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SOP feacal sample

This document outlines the standard operating procedure for the collection, transportation, processing, and culture of faecal specimens for microscopic examination. It details the methods for specimen collection, transport media, microscopy, culture techniques, and result interpretation, ensuring quality control and assurance throughout the process. The document serves as a comprehensive guide to ensure accurate and reliable results in the examination of enteric pathogens.

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10 views4 pages

SOP feacal sample

This document outlines the standard operating procedure for the collection, transportation, processing, and culture of faecal specimens for microscopic examination. It details the methods for specimen collection, transport media, microscopy, culture techniques, and result interpretation, ensuring quality control and assurance throughout the process. The document serves as a comprehensive guide to ensure accurate and reliable results in the examination of enteric pathogens.

Uploaded by

dmanneme
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© © All Rights Reserved
We take content rights seriously. If you suspect this is your content, claim it here.
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SOP for Faecal specimen microscopic examination & culture

Purpose :
1. To establish standardised procedure for collecting, transporting and
processing of faecal specimen for microscopic examination & culture.
2. To ensure quality of microscopic examination & culture results.
Scope:
1. Specimen collection, transportation, storage and processing
2. Culture & incubation
3. Result interpretation & reporting
4. Quality control & quality assurance

Specimen collection and transport :


• A small quantity of solid/semisolid stool or one third of the container in case
of watery stool is collected in a sterile screw-capped disposable 40 ml
container.
• A rectal swab is not recommended as the material obtained is never
adequate for all the tests or for inoculating all the media used for culture.
(Rectal swab is collected by inserting a sterile swab approximately 1 inch
into anal canal and gently moving from side to side to sample the anal
crypts. Faecal matter should be evident on the swab. It is transported in
transport medium.)
• The sample should be collected preferably prior to initiation of antibiotics in
the container directly, taking care not to soil the outside of the container.
Samples should not be collected from bedpan or diaper.
Transport:
• The sample should be immediately transported to the laboratory on
collection within 1 hour.
• If there is a delay in transporting faecal specimens or if samples need to be
sent by post, one of the following transport media may be employed:
♦ Phosphate buffered glycerol saline solution
♦ Stuart’s transport medium
♦ Cary and Blair transport

Processing:
1.Microscopy:
 For all watery faeces samples, whether the doctor orders or not,
examines a hanging drop (HD) immediately to see darting motility of
bacilli.
 Wet mount preparations (saline & Iodine): to make a note on presence
of pus cells, RBCs, parasitic ova, cyst, trophozoites & larvae

2.Culture and Isolation


Commonly encountered enteric pathogens and potential pathogens include
Salmonella, Shigella, V. cholerae, Diarrhoegenic E. coli.
• Routine media to be included are MA, XLD or DCA, and Selenite F broth.
Note: BA, TCBS & APW are added if HD is suggestive of V.cholerae or on
request
♦ Place a loopful of the specimen over a small area of each plate, then flame
the loop, and streak from the inoculated area over the entire plate.
♦ Inoculation on DCA: 1 in 10 dilution of specimen in saline is streaked with
the help of triangular loop to get maximum isolation of colonies.
• If V. cholerae is suspected, a TCBS and alkaline peptone water (APW)
medium with a pH 8.4 to 8.6 are also inoculated. After 4 hours of incubation at
37°C, a drop is taken from the surface of the APW is examined microscopically
for darting motility. A subculture is also made on BA and TCBS.
• Place 1-2 ml or 1 g of faecal suspension into a tube containing selenite F
broth, or pick up an amount approximately the size of a pea and emulsify it in
the selenite F broth.
• For all samples, media incubated are examined after 18 hours and a
subculture is done immediately from the selenite F broth onto a DCA plate.

Colony characteristics on different media


Desoxycholate citrate agar and MacConkey’s agar:
1. DCA is examined at the end of 24 hours and again after 48 hours.
2. Colonies of all non-lactose fermenters (NLF) may look similar: reliance should
not be placed on colony characteristics for differentiation. Commonly isolated
microorganisms are Shigella, Salmonella and Vibrio. Occasionally 48 hours
incubation may be needed for the NLFs to appear.
3. E.coli, Klebsiella and Enterobacter will look pink being lactose fermenters.
Generally suppressed on DCA.
Xylose lysine deoxycholate medium :
• Shigella, Salmonella and NFGNB: Small pink colonies with or without black
centers (Salmonella).
• Coliforms: yellow colonies, or yellow with black centres (Citrobacter freundii
and Proteus vulgaris)
Thiosulphate citrate bile salts sucrose (TCBS) agar:
• V. cholerae O1 and V. cholerae O139 produce flat yellow disk-like colonies
due to the fermentation of sucrose in the medium.
• V. parahaemolyticus produces green colonies.

Biochemical tests:
 Mannitol motility medium: Stab down the centre of the medium,
reaching the bottom
 TSI: Stab into the butt and streak along the surface.
 Indole test in Peptone water broth.
 Citrate utilization test: Inoculate lightly from a young culture over the
entire surface of the slant of Simmon’s citrate agar using a straight wire.
Incubate at 37ºC for 1-2 days.
 Lysin Iron Agar (LIA): For black centre colonies on XLD & DCA, stab the
butt and streak the slope. Examine after overnight incubation.
Reporting :
For negative culture—” No Enteropathogen Grown”
Release of Report: After authentication by signatory authority
Quality Control:
 Media quality –quality & sterility testing of plating media and
biochemical tests
 Instrument calibration—Daily checking of incubator temperature
 Reagent quality—checking expiry date of antibiotic discs, dehydrated
culture media and reagents

Reference: Standard Operating Procedures Bacteriology. Antimicrobial


Resistance Surveillance and Research Network. 2nd Edition, 2019. Indian
Council of Medical Research, New Delhi, India.

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