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Microbiology
Lab - Biochemical Tests (Part I)
| Question | Answer |
|---|---|
| 7 things to know about biochemical tests (part 1) | 1. name of test/medium 2. ingredients in medium for test 3. how to inoculate/incubate medium for test 4. how to perform test (reagents/procedures used) |
| 7 things to know about biochemical tests (part 2) | 5. how to read test results (how +/- test results look) 6. chemical reaction during test process 7. what results mean in terms of metabolic processes of the organism (metabolic pathway/enzyme indicated) |
| What are the 6 tests conducted (03/02/2010)? | 1. catalase test 2. gelatine liquefaction test 3. oxidase test 4. phenol red carbohydrate fermentation test 5. methyl red & voges-proskauer tests 6. nitrate reduction test |
| What is the purpose of the catalase test? | It's a procedure to detect the presence of the catalase enzyme that enables bacteria to live in presence of oxygen. In this test, it's all about getting rid of hydrogen peroxide |
| What is the medium used in the catalase test? | Nutrient agar |
| How do you inoculate the medium in the catalase test? | By streaking and incubating @ 35 degrees C |
| How do you perform the catalase test? | Drop hydrogen peroxide onto slide to check for bubbling |
| How do you read the catalase test? | If bubbling occurs, it means the bacteria has the catalase enzyme (POSITIVE!) |
| What happens chemically in the catalase test? | 2 H2O2 ---- catalase ----> 2H2O + O2 What does the results of the catalase test mean in terms of metabolic processes? |
| What is the purpose of the gelatin liquefaction test? | It's a procedure to test if an organism can break down gelatin and produce gelatinases |
| What is the medium used in the gelatin liquefaction test? | Nutrient gelatin |
| What ingredients are in nutrient gelatin? | Gelatin |
| How do you inoculate the medium in the gelatin liquefaction test? | By stabbing nutrient gelatin tube with a needle and incubating at 35 degrees C |
| How do you perform the gelatin liquefaction test? | Allow the nutrient gelatin to get COLD (refrigerate for ~1.5 hours) before reading |
| How do you read the gelatin liquefaction test? | Solid = negative, Liquid faction = positive |
| What is the purpose of the oxidase test? | Test to see if bacteria contains the respiratory enzyme cytochrome c oxidase |
| What is the medium used in the oxidase test? | Trypticase soy agar plate |
| What ingredients are in the trypticase soy agar plate? | Enzyme tryptin from pancrease, no carbohydrates |
| How do you inoculate the medium in the oxidase test? | By streaking soy agar plate and incubating at 35 degrees C |
| How do you perform the oxidase test? | By adding the chromogenic reducing agents to the bacteria |
| What is the name of the chromogenic reducting agent called in the oxidase test? | N,N tetramethyl-p-phenylenediamine dihydrocholoride |
| How do you read the oxidase test? | Purple - POSTIVE, No color change = NEGATIVE |
| What is the purpose of the cytochrome c oxidase enzyme? | associated to electron transport chain. if oxidase negative = no electron transport OR have electron transport but no cytochrome-c |
| What is the purpose of the carbohydrate fermentation test? | Tests to see if bacteria can ferment certain carbohydrates (mainly sugar) |
| What is the definition of fermentation? | the process by which organisms take carbohydrate, breaks it down, and produce acid products |
| What kind of medium is used in the carbohydrate fermentation test? | Phenol red carbohydrate broths |
| What ingredients are in phenol red carbohydrate broths? | 1. carbohydrate 2. peptones (digestive proteins) 3. phenol red - pH indicator, changes color according to pH "yellow" <= 6.8 |
| How do you inoculate the phenol red carbohydrate broths? | Loop inoculation and incubate @ 35 degrees C |
| What are the 3 specific phenol red carbohydrate broths used? | Phenol red 1. glucose 2. lactose 3. sucose |
| How do you read the fermentation test? | "Yellow" = fermented (POSITIVE) |
| What is the purpose of the Methyl Red Test & Voges-Proskauer Test? | To detect 2 different types of fermentation |
| What medium is used in the Methyl Red Test & Voges-Proskauer Test? | MRVP medium |
| What ingredients does MRVP medium contain? | 1. glucose carbohydrates 2. peptones |
| What is the purpose of the MR test? | looks for mixed acid fermentation (lowers pH a LOT) |
| What is the purprose of the VP test? | looks for 2-3 butanediol fermentation |
| How is the MRVP medium inoculated? | Loop inoculation and incubate @ 35 degrees C |
| How do you perform the MR test? | add 5 drops of methyl red (pH indicator) |
| How do you read the results of the MR test? | "RED" when acidic, pH <= 4.4 |
| How do you perform the VP test? | Add 10 drops of Barritts A and 10 drops of Barritts B, mix frequently, wait 1.5 hours |
| What is Barritts A and Barritts B? | alpha naphthol & calcium hydroxyl |
| How do you read the results of the VP test? | "RED" color is positive |
| T/F in the MRVP test, there is usually one positive and one negative | True, but both can be negative as well |
| What is the purpose of the nitrate reduction test? | test bacteria to see if it reduces nitrate to nitrite |
| What does "reduction" mean? | Accepting electrons (opposite of oxidation) |
| What kind of medium is used in the nitrate reduction test? | Nitrate broth |
| How do you inoculate the nitrate broth? | Loop inoculation, incubate @ 35 degrees C |
| How do you perform the nitrate reduction test? | Add 2 reagents 1. Nitrate A (sulfamilic acid) 2. Nitrate B (dimethyl alpha naphthylamine) to tubes |
| How do you read the nitrate reduction test? | If "RED" ==> POSITIVE |
| What do you do when the nitrate reduction test shows up as NEGATIVE? | Confirm the negative by addding Nitrate reagent C (zinc dust) |
| What does zinc dust do? | Catalyzes reduction of nitrate to nitrite |
| How do you read the results of nitrate reagent C? | If "RED" ==> NEGATIVE, else "NO COLOR" = POSITIVE |
| What is the chemical reaction in nitrate reduction? | NO3 (nitrate ion) --(remove water)--> NO2 (nitrite) --(remove water)-->N2 (gaseous nitrogen) |
Created by:
lophung
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