do a 300 words minimum lab report.

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Sulfur / Indole / Motility deeps, i.e. SIM deeps

SIM deeps: SIM is a medium, NOT a test. Actually, we can conduct 3 tests with this single medium: 1. sulfur reduction 2. release of indole from utilization of Tryptophan and 3. motility

We will conduct the Indole test as well as the sulfur reduction (H2S production) test using SIM deeps. Many medical labs use SIM deeps (Sulfur Indole Motility) in conjunction with TSI, urea and Simmons citrate media for characterization and identification of enteric bacteria. To inoculate, stab 1 SIM deep using your needle exactly as you did when inoculating a motility deep in one of our previous exercises. Incubate for 24-48hrs. at 37oC. Add 3-5 drops of Kovacs reagent and set aside for 5 minutes. Examine the top layer of the tube for red color which indicates a positive Indole test.

Sulfur reduction: As discussed in the TSI exercise, the ability to generate hydrogen sulfide (H2S) can be used to distinguish the enteric genera. Thiosulfate is present in SIM media which is reduced to hydrogen sulfide (H2S) during anaerobic respiration. H2S combines with Ferric iron in this medium, and precipitates as black FeS. The black compound is obvious in an incubated tube.

Sulfur negative: Enterobacter, E. coli, Klebsiella, Shigella, and all the non-fermentors

positive: Proteus, Salmonella, Citrobacter SEE IMAGE: Sulfur reduction: left positive, right negative

Indole: Some bacteria are capable of using the amino acid tryptophan as a carbon and energy source during the process of aerobic respiration, releasing indole as a by-product. Being produced primarily at the top of the tube, as well as accumulating there due to its non-polar nature, indole will be concentrated on the top of the media where it can be detected by Kovac's reagent (paradimethylamino-benzaldehyde in HCl and amyl alcohol). Before using the tryptophan, bacteria split it into 3 parts:

a) indole (see structure)

b) an amine group -NH2

c) pyruvate which is respired by the cell

If a reagent such as Kovac's is added to indole, a thin layer of red color will develop at the top of the deep indicating the presence of indole. The color may become obvious in a few seconds, or it may take a minute or 2. Of our cultures, only E. coli and Proteus are indole positive.

Indole negative: Enterobacter, Klebsiella, Salmonella, Shigella, Citrobacter and all the non-fermentors

positive: Proteus, E. coli SEE IMAGE: Indole production from tryptophan utilization: left negative, right positive

Motility: As said above, SIM is a soft agar medium, and thus, a motility medium. Our non-motile Gram negative rods include Shigella, Klebsiella and Acinetobacter.

Motility negative: Shigella, Klebsiella, Acinetobacter

positive: Enterobacter, E.coli, Salmonella, Citrobacter, Aeromonas, Alcaligenes, Pseudomonas

SEE IMAGE: Motility: left non-motile, right motile

The tests described above for the characterization of enteric Gram negative rods represent only a few of the options available. Additional selective & differential media exist including: Hektoen enteric agar (HE), Salmonella Shigella agar (SS), Leifson’s deoxycholate-citrate agar (DCA), Wilson and Blair’s brilliant-green bismuth sullphite agar (BBSA) or simply brilliant green agar, Xylose lysine deoxycholate agar (XLD), Kligler’s iron agar (KIA), and bile esculin agar.

Additional biochemical tests include Lysine and Ornithine decarboxylation, Urea hydrolysis, orthonitrophenyl-b -D-galactopyranoside (ONPG) for beta-galactosidase production, phenylalanine deaminase, lysine decarboxylase and other amino acid modification, nitrate reduction, denitrification, and fermentation characteristics based upon various carbohydrates.

NOTES:

* Other than the use of MR of differentiate Listeria (MR+) from Corynebacterium (MR-), we will only

use these tests for Gram negative rods.

* All of the non-enteric Gram negative rods are negative for all of these tests except for motility.

* Remember that the initial weak red color for MR on Klebsiella is a false positive – the color will fade

over time (somewhere between a few minutes to 24 hours).

* Use Kovac’s reagent with caution. If Kovac’s reagent comes in contact with your skin wash

immediately and liberally with soap and water.

* Positive sulfur results mask motility results in SIM deeps. This is not an issue for us due to the fact

that our only non-motile Gram negative rods (Klebsiella, Shigella and Acinetobacter) are also sulfur

negative.