LABORATORY

DIAGNOSIS OF
INFECTIOUS
DISEASES
Roll no.s-21-30
2nd Year MBBS
STEPS
PRE ANALYTICAL PHASE
ANALYTICAL PHASE
POST ANALYTICAL PHASE
PRE ANALYTICAL PHASE
 BLOOD CULTURES
• One blood culture usually consists of blood from a single
venepuncture inoculated into two separate bottles (one
aerobic, one anaerobic)

• Optimal blood-to-broth ratios are 1:5 to 1:10.

• Sodium polyanethol sulfonate (SP S)- 0.025 to 0.05%-

is added as an anticoagulant, an antlphagocytic agent that
inactivates complement, and a neutralizing agent to
inhibit effects if many antimicrobial and antibacterial
factors in blood.
RECOMMENDED TOTAL VOLUME
AND NUMBERS OF BLOOD
CULTURES
 TIMING OF BLOOD
CULTURES

 Acute sepsis or another condition (osteomyelitis, meningitis,pneumonia or

pyelonephritis): two blood cultures of maximum volume consecutively from
different anatomical sites before starting therapy.

 Pyrexia of Unknown Origin, Subacute Bacterial Endocarditis or other continuous

bacteraemia or fungemia: maximum of three blood cultures with maximum
volume.

 Patients on antimicrobial therapy : Sample is drawn when antimicrobial agents are

at their lowest concentration.
 COLLECTION FROM
VENEPUNCTURE
• A different venepuncture site for each blood culture is selected.

• If blood for culture is required to be drawn through a port in an indwelling

catheter, the second culture must be from a peripheral site.

• Vigorous cleaning is done with 70% isopropyl or ethyl alcohol. Allowed to dry.

• Swab or wipe concentric circles of 1 to 2% tincture of iodine, moving outward

from centre of the site.

• Allow the iodine to dry and avoid touching the site.

•First the aerobic bottle is inoculated and then the anaerobic bottle.
 COLLECTION FROM IV
CATHETERS
• Comparison of cultures that are drawn through an indwelling intravenous
catheter and through a peripheral site may be useful for diagnosis of
catheter related sepsis.

• Bottles are labelled with patient name, site, date and time of draw.

• Using two separate alcohol preps, catheter hub connection is scrubbed for
15 sec with 70% alcohol. Air dry.

• Wear gloves, disconnect tubing or cap of catheter and attach syringe to

collect discard blood ( 3ml for adults and 0.2ml for peadeatric patients),
which is not used for culture.

• Using a new syringe, blood for culture is collected through the hub. Quickly
reconnecting tubing.
 SPECIMEN TRANSPORT

• Blood cultures should not be refrigerated. They are generally held at

room temperature until processed, for a maximum of 4 h.
 REJECTION CRITERIA

• Blood cultures that are received unlabelled.

• If the tube or bottle is cracked or broken.

• Labelled blood cultures are rejected even if medium is expired, or

number of bottles is insufficient, or bottles are received >12h after
collection.
 LOWER RESPIRATORY
TRACT CULTURE

• EXPECTORATED SPUTUM
• INDUCED SPUTUM:

Used for Pneumocystis carinii and Mycobacterium tuberculosis.

Patient’s mouth is rinsed thoroughly with sterile water or saline.

Using an ultrasonic nebulizer, patient is made to inhale approximately

20-30 ml of 30% NaCl.

• TRACHEOSTOMY AND ENDOTRACHEAL ASPIRATES:

The specimen is aspirated into a sterile sputum trap or leak proof

cup.

Tracheostomy aspirate should not be cultured unless clinical

pneumonia is present (fever and infiltrates).

Tracheostomy is followed by colonization within 24h of insertion, and

results may not correlate with disease
• BRONCHOSCOPY SPECIMENS:

Include BAL samples, bronchial washings, transbronchial biopsy

specimens.

Culture BAL samples quantitatively or semiquantitatively for bacterial

pathogens.
• SPECIMEN TRANSPORT:

Specimens are collected in leakproof cups and labelled with

source of material.

Specimens are labelled with demographic information, date and time

of collection, and site of collection.

Provisional diagnosis should be mentioned for proper evaluation of

the cultures.

Appropriate tests should be requested. Anaerobic

cultures should be limited to specimens that are not contaminated with
upper respiratory microbiota (eg. Biopsy samples) and only when
aspiration pneumonia or similar disease is being considered.

Specimens should be stored at 2 to 8°C until cultures can be submitted

or processed.
• REJECTION CRITERIA:

Repeat cultures at intervals of less than every 48 h.

24h sputum collection

Contaminated sputum and endotracheal specimens per Gram stain

rejection criteria

Saliva

Nasal washes and aspirates or swabs of nares

Throat specimens

Specimens for anaerobic culture, except transtracheal aspirates, ples,

pleural fluid or other uncontaminated specimens.

For specimens delayed in transit time more than 2 h without refrigeration.

 URINE CULTURES
• CLEAN VOIDED MIDSTREAM URINE COLLECTION:

Females- labia are held apart, vulva is washed from front to back Y

Circumcised males- no preparation.

Uncircumcised males- foreskin is retracted, glans penis is washed

Patient is asked to collect voided urine directly into a disposable leak

proof container, instructing the patient to not halt and restart the urine
stream for a “midstream” collection but preferably move the container
into the path of already voiding urine.

• CATHETER URINE:

Using a needle and syringe, urine is collected through the catheter port
after cleaning with alcohol.

Urine obtained from a catheter bag should not be sent.

• SPECIMEN TRANSPORT:

Urine should be transported to the lab after collection or, if urine cannot
be delivered within 2 h after collection, can be refrigerated for up to 24h.

• REJECTION CRITERIA:

No evidence of refrigeration and the specimen is >2 h old.

Collection time and method of collection have not been provided.

24-h urine collections Foley catheter tips

Urine from the bag of a catheterized patient Specimens that
arrive in leaky containers

Except for suprapubic bladder aspirates, reject specimen

requests for anaerobic culture.
 WOUND & SOFT TISSUE
CULTURES

• Specimen should be collected prior to initiation of therapy and only from

wounds that are clinically infected or deteriorating or that fail to heal over a
long period.
• Skin or mucosal surfaces should be cleansed.
Closed wounds and aspirates- disinfected with 2% chlorhexidine or 70%
alcohol followed by iodine solution (1 to 2% tincture of iodine or a 10%
solutionof povidine-iodine)

• Open wounds should be debrided, and thoroughly rinsed with sterile saline
prior to collection.

• Viable infected tissue should be sampled, rather than superficial debris.

• Swab collection should be avoided if aspirates or biopsy samples can be

obtained.
• CONTAINERS:

Anaerobe transport vial for small tissues.

Sterile cup for large tissues with nonbacteriostatic saline on a gauze pad to keep
moist.
Syringes
Broth culture medium in small sterile snap -top microcentrifuge tubes for FNA
Swabs (ideally, submit two, one for Gram stain and one for culture)

SPECIMEN COLLECTION:

Cold abscesses- infected material is aspirated with needle and syringe.

Open wounds- superficial area is cleansed with sterile saline. Overlying debris is
removed with scalpel and swabs or sponges. Biopsy or curette sample is
collected from base or advancing margin of lesion.

Pus- deepest portion of the lesion or exudate is aspirated with a syringe and
needle

Tissue and biopsy samples- 3- to 4-mm biopsy samples are collected, avoiding
necrotic areas.
• SPECIMEN TRANSPORT:

Aspirates and tissues should be delivered to the lab within 30 min

Tissues should be kept moist to preserve organism viability

Specimen should not be refrigerated or incubated.

• REJECTION CRITERIA:

Specimens in container with formalin.

If numerous squamous epithelial cells are present on Gram stain

Swabs that have been delayed in transit more than 1 h if they are not
in some transport system.
 
For multiple requests (AFB, fungal, bacterial and viral) but little specimen.
 FAECAL CULTURES FOR
AEROBIC PATHOGENS
Patient should pass stool into a clean, dry pan.
• Transfer at least 5 ml of diarrhoeal stool, 1 g of material or a walnut sized
portion of stool.

• Container
• Clean, leak proof container with a tight fitting lid or
• Buffered glycerol saline
• Modified Cary-Blair medium
• Stool enrichment broths
• Anaerobic transport tube for C. difficile culture

• Rectal swabs
Pass the tip of a sterile swab approx. 1 in. beyond the anal sphincter
Carefully rotate the swab to sample the anal crypts, and withdraw the swab.

• Timing:
Submit the specimen during acute stage of infection (usually 5 to 7 days)
 ANAEROBIC CULTURE
• The best specimen is obtained by using a needle and syringe.

• Tissue samples and biopsy samples are also very good specimens.

• The least desirable specimen is collected by swab

 FUNGAL CULTURE
• Respiratory specimens- Sputum, tracheal secretions and BAL fluid.

• CSF
• Blood
• Tissue, Bone Marrow and Body Fluids

• Skin, Hair and Nail-

Skin scraping.
Nail clippings should be taken from discoloured, dystrophic or brittle
parts of nails.
Hairs are removed by plucking them with forceps.
• Urine
• Vaginal Secretions
• Stools
• Eye 
 FAECAL SAMPLE
EXAMINATION FOR PARASITES
• All faecal specimens should be collected prior to the administration of
antibiotics or antidiarrhoeal agents.

• Use of mineral oil, bismuth, and barium prior to faecal collection

should be avoided.

• Faecal specimen is collected in a clean, wide mouthed container and

transfer it to a container with a tight-fitted lid.
ANALYTICAL PHASE
 BLOOD CULTURE
•Macroscopic signs of growth caused by organisms commonly encountered in blood
culture.
• Haemolysis- Streptococci, Staphylococci, Listeria spp, Clostridia,
Bacillus
•Turbidity- Aerobic GNB, Staphylococci, Bacteroides.

•Gas formation: Aerobic GNB, anaerobes

•Pellicle formation- Pseudomonas spp., Bacillus spp., yeast cells
•Clotting- Staphylococcus aureus
•Visible colonies (“puffballs”)- staphylococcus, streptococci

•MICROSCOPY:
- A thin smear is Gram stained from the broth or agar ly when suggestive
of growth 24h for optimal pt. care.

•SUBCULTURED:to agar media and biochemical tests based on Gram

stain results
 LOWER RESPIRATORY
TRACT CULTURES
• INOCULATION:

Most purulent or most blood-tinged portion of the specimen is

selected.

Incase of bronchoscopy, quantitative cultures are performed

on BAL samples.

Gram stain is prepared.

Using a sterile swab, stick or pipette, inoculate specimen on

blood agar, chocolate agar, and MacConckey agar.

• INCUBATION:
Plates are incubated at 35 to 37°C in 5% CO, for a minimum
of 48 h.
• DIRECT TESTS- Gram stain; note cells & bacteria.

• CULTURE EXAMINATIONS:

Plates are observed at 24h

• Plates are incubated for an additional 24 to 48 h, which is useful to
detect moulds and slow growing, fastidious gram negative rods.
•Gram stain result is used as a guide to interpreting the culture. 
Presence of inflammatory cells and bacteria is used in deciding the
extent of processing the culture

If the culture does not match the smear results, the smear is lewed a
second time.
Organisms present in significant amounts are identified, defined as
colony types that are not considered part of normal respiratory
microbiota and are present in following amounts:

•Large numbers in the second or greater quadrant of the plate

• >10^4 in a BAL sample

• Any amount of selected pathogens in a patient with cystic
fibrosis
• Small amount of selected pathogens in the culture that are
consistent with an etiological agent seen in the Gram stain
associated with inflammatory cells.
• Colonies in the first quadrant of the plate, only if there is little or no
other microbiota on the plate and smear suggests inflammation.

• Subcultured on blood or chocolate agar to obtain isolated colonies

for accurate identifications from mixed cultures.
 URINE CULTURES
• MICROSCOPIC & OTHER METHODS:
Gram stain is useful in rapidly determining the type and count of bacteria
and cells in urine and should be performed on request.

Detection of pyuria- Detect by either Gram stain or urinalysis, from

examination of freshly collected uncentrifuged urine. Pyuria with a WBC
count of >10/micro I (or >5 per high-power field in conventional
• urinalysis) has a specificity of 90% for predicting CA-UTI with >10^5
CFU/ml but a sensitivity of only 37%.

• CULTURE METHODS:

Only streak the blood plate for colony count. Other plates should be in
quadrants for isolation of colonies, rather than for colony count, to
minimize delays in obtaining isolated colonies and jive culture results due
to antimicrobial inhibition.

• Inoculation methods: Calibrated-loop method, Pipette inoculation

• INCUBATION:

In ambient air overnight at 35 to 37°C

If convenient, blood agar and CNA is incubated in 5% CO, to enhance growth of

gram- positive organisms

• EXAMINATION OF CULTURE MEDIA:

Cultures that have been incubated overnight are examined but a final reading is
made at 18 h.

For positive cultures, culture media is examined for quantity and morphological
type of organisms present

Normal urogenital flora is not identified to the genus or species level

Identification is done always to the species level if oxidase positive and indole
positive, since such organisms are pathogens regardless of the count
(Aeromonas and Vibrio spp.)
 WOUND AND SOFT TISSUE
CULTURE
• INOCULATION:

Anaerobic culture is performed first, preferably in an anaerobic

chamber. Incubate immediately.

Smear is always prepared after culture is inoculated.

• GRAM STAIN:

Relative numbers of WBCs, Epithelial cells, Bacterial and Fungal

morphotypes are recorded.

• CULTURE WORKUP:

Plates and broths are read daily.

• Generally up to three microorganisms are identified if any of the
following is true:
• % PMNs are present on direct smear
• % Specimen was collected from a normally sterile site
• The specimen was of good quality (eg. No or few epithelial cells
present)
• The organism was seen on direct smear
 
• Any number of microorganisms that only grow on chocolate agar,
and not on blood agar ( N. gonorrhoea, Haemophilus, and
Francisella) are identified

• Microorganism is identified and AST is performed

FAECAL CULTURE-AEROBIC
• Wet preparations are performed for faecal leukocytes from fresh
stools.

• CULTURE METHODS:

Inoculation of media- Swab is rolled over one small area of blood

agar and Mac Conckey media and streaked in quadrants for isolated
colonies. Use larger amounts of specimen for HEK, XLD and SS.
agars.

• BIOCHEMICAL TESTS:
Salmonella and Shigella somatic (O) antigens are tested for jon when
the screening biochemical tests fit.
 ANAEROBIC CULTURE
• MACROSCOPIC EXAMINATION:

Presence of blood, purulence, necrotic tissue, foul odour, and sulphur granules.

• SPECIMEN PREPARATION:

Purulent specimens are vortexed grossly in the anaerobic transport vial to ensure even
distribution of microorganisms.

Bone or tissue are grounded with approximately 1ml of liquid medium to make a thick paste.

Swabs are wringed out in 0.5ml of liquid medium and then treated as a liquid specimen

Large voume of nonpurulent material is centrifuged. Sediment is inoculate the media and to
prepare the Gram stain
• INOCULATION OF MEDIA:

Brucella agar with 5% sheep blood supplemented with vitamin K and

hemin for isolation of most organisms

Phenylethyl alcohol (PEA)- sheep blood agar for the inhibition of

enteric and other facultatively anaerobic gram-negative bacilli that
may overgrow the anaerobes. PEA also reduces the spreading or
swarming characteristic of some anaerobes.

Kanamycin-Vancomycin-lakedblood agar for selection of pigmented

Prevotella and other Bacteroides spp.

Bacteroides bile esculin agar

Chopped meat broth or THIO (supplemented with Vit K & in)

Preduced anaerobically sterilized (PRAS) media

• INOCULATION PROCEDURE:

Prepared specimen is transferred onto the appropriate aerobic and

anaerobic media, liquid medium and a slide for Gram stain.

• INCUBATION:

• MICROSCOPIC EXAMINATION:
Gram staining: Reveals the types and relative numbers of
microorganisms and host cells present and serves as a QC measure
for the adequacy of anaerobic techniques.
 FUNGAL CULTURE
• INOCULATION:

The number of media inoculated may be dependent on the

specimen type and amount.
Both non-selective (SDA and BHI) and selective media should be
included, and specialized media may be required for specimens
in which difficult-to-grow etiological agents are suspected.

• INCUBATION:

Fungal cultures are incubated at 30°C for a minimum of 4weeks.

Plates should be examined daily for first 7 days and at least twice
per week thereafter
 FAECAL SAMPLE-
PARASITES
• MACROSCOPIC EXAMINATION:

Consistency

Surface of faecal specimen for the presence of blood and mucus.

Areas of blood or mucus are sampled for examination of trophic

amoeba

• MICROSCOPIC EXAMINATION:

Saline and iodine wet mounts are prepared.

Examined under 10x. Use 40x objective for more detailed study.

Floatation and concentration methods are used for the recovery of all
protozoa, eggs and larvae present.
MOLECULAR TECHNIQUES
 ANTIBIOTIC
SUSCEPTIBILITY TESTING
• Only isolates producing an infection should be tested.
• In most situations a disk diffusion is adequate to guide clinical therapy
• Choice of antimicrobial agents and the susceptibility pattern is guided
by recommendations of CLSI
• Only antimicrobial agents appropriate for the infection should be
included in a report.
• Antimicrobial agents that do not penetrate into a site should not be
reported for organisms isolated from that site.
• Commercial systems include Vitek which uses a computer assisted
analysis of growth in rds to calculate MIC.
POST ANALYTICAL PHASE
 BLOOD CULTURES
•REPORTING RESULTS:

For “No growth cultures”, length of incubation is indicated: “No growth after x
days of incubation” for both preliminary and final reports.

•POSITIVE CULTURES:

Gram stain results of all positive cultures are reported immediately.

For single positive cultures with microorganisms generally considered skin

contaminants, only minimal identification is performed and AST is not done.
Reported as “one set of two positive. Isolation does not necessarily mean
infection. No susceptibility tests performed. Contact laboratory for further
information.
 
• INTERPRETATION:

The report of a positive culture generally means that the patient is

bacterimic. However, skin microbiota may infect the culture,
causing a false-positive result or pseudobacteremia. 
•Mixedicultures can be present and account for a significant number
of bacteremias.

Performance and reporting of AST are critical for timely patient care
and increase chance of appropriate therapy and cure.
 LOWER RESPIRATORY
TRACT CULTURES

• REPORTING RESULTS:

Gram stained smear is reported. Rare (<1 in 20 fields) numbers of

bacteria seen in the smear are not reported.

Reported as: “No growth of pathogens or normal upper respiratory

tract organisms” if there is no growth on any plates.

Positive reporting-
Preliminary and final results are reported as “Isolates consistent with
microorganisms encountered in URT” if no pathogens isolated.

All pathogens reported and AST is performed.

• INTERPRETATION:

A positive culture with S. pneumoniae or H. influenzae generally

indicates infection with that organism, although carriage may lead
to false positive results.

A positive culture with a predominant gram-negative rod or

S. aureus generally indicates infection with that organism if smear
suggests an infectious process involving the corresponding
morphology.

A negative culture cannot rule out infection

 URINE CULTURES
• REPORTING RESULTS:

Gram stain results for bacteria & cells per Gram stain are reported.

Negative results:
• “If no growth is observed on all media, report “ No growth after 48H”

Positive results:
If only urogenital or skin microbiota is observed, report as such.

“Mixed Cultures are reported with the count in CFU per millilitre,
wed by “Multiple bacterial morphotypes present; possible
contamination; suggest appropriate recollection, with timely delivery
to the laboratory, if clinically indicated.
• INTERPRETATION:

A mixed culture in an uncomplicated outpatient population likely

indicates contamination.

Low levels (<104/ml) of organisms commonly found on the skin

and external and internal genitalia are considered to be
contaminants, but in selected circumstances, a count of
Enterobacteriacae of 10^2 CFU/ml or more, especially for
Salmonella, can be considered significant.

 Performance of AST directly from the urine specimen is not

recommended.
 WOUND AND SOFT TISSUE
INFECTIONS
• REPORTING RESULTS:

Report gram stain results as soon as possible, generally within 1h for

specimens from critical sites.

Report all negative cultures as “ no growth in-- --days”

Report individually those organisms that are always considered

pathogenic with enumeration, using preliminary identification initially
and the genus and species as the final identification.

Due to their known virulence factors, indicate the presence of the

following species: Beta-haemolytic Streptococci, S. aureus, P.
aeruginosa, Clostridium perfringens, Pigmented anaerobes etc.
• Interpretation:

Performance of AST is not indicated in cases of mixed microbiota

indicative of infection of the abdominal cavity with bowel contents.
•Treatment should include broad-spectrum coverage for normal
intestinal microbiota.

• Use of the Gram stain can improve the accuracy of evaluating the
importance of each potential pathogen. Organism present in the
Gram stain of an appropriately collected specimen correlate with >
10^5 organisms per g of tissue.
• Microbial load in an acute wound can predict delayed healing or
infection.
• Many wound infections are polymicrobic, and the isolation of an in
culture may or may not correlate with infection of the wound.
 FAECAL CULTURE FOR
AEROBIC PATHOGENS
• REPORTING RESULTS:

If gram negative enteric microbiota are not present in the cultures, add a
comment “no normal enteric gram-negative rods isolated”.

Positive cultures: report presumptive presence of any enteric pathogens with or

without quantitation. Report the pathogen with the preliminary designation as
“probable” until the identification is confirmed by both biochemical testing and
serology.

Report AST on Shigella, Aeromonas, Plesiomonas, Edwardsiella, Vibrio, Yersinia

and selected cases of Salmonella.

• INTERPRETATION:

The isolation of a stool pathogen may not identify the cause of disease.

Ex: Salmonella is present in carrier state, without the disease.

 FUNGAL CULTURES
• REPORTING AND INTERPRETATION OF YEAST:

Nitrate assimilation test for yeast speciation

Sporulation

Carbohydrate fermentation tests

• REPORTING AND INTERPRETATION OF MOULD:

Reporting the growth of a mould as soon as such growth is evident but before
identification structures are formed is important where immunocompromised
patients are concerned.

But is normally necessary in cases of subcutaneous infection of immunocompetent

patients.
 PARASITIC INFESTATIONS
• To ensure the recovery of parasitic organisms that are passed
intermittently and in fluctuating numbers, the examination of a
minimum of three specimens collected over a 7- to 10- day period is
recommended.

Report the presence of adult helminths Morphology and size are

usually adequate for identification of pinworm and Ascaris adults and
tapeworm proglottids.

Report the presence of blood on or in the faecal sample.

THANK YOU