Pyruvate Broth Test Introduction
Many of the biochemical tests are used to detect the ability of the organism to utilize the organic and chemical substances in the medium, one such biochemical test is Pyruvate broth test.
Biochemical tests help us to identify the species of bacteria on the basis on their ability to differentiate the biochemical activities of the various species of bacteria.
As bacterial physiology differs from one species of the organism to the another. Thus, ability of the bacteria to form the organic compounds which helps to metabolize the certain compounds of the carbohydrates and the other related compounds that is widely used for identifying the microorganism.
One such test is pyruvate test, which tests the ability of the specific species to the substrate pyruvate.
What is Pyruvate Broth Test?
Pyruvate is considered as the conjugate base of pyruvic acid, which is used as a key in intermediating the biological processes.
Pyruvate is generally producing as the end product of the process of glycolysis which acts as a connective link between various biochemical processes including gluconeogenesis, fermentation, cellular respiration, fatty acid synthesis etc.
However, by accompanying these processes, pyruvate plays a key role in providing energy to the living cells through Krebs cycle, which is one of the metabolic pathways in all living cells.
In Krebs cycle, Pyruvate is decarboxylated into acetyl coA which is then converted into acetyl CoA.
Pyruvate broth is usually used to determine the ability of the microorganism to utilize the pyruvate.
The presence of this test substrate pyruvate helps us to differentiate the characteristics of an organism which metabolize the pyruvate and other which does not have the capability to metabolize.
This characteristic is useful in differentiating the organisms such as Enterococcus faecalis from Enterococcus faecium.
Here pyruvate broth is made up of pancreatic digest that includes casein and the yeast extracts which provides the bacteria healthy supplements like amino acids, vitamins and other necessary growth factors, generally phosphate is added here in the broth to maintain the stable pH needed for the culture medium.
Due to the pyruvate breakdown it results in the change in pH to the acidic form in the culture.
Pyruvate is one of the active substrates where, the organisms can utilize it and breakdown the substrate thus generating the host of metabolic acids, and it is detected by the pH indicator known as Bromothymol blue.
At the time of acid generation, the culture medium changes its color from a blue green to yellow.
Pyruvate Broth Test Objective
The main aim of the test is to determine the ability of an organism to utilize the pyruvate to synthesis the acidic products.
Pyruvate Broth Test Principle
To perform this test, pyruvate broth test is added to the culture broth, which helps us to determine the capability of the organism to utilize pyruvate, thus results in formation of the metabolic acids.
Acid produces as a result of metabolic waste when pyruvate broth is being inoculated with bacteria so that they are capable of metabolizing the pyruvate.
The acid production decreases the pH of the medium which results in change in color from.
Bromothymol blue is used as an indicator as it one of the acid base indicators which suits the medium.
It generally has a greenish blue at an alkaline pH and during acid production, it changes its color to yellow indicating the fermentation in the pyruvate.
Thus, yellow color is one of the indicatives of the positive reaction of the fermentation.
Pyruvate Broth Test Reagents
Pancreatic digest of casein of about 10 gram per Liter
Sodium pyruvate – 10 grams
Yeast extract of about 5 grams
Potassium hydrogen phosphate- 5 grams
Sodium chloride – 5 grams
Indicator- Bromothymol blue- 40 grams/liter
Pyruvate Broth Test Procedure
Before performing the procedure, initially the medium is allowed to reach the temperature.
Then the direct inoculum is prepared overnight so that it can be inoculated into the broth.
Initially, the pH of a medium was set at 7.3 and then the pyruvate broth is inoculated with an organism cultured for about 18 to 24 hours overnight from the Todd-Hewitt broth culture for blood agar.
The tube is then incubated in an ambient air for 24 to 48 hours at a temperature of about 35ºC.
The culture should be observed continuously for a color change. Usually, the tubes are observed maximum for about 5 days.
Pyruvate Broth Test Result
A change in color of the broth from greenish-blue to yellow, indicates a positive control for pyruvate broth test.
Whereas, if there is no color change then it results in a negative result.
In pyruvate broth test, positive control is usually determined for Enterococcus faecalis, and the negative control is determined for two species namely, Enterococcus faecium and Staphylococcus bovis.
Pyruvate Broth Test Uses
Pyruvate broth test is usually used in differentiating the species of Enterococcus faecalis from the Enterococcus faecium.
This test can also be used as a part of identification of the other organisms which have the capability to utilize pyruvate.
Pyruvate Broth Storage and Shelf Life
Usually, pyruvate broth is stored in an upper right position at a temperature of about 4 to 8ºC. It is also important that the broth should not be placed in a direct sunlight.
By following these conditions, the broth has a shelf life of about 26 weeks, calculated from the date of manufacture.
Pyruvate Broth Test Citations
- Susceptibility of anaerobic bacteria to sulfamethoxazole/trimethoprim and routine susceptibility testing. Antimicrob Agents Chemother . 1978 Sep;14(3):384-90.
- Identification of Enterococcus spp. with a Biochemical Key. Appl Environ Microbiol. 1999 Oct; 65(10): 4425–4430.
- Development of a blood-free Campylobacter medium: screening tests on basal media and supplements, and the ability of selected supplements to facilitate aerotolerance. J Appl Bacteriol . 1983 Feb;54(1):115-25.