General Biochemical Tests

Identifying Unknown Bacteria


This lab should help give you the background information and techniques you will need to successfully perform general biochemical tests in order to help identify unknown bacterial samples. The micro lab website, your textbook, the web and assorted books available in lab will be the reference materials necessary for you to successfully complete the next several weeks of lab work.

  • You will perform general biochemical tests on an unknown organism.
  • For each biochemical test you perform, make sure to record the following in your lab book:
    • What does a positive test result look like?
    • What is the biochemical basis of the test?

Lab Procedure

We have included the basic procedure for doing each biochemical test in the table below. You will find more specific procedures for each biochemical test on the following pages. More complete information on selective & differential media can be obtained by consulting the Difco manuals in lab. You will need to look up the individual test for a more detailed description, including the biochemical basis of each test.

Table 1: Brief Description of general tests and probable results.

Test Brief Instructions Probable Results
TSA/BHI Staphs/Enterics on TSA; Streps on BHI Determine macromorphology
Gram Stain To confirm culture purity Staphs/Streps (Gram+), Enterics (Gram-)
Motility Stab with a needle straight in and straight out of the center of the tube half way down.
Incubate for 24 hours at 37°C.
Staphs/Enterics in O2; Streps in CO2.
Motile organisms have obvious growth away from inoculation area; Non-motile organisms grow only in inoculation area.
McFarland Standard Dilute your organism in a tube of sterile water to obtain a turbidity equivalent to a 0.5 McFarland test standard. Hold your diluted tube and the 0.5 McFarland test standard against the black-lined McFarland reference card to accurately rate the turbidity.
FTM Use a sterile transfer pipette to add 1 mL of your McFarland standard organism in the middle of the tube. Cap tightly; do not jostle. Incubate for 24 hours at 37°C. Strict aerobes will grow near the top of the media; Facultative anaerobes will grow throughout; Strict anaerobes will grow near the bottom.
Catalase Transfer a well-isolated colony to a clean slide & add 1 drop of 3% H2O2. Do not reverse the order & do not mix. Observe for immediate bubble formation. Staphs/Enterics are catalase positive; bubble formation should occur. Note: do not take colony from a blood plate.
Oxidase Add a few drops of oxidase reagent onto Whatman filter paper. Smear with a loop-full of organisms. Use colonies from low glucose, non-selective media.  Look for appearance of purple color within 30 seconds.