MEDIO OF: RATIONALE, PREPARATION, USES AND LIMITATIONS - BIOLOGY - 2024

The OF or glucose fermentation medium is a semi-solid agar specially designed for the study of the oxidative and fermentative metabolism of carbohydrates in an important group of microorganisms other than Enterobacteriaceae, called non-enteric Gram negative bacilli.

It was created by Hugh and Leifson; These researchers realized that conventional means of studying acid production from carbohydrates were not suitable for this specific group of bacteria.

A. Commercial basal OF medium. B. Tubes with OF medium seeded. Source: A and B Photos taken by the author MSc. Marielsa Gil.

This is because non-enteric Gram negative rods generally produce low amounts of acids, unlike Enterobacteriaceae.

In this sense, the OF medium has special characteristics that can detect the small amounts of acid formed, both by oxidative and fermentative routes. These differences are related to the amount of peptones, carbohydrates and agar.

This medium contains less peptones and a higher concentration of carbohydrates, thus reducing the products that alkalize the medium as a result of protein metabolism and increasing the production of acids due to the use of carbohydrates.

On the other hand, the decrease in the amount of agar favors the spread of the acid produced throughout the medium, in addition to allowing us to observe motility.

The OF medium consists of peptone, sodium chloride, bromothymol blue, dipotassium phosphate, agar, and a carbohydrate. The most common carbohydrate is glucose but others can be used according to the one you want to study, such as lactose, maltose, xylose, among others.

Basis

Like any culture medium, the OF medium must contain nutritive substances that guarantee bacterial growth; these substances are peptones.

For its part, the carbohydrate provides energy and at the same time serves to study the behavior of the microorganism against it, that is, it allows the bacteria to be classified as an oxidative, fermentative or non-saccharolytic organism.

OF medium contains a 1: 5 peptone / carbohydrate ratio as opposed to 2: 1 conventional media. This ensures that the amount of alkaline amines formed from the degradation of peptones does not neutralize the formation of weak acids.

On the other hand, the medium contains sodium chloride and dipotassium phosphate. These compounds osmotically stabilize the medium and regulate the pH respectively. Bromothymol blue is the pH indicator, which turns the color of the medium from green to yellow with the production of acid.

Some microorganisms can use carbohydrates through the oxidative or fermentation routes, while others do not take either route.

This depends on the characteristics of each microorganism. For example, some strict aerobic microorganisms can oxidize certain carbohydrates, and facultative anaerobes can oxidize and ferment depending on the environment around them, while others do not oxidize or ferment carbohydrates (asacarolytic).

Finally, there is a modification of the OF medium recommended by the CDC that contains a special OF base with phenol red as an indicator.

Oxidation process

The glucose oxidation process does not require the phosphorylation of glucose, as does the fermentation process. In this case the aldehyde group is oxidized to a carboxyl group, resulting in gluconic acid. This in turn is oxidized to 2-ketogluconic.

The latter either accumulates or breaks down to two molecules of pyruvic acid. This system requires the presence of oxygen or some inorganic compound as the final electron acceptor.

The production of acids by this route is weaker than that obtained by the fermentation route.

Fermentation process

For glucose fermentation to occur by any of the available routes, it must first be phosphorylated, becoming glucose-6-phosphate.

Glucose fermentation can take several routes, the main one being the Embden-Meyerhof-Parnas route, but they can also take the Entner-Doudoroff route, or the Warburg-Dickens hexose monophosphate route, also known as the of the degradation of pentoses.

The route chosen will depend on the enzymatic system that the microorganism possesses.

Via Embden-Meyerhof- Parnas

In the fermentation of glucose through the Embden-Meyerhof-Parnas route, it is split into two triose molecules, to later be degraded to various carbon compounds, until the formation of glyceraldehyde-3-phosphate. From there an intermediate substance originates, which is pyruvic acid.

From there various types of mixed acids will be formed that can vary from one species to another.

This system occurs in the absence of oxygen and requires an organic compound as the final electron acceptor.

Entner-Doudoroff pathway

In the fermentation of glucose via the Entner-Doudoroff pathway, glucose 6-phosphate becomes glucono-ᵼ-lactone-6-phosphate and from there it is oxidized to 6-phosphogluconate and 2-keto-3-deoxy-6- phosphogluconate, to finally form pyruvic acid. This pathway does need oxygen for glycolysis to occur.

Pentoses degradation pathway or Warburg-Dickens Hexoxa monophosphate pathway

This route is a hybrid of the 2 above. It begins similar to the Entner-Doudoroff pathway, but later glyceraldehyde-3-phosphate is formed as a precursor of pyruvic acid, as occurs in the Embden-Meyerhof-Parnas pathway.

Preparation

To weigh:

2 g of peptone

5 g of sodium chloride

10 g of D-glucose (or the carbohydrate to be prepared)

0.03 g of bromothymol blue

3 gr of agar

0.30 g of dipotassium phosphate

1 liter of distilled water.

Mix all the compounds except the carbohydrate and dissolve in 1 liter of distilled water. Heat and shake until completely dissolved.

On cooling to 50 ° C, 100 ml of 10% glucose (filtered) is added.

Aseptically distribute 5 ml of OF medium into cotton-capped test tubes and autoclave at 121 ° C, 15 pounds pressure for 15 minutes.

Allow to solidify in a vertical position.

The pH of the medium should be 7.1. The color of the prepared medium is green.

Store in a refrigerator.

Applications

The OF medium is a special medium for determining the metabolic behavior of a microorganism against a carbohydrate. Especially for those that form little, weak or no acids.

Sown

For each microorganism, 2 OF tubes are needed, both must be inoculated with the microorganism to be studied. The colony is taken with a straight handle and a puncture is made in the center of the tube without reaching the bottom; Several punctures can be done, as long as there is no interest in observing motility.

A layer of sterile liquid petroleum jelly or sterile melted paraffin (approximately 1 to 2 ml) is added to one of the tubes and labeled with the letter "F". The other tube is left original and labeled "O". Both tubes are incubated at 35 ° C and observed daily for up to 3 to 4 days.

Interpretation

Metabolism and gas production

Table: Classification of microorganisms according to their behavior in open (oxidative) and closed (fermentative) OF tubes

Source: Made by the author MSc. Marielsa gil

The gas is observed with the formation of bubbles or displacement of the agar.

It should be noted that an organism that only oxidizes glucose but does not ferment it, will not be able to ferment other carbohydrates, in any case it will only oxidize it. Therefore, in this situation the sealed tube for the study of other carbohydrates will be omitted.

Motility

Additionally, motility can be seen in the OF medium.

Positive motility: growth that is not limited to the area of ​​inoculation. There is growth towards the sides of the tube.

Negative motility: growth only in the initial inoculum.

QA

The following strains can be used as quality controls: Escherichia coli, Pseudomonas aeruginosa and Moraxella sp. The expected results are:

1. coli: Glucose fermenter (both yellow and sparkling tubes).
2. aeruginosa: Glucose oxidizer (open yellow tube and green or blue seal).
3. Moraxella sp: Not saccharolytic (green or blue open tube, green sealed tube).

Limitations

-Some microorganisms cannot grow in OF medium. In these cases, the test is repeated but 2% serum or 0.1% yeast extract is added to the medium.

-Oxidation reactions are often only observed close to the surface and the rest of the medium can remain green, in the same way it is taken as positive.

References

1. Koneman E, Allen S, Janda W, Schreckenberger P, Winn W. (2004). Microbiological Diagnosis. 5th ed. Editorial Panamericana SA Argentina.
2. Forbes B, Sahm D, Weissfeld A. (2009). Bailey & Scott Microbiological Diagnosis. 12 ed. Editorial Panamericana SA Argentina.
3. Mac Faddin J. (2003). Biochemical tests for the identification of bacteria of clinical importance. 3rd ed. Editorial Panamericana. Buenos Aires. Argentina.
4. Francisco Soria Melguizo Laboratories. 2009. OF Glucose Medium. Available at:
5. Conda Pronadisa Laboratories. OF glucose medium. Available at: condalab.com
6. BD Laboratories. 2007. OF Basal Medium. Available at: bd.com