Lab 3 Stool Cultures Flashcards
Which organisms cause GI infections?
Most common causative bacteria for GI infections (pathogens):
1. Salmonella spp.,
2. Shigella spp.,
3. E. coli O157,
4. Campylobacter spp.,
5. Yersinia spp.,
6. Aeromonas spp. (controversial, some labs do not culture for it)
–> are the ones that we mostly look for in routine cultures.
- Vibrio spp. are pathogens too but because they are not frequent in North America, they are investigated after a specific request.
Which is the typical “normal flora” for the GI tract?
GI normal flora includes:
1. Majority of anaerobic bacteria
2. Common facultative anaerobes:
- E. coli and
- other Enterobacterales (sometimes referred as “coliforms”),
- Enterococcus and - Streptococcus.
What amount should be collected for a stool culture to be performed? Container?
Approximately 1 to 2 grams of feces should be collected in a CLEAN, wide-mouthed container with a tight lid.
How should rectal swabs be transported?
Rectal swabs should be placed in a transport system containing modified Stuart’s medium, preferably with charcoal (charcoal neutralizes the toxic effects of fatty acids produced by some bacteria – Shigella are particularly susceptible).
What can be done if you need to delay planting a stool specimen?
If there is a delay in planting, specimens should be refrigerated at 4º to 6º C. Some labs recommend a modified Carey- Blair or other transport medium, which contains buffers; however, toxin testing cannot be done on these if needed.
How soon should an unpreserved stool specimen be processed?
Ideally, unpreserved stools should be processed 30 min after collection (but <2 hrs is ok), or go into a transport medium (Shigella is mostly compromised).
Note: Added <2 hrs from lecture notes.
What general contaminates (not other bacteria that is) are not acceptable in specimens?
Specimens contaminated with urine, barium or toilet paper are unacceptable.
Can the lab accept stool from patients that have been in the hospital for over 3 days?
No
What preservatives or transport mediums are not acceptable for stool specimens for culture?
Note acceptable are:
1. Specimens for bacterial investigation collected in a preservative for parasites (SAF)
2. Stool specimens in a transport medium with yellow phenol red indicator, indicating failure of the buffering system to maintain a neutral pH, and thus death of some organisms, especially Shigella
Are dry rectal swabs acceptable?
No
What do you do with a non-acceptable stool specimen?
IF UNACCEPTABLE SPECIMENS ARE RECEIVED,
1. A REJECTED SPECIMEN REPORT SHOULD BE SENT TO THE PHYSICIAN, CLINIC OR WARD.
2. HOLD THE SPECIMEN AT 4º to 6º C WHILE WAITING TO DISCARD IT IN CASE IT IS IRREPLACEBLE AND CRITICAL FOR THE PATIENT.
What is the traditional media for stool cultures?
BA
MAC
Sorbitol-MAC (SMAC)
XLD (could be Hektoen)
Campylobacter agar
Selenite Broth
When is TCBS agar used?
TCBS agar is used if the Dr. is looking for Vibrio spp. and it should be requested on the requisition.
What descriptions for appearance are used for stools?
Liquid, solid, watery, bloody, ‘rice-water.
Do you flame in-between quadrants after planting the stool sample?
Yes, when streaking for isolation.
What are the incubation requirements for most plates, campy plates?
- Incubate plates and enrichment broth at 35C for 18 to 24 hours.
- Incubate campy plates in a jar with a campy pack for microaerophilic incubation at 42C.
When and to what do you sub from the selenite broth?
After 18 hours the selenite broths need to be subbed to an XLD agar with a loopful.
When do you assign an isolate number?
If a pathogen is suspected, assign an isolate number.
How do you enumerate the amount of a pathogen on a plate for stool cultures?
Semi-quantitatively as follows:
1+, growth in 1st quadrant only, ignoring a few colonies in the second.
2+, growth up to second quadrant, ignoring a few colonies in the next quadrants.
3+, growth up to 3rd, typ.
4+, growth up to 4th.
What organisms are you looking for on MAC and XLD?
For stool cultures on MAX & XLD you are looking for:
- Salmonella
- Shigella
- Yersinia
What do salmonella, shigella, and yersinia look like on MAC?
Salmonella,Shigella, and Yersinia:
They are NLFs and look like yellow or clear colonies on MAC.
What do salmonella, shigella, and yersinia look like on XLD?
Salmonella,Shigella, and Yersinia on XLD look like the color of the media or black as they are NLF.
From module in Micro 1 it says on XLD: Salmonella appears red with black centers and Shigella appears red.
What do lactose fermenters (LF) look like on XLD?
LF bacteria look like yellow colonies on XLD (enteric flora typically).
What morphology appearance is suspect of Yersinia (on XLD I think - confirm)?
Yersinia might appear slightly pink and is typically tiny at 18 hours. This should make you suspect Yersinia in the absence of CIN media.
What tests are included in a enteric (biochemical screen)?
Enteric screen tests from MAC or XLD:
1. TSI
2. SIM (maybe)
3. Urea
4. BA check plate (matter of procedure)
If the enteric screen falls into a pathogen profile for Salmonella, Shigella or Yersinia, then perform that API 20 E test for full identification.
Perform serotyping as necessary.
What pathogenic organism are you looking for on the BA plate?
Aeromonas spp.
What characteristics are you looking for to make you suspect Aeromonas spp. on the BA plate? What other tests support?
- Beta-hemolysis
Supporting tests:
- Gram stain if GNB.
- Oxidase, if pos.
If oxidase neg then Aeromonas spp. can be ruled out and BA is not needed anymore.
What organism are you looking for on the Sorbital-MAC (SMAC)?
On SMAC looking for E. coli O157 which is Sorbitol neg compared to other strains that are Sorbitol positive.
What do non-sorbitol fermenting colonies look similar to?
They look similar to NLF colonies, i.e. colourless whereas LF will be pink (e.g. other E. coli).
See Module 4 from Micro 1, p16.
What is the purpose of Hektoen Enteric agar?
It is a differential and selective agar for isolation of Salmonella and Shigella spp. from other enteric organisms.
What do you do if you suspect E.coli O157 on S-MAC?
Suspicious colonies should be subbed, and screened with a serology test for E.coli O157, then fully ID.
What does the colonial morphology look like for Campylobacter spp. on Campy plates?
Campylocbactor on campy plates look:
Small-tiny, translucent grey-silver/shiny mucoid and irregular.
Why might you need to use a magnifying lamp to look for Campylobactor on a campy plate?
They may grow very pinpoint at 24 hours so use the magnifying lamp.
If no growth, re-incubate for another 24 hrs.
What is the next test if you suspect Campylobactor on your campy plate? And why?
- Gram stain. They are small, curved GNB that resemble gull wings or commas.
- Catalase - weak positive
- Oxidase - weak positive
After this, ID is confirmed.
What do you do if there are no good isolated colonies on the campy plate?
Sub-suspicious colony to BA or another campy plate in microaerophilic environment at 42C before continuing ID.
Can you perform AST for Campylobactor?
No, AST methods are not available for Campylobacter.
What Campylobacter species are most culture protocols designed to recover?
C. jejuni
and other most common strains.
Do you recovery Campylobacters from non-fecal specimens?
Not without a special request made to the microbiologist (as it would require a different atmosphere).
What do Vibrio spp. colonies look like on a TCBS plate?
Vibrio spp. is suspected if colonies are green or yellow colour.
Yellow - Vibrio cholera
Green - V. parahaemolyticus
What next tests are performed if Vibrio spp. is suspected on a TCBS plate?
Next tests if Vibrio spp. suspected are:
1. Smear for gram stain
2. Oxidase and spot indole tests (Vibrio are oxidase pos!)
What do you do if you have no isolated colonies on the TCBS plate?
If no isolated colonies, sub to a BA & Mac (w/o CV), repeat oxidase & Spot Indole.
Fully ID with API20 E or APINE.
Why is no susceptibility testing done during our lab?
Because most enteric organisms do not require AST routinely.
Why are pathogens stocked after reporting results? And how?
They are stocked in the lab at -70/80C in case more testing is needed in the future, and to be sent to the provincial lab for further serological testing (Salmonella and Shigella).
Do you report what you don’t find?
Yes, because the media used is looking for specific pathogens you report what is not found.
For example: Report “No Salmonella, Shigella, Yersinia, Aeromonas or Campylobacter isolated.”
Basically all organisms that are looked for in the routine culture are mentioned.
**NOTE: Do not report “No enteric pathogens” as different labs look for different pathogens.
Look at samples of reporting in Lab notes (see One Note or Learn).
