Use the quadrant steak method to isolate the unknown bacteria so it can be used for biochemical testing.
Sterilize the inoculating loop by passing it through a Bunsen burner flame or inside a bacteria incinerator. Once sterilized place the loop into the tube containing the unknown bacteria sample. Place the inoculating loop on one section of a nutrient rich agar petri dish plate; don't spread the sample over the entire plate. Resterilize the loop and instead of getting bacteria from the sample, spread some of the bacteria from the region just plated, known as quadrant one. Once a new area of the plate is streaked (this will be quadrant two), repeat steps for a third and fourth quadrant. The fourth quadrant should take up the most space on a plate since this is where more isolated colonies of the bacteria will grow.
Once the bacteria sample seems dry on the plate, place the lid back on the petri dish, invert it and store for incubation. If it is not known which temperature the bacteria grows at, streak two petri plates and place one in the incubator (37 degrees C) and one on the lab shelf (for room temperature).
After incubation, isolated colonies of the unknown bacteria should be present in the fourth quadrant. If the known bacteria material contains more than one strain of bacteria, different colony morphology may be seen such as red or yellow colonies. Take the sterilized inoculation loop and place it on an isolated colony. Once it adheres to the loop place the colony in a nutrient rich broth tube. Place the tube cap on and incubate in the same location the plate was incubated. The bacteria in the broth tube should just be one bacteria strain since just those colonies were taken off the plate.
The broth tube of isolated bacteria now can used for further biochemical tests. However, different tests are used for different bacteria, such as Gram + and Gram - bacteria. Use the Gram stain according to the instructor's procedure to determine what the unknown bacteria is. Gram + bacteria have a thicker cell wall and stain purple; Gram - have a thinner cell wall and stain pink.
Inoculate different types of biochemical tests to determine characteristics of the bacteria strain. For example, if the bacteria is spread on an Eosin Methylene Blue plate and the bacteria is purple in color this indicates it can ferment lactose; if it does not it will appear pink on the plate. Using this and other biochemical tests can help narrow down what bacteria is in the unknown sample.