The procedures to use in culturing micooganisms depend on which types of organisms you wish to study.
To culture bacteria, the following media should be used to prepare agar plates:
Growth Media: 1/10-strength Trypticase Soy Agar (TSA) Media:
Ingredients:
2 g Trypticase Soy Agar
7.5 g Bacto Agar
500 ml distilled water
Mix the ingredients, autoclave for 20 minutes, and pour into sterile petri dishes.
Plate out the bacteria using a 10-7 dilution starting with 5 g dry weight of the compost in 45 ml of an autoclaved .06M NaHPO4/NaH2PO4 buffer of 7.6 pH. (approximately 4:1 dibasic:monobasic). Put this first dilution in a blender for 40 sec. at high speed.
Perform serial dilutions to 10-7 and add 0.1 ml of the dilution per plate. Incubate at 28C for 4 days.
Count the colonies as colonies per unit after 4 days. Prepare slides of specific colonies the same day.
Growth Media: 1/50-strength TSAPoly B
Ingredients:
0.4 g Typticase Soy Agar
10.0 g Bacto Agar
500 ml distilled water
10 mg Polymixin B in 10 ml 70% Ethanol
Mix the first 3 ingredients, autoclave for 20 minutes, and cool to room temperature. Add the antibiotic and pour into sterile petri dishes.
Plate out the actinomycetes using a 10-7 dilution starting with 5 g dry weight of compost in 45 ml of the autoclaved buffer. Put this first dilution in a blender at high speed for 40 sec.
Perform serial dilutions to 10-7 and add 0.l ml of the final dilution to each plate.
Incubate the plates at 28C for 14 days.
Take counts and samples of actinomycetes colonies after 14 days. Many of the colonies will look powdery white. However, some may take on a rough appearance and produce a variety of pigments.
Note: If you are comparing mesophilic compost to thermophilic compost, you should prepare double the usual number of plates so that you can incubate plates at both 28C and 50C.
Growth Media: 1/3-strength PDARP
Ingredients:
6.5 g Potato Dextrose Agar
5.0 g Bacto Agar
500 ml distilled water
15 mg Rifampicin in 10 ml Methanol
15 mg Penicillin G in 10 ml 70% Ethanol
Mix the first 3 ingredients, autoclave for 20 min. and cool to room temperature. Add the antibiotics and pour into sterile petri dishes.
Plate out the fungi using a 10-4 dilution starting with 5 g dry weight of compost in 45 ml of the autoclaved phosphate buffer. Put this first dilution in a blender at high speed for 40 sec.
Perform serials dilutions to 10-4 and add 0.l ml of the final dilution to each plate.
Incubate the plates at 28C for 3 days.
Take counts and samples of fungal colonies at 3 days.
Preparing Slides of Microorganisms
These protocols were written by Elaina Olynciw, biology teacher at A. Philip
Randolph High School, New York City, while working in the laboratory of Dr.
Eric Nelson at Cornell University as part of the Teacher Institute of Environmental
Sciences.
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