INTRODUCTION:

In this exercise you will be working with a partner to observe positive and negative results of two biochemical tests. Biochemical reactions are, of course catalyzed by enzymes. Subtrates are converted into products. Sometimes, reagents must be added after incubation of the organism to detect the presence of the product. Other time, reagents are included in the medium. Since each enzyme is a protein which is coded for by a gene, biochemical reactions actually give us partial genetic profiles of bacteria. These biochemical tests can provide information which can be very useful in identifying bacteria.

You and your partner will perform each biochemical test on two different organisms; one which will produce a positive result and one which will provide a negative result. A description of these tests and the necessary media follows.

CATALASE

medium: TSA (plate or slant)
NOTE: STOCK PLATE CAN BE USED

substrate: hydrogen peroxide (not in the medium)
products formed: oxygen
reagent: one drop of 3% hydrogen peroxide is added to one colony on a glass slide (DISCARD IN BEAKER OF DISINFECTANT) or on a plate you can discard
positive reaction: BUBBLES observed
negative reaction: no bubbles observed
purpose of the test: determine the presence of the enzyme catalase (converts H2O2 to H2O and OXYGEN)

OXIDASE

medium: TSA plate (a fresh stock plate may be used)
substrate: none (reagent gives electrons to cytochrome c and becomes oxidized)
products formed: none
reagent: oxidase test reagent (a swab is used to transfer bacteria to a filter paper to which the reagent has already been applied)
positive reaction: the organism turns DARK PURPLE color in less than 30 seconds
negative reaction: no color (or only slight color) change in 30 seconds
purpose of the test: detect the presence of the plasma membrane bound cytochrome c (an electron transport protein)