In the differential determination of enterobacteria and some vibrios, the Voges-Proskauer reaction occupies a special place. The test is based on the ability of bacteria to ferment glucose to form acetoin.
The essence of the research process
In microbiology, the Voges-Proskauer reaction is often used to differentiate within the Yersinia family of enterobacteria (including the pathogens of pseudotuberculosis and enterocolitis), Escherichia coli, and spore-forming aerobes. The result is visualized by coloring the medium when certain reagents are added.
This test belongs to the IMViC series (abbreviation of Indol, Methyl Red, Voges-Proskauer i Citrate) - a group of identification tests including differential definitions:
- with indole, based on the breakdown of tryptophan into indole, used in the presence of a Kovacs or Ehrlich reagent;
- usingmethylroth, or methyl red, which detects a given pH as a result of glucose metabolization;
- Voges-Proskauer reactions for the detection of acetyl-methylcarbinol;
- utilization of citrate with color change as a result of alkalization of the medium.
The essence of the process is the visualization of the presence of diagnosed bacteria due to the interaction of acetoin formed by them with caustic potash in the presence of oxygen. Acetyl-methylcarbinol is oxidized to diacetyl, which forms a bright red or pink compound. Increase the sensitivity of the test by introducing alpha-naphthol before adding caustic potash.
Test setting
The formulation of the Voges-Proskauer reaction involves the preliminary cultivation of microorganisms. A pure culture is sown on Clark's differential diagnostic medium, a variation of which is Clark's broth (without the addition of agar-agar). Either ready-made medium is used, or prepared independently. Ingredients include:
- 5g peptone;
- 5g glucose;
- 5g dibasic potassium phosphate;
- 1L distillate.
During the study, the culture is inoculated with a sterile bacteriological loop into a liquid medium. Quantity - 5 ml in a test tube plus control. Incubation is carried out at a temperature of 35-37 degrees for two days. Next, a study of the Voges-Proskauer reaction is carried out in stages:
- 2, 5 ml of broth culture is transferred to a sterile tube.
- Add six drops of alpha-naphthol (5% alcohol solution).
- Add 40%a solution of caustic potassium in an amount of 0.1 ml, or two drops.
- Stirring is carried out by gently shaking the test tube.
- Evaluate the result after 15 minutes from the start of the experiment.
An alternative test method is overnight incubation, the time of which is reduced to 18 hours or up to a day. In addition to this method, an express test is also used: a culture is introduced in a loop into 2 ml of the medium, incubated for about four hours, then reagents are added in an equal amount of 2-3 drops, mixed and the result is evaluated after ten minutes.
The control is one of the causative agents of pneumonia Klebsiella pneumoniae - strain atcc 13883.
Evaluation of the result
After the required time after adding the reagents (from 5 to 15 minutes), a cherry-red color should be observed with a pronounced positive reaction, red and pink - with a weak positive reaction. No change is recorded as a negative result.
Excess of caustic potash in a solution can give an imitation of a positive reaction, staining in a copper color. In this case, the negative response of the tested colony should be recorded. Also, copper staining appears in cases where the assessment is given an hour after the introduction of the reagents.
When evaluating the result, it must be taken into account that prolonged cultivation of the studied microorganisms (more than three days) leads to acidification of the medium, which can lead to an incorrect outcome of the study. The reaction may be mildly positive orfalse negative.
Requirements for test reagents
Voges-Proskauer reagents must meet requirements such as correct concentration, high purity and stability, which is achieved through proper storage.
Ready-to-use test kits typically contain reagents for 100 or more uses and come in plastic or tinted glass vials. Quality is regulated by the relevant documentation.
