Oxidative Fermentative test test: Introduction, Principle, procedure ,results

Oxidative Fermentative (OF) Test: Introduction, Principle, Procedure ,Results Interpretation and Limitations

Oxidative-Fermentative (OF) Test determines if certain gram-negative bacilli metabolize glucose by fermentation or aerobic respiration (oxidatively). During the anaerobic process of fermentation, pyruvate is converted to a variety of mixed acids depending on the type of fermentation. The high concentration of acid produced during fermentation will turn the indicator, bromthymol blue present in OF basal medium from green to yellow in the presence(oxidatively) or absence of oxygen (fermentatively) .Some nonfermenting gram-negative bacteria metabolize glucose using aerobic respiration and therefore only produce a small amount of weak acids during glycolysis and Krebs cycle. The decrease amount of peptone and increase amount of glucose helps the detection of weak acids so produced. Dipotassium phosphate buffer present in the medium is useful to further promote acid detection. It is also useful to test nonsacchrolytic (have no ability to use the carbohydrate in the media) activities of organisms.

Introduction of Oxidative Fermentative Test

OF stands for Oxidative Fermentative test. The OF basal media was developed by Hugh and Leifson in 1953. OF Basal Medium is recommended for the detection of oxidation or fermentation of carbohydrates by bacteria. This basal medium is useful to aid in the identification of gram-negative bacteria on the basis of their ability to oxidize or ferment a specific carbohydrate. As compared to other OF Media, Hugh and Leifson’s formula employs a low peptone/carbohydrate ratio and a minimal amount of agar i.e. 2% from 3%. The decreased ( 2% from 11%) amount of peptone reduces the formation of alkaline amines which can ultimately mask the small quantities of acid that may be produced from oxidative metabolism. The increased carbohydrate (1% from 0.5%) results in an increase in the amount of acid that may be formed that can be detected by the bromthymol blue indicator. The small amount of agar added to the medium provides a semi-solid structure that concentrates the acid at the point of reaction, thereby facilitating the visual interpretation of the pH shift. Proper performance of the OF test requires an organism to be inoculated into two tubes of each OF Medium. Once inoculated, one tube is overlaid with mineral oil or melted paraffin. The other tube is left open to the air. Oxidative utilization of the carbohydrate will result in acid production (yellow) in the open tube only. Fermentative utilization of the carbohydrate will result in acid production (yellow) in both the open and closed tubes. Acidic changes in the overlaid tubes are considered to be a result of true fermentation, while acidic development in the open tubes is due to oxidative utilization of the carbohydrate present. Asaccharolytic (non-fermenter and non-oxidizer) organisms will not produce acid in either tube.

Principle of the Oxidative Fermentative Test

This test determines if certain gram-negative bacilli metabolize glucose by fermentation or aerobic respiration (oxidatively). During the anaerobic process of fermentation, pyruvate is converted to a variety of mixed acids depending on the type of fermentation. The high concentration of acid produced during fermentation will turn the indicator, bromthymol blue present in OF basal medium from green to yellow in the presence(oxidatively) or absence of oxygen (fermentatively). Some nonfermenting gram-negative bacteria metabolize glucose using aerobic respiration and therefore only produce a small number of weak acids during glycolysis and Krebs cycle. The decrease amount of peptone and increase amount of glucose helps the detection of weak acids so produced. Dipotassium phosphate buffer present in the medium is useful to further promote acid detection. It is also useful to test the non-saccharolytic (have no ability to use the carbohydrate in the media) activities of organisms.

Composition of the OF basal medium

Constituents of Hugh and Leifson’s OF basal medium are as follows:

Sodium chloride: 5.0 g
Pancreatic digest of Casein:2.0 gm
Di-potassium phosphate : 0.3
Bromothymol blue:0.08 g
Agar: 2.0 g
Distilled water: 1000 ml

Adjust final pH 6.8 +/- 0.2 at 25ºC. After autoclave 121°C for 15 minutes, add OF media with carbohydrate contains 10.0gm/L of specific carbohydrate i.e. dextrose, maltose, lactose, sucrose, etc.

Requirements for Oxidative Fermentative Test

Test organisms ( Gram-negative bacilli) pure well-isolated colonies from an 18-24-hour culture
OF basal media tubes sterile mineral oil or liquid paraffin or sterile melted petrolatum
Bunsen burner
Inoculating wire
Incubator

Quality control strains

Glucose Fermenter: Escherichia coli
Glucose oxidizer: Pseudomonas aeruginosa
Nonsaccharolytic: Alcaligenes faecalis

Procedure of Oxidative fermentative test

Bring the basal media to equilibrate to room temperature.
Take pure well-isolated colonies from an 18-24 hour culture.
For each test organism, inoculate two basal media tubes and inoculate by stabbing the agar to approximately 1/4 inch from the bottom.
Apply with 1 cm layer either sterile mineral oil or liquid paraffin or sterile melted petrolatum to one of each two tubes. Tighten the cap of the overlaid tube, and loosen the cap of the non-overlaid tube.
Incubate both tubes aerobically at 35ºC for 48 hours (Slow-growing bacteria may take 3 to 4 days before results can be observed).
Examine tubes daily.
A control tube of OF Base Medium should be inoculated and incubated in parallel with the OF tests.

Observations

Notice for Acid production in the medium by the appearance of a yellow color. In the case of oxidative organisms; color production may be first noted near the surface of the medium.

Interpretation of Result for OF Test

A positive carbohydrate utilization test is indicated by the development of yellow color in the medium.

A negative carbohydrate utilization test is indicated by the absence of a yellow color (media remains green or turns blue).

The method of metabolism is determined as follows:

Fermenter: Acid production on both i.e. open and overlaid tubes. The acid produced changes the pH indicator, bromothymol blue, from green to yellow

Oxidizer: Acid production in the open tube (aerobic) and not in the oil-covered tube (anaerobic) indicates an oxidizer.

Non-saccharolytic or non-utilizer or Negative oxidative and fermentative organisms: The negative result is indicated by no color change in the oil-covered tube and in some cases an increase in pH (pH 7.6) changing the bromothymol blue from green to blue at the top of the open tube. The increase in pH is due to amine production by bacteria that break down the peptone (protein) in the medium.

Result of quality control strains

Escherichia coli: Glucose Fermenter
Pseudomonas aeruginosa: Glucose oxidizer
Alcaligenes faecalis: Nonsaccharolytic:

Limitations of Oxidative Fermentative Test

It is recommended that biochemical, immunological, molecular, or mass spectrometry testing be performed on colonies from pure culture for complete identification.
Bacteria that only oxidize dextrose will not ferment any other carbohydrate. Other carbohydrates will only be oxidized. The covered tube, therefore, may be omitted when determining other carbohydrate utilization of such organisms.
Some organisms do not grow in this OF the Basal Medium. It may be necessary to use another basal medium containing dextrose to confirm the negative reaction.
Some mineral oils are acidic in nature and therefore may cause erroneous results.

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