Pyruvate Broth Test- Principle, Procedure, Results, Uses

The pyruvate broth test is a biochemical test that can serve two main objectives:

• To assess the ability of bacteria to utilize pyruvate and produce metabolic acid.
• To aid in the differentiation of Enterococcus faecalis from Enterococcus faecium.

Pyruvate is a key intermediate in several metabolic pathways, such as glycolysis, fermentation, and the citric acid cycle. Some bacteria can use pyruvate as a sole carbon and energy source and produce organic acids as the end products. These acids lower the pH of the medium and can be detected by a color indicator. Other bacteria cannot utilize pyruvate or produce neutral or alkaline compounds instead of acids.

Enterococcus faecalis and Enterococcus faecium are two closely related bacterial species that belong to the genus Enterococcus. They are gram-positive, catalase-negative, facultatively anaerobic cocci that are commonly found in the human gastrointestinal tract and can cause various infections, such as urinary tract infections, endocarditis, bacteremia, and wound infections. They are also resistant to many antibiotics and can pose a serious threat to public health.

The pyruvate broth test can help to distinguish between these two species based on their ability to utilize pyruvate and produce acid. Enterococcus faecalis can ferment pyruvate and produce lactic acid and oxaloacetic acid, which turn the medium yellow. Enterococcus faecium cannot ferment pyruvate and does not produce acid, so the medium remains greenish or slightly yellowish-green.

The pyruvate broth test is a simple, inexpensive, and reliable method to identify Enterococcus faecalis and differentiate it from Enterococcus faecium. It can also be used to test other bacteria that can utilize pyruvate and produce acid, such as Streptococcus bovis. However, it is not a definitive test and should be confirmed by other biochemical tests.

The principle of pyruvate broth test is based on the ability of some bacteria to utilize pyruvate and produce organic acids as the end products of their metabolism. Pyruvate is a key intermediate in the glycolysis pathway and can be converted to various compounds depending on the enzyme systems present in the bacteria. Some bacteria, such as Enterococcus faecalis, can use pyruvate as a sole carbon source and produce lactic acid and oxaloacetic acid; whereas, some bacteria, such as Enterococcus faecium, cannot utilize pyruvate or do not produce acid as the end product.

The pyruvate broth medium contains sodium pyruvate as the main substrate and bromothymol blue as the pH indicator. Bromothymol blue is a dye that changes color depending on the acidity or alkalinity of the solution. At neutral pH (around 7), it is greenish-blue; at acidic pH (below 6), it is yellow; and at alkaline pH (above 7.6), it is blue. When bacteria grow in the pyruvate broth medium and produce acid, they lower the pH of the medium and cause the color change of bromothymol blue from greenish-blue to yellow. This color change indicates a positive result for pyruvate utilization and acid production. On the other hand, if bacteria do not grow or do not produce acid in the medium, there is no change in the pH or color of bromothymol blue. This indicates a negative result for pyruvate utilization and acid production.

The pyruvate broth test is useful for differentiating Enterococcus faecalis from Enterococcus faecium, which are two closely related bacterial species that cause various infections in humans and animals. Enterococcus faecalis is positive for pyruvate utilization and acid production, while Enterococcus faecium is negative for both. The pyruvate broth test can also differentiate Streptococcus bovis from other streptococci, as Streptococcus bovis is negative for pyruvate utilization and acid production.

To perform the pyruvate broth test, you will need the following items:

• Culture Medium: Pyruvate broth is the culture medium used for this test. It is a low-nutrient medium that does not contain any added carbohydrates. It consists of pancreatic digest of casein, yeast extract, sodium pyruvate, sodium chloride, dipotassium phosphate and bromothymol blue indicator. The final pH of the medium is 7.3 ±0.2 at 25°C. The medium is prepared by dissolving the components in water, heating to boiling, dispensing in test tubes and autoclaving.
• Reagents: No extra reagents are required for this test.
• Equipment: You will need personal protective equipment (PPE) and other general laboratory materials such as inoculating loop, incubator, test tube rack, etc.
• Test Organism: You will need a sample of bacteria that you want to test for pyruvate utilization and acid production. You can use a sample from a fresh (18 to 24 hours old) culture on blood agar medium or any other suitable medium. The sample should be well-isolated and pure.
• Positive Control: You will need a positive control organism that can utilize pyruvate and produce acid. Enterococcus faecalis ATCC 29212 is a recommended positive control for this test. It will result in a yellow color change in the medium within 24 hours of incubation.
• Negative Control: You will need a negative control organism that cannot utilize pyruvate or does not produce acid. Enterococcus faecium ATCC 6569 or Streptococcus bovis ATCC 9809 are recommended negative controls for this test. They will grow in the medium but the color will not change even after 48 hours of incubation.

To prepare pyruvate broth, you need to follow these steps:

• Measure the appropriate amount of pyruvate broth powder (or the media components) according to the manufacturer’s instruction (35.0 grams per 1000 mL for above) and add the mixture in a conical flask (or glass bottle) with 1000 mL water.
• Stir well using a magnetic stirrer or manually and heat to boiling so that all the components dissolve completely in water.
• Dispense 5 mL (or desired volume) of the broth in test tubes and loosely put on the cap or cotton plug.
• Autoclave the test tubes at 121°C and 15 lbs pressure for 15 minutes and let them cool to below 40 – 45°C before inoculation.

• Using a sterile inoculating loop, pick up well-isolated colonies of sample bacteria from fresh (18 to 24 hours old) culture (sample from blood agar medium is preferred) and inoculate the broth.
• Incubate the tubes aerobically for 24 hours at 35±2°C and observe for color change (yellow color formation).
• If no color change is observed, re-incubate the medium for the next 24 hours and observe for color change.

The result of the pyruvate broth test is based on the color change of the medium due to the production of acid by the bacteria. The bromothymol blue indicator in the medium changes color from greenish-blue to yellow when the pH drops below 6.0. The color change indicates that the bacteria can utilize pyruvate and produce organic acids as the end product of their metabolism.

• A positive result is indicated by the change of color of the broth from greenish to yellow. This means that the bacteria can utilize pyruvate and produce acid. Enterococcus faecalis is an example of a positive test organism.
• A negative result is indicated by no change in the color of the medium or slight yellowish-green color formation due to a weak reaction. This means that the bacteria cannot utilize pyruvate or do not produce acid as the end product. Enterococcus faecium and Streptococcus bovis are examples of negative test organisms.

The result should be interpreted after 24 hours of incubation. If no color change is observed, the medium should be re-incubated for another 24 hours and observed again. A negative result should not be reported until after 48 to 72 hours of incubation, as some strains may be slow growers and late fermenters.

The following table summarizes the result and interpretation of the pyruvate broth test:

To ensure the validity and accuracy of the test results, it is important to use quality control strains along with the test organisms. Quality control strains are bacteria that are known to give either positive or negative results for the test and can be used to check if the medium and the procedure are working properly.

For the pyruvate broth test, the recommended quality control strains are:

• Positive Control: Enterococcus faecalis ATCC 29212
• Negative Control: Enterococcus faecium ATCC 6569 or Streptococcus bovis ATCC 9809

Enterococcus faecalis ATCC 29212 is a strain of bacteria that can utilize pyruvate and produce acid, resulting in a yellow color change in the medium within 24 hours of incubation. This strain should be inoculated in a separate tube of pyruvate broth and incubated under the same conditions as the test organisms. If the medium turns yellow, it indicates that the medium is capable of detecting acid production and the procedure is performed correctly.

Enterococcus faecium ATCC 6569 or Streptococcus bovis ATCC 9809 are strains of bacteria that cannot utilize pyruvate or do not produce acid as the end product, resulting in no color change in the medium even after 48 hours of incubation. These strains should also be inoculated in separate tubes of pyruvate broth and incubated under the same conditions as the test organisms. If the medium remains greenish or slightly yellowish-green, it indicates that the medium is not contaminated with carbohydrates and the procedure is performed correctly.

If the quality control strains do not show the expected results, it may indicate that there is a problem with the medium, the inoculation technique, or the incubation conditions. In such cases, the test should be repeated with fresh medium and quality control strains.

• Make sure that the medium doesn`t contain free carbohydrates because the test organisms may ferment carbohydrates and produce acid which will give a false positive result.
• Don`t report negative until after 48 to 72 hours of incubation. Some strains may be slow growers and late fermenters.
• A slight color change to yellowish green should be reported as a negative result.

Pyruvate broth test is used to identify Enterococcus faecalis (positive test) in clinical isolates and differentiate it from similar bacterial species, Enterococcus faecium (negative test).

Enterococcus faecalis and Enterococcus faecium are gram-positive, catalase-negative, facultatively anaerobic cocci that belong to the genus Enterococcus. They are part of the normal flora of the human gastrointestinal tract and can cause opportunistic infections such as endocarditis, urinary tract infections, bacteremia, and wound infections. They are also known to be resistant to many antibiotics and disinfectants, making them difficult to treat and control.

Pyruvate broth test is one of the biochemical tests that can help in the identification and differentiation of these two species. Enterococcus faecalis can utilize pyruvate and produce acid, resulting in a yellow color change in the medium. Enterococcus faecium cannot utilize pyruvate or does not produce acid, resulting in no color change or a slight yellowish-green color in the medium.

Pyruvate broth test is a simple, rapid, and inexpensive method that can be performed in any microbiology laboratory. It can provide useful information for the diagnosis and treatment of enterococcal infections. However, it is not sufficient for the complete identification of the bacteria and should be confirmed by other tests such as 6.5% NaCl broth test, bile esculin test, or molecular methods.

• Pyruvate broth test has a very limited application – it is suitable only for Enterococcus and Streptococcus spp. Other bacteria that can utilize pyruvate and produce acid may also give a positive result, which can lead to false identification. Therefore, this test should not be used as a sole criterion for the identification of bacteria.
• Pyruvate broth test does not give complete identification and requires other biochemical test results to confirm the bacterial identity. For example, Enterococcus faecalis and Enterococcus faecium are both positive for catalase test, bile esculin hydrolysis test, and growth in 6.5% NaCl broth. To differentiate them, other tests such as vancomycin susceptibility test, motility test, or PCR-based methods are needed.
• Pyruvate broth test may not be reliable for some strains of bacteria that are slow growers or late fermenters. Some strains may take longer than 48 hours to produce acid from pyruvate, which can result in false negative results. Therefore, it is important to incubate the tubes for up to 72 hours and observe for any color change before reporting negative results.
• Pyruvate broth test may not be sensitive enough to detect weak acid production by some bacteria. A slight color change to yellowish green may indicate a weak positive result, but it should be reported as negative according to the standard protocol. This may lead to false negative results and misidentification of bacteria.

Pyruvate broth test is a simple and rapid test that can be used to differentiate Enterococcus faecalis from Enterococcus faecium based on their ability to utilize pyruvate and produce acid. This test can be useful for the identification and characterization of these bacteria, which are clinically important as they can cause various infections such as urinary tract infections, endocarditis, bacteremia, and wound infections. Pyruvate broth test can also be used to detect other bacteria that can ferment pyruvate, such as Streptococcus bovis, which is associated with endocarditis and colorectal cancer. Pyruvate broth test does not require any additional reagents or equipment, and can be easily performed and interpreted by observing the color change of the medium. Pyruvate broth test can also be combined with other biochemical tests to confirm the bacterial identity and provide more information about their metabolic capabilities.

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