Jakes Microbiology LAB #2
| A | B |
|---|
| Is the Oxidase test Differential or Selective? | Differential |
| Is the Citrate test Differential or Selective? | Differential |
| Is the Phenylethyl alcohol test Differential or Selective? | Selective. |
| Is the Eosin Methylene blue agar Differential or Selective? | Both |
The active substrate in oxidase reagent, N,N,N,N- tetramethyl-p-phenylenediamine dihydrochloride, acts as an artificial electron acceptor for the enzyme oxidase and is oxidized to form the colored compound Wurster's blue. Wurster's blue is a purple compound that is readily visible and signifies a positive reaction.
The oxidase reagent contains a chromogenic reducing agent, which is a compound that changes color when it becomes oxidized. If the test organism produces cytochrome oxidase, the oxidase reagent will turn blue or purple within 15 seconds.
What is the purpose of the oxidase test? used to identify bacteria containing the respiratory enzyme cytochrome C oxidase.
The IMViC tests are a group of individual tests used in microbiology lab testing to identify an organism in the coliform group. The presence of some coliforms indicate fecal contamination. The term "IMViC" is an acronym for each of these tests.
1. OXIDASE TEST BY, Dr.M.Malathi. INTRODUCTION; ï‚¢ It is a biochemical test ï‚¢ Used for the identification of bacteria. 3. PRINCIPLE: ï‚¢ Cytochromes are the iron containing hemoproteins .
Test Procedure
- Dilute your organism in a tube of sterile water to obtain a turbidity equivalent to the 0.5 McFarland test standard.
- Using a sterile 1mL pipette, place 1 mL of organism into the middle of the tube.
- Cap tightly; do not jostle.
- Incubate for 24 hours at 37°C.
Add one drop of hydrogen peroxide and look for bubbles. Bubbles are a positive result for the presence of catalase. If no bubbles form, it is a negative result; this suggests that the organism does not produce catalase. Dispose of the slide in a slide disposal bin and any toothpicks in a biohazard bin.
Principle. Oxidase Test is based on the principle that certain bacteria produce indophenol blue from the oxidation of dimethyl-p-phenylenediamine and α-naphthol. In presence of the enzyme cytochrome oxidase (gram-negative bacteria), the N,N-dimethyl-p-phenylenediamine oxalate and α-naphthol react to indophenol blue.
The oxidase test is used to determine if an organism possesses the cytochrome oxidase enzyme. The test is used as an aid for the differentiation of Neisseria, Moraxella, Campylobacter and Pasteurella species (oxidase positive). It is also used to differentiate pseudomonads from related species..
-By adding oxidase test reagent (tetraethyl-p-phenylenediamine dihydrochloride) to a bacterial culture growing on a plate medium (the surface is exposed to air, providing oxygen for aerobic cellular respiration).
In the oxidase test, the reagent becomes the source of the electrons. It is a chromogenic reducing agent (CRA). When this happens it will change color even when no cytochrome c oxidase is present. Thus, it is very important that you read this test within 20 seconds of applying the reagent.
catalase test. Tests for the presence of catalase enzyme that converts hydrogen peroxide (H2O2) into water and oxygen gas. oxidase test. Used to determine if a bacterium has enzyme cytochrome oxidase. The reagent is colorless if negative and turns blue/purple when oxidized.
The oxidase test is a key test to differentiate between the families of Pseudomonadaceae (ox +) and Enterobacteriaceae (ox -), and is useful for speciation and identification of many other bacteria, those that have to use oxygen as the final electron acceptor in aerobic respiration.
Enterobacteriaceae are typically oxidase negative, meaning they either do not use oxygen as an electron acceptor in the electron transport chain, or they use a different cytochrome enzyme to transfer electrons to oxygen.
-The reagents of this test are unstable, therefore they may oxidize independently shortly after becoming moist. Therefore it is important to read within 30 seconds.
What happens to the oxidase reagent after 20 seconds? The reagent will oxidize independently shortly after becoming moist, giving a false positive. This can happen before or after the 20 seconds because the reagents are unstable and could oxidize independently, but the oxidase test usually works best in 20 seconds.
An exoenzyme, or extracellular enzyme, is an enzyme that is secreted by a cell and functions outside that cell. Bacteria and fungi also produce exoenzymes to digest nutrients in their environment, and these organisms can be used to conduct laboratory assays to identify the presence and function of such exoenzymes.
N,N,N′,N′-tetramethyl-p-phenylenediamine dihydrochloride (TMPD) is an oxidizable compound that serves as a reducing co-substrate for heme peroxidases. It is also used as a test reagent in microbiology for the classification of cytochrome oxidase positive aerobic microorganisms.
E. coli bacteria are among the few species of lactose (LAC)-positive, oxidase-negative, gram-negative rods that are indole positive.
The medium used with this reagent is either broth containing tryptophan, motility- indole-ornithine agar, or sulfide-indole-motility agar (SIM).
