.webp "Pyruvate Broth Test: Overview of Principle, Procedure, Results, and Applications")
Table of Contents
- Introduction
- Objectives
- Principle of Pyruvate Broth Test
- Requirements for Pyruvate Broth Test
- Procedure of Pyruvate Broth Test
- Result and Interpretation of Pyruvate Broth Test
- Quality Control
- Precautions
- Applications of Pyruvate Broth Test
- Limitations of Pyruvate Broth Test
Introduction to Pyruvate Broth Test
- The Pyruvate Broth Test is a biochemical assay employed to evaluate bacterial capability in utilizing pyruvate and generating metabolic acids. Pyruvate, derived from glycolysis, serves as a precursor for various metabolic reactions, yielding diverse products like metabolic acids, ethanol, or acetyl CoA.
- Bacteria exhibit differential pyruvate metabolism, yielding distinct end products. To discern between pyruvate-fermenting and non-fermenting bacteria, the pyruvate broth test is utilized, particularly for distinguishing Enterococcus spp.
- This test involves a nutrient-deficient broth supplemented with sodium pyruvate. Without available carbohydrates, bacteria prioritize pyruvate metabolism over glycolysis. The resulting metabolic end products dictate test outcomes, aiding in bacterial identification and classification.
Objectives
- To evaluate bacterial capacity for utilizing pyruvate and generating metabolic acid, aiding in distinguishing between Enterococcus faecalis and Enterococcus faecium.
Principle of Pyruvate Broth Test
- Some bacteria possess the ability to metabolize pyruvate, yielding organic acids such as lactic acid and oxaloacetic acid, while others lack this capability or do not produce acid as an end product.
- Acid-producing bacteria cause a decrease in the pH of the medium, which is detected by the bromothymol blue indicator.
- In acidic conditions, the bromothymol blue indicator transitions from greenish-blue to yellow, resulting in a yellow-colored medium.
- This color change serves as a positive indication of bacterial pyruvate utilization and acid production.
Requirements for Pyruvate Broth Test
a. Culture Medium
Pyruvate broth serves as the medium for this test, characterized by its low-nutrient composition and absence of added carbohydrates.
Composition of Pyruvate Broth per 1000 mL:
- Pancreatic Digest of Casein: 10.00 grams
- Yeast Extract: 5.0 grams
- Sodium Pyruvate: 10.0 grams
- Sodium Chloride: 5.0 grams
- Dipotassium Phosphate: 5.0 grams
- Bromothymol Blue: 0.04 grams
- Final pH: 7.3 ±0.2 at 25°C
Preparation of Pyruvate Broth
- 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 the above) and add the mixture to a conical flask (or glass bottle) containing 1000 mL of water.
- Stir the mixture well using a magnetic stirrer or manually and heat it to boiling to ensure complete dissolution of all components in water.
- Dispense 5 mL (or desired volume) of the broth into test tubes and loosely cover them with caps or cotton plugs.
- Autoclave the test tubes at 121°C and 15 lbs pressure for 15 minutes, then allow them to cool to below 40 – 45°C before inoculation.
b. Reagents
- No additional reagents are necessary.
c. Equipment
- Test tubes
- Incubator
- Weighing Machine
- Autoclave
- Bunsen burner
- Inoculating loop
- PPE and other general laboratory materials
d. Test Organism (Sample Bacteria)
- Positive Control: Enterococcus faecalis ATCC 29212
- Negative Control: Enterococcus faecium ATCC 6569 or Streptococcus bovis ATCC 9809
Procedure of Pyruvate Broth Test
- Using a sterile inoculating loop, select well-isolated colonies of the sample bacteria from a fresh culture (preferably from blood agar medium) and inoculate the broth.
- Incubate the tubes aerobically at 35±2°C for 24 hours and observe for any color change, particularly the formation of yellow color.
- If no color change is observed, re-incubate the medium for an additional 24 hours and reevaluate for any color change.
Result and Interpretation of Pyruvate Broth Test
- A positive result is signified by a transition in the color of the broth from greenish to yellow.
- A negative result is indicated by the absence of any change in the color of the medium, or occasionally, by a faint yellowish-green color formation indicative of a weak reaction.
Quality Control
- Enterococcus faecalis ATCC 29212 yields a yellow-colored medium within 24 hours of incubation under ambient conditions.
- Enterococcus faecium ATCC 6569 exhibits growth, but the medium remains unchanged in color even after 48 hours of incubation.
Precautions
- Ensure that the medium does not contain free carbohydrates, as the test organisms may ferment carbohydrates, producing acid and leading to a false positive result.
- Delay reporting negative results until after 48 to 72 hours of incubation, as some strains may exhibit slow growth and fermentation.
- A slight color change to yellowish-green should be interpreted as a negative result.
Applications of Pyruvate Broth Test
The Pyruvate Broth Test serves as a method to identify Enterococcus faecalis (positive test) in clinical isolates and distinguish it from similar bacterial species such as Enterococcus faecium (negative test).
Limitations of Pyruvate Broth Test
- Its application is highly restricted, primarily suitable for Enterococcus and Streptococcus spp.
- However, it does not provide comprehensive identification and necessitates additional biochemical test results to confirm bacterial identity.
