Do a lab summary of minimum 300 words.
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Note: these tests are unique because the organism is not grown on a particular medium prior to viewing the result of the test. These tests are performed immediately, therefore fresh cultures MUST be used. In regard to culture prep for this lab, it is best to use cultures grown on plates (TSA) rather than on slants.
Cytochrome C oxidase test
All respiring organisms have one or more enzyme/cytochrome complexes at the terminal end of their electron transport system (ETS). These complexes, referred to as terminal oxidases, function to pass electrons from the ETS to the terminal electron acceptor. Most organisms (including you and me) possess cytochrome C oxidase as one of their terminal oxidases. The oxidase test allows us to determine if the organism being tested possesses cytochrome C oxidase (considered an "oxidase positive" organism). This test is commonly used in diagnostic Microbiology to differentiate between 2 families of Gram negative rod shaped bacteria; the Enterobacteriaceae which are oxidase negative and the Pseudomonadaceae which are oxidase positive. For our purposes, all of the enteric species and Acinetobacter are oxidase negative, whereas Pseudomonas, Alcaligenes and Aeromonas are oxidase positive. The test is based upon the fact that cytochrome C oxidase will oxidize (remove electrons from) a chemical mixture of alpha-napthol and dimethyl-p-phenylenediamine causing it to turn from colorless or slightly blue to a deep blue color. This chemical mixture is otherwise known as "oxidase reagent".
It is critical that you start with fresh cultures (~24 hrs.) of the organisms to be tested. Collect enough cells from an isolated colony (if possible) on a sterile cotton swab so that you can see the growth - maybe a spot about as big as the lead of a pencil or a mustard seed. Place a drop of fresh oxidase reagent (mixed immediately before conduction the test) directly on the growth. A color change of the cells (NOT the swab) to dark blue within 10 seconds constitutes a positive reaction for the oxidase test. Color changes after an extended time period are considered negative reactions. You can also conduct this test by spreading the cells on a small piece of filter paper to which you add the drop of oxidase reagent. See image “Cytochrome C oxidase: left positive.”
NOTES:
* FRESH cultures must be used for this test.
*We will only use this test to differentiate Gram negative rods.
* Oxidase reagent is highly photosensitive and must be sheltered from the light. You must use fresh
reagent mixed in distilled water. Dark purple reagent is no good. Make some more.
* A result is only considered positive if a purple color change (of the CELLS, NOT the swab) occurs
within 10 seconds!
Catalase test
Although aerobically respiring organisms rely on oxygen as a terminal electron acceptor, oxygen causes problems for some organisms. During aerobic respiration, oxygen is reduced to water by the following reaction: O + 2H+ + 4 electrons H2O. The partially reduced forms of oxygen (oxygen with 1, 2 or 3 added electrons) are types of free radicals called reactive oxygen species (ROS). If not detoxified, these ROS will “freely” remove electrons from the first thing they find, perhaps proteins or even nucleic acids (mutation?). This is bad! Aerobically respiring organisms have 2 enzymes that protect them from these ROS. Superoxide dismutase (SOD) takes 2 molecules of superoxide (O with 1 added electron) adds them to 2 protons to yield hydrogen peroxide (2 O- + 2H+ H2O2). Hydrogen peroxide is also an ROS, but never fear. The second enzyme, catalase, turns peroxide into harmless oxygen and water by the following reaction: 2H2O2O2 + 2H2O.
The catalase test is used to determine whether an organism that is growing in air is doing so by the process of aerobic respiration (including the obligate aerobes and facultatives) or if that organism is an aerotolerant anaerobe like Streptococcus. Organisms capable of aerobic respiration are catalase positive and those that are not are catalase negative. In a way, catalase is a variation of the oxygen requirements test, and it is highly . Although this test can be used on all kinds of bacteria, we will only use it to separate our Gram positive cocci into 2 groups: Staphylococcus species are catalase positive and Streptococcus species are catalase negative.
Start with fresh cultures (~24 hrs.) of the organisms to be tested. Test a Staphylococcus species as a catalase positive control and a Streptococcus species as a negative control. Using your loop, place a healthy wad of cells on opposite ends of a microscope slide, Staph on one end and Strep on the other. Spread the cells out to the size of a BB. Immediately (before the cells dry out) place 1 drop of fresh H2O2 on each spot of cells. If the mixture bubbles vigorously, the culture is catalase positive. If not, the culture is catalase negative. The bubbling is due to released oxygen gas. See image “Catalase: right positive.”
NOTES:
* FRESH cultures must be used for this test.
* We will only use this test to differentiate Gram positive cocci.
* DO NOT add water to the cells on the slide. You are NOT making a smear for Gram stains here.
* Add the peroxide quickly after placing the cells on the slide. DO NOT let the cells dry up.
