Re: How can the Lactobacillus bulgaricus, Streptococcus thermophilus,
Posted By: Jorge Galazzo, Sr.Sci. Molecular Diversity-Fermentation, Microcide Pharmaceuticals, Inc. Moutain View, CA
Date: Thu Feb 13 17:11:20 1997
Message ID: 853531367.Mi
In order to determine the number of bacteria in a cup of yogurt you will need to aliquot very carefully your yogurt -that is take a representative sample of the yogurt you are studying-. Then, you will need to plate this sample onto agar plates. However, if the sample contains too many microorganisms (more than 300 per plate) the microorganisms will be too crowded and will cover the entire plate. For this reason, samples need to be diluted.
What follows is a simple protocol you may want to follow. I'm assuming you have access to a microbiology lab at your school, and that your instructor will help you with some of the basic techniques involved.
- Petri dishes with plate count agar*
- Sterile screw-capped tubes with 9 ml sterile saline
- 1 ml pipetes
- Glass spreaders
- Incubator set at 37 degrees C
*Plate count agar: casein peptone, 10 g; tryptone, 5 g; yeast extract, 5 g; glucose, 10 g; agar, 15 g; distilled water, 1000 ml. Autoclave to sterilize and pour into sterile Petri dishes.
- Take a representative sample of the yogurt you are studying (1 ml) and dilute that sample in into 9 ml of sterile saline. This is your initial yogurt sample.
- Do serial dilutions of this initial yogurt sample by pipeting 1 ml into 9 ml of sterile saline (1:10), then pipete 1 ml of this new solution into another 9 ml of sterile saline (1:100). Repeat procedure to get 1:1000 and 1:10000 dilutions.
- Pipet 0.1 ml of the 1:100, 1:1000, 1:10000 dilutions of the initial sample onto appropriately labeled Petri dishes with plate count agar.
- Using a sterile glass spreader, spread the 0.1 ml of suspension over the entire surface of the agar.
- Incubate the plates in an inverted position in the incubator for 24 to 48 hr.
- Count all colonies on plates exhibiting 50-150 colonies.
- Calculate CFU/ml in the original 1 ml of yogurt. (CFU: colony forming units)
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