Pure Culture Techniques ( Jackie Reynolds )


Most specimens (from animal tissue, plant tissue, or environmental samples) will be mixed, with a variety of bacteria (or other microorganisms). A single gram of feces, for example, has over 1010 bacteria and that gram would have over 20 different bacterial species.

Particularly in a medical setting, where a patient’s body fluid or tissue (skin, blood, spinal fluid, urine) is sent to the microbiology lab for analysis, the specimens will most often be mixed. In order to identify the bacteria and run antibiotic susceptibilities on the causative agent of the infection, the microbiologist must be working with a pure culture of the bacterium. This exercise begins with a mixed culture of bacteria and will end with, hopefully, pure cultures of the 2 bacteria. Luckily, the 2 bacteria being used look different from each other when growing on agar plates. Two different types of agar plate isolation techniques will be used—streak and pour. Each method has advantages and disadvantages, and particular uses. The same mixed culture will be used for both methods. Another technique, called the spread plate, is used commonly for counts, as is the pour plate technique. It uses pre-made agar plates, with the fluid inoculum being placed on top of the agar medium. The inoculum is then spread around on the plate with a bent glass rod. That procedure will not be done today. This also gives you a chance to see how agar plates are made. Some of the TSA plates are already made: others will be made by you. This requires that liquefied agar medium (sitting in a water bath) be kept above the solidification temperature. Agar’s solidification temperature is 42 degrees C, but its liquefaction temperature is 100 degrees C. When sterilized in an autoclave or boiled, the medium will stay liquid until the temperature gets to 42 C. When that happens, the medium will solidify very fast. If it solidifies on you before you finish making your cultures, it has to be disposed of (if not used yet, it can be placed back in the sterile media racks).


ALWAYS check agar plates carefully to make sure that there are no mold or bacterial contaminants on the plate: if so, discard the plate in the autoclave bag. If you see water running on the agar plate, you can do 2 things:

• Place the agar plate upside down in the 37C incubator with the top cracked.

• Get another agar plate.

Source: http://delrio.dcccd.edu/jreynolds/microbiology/2420/files/pure%20cultures.pdf

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