Pyruvate Broth Test Principle and Procedure

In this pyruvate broth test principle and procedure post we have briefly explained pyruvate broth test principle, objectives, requirements, pyruvate broth test procedure, uses and limitations.

Pyruvate Broth Test Principle and Procedure

Pyruvate Broth test is used to test an organism’s capacity to utilise the pyruvate substrate. The presence of the test substrate pyruvate enables for the distinguishing of species that can and cannot metabolise pyruvate.

This characteristic is helpful in individual between Enterococcus faecalis and Enterococcus faecium.

Principle

Pyruvate broth comprises pancreatic digest of casein and yeast extract, which supply amino acids, vitamins, and other essential growth factors to the bacteria.

Because the breakdown of pyruvate causes an acidic shift in the pH of the broth, phosphate is given to help maintain a constant pH.

The active substrate is pyruvate, which can be broken down by organisms that can use it, resulting in a variety of metabolic acids that can be detected using the pH indicator bromthymol blue. When enough acid is produced, the medium changes colour from blue-green to yellow.

Materials Required

Inoculum

It’s best to use a fresh inoculum produced in Todd Hewitt broth. A little loopful of growth from a blood agar plate could also be employed.

Media per Litre

Pancreatic digest of casein: 10.0 g

Yeast extract: 5.0 g

Sodium chloride: 5.0 g

Dipotassium phosphate: 5.0 g

Bromthymol blue: 0.04 g

Sodium pyruvate: 10.0 g

pH: 7.3 ± 0.2

Procedure

1. Inoculate the pyruvate broth lightly with an organism cultured for 18 to 24 hours from an overnight Todd-Hewitt broth culture or blood agar.

2. Incubate the tube in ambient air for 24-48 hours at 35°C. Incubation time for fastidious strains is 14 days.

3. Keep an eye out for a yellow reaction. Tubes should be examined every day for up to 5 days.

Results

A colour change in the broth from greenish-blue to yellow indicates a favourable pyruvate utilisation outcome in pyruvate broth test.

No colour change and a greenish-blue tint in the resultant broth suggest a negative pyruvate utilisation result in pyruvate broth test.

Quality Control

The following organisms are used to determine the performance of the final medium after it has been checked for correct pH, colour, depth, and sterility. After 48 hours at 35°C, the tubes are cultured and analysed.

Each fresh batch of produced medium is subjected to quality control testing. As positive and negative controls, E. faecalis strain SS-1273 and S.sanguinis strain SS-910 were utilised.

Applications

1. The test helps distinguish between Enterococcus faecalis (positive) and Enterococcus faecium (negative). It can be used to identify several other species that can utilise pyruvate as part of an identification test.

Further Readings

Reference