The isolation and identification enteric pathogens (Salmonella/Shigella and determination of their
Specimen Sorting and numbering
Interpretation of Culture
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*Under supervision only
Safety wear gloves
Reception – stool specimens are receive in the laboratory on a suitable wide-mouth non-sterile container (mono wax) or in the form of a rectal swab. Specimen should promptly be delivered to the laboratory for processing.
Inoculating loops, Dexoxycholate citrate Agar (DCA) or Salmonella Shigella Agar (SS),MacConkey with crystal violet, Selenite F broth, Triple Sugar Iron (TSI)Agar. Urea Agar slope peptone water.
Note the presence of mucus and blood
Note: (i) technologist to see Biochemical reaction & sensitivity plate before signing
(ii) isolates are to be kept on slopes. (for storage)
1. Inoculate a loopful of the stool specimen (if loose or watery) or a peanut size if
formed) on to DCA/SS Agar and MacConkey Agar (with 1 crystal violet). Also
inoculate Selenite F broth with about 2 – 3 times the size of sample used for solid
media. Incubate the seeded media overnight aerobically at 35 -37oC
EXAMINATION AND IDENTIFICATION
Routine identification of suspected Salmonella and shigella isolates:
1. UREASE TEST using a sterile straight wire pick an individual clourless smooth colony with or without black spot in the centre on the DCA/SS and inoculate a tube of Peptone water and Urea Agar slope.
2. Incubate at 35-37oC up to 4 hours examining at hourly intervals for a pink colour throughout the urea agar slope.
3. If urease test is negative at 4 hours incolate a tube of TSI/KIA with a sterile straight wire from the peptone water tube or similar smooth colony stabbing first the butt and then streaking the slope. Close the tube with a loose-fitting cap and incubate at 35-37oC overnight.
4. PURITY PLATE
inculate Macconkey plate with same colony as purity plate for serology.
5. ENRICHMENT BROTH
Sub-culture the Selenite F broth culture on a plate of DCA/SS Agar and follow up as above on Day 3. this is necessary if no colonies resembling Salmonella spp are seen on the primary culture plates. Discard selenite F. broth.
1. Examine the TSI/KIA Urea Agar slope, iodole etc for reactions
suggestive of Salmonella or Shigella.
2. Test serologically and isolate giving reactions suggestive ofSalmonella
technique. Subculture on MacConkey for purity to make sure you are
dealing with non-lactose fermented.
3. Perform sensitivity test on the isolate using appropriate antibiotic
discs. Include a control E. coli strain on a separate sensitivity test agar
For stool specimens pf infants up to 2 years inoculate one of the Lactose fermenting rose-pink colonies on MacCoonkey plate into Peptone water, kosers citrate, Urea Agar slope and TSI for the isolation of E. coli. If E. coli is isolated test serologically for the appropriate E. coli stains (ETEC,EIEC, EPEC or EHEC)
Summary of the laboratory examination of Raecal Specimens
DAY 1 Culture
Primary Plating Media SS Agar or DCA
MacConkey with Crystal violet (children less than 2 years)
Enrichment: Selenite F
1 Examine SS/DCA culture for NLF’s
2. Inoculate Peptone water & Urea
If no Urease production after 4 hrs
Inoculate 1. TSI/KIA incubate at 35oC – 37oC.
2. MacConkey Agar for Purity and serology.
3. Also subculture the selenite F culture on SS/DCA. This is important if there is not
growth suggestive of Salmonella or shigella on the Primary Culture.
If suspected, cholera specimens should be sent to the Public Health Reference Laboratory
1. Report the type of Salmonella or Shigella spp isolated with the sensitivity on Day 4
2. If there was no suggestive isolate report as:
No Salmonella or Shigella isolated.
BY BMS DZAMESI