Biochemical Characterization of Bacteria

1. LECTURE 9: Biochemical Characterization of Bacteria Microbiology and Virology; 3 Credit hours Atta-ur-Rahman School of Applied Biosciences (ASAB) National University of Sciences and Technology (NUST)

2. Indole Test Principle: • Demonstrates the ability of certain bacteria to decompose the amino acid tryptophan present in peptone water to indole. • Indole is then tested for by adding few drops of Kovac’s reagent which gives a pink ring in the presence of indole.

3. Indole Test • Procedure: • The organism is inoculated in peptone water and after incubation at 37 degree for 24 hours, Kovac’s reagent is added. • Interpretation: • If a pink ring is produced, the organism is indole +Ve (E. coli). • If a yellow ring is produced, the organsim is indole –Ve (Klebsiella).

4. Voges-Proskauer’s reaction (VP) Principle: • Some bacteria ferment glucose with production of acetyl methyl carbinol. Procedure: • Bacteria is grown in glucose phosphate peptone water for 48 hours. • Then KOH is added to test for acetyl methyl carbinol (acetoin) formation. Interpretation: • If an eosin pink color is produced, VP reaction is +Ve.

5. acetyl methyl carbinol acetoin dehydrogenase

6. Voges-Proskauer’s reaction (VP) Eosin pink color

7. Citrate Utilization Test • Bacteria are inoculated on a medium containing sodium citrate and a pH indicator such as bromothymol blue. • The medium also contains inorganic ammonium salts, which are utilized as sole source of nitrogen. • Use of citrate involves the enzyme citritase, which breaks down citrate to oxaloacetate and acetate. • Oxaloacetate is further broken down to pyruvate and carbon dioxide (CO2). • Production of sodium bicarbonate (NaHCO3) as well as ammonia (NH3) from the use of sodium citrate and ammonium salts results in alkaline pH. • This results in a change of the medium’s color from green to blue.

8. Citrate Utilization Test Sodium Citrate + amonia salt Sodium bicarbonate + Amonia gas + Alkaline pH Change of the medium’s color from green to blue is positive test Change of the medium’s color from green to blue is positive test

9. Urea Utilization Test • This medium is especially recommended for the differentiation of Proteus species from Salmonella and Shigella species in the enteric infection diagnosis, based on urea utilization. • Gram negative enteric bacilli are unable to utilize urea. • Urea Broth becomes alkaline as the utilization of urea by the organisms liberates ammonia during the incubation, indicated by pink red colour. • A medium without urea serves as negative control to rule out false positive results. • Also, all urea test media rely on the alkalinity formation and so they are not specific for determining the absolute rate of urease activity.

10. Urea Utilization Test Pink red colour is positive test

11. Sugar Fermentation Test • Carbohydrate fermentation tests detect the ability of microorganisms to ferment a specific carbohydrate. • Fermentation patterns can be used to differentiate among bacterial groups or species. • For example, all members of the Enterobacteriaceae family are classified as glucose fermenters because they can metabolize glucose anaerobically • Within this family however, maltose fermentation differentiates Proteus vulgaris (positive) from Proteus mirabilis (negative

12. Sugar Fermentation Test • Phenol red is commonly used as a pH indicator in carbohydrate fermentation tests because most of the end-products of carbohydrate utilization are organic acids

13. Sugar Fermentation Test Peptone media with phenol red indicator. From left to right: uninoculated tube; Escherichia coli, a glucose fermenter with gas production (visible air bubble in the inverted Durham tube); Shigellasonnei, a glucose fermenter without gas production (no visible air bubble in the inverted Durham tube); Pseudomonas aeruginosa, non fermenter.

14. Sugar Fermentation Test Peptone media with bromocresol purple indicator. From left to right: uninoculated tube; E. coli, glucose fermenter with gas production (visible air bubble in the inverted Durham tube); S. sonnei, glucose fermenter without gas production (no visible air bubble in the inverted Durham tube); P. aeruginosa, nonfermenter

15. Analytical profile index (API) Principle: • It is composed of a plastic strip with cupules containing dehydrated substances. Each cupule has a small hole at the top. Procedure: • A saline suspension of the test organism is dropped in the cupules. • The strip is covered with a lid and placed in a humidified plastic chamber and incubated at 37 degree for 24-48 hours. Interpretation: • Biochemical profiles are determined by reading the color change and interpret according to the available charts. • These are then converted to numerical codes which will be read from a profile index to identify the bacteria.

16. Analytical profile index (API)

18. Analytical profile index (API)