and numbers of blood cultures

Age Body weight Volume (to be divided into 2

bottles)
Neonates to 1 year <4 Kg 0.5 -1.5 ml
Children <40 Kg 10-20 ml
Adolescents and adults >40 Kg 30-40 ml
Adult and old age >50 Kg 30-40 ml (Separate
venipuncture site)

Pediatric patient: 6-10 ml in 2 bottles

A properly collected paired sample need not to be repeated up to 5 days
For infective Endocarditis: 3 sets within 30 min period before antibiotic therapy- if
negative at 24 hours- obtain two more sets  total 5 sets overall.

Dilution of samples
Dilution is 1:5 to 1:10 in broth

Bottle (Vol.) Broth (Vol.) Blood (Vol.)

Pediatric patients 50 ml 30 ml 3-5 ml
(<12 years)
Adult patient (>12 100 ml 50 ml 5-10 ml
years)

Broth /Culture media used in bottles:

i) Brain heart infusion broth (BHIB)

ii) Trypticase Soy broth
iii) Bile broth
iv) Bile streptokinase broth
v) Saponin broth
vi) Liquoid broth
 vii) Robertson’s cooked meat media
viii) Thioglycolate broth
ix) Brucella broth with 6% sorbitol & 10% sucrose
x) Columbia broth
xi) Castaneda Medium (Biphasic)

Timing of blood collection:

- Before starting of antibiotic therapy always

- If patient receiving antibiotic already- just before the next dose of antibiotic

Transport: Blood sample need to be transported immediately to the laboratory as early as possible.
If any unavoidable delay, bottle must not be refrigerated, rather to be kept in incubator (370C) or
at room temperature.

Collection of CSF:

A) Lumbar puncture

- The patient lies on his or her side with knees flexed and back arched to separate the lumbar
vertebrae.
- The patient is to be surgically draped and an area of skin overlying the lumber spine is
disinfected as described early (Spirit-iodine-spirit).
- Cap, face mask, gown and gloves for physician drawing CSF are useful adjuncts to
infection prevention.
- The space between the lumbar vertebrae (L3-L4, L4-L5, or L5-S1 interspace) is to be
palpated with the sterile gloved forefinger.
- The spinal needle is to be inserted carefully between the spinous process, through intra-
spinous ligaments into the spinal canal.
- When the subarachnoid space is reached, the stylet is to be removed; spinal fluid will
appear in the needle hub.
- The hydrostatic pressure may be measured with a manometer.
- The CSF is to be collected into five calibrated sterile labeled tubes.
- Physicians should be instructed to sequentially collect 2.0 ml of CSF each into three sterile
calibrated tubes if only routine chemistry (total protein and glucose), bacteriology (culture
& susceptibility), and hematology (cell count) are required.

B) Ventricular shunt fluid

The reservoir site is to be cleaned with antiseptic solution and alcohol prior to removal of fluid to
prevent introduction of infection.
Fluid is to be removed by aspiration of CSF from the Ommaya reservoir or by collection from the
ventricular drain or shunt.
CSF is to be collected into a minimum of three sterile calibrated tubes if only routine chemistry
(total protein and glucose, tube no. 1), bacteriology (culture & susceptibility, tube no. 2), and
hematology (cell count, tube no. 3) are required.
An initial CSF sample should be collected prior to antimicrobial therapy for highest diagnostic
sensitivity.
Transport: CSF sample need to be transported immediately to the laboratory as early as possible.
CSF should not be refrigerated.

Collection of Body fluids from sterile sites:

- Body fluids from sterile sites should be collected by percutaneous aspiration for pleural,
pericardial, peritoneal, amniotic, and synovial fluids.
- The patient is to be surgically draped and an area of skin overlying the lumber spine is
disinfected as described early (Spirit-iodine-spirit).
- Cap, face mask, gown and gloves for physician drawing CSF are useful adjuncts to
infection prevention.
- Aseptically percutaneous aspiration with syringe and needle is to be performed to obtain
pleural, pericardial, peritoneal, or synovial fluid.
- Immediately a portion of the joint fluid or peritoneal fluid collected from patients with
CAPD or SBP is to be placed into aerobic and anaerobic blood culture bottles, retaining
some (0.5 ml) in syringe for Gram stain and direct plating.

Transport: Sample need to be transported immediately to the laboratory as early as possible. It

should not be refrigerated.

Collection of Pus:
a) For open wounds:

- Hands are cleaned properly and gloved.

- The wound should be debrided first to remove slough.
- All superficial exudates should be removed.
- Then the area along with surrounding skin or mucosal surface should be cleansed
thoroughly with sterile saline soaked gauze piece.
- Pus sample may be collected from the deepest part of the wound using a sterile swab stick
and should be placed immediately into sterile test tube.
- The ideal way is to aspirate pus from the deepest part of wound using a sterile disposable
plastic syringe and needle or using an approach through nearby decontaminated skin.
- Swab should always be sent in duplicate.
 b) For Closed wounds:

- Hands are cleaned properly and gloved.

- The area should be disinfected with 2% chlorhexidine or 70% alcohol followed by an
iodine solution [1 to 2% tincture of iodine or a 10% solution of povidone-iodine (1% free
iodine)]. Iodine should be removed with alcohol prior to specimen collection.
- Pus should be aspirated from the deepest part of wound using a sterile disposable plastic
syringe and needle.

c) For tissue samples:

- Hands are cleaned properly and gloved.

- Skin preparation should be done as described above
- Tissue biopsy samples should be collected from areas within and adjacent to the area of
infection. Large enough tissue samples should be collected to perform all of the tests
required (i.e., 3 to 4 mm biopsy samples).

d) Special mention:

- For anaerobic culture, the aspirated material should be discharged into an anaerobic
transport vial and promptly sent to the laboratory for processing.
- A separate piece of tissue should be submitted in a sterile tube containing anaerobic
medium.
- The syringe with the needle should not be recapped, as this also represents a needle stick
hazard; rather, following the discharge of the specimen into a transport container, the
needle and syringe should be discarded.
Transport: Sample need to be transported immediately within 30 mins to the laboratory or as
early as possible. It should not be refrigerated or incubated.

Collection of genital tract specimens:

a) Urethral Specimens from Male:
- Exudate may be expressed from the urethral orifice by gently applying circumferential
pressure to the penis; if material is not readily obtained, the tip of a narrow-diameter cotton,
rayon, or Dacron swab on a plastic or aluminum shaft may be inserted 3 to 4 cm into the
anterior urethra. The swab should be left in place for a few seconds to allow the fibers to
become saturated with the exudate.
- If a culture for C. trachomatis is being obtained, the swab should be rotated 360 degrees to
dislodge some of the epithelial cells.
- First portion of early morning urine samples may be collected.
 b) Genital Specimens from female:
- In females with signs and symptoms of acute genital infection, samples are most commonly
obtained from the uterine cervix. Cervical specimens are collected with the aid of a
speculum after removal of the cervical mucus with a large swab. A smaller swab with a
plastic shaft and a Dacron or polyester tip is recommended for obtaining the specimen. The
tip of the swab is inserted a few millimeters past the cervical os, rotated firmly to obtain
both exudate and cervical cells, and removed, taking care not to touch the lateral walls of
the vaginal canal.
- Urethral specimen should be collected with first portion of urine sample.
- Vaginal secretions can be aspirated or collected with a swab. Specimens from the upper
genital tract are difficult to obtain from below without contaminating the specimen with
vaginal microbiota.
- Endometrial specimens are best obtained by inserting the swab through a narrow-bore
catheter that has been introduced into the cervical canal to minimize the chance of
contaminating the specimen from secretions of the cervical os or the vaginal canal.
- For specimens from the fallopian tubes and ovaries, laparoscopy or surgical approaches are
necessary.

c) Collection of Specimens from Genital Ulcers:

- Ulcers should be cleaned to remove surface debris and contaminating bacteria. A scalpel
blade should be used to abrade the base of the ulcer and transfer the material onto a glass
slide. For culture, either a washing of the base or a cotton swab may be used.
- If intact vesicles are present, a swab may be used to collect fluid.

 Transport: Sample for bacterial culture may be placed in Amies’ or Stuart’s transport
media and need to be transported immediately to the laboratory as early as possible. It
should not be refrigerated. Swab for viral samples should be kept in viral transport media
(VTM) and to be transported immediately.

Collection of Ocular samples:

Conjunctivitis is usually diagnosed with a swab of the affected conjunctiva, which can be placed
in an appropriate transport medium. All other infections are appropriately collected by an
ophthalmologist. Keratitis is addressed by scrapings of the affected lesion; if a bacterial or fungal
etiology is suspected, material is often inoculated directly onto appropriate media by the clinician.
Specimens for the diagnosis of endophthalmitis must be collected surgically.