do a 300 words minimum lab report.

Many species of medically important enteric Gram negative bacteria exist. Furthermore, most have similar cellular and colonial morphologies making identification difficult. For this reason, much emphasis has been placed on characterization and identification of enteric species. More than any other group of bacteria, enterics are characterized and identified on the basis of carbohydrate utilization (what sugars they eat and how they eat them) and other biochemical characteristics.

We have already discussed some commonly used methods of characterizing enteric bacteria:

1) the oxidase test distinguishes between 2 families of Gram negative rods

2) MacConkey agar determines the ability (or inability) to ferment lactose

3) EMB agar does the same as MacConkey, but quantitates relative amount of acid produced

4) Simmon's citrate agar determines the ability to utilize citric acid

5) carbohydrate fermentation broth characterizes fermentation profiles (growth, acid, gas)

6) TSI looks at 3 carbohydates at once, as well as H2S production, and peptone utilization, all in 1 tube!

Most labs use 1 or more of the methods summarized above. Now lets look at some additional methods used to characterize enterics, and some other Gram negative rods as well. A group of tests used for years (and still used in some labs) to characterize enterics is known as the "IMViC" series. IMViC is an acronym which stands for Indole, Methyl red, Vogues Proskauer, and Citrate. The IMVIC series is NOT a single medium. We will conduct the Methyl red test using MR/VP broth, the Indole test using SIM deeps, and the sulfur reduction test (H2S production) also using SIM deeps.

Methyl red / Vogues Proskauer (MR/VP): Some enteric bacteria can be separated into 2 groups based upon the end-products of glucose fermentation. One group, which contains the genera Escherichia, Proteus, Salmonella, Shigella and Citrobacter, conduct a mixed-acid-type fermentation meaning that they produce a variety of acidic fermentation products. A mixed-acid fermentation equates to a methyl red positive result, and a Vogues Proskauer negative result (MR+/VP-). The genera Klebsiella and Enterobacter produce a pH neutral fermentation product called butanediol, which equates to a methyl red negative result, and a Vogues Proskauer positive result (MR-/VP+). Non-enteric Gram negative rods (the Gram negative non-fermentors) will give negative results on both MR & VP reactions due to the fact that they do not ferment glucose. Although this test is used primarily for enteric Gram negative rods, it can also be used to distinguish the Gram positives rods Listeria (MR+) from Corynebacterium (MR-).

MR/VP broth is a defined medium containing glucose as a source of carbon and energy. To conduct the test, inoculate 1 tube of MR/VP broth with the organism as you would inoculate a tube of carbohydrate fermentation broth, and incubate at 37oC for 24 hours. If you want to conduct the MR and the VP tests, pour half of the broth into a second clean tube following the incubation period. For the MR test, add 3 or 4 drops of methyl red reagent to the broth and swirl. If the organism is a mixed-acid fermenter (MR+), the broth will turn red in color due to the acidic pH. The red color will probably develop immediately, but may take 5 or 10 minutes.

NOTE: Klebsiella will often give an apparently weak MR positive reaction immediately upon adding the MR reagent but the orange-red color will fade over time (a few minutes to several hours). This is a false positive reaction. SEE IMAGE: Methyl red: left to right: positive, weak positive, negative

MR negative: Klebsiella (watch out! see NOTES), Enterobacter

MR positive: Escherichia, Proteus, Salmonella, Shigella, Citrobacter

For the VP test, add 4-6 drops of VP “reagent A” (potassium hydroxide). Thump the tube for ~10 seconds to mix the reagent with the culture. Next add 2 drops of VP “reagent B” (alpha napthol). Cover the tube with a piece of parafilm or something else to prevent spillage. Now shake this tube vigorously to oxygenate the solution. Shake for at least 1 minute. Set the tube in the rack and wait 15 minutes. The development of red color in the tube indicates a positive VP reaction. Of our 6 enteric organisms only E. aerogenes and K. pneumoniae are MR-/VP+. The remaining 5 are MR+/VP-. Take caution while shaking as the VP reagents cause skin irritation, and can damage clothing.

For our purposes, there is no need to run the VP test. All of our enterics that are MR + are VP- and vice versa. This is good considering the fact that VP reactions are not terribly consistent and the VP reagents are unpleasant to work with.