Objectives of Pyruvate Broth Test
- To determine the ability of an organism to utilize pyruvate.
- It aids in the differentiation between Enterococcus faecalis and Enterococcus faecium.
Principle of Pyruvate Broth Test
In the identification of microorganisms, the widely used method is the ability of bacteria to form organic compounds by metabolizing certain carbohydrates and related compounds. Pyruvate broth is a carbohydrate-free, nutrient-limited medium. Pyruvate Broth contains casein peptone, sodium chloride, yeast extract, dipotassium phosphate, sodium pyruvate, and bromothymol blue. Casein peptone and yeast extract provide the nutrients and growth factors necessary for growth and the sodium chloride maintains osmotic balance. Sodium pyruvate is added as the fermentable ingredient and dipotassium phosphate is added as a buffering system. Bromothymol blue is the acid-base indicator and it is greenish-blue at an alkaline pH, and shifts to yellow when acid is produced during the fermentation of the pyruvate. Pyruvate is the active substrate and those organisms that can utilize pyruvate can breakdown the substrate generating metabolic acids which is detected by the pH indicator bromothymol blue. Acid production causes a decrease in pH which results in a color shift in the medium. When sufficient acid is generated the medium changes from a blue-green coloration to yellow. No color change in the medium is indicative of the negative reaction.
Media (Pyruvate broth)
Pancreatic digest of casein (10 gm/L),
sodium pyruvate (10 gm/L),
yeast extract (5 gm/L),
dipotassium Phosphate K2HPO4 (5 gm/L),
NaCl (5 gm/L),
bromothymol blue (0.04 gm/L),
pH 7.3 +/- 0.2 at 25ºC.
Procedure of Pyruvate Broth Test
- Inoculate the pyruvate broth tubes with an 18-24 hour culture of the organism.
- Incubate tubes at 35°-37°C in ambient air.
- Examine tubes daily for up to 5 days for the color change in the medium.
Results of Pyruvate Broth Test
Positive test: Color change of broth from green to yellow
Negative test: No color change
Weak reaction: yellow-green coloration and should be regarded as negative
Positive: Enterococcus faecalis ATCC29212
Negative: Enterococcus faecium ATCC 6569
Negative: Streptococcus bovis ATCC9809
- Tille P.M. 2014. Bailey and Scott’s diagnostic microbiology. Thirteen editions. Mosby, Inc., an affiliate of Elsevier Inc. 3251 Riverport Lane. St. Louis. Missouri 63043
- MacFaddin, JF. Biochemical Tests for the Identification of Medical Bacteria, 3rd ed. Philadelphia: Lippincott Williams & Wilkins, 2000.