Biochemical tests

1. Biochemical tests

2. Tests To Know • Case Study Tests • Methyl Red/ Voges Proskauer • Indole • Citrate • H2S production in SIM • Urea hydrolysis • Motility • Lactose fermentation • Sucrose fermentation • Glucose fermentation & gas production Mohammed Laqqan

3. Introduction • Microbial metabolic processes are complex, but they permit the microbiologist to distinguish among microorganisms grown in culture. Many clinical pathogens can be identified by inoculating pure cultures into media that contain one or more specific biochemicals. • The biochemical reactions that take place can then be determined by relatively simple indicator reagents, included in the medium or added to the culture later. • Some bacteria ferment carbohydrates, producing acidic, alcoholic, or gaseous end products. • Many different species are distinguished on the basis of the carbohydrates they do or do not attack, as well as by the nature of end products formed during fermentation. Mohammed Laqqan

4. The nature of products formed in amino acid metabolism also provides information as to the identification of bacterial species. • The production of visible pigments distinguishes certain types of bacteria (pseudomonas, serratia). • Working with pure cultures freshly isolated from clinical specimens, the microbiologist uses a carefully selected battery of special media to identify their outstanding biochemical properties. Mohammed Laqqan

5. Fermentation of carbohydrates • Carbohydrates are complex chemical substrates which serve as energy sources when broken down by bacteria and other cells. They are composed of carbon, hydrogen, and oxygen (with hydrogen and oxygen being in the same ratio as water; [CH2O]) and are usually classed as either sugars or starches. • Facultative anaerobic and anaerobic bacteria are capable of fermentation, an anaerobic process during which carbohydrates are broken down for energy production. • A wide variety of carbohydrates may be fermented by various bacteria in order to obtain energy and the types of carbohydrates which are fermented by a specific organism can serve as a diagnostic tool for the identification of that organism. Mohammed Laqqan

6. We can detect whether a specific carbohydrate is fermented by looking for common end products of fermentation. When carbohydrates are fermented as a result of bacterial enzymes, the following fermentation end products may be produced: • Acid end products. • Acid and gas end products. Mohammed Laqqan

7. Carbohydrate Utilization • Bacteria produce acidic products when they ferment certain carbohydrates. The carbohydrate utilization tests are designed to detect the change in pH which would occur if fermentation of the given carbohydrate occurred. Acids lower the pH of the medium which will cause the pH indicator (phenol red) to turn yellow. If the bacteria do not ferment the carbohydrate then the media remains red. If gas is produced as a by product of fermentation, then the Durham tube will have a bubble in it. • The carbohydrate tests are the: • Glucose (Dextrose) test • Lactose test • Sucrose test • All carbohydrate test media should be inoculated with the transfer loop. Left tube shows less acid formation than far right tube, but gas is still made Center shows no carbohydrate utilization to produce acid or gas. Right tube shows acid was produced as evidenced by the yellow color, and gas was made (look at the bubble in the Durham tube) Mohammed Laqqan

8. MR-VP tests • This test is used to determine two things. • The MR portion (methyl red) is used to determine if glucose can be converted to acidic products like lactate, acetate, and formate. • The VP portion is used to determine if glucose can be converted to acetoin. • These fermentation tests are used to differentiate between certain intestinal bacteria called coliforms. • The medium contains dextrose as the carbohydrate source. Some coliforms will ferment the dextrose to acid products that will cause the pH to drop below pH 5. This is called mixed acid fermentation. After incubation the addition of methyl red, a pH indicator which turns red below pH 4.4, will indicate whether such fermentation has occurred. • Other coliforms will convert dextrose to less acidic products such as ethanol or butanediol. These bacteria are negative in the methyl red test. Mohammed Laqqan

9. Butanediol fermentation is demonstrated by the Voges-Proskauer test which measures the presence of acetoin (acetyl methyl carbinol), a precursor to butanediol. • This test uses the same medium as the methyl red test and both tests are usually performed in parallel. • Barritt's reagents, alpha-naphthol and potassium hydroxide, are added to a 48 hour culture and the tube is shaken to aerate the solution. The development of a pink or red color after agitation is a positive reaction for the production of acetoin. Mohammed Laqqan

10. How to Perform Tests: Inoculate 2 MR-VP broths with inoculating loop. One tube of culture will be used to conduct the MR test, the second tube serves as the VP test. After 48 hours of incubation, add a few drops of MR to one tube, and VP reagents to the other tube. • Properties they test for: Both tests are used to help differentiate species of the family Enterobacteriaceae. • MR—tests for acid end products from glucose fermentation. • VP—tests for acetoin production from glucose fermentation. • Media and Reagents Used: • MR-VP Broth • Methyl Red indicator for acid • Voges Proskauer reagents—A: 5% Alpha-Naphthol, B: Potassium Hydroxide, Mohammed Laqqan

11. Results • MR (methyl red) test: Methyl red is added to the MR tube. A red color indicates a positive result (glucose can be converted into acidic end products (indicating pH below 6) . A yellow color indicates a negative result, glucose is converted into neutral end products (indicating no acid production) Mohammed Laqqan

12. VP (Vogues Proskauer) test: First alpha-napthol (also called Barritt’s reagent A) and then potassium hydroxide (also called Barritt’s reagent B) are added to the VP tube. The culture should be allowed to sit for about 15 minutes for color development to occur. If acetoin was produced then the culture turns a red color (positive result); if acetoin was not produced then the culture appears yellowish to copper in color (a negative result). Mohammed Laqqan

13. End of lecture Mohammed Laqqan