The methyl red (MR) test, at its core, is all about the detection of acid during glucose fermentation.
It also assesses the stability of an old culture by measuring for a pH level of 4.5 pH or below, which will be indicated by the color shift of the solution towards the termination of a period of incubation.
We have Clarks and Lubs to thank for the process we’ll be discussing today, as they developed the MR-VP broth that facilitates both the MR and VP observations that can be undertaken using a singular inoculated medium via aliquoting said medium into separate test tubes.
The MR Test: Principle
Certain bacteria are capable of processing glucose in such a way that it is converted into a very stable acid. Such acids include, but are not limited to, formic acid, lactic acid, and acetic acid.
The chain of events that lead to the stable acids goes a little something like this:
- Bacteria metabolizes the glucose.
- The glucose is gradually converted into pyruvic acid.
- Metabolization continues via a “mixed acid pathway”.
- A stable acid is created.
Now, the specific type of stable acid that we end up with depends on a couple of factors, namely, the bacteria’s enzymatic pathways, and, of course, the species of bacteria.
But we will know for sure that it is a stable acid when the methyl red broth shifts from its standard yellow hue, to a vivid red. This tells us that the resulting acid has a pH score of 4.5 or less.
This is all accomplished by germinating the chosen bacteria in Clarks and Lubs’ infamous broth, one of the key ingredients of which is glucose.
Should our chosen bacteria be capable of producing a stable acid, as already established, we’ll end up with that lovely red color when the methyl red is added to the broth.
The reason the pH level of the culture medium drops so significantly in this experiment is down to the sheer quantity of acids produced when bacteria metabolizes glucose.
For example, once the glucose is metabolized in the mixed acid path way of bacteria, approximately 4 mol of acids are produced, as opposed to a combined 3 mol of other by-products (ethanol, CO2, H2) for every mol of glucose.
Methyl Red Test: Media And Reagents
MRVP Broth (pH 6.9)
Broth recipe (per l of deionized H2O):
- 7.0 gm of buffered peptone
- 5.0 gm of glucose
- 5.0 gm of dipotassium phosphate
Methyl Red Solution (0.02%)
MR solution recipe:
- Prepare 300 ml of 95% ethyl alcohol
- Take 0.1 g of your methyl red and dissolve it in the alcohol.
- Add distilled water until the solution reaches a volume of 500 ml.
- Decant solution into a brown bottle, and store it in a safe location with temperature between 4–8 °C. This solution is known to be stable for up to a year, so there’s no rush to get this experiment done.
Methyl Red (MR) Test: Procedure
Let’s break this experiment down into small, detailed stages, so you can nail it first time around.
- Step 1 – Give your medium time to equilibrate in a room temperature environment. This must be done before inoculation (the introduction of microorganisms into conditions that facilitate reproduction).
- Step 2 – Choose organisms from a pure culture (18–24 hours), then inoculate the equilibrated medium. Don’t go too heavy with the inoculation; keep it light.
- Step 3 – Aerobically incubate the medium for 24 hours at a temperature of 37 degrees C.
- Step 4 – Once 24 hours have elapsed, grab a fresh test tube and aliquot 1 ml of the incubated broth into it.
- Step 5 – With the remaining broth, repeat step 3.
- Step 6 – Use a dropper to add 3 drops of methyl red to the test tube holding the aliquot.
- Step 7 (the exciting part) – Watch immediate color shift (if stable acids have successfully been cultivated). Of course, if the bacteria has failed to create a stable acid, or perhaps a mistake has been made at some point in the experiment, the methyl red will not change hue, giving us a negative reaction and a faintly yellow coloring.
Methyl Red (MR) Test: Interpreting Your Results
Obviously, we know what colors indicate a positive or negative result:
Positive reaction = A vivid red color (occurs immediately after drop)
Examples of bacteria that we know will give a positive reaction include E. coli and Yersinia sps.
Negative reaction = A faint yellow color
Examples of bacteria that we know will give a negative reaction include Klebsiella pneumoniae, and enterobacter aerogenes.
… But here’s the thing. These aren’t the only possible outcomes. You may also encounter what is known as a “weak positive”. A weak positive result will show a faint orange color. It will neither remain completely yellow, nor will it turn totally red.
Not to worry, though; this is why we saved the remaining broth and left it to incubate. The orange indicates that the process was going well, but perhaps you went a little too light in the inoculation stage.
Leaving the broth for a further 4 days will give it time to mature. Then you can repeat the process, and with any luck, you’ll get that super vivid red positive result you were hoping for the first time around.
Why Is The MR Test Used?
The MR-VP test was initially developed as a means of distinguishing between members of the bacterial family, Enterobacteriaceae.
These days, however, it has taken on more of a broad scope and is utilized to study members of many other families of bacteria, one of which is the Actinobacteria family.
The MR Test: Quality Control
- MR Negative — Klebsiella pneumoniae ATCC 13883
- MR positive — Escherichia coli ATCC 25922
Does The Methyl Red Test Have Any Limitations?
Although the MR test is a fantastic and effective experiment, it’s not without its limitations, so before you start your own methyl red test, it’s worth considering the following:
- For unequivocal classification, it’s recommended that further biochemical tests are carried out on the pure culture. A methyl red test is a great starting point but shouldn’t be solely relied upon.
- It’s quite a slow moving experiment. You have to incubate the inoculated broth for at least 24–48 hours, otherwise, you may end up with a false-positive.
- You have to be very accurate with the inoculation. Inoculating too heavily will actually impede bacterial germination, leading to invalid results.
- Sometimes, 48 hours isn’t a long enough incubation period. In some instances, you could be waiting up to 5 days before you’ll get a conclusive result. If you’re looking for a quick answer to your inquiry, I’m afraid you’re probably out of luck.
That about covers all bases, I feel. The methyl red (MR) test can be used to classify species of bacterial organisms within large families, and now you know how it’s done, you can give it a try.
This is a fantastic, beginner experiment you can do to get people interested in chemistry.
Yes, it’s a slow burner, but in a classroom setting, the incubation period can really build suspense and get students even more invested in the eventual outcome. What’s more, as it’s such a visual experiment, the results can be extremely rewarding.
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