Objective: To determine the organism's oxygen requirement.
1. Inoculate 5 ml of BHI broth with your unknown organism and incubate overnight. We have found that broth cultures provide much more accurate results than using inoculum from a plate. However, if you are inoculating from a plate, make sure you use a very light inoculum.
2. Obtain a thioglycollate tube and make sure that it does not have more than 20% of the medium in pink color. This may happen due to oxidation of the top layer of the medium. To restore anaerobic conditions, such a tube should be placed in boiling water for 10 minutes and then cooled to room temperature. If you do not see any pink color against a white background, the tube is good to use.
3. Use a sterile narrow thin needle (rather than a thick one), insert into your culture broth and slowly stab a thioglycollate tube to the bottom. Carefully remove the needle along the same stab line. Do not shake the tube or move the needle around, or you will introduce extra oxygen into the medium. The needle should reach all the way to the bottom of the tube.
4. Incubate the tube at 30°C (without any regard to the optimum temperature requirement of your species) for 24 hours before reading the tube.
-- Aerobe: band of growth on the top of the tube. Some species have a tendency to grow very rapidly in thioglycollate tube so that the growth covers a rather thick band from the top and extends to the line of stab where there is oxygen available (brought in by the needle). So it is best to look at the bottom 1-cm of the tube and if it is clear with no growth whatsoever, then you can be sure that you have an aerobe.
-- Microaerophile: band of no growth at the top, then a band of growth extending a short distance down proceeded by no growth to the bottom. The bottom 3-cm of the tube should be clear of any growth.
-- Facultative Anaerobe: growth can occur either throughout the tube or can begin at some point below the surface and extend all the way to the bottom, even in the 1-cm bottom of the tube.
-- Anaerobe: growth only at the bottom fifth of the tube.