GI Specimen Processing

Order for Inoculating GIT tubes

1. MAC (small line)

2. BA (small line)

3. TSI (down to 1/4" from bottom in straight line, then streak surface)

4. ONPG-PAM (down to 1/4" from bottom in straight line)

5. TSB (swish around)

Then return to MAC and BA to streak out the quadrants.

Spot Oxidase

1. Put filter paper on microscope slide

2. Add a drop of oxidase reagent to the filter paper

3. Let dry for 1-2 minutes (IMPORTANT: must let dry for at least 1 minute, otherwise it may give a false negative result)

4. Get a colony on an applicator stick. Press and hold it onto the paper for 5 sec.

5. Read results (positive: blue)

Other Notes

Serology: perform from TSI or BApp (cannot use anything that's selective)

• Serology may take a while to show agglutination - use a thick/milky suspension and rock card back and forth for 1-2 minutes (may need to wait the full 2 minutes)

Indole: use TSB tube (make sure you're using correct reagent - Kovacs!) or spot indole from BApp. If doing motility, take drop before adding reagent.

Motility darkfield: use TSB sample or colony from BApp with saline drop

ONPG: Check ONPG and motility first, then add ferric chloride.

Check for purity from MAC plate.

Biochemical Testing

Test	Abbreviated Procedure	Positive Result	Negative Result
Motility		Presence of organism throughout tube (turbid/cloudy throughout)	Organism only present along inoculation line
Motility (Darkfield)	Place drop from TSB tube onto slide and coverslip. OR Place drop of saline on slide, take colonies from BA plate, mix, and coverslip.	Movement seen. While fluid motion may cause most organisms to move in one direction, it may be helpful to look for organisms that are moving in other directions as this indicates actual motility.	No significant movement. NOTE: Be careful as Brownian motion is not indicative of motility. Additionally, movement in one direction due to the movement of fluid is not indicative of motility either.
TSI	N/A
ONPG	N/A	Any yellow colour	No colour change. Straw/brown colour.
Indole	Add Kovac's Reagent to TSB tube	Pink/red ring	Brown or yellow
PPD	Add Ferric Chloride	Dark green (forest green)	Yellow, orange, or brown

Organisms

very tiny clear things on plate - bile salts precipitating out (likely that your plate has been refrigerated)

Edwardsiella are always indole + but otherwise appear to be Salmonella (which is indole -)

Shigella may appear similar to Salmonella, but is non-motile.

Proteus is PPD + (organisms of interest are all PPD -)