In the differential definition of enterobacteria and some vibrios, a special place is occupied by the Voges-Proskauer reaction. 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 family of enterobacteria yersinia (including pathogens of pseudotuberculosis and enterocolitis), Escherichia coli, spore-forming aerobes. The result is visualized by color staining the medium with the addition of certain reagents.
This test belongs to the IMViC series (abbreviation 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, is used in the presence of a Kovac or Erlich reagent;
- using a methylroth, or methyl red, detecting a given pH as a result of glucose metabolism;
- Foges-Proskauer reactions to the detection of acetyl-methylcarbinol;
- disposal of citrate with a 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 the acetoin formed by them with potassium hydroxide 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 the introduction of alpha-naphthol prior to the addition of caustic potassium.
Test setting
The formulation of the Voges-Proskauer reaction involves the preliminary cultivation of microorganisms. Pure culture is seeded on the differential diagnostic environment of Clark, a variation of which is Clark broth (without adding agar-agar). Either a ready-made medium is used, or prepared independently. The composition includes:
- 5 g peptone;
- 5 g glucose;
- 5 g of dibasic potassium phosphate;
- 1 liter of distillate.
During the study, the culture is inoculated with a sterile bacteriological loop in a liquid medium. Amount - 5 ml in vitro plus control. Incubation is carried out at a temperature of 35-37 degrees for two days. Next, the Foges-Proskauer reaction is studied stepwise:
- 2.5 ml of culture in the broth is transferred into a sterile tube.
- Make six drops of alpha-naphthol (5% alcohol solution).
- Add a 40% solution of potassium hydroxide in an amount of 0.1 ml, or two drops.
- Stirring is done by gently shaking the tubes.
- Evaluate the result after 15 minutes from the start of the experiment.
An alternative test method is night 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 loop is introduced into a 2 ml 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.
Score
After the required time after adding 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. No change is recorded as a negative result.
Excess caustic potassium in solution can give an imitation of a positive reaction, staining in 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 one hour after the introduction of the reagents.
When evaluating the result, it must be taken into account that long-term cultivation of the studied microorganisms (over three days) leads to acidification of the medium, which can lead to an incorrect outcome of the study. The reaction can be mild positive or false negative.
Test reagent requirements
The reagents for the Voges-Proskauer reaction must meet the requirements, such as the correct concentration, high degree of purification and stability, which is achieved by observing storage rules.
Ready-made test kits contain, as a rule, reagents for 100 or more applications and are in plastic or darkened glass bottles. Quality is regulated by relevant documentation.