Stool culture is demanded in the bacteriology laboratory as method for detecting and diagnosing enteric bacterial infections (i.e. infections caused by pathogens in the Enterobacteriaceae family e.g. Salmonella species and Shigella species) that lead to enteric fever, diarrhea and dysentery.
AIM: To isolate and identify pathogenic bacteria from feacal specimen culture as an aid in the diagnosis of gastrointestinal infections.
MATERIAL/APPARATUS: Stool specimen, salmonella shigella agar (SSA), MacConkey agar (MCA), Bunsen burner, inoculating loop, incubator, grease pencil.
SSA = Salmonella-Shigella agar
MCA = MacConkey agar
- Label the SSA and MCA plates using a grease pencil.
- Using a sterilized inoculating loop, collect a speck or loopful of the stool specimen.
- Inoculate it on the SSA and MCA plates respectively. Note: The inoculating loop should be flamed or sterilized intermittently as the streaking continues.
- Incubate both plates in the incubator at 37oC overnight.
- After incubation, bring out the plates and look for colonies of Shigella or Salmonella on the SSA plate, and colonies of Escherichia coli and other lactose fermenting organisms on the MCA plate.
- Subculture suspect colonies of bacteria to a new SSA and MCA plates in order to obtain a pure culture – which is the basis for any meaningful identification of a bacteria. The above procedure should be followed aseptically. But this time, the stool specimen is not used but the isolated organism in both the SSA and MCA plates.
- Perform biochemical testing on the isolated pure culture organisms in order to identify the isolate organisms. The biochemical tests to perform are: urease test, indole test, citrate test, and sugar test using triple sugar iron agar.
- Perform antimicrobial susceptibility test only when a pathogen is successfully isolated and identified.
NOTE: If the stool specimen is very thick that obtaining a loopful of it will be difficult, make a thick suspension of it in about 1ml of sterile peptone water. Take a loopful from the stool specimen in the peptone water tube and perform your inoculation.
SSA plate is for the isolation of Salmonella and Shigella. Both are non – lactose fermenters (NLF), and appear as a pale or colourless colonies on the SSA plate. Xylose – lysine deoxycholate (XLD) agar can also be used in place of SSA as both perform almost the same function.
MCA PLATE is for the isolation of lactose fermenters (LF) like E. coli. E. coli and other lactose fermenting organisms appear as pinkish colonies on the MCA plate.
REPORTING OF THE RESULT:
Examine the plates and report the cultures. The culture of a stool specimen, and subsequent isolation and identification of any pathogen present may take about 3 – 4 days before any final conclusions can be reached. Depending on whether any meaningful isolation and identification was made or not, stool culture results can be reported in any of the following ways:
- Cultures yield no significant growth.
- Cultures yield no bacteria growth.
- Cultures yield Enteropathogenic coli. Before you can confidently say that cultures yield a particular organism, you must have carried out all the necessary biochemical tests and serotyping required for identifying a pathogenic bacterium. It is advisable to stick to and operate by the standard operating procedures (SOP’s) of the laboratory in which you are doing the work, as the SOP’s for laboratories vary. Also, familiarization with the different types of bacteria implicated in causing intestinal infections and how they can be identified and differentiated from each other is an advantage.