MICROBIOLOGY- STOOL CULTURE AND SENSITIVITY


STOOL CULTURE

PRINCIPLE:

The isolation and identification enteric pathogens (Salmonella/Shigella and determination of their

 Personnel
Student Technician
Laboratory Assistant
Rotating
Technician
Technician
Technologist
Specimen Sorting and numbering
Culture

Interpretation of Culture

     *
   
     *

        *
Identification Tests
     
     *
Sensitivity Tests

     *
      
Reporting





*Under supervision only

Safety    wear gloves
              Open bench

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.

Equipment/media
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.

Macroscopic examination:
Note the presence of mucus and blood



CULTURE
Note:      (i)    technologist to see Biochemical reaction & sensitivity plate before signing
                       of report.
               (ii)    isolates are to be kept on slopes. (for storage)
DAY 1

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

DAY 2

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.
       


DAY 3

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
                                plate.

ADDITIONAL TESTS

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

DAY 2

1      Examine SS/DCA culture for NLF’s
        Salomnella species
        Shigella species

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.
  
Cholera
If suspected, cholera specimens should be sent to the Public Health Reference Laboratory

Reporting
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

Comments

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