METHYL RED (MR) TEST (Mixed Acid Fermentation)

          

Objective:
 -  To determine whether the organisms performs mixed acid fermentation by using glucose.
 - To identify the bacteria producing stable acids by fermentation.
 -  To differentiate between all glucose oxidizing enteric organisms.

Principle:

            The glucose is the major substrate oxidized by all enteric organisms for energy production. The end products of this process vary depending on the enzymatic pathways present in the bacteria. The pH indicator methyl red detects acid range as a result of acid end products. This test is value to differentiate Escherichia coli and Enterobacter aerogenes.  E.coli (Mixed acid fermenters) produces a mixture of fermentation acids such as lactic, acetic, succinic and formic acids and thus acidifies the medium. E. aerogenes (Bautanediol fermenters) forms neutral end products like butanediol, acetoin so the pH is elevated to approximately 6.

            The pH is indicates by methyl red indicator, it turns red color in the pH range of 4. At  a pH of 6 still acidic but a lower hydrogen ion concentration. The indicator turns yellow which is a negative test.   

Materials required:

Cultures : E.coli, Klebsiella sp., and E. aerogenes

Medium :  MR-VP broth

Indicator : 0.02% methyl red reagent

Procedure : 
  -   Inoculate one loopful of test culture into the test tubes of MRVP broth.
 - The tubes are incubating at 37° C for 24 hrs.
  - After Incubation add 5-6 drops of methyl red reagent to the culture tubes and thoroughly mixed.
 - To determine and record the result based on color change.

Observation:
-Red color formation in  tube with E.Coli
-Yellow color in Klebsiella sp., and E. aerogenes tubes.

Result :
 - E.coli  : MR  Positive 
  - E. aerogenes  and Klebsiella sp.., Gives Negative Result in Methyl Red test. 

Interpretation:

            When microbes ferments glucose releasing acids, the hydrogen ion concentration increases i.e., the pH goes down.  The pH indicator methyl red is yellow above pH 6.3 and red below 4.2 pH.  E. coli  Shows a red color indicating acidic nature of the medium due to acidic end products.   The organisms like Klebsiella sp., and E. aerogenes are not producing acid end products so lack of color change and maintains yellow color.

MR Positive Organisms:

E.coli, Salmonells Sp.,

MR Negative Organisms:

Klebsiella Sp., E. aerogenes and Pseudomonas sp., 

MR/VP medium Composition:

Peptone    5g

Dipotssium hydrogen phosphate    5g

Glucose (10% solution)         50 ml

Distilled water 1000ml           

Methyl Red : 
Methyl red    0.1g
ethanol   300 ml
distilled water 200ml