Safety

General Safety Guidelines
Microbiology Safety Guidelines

Procedure 1

1. Mass 1.0 g of soil for each sample to be tested.

   Transfer each sample to 9 cm³ of sterile water. This is a 1/10 dilution. SHAKE VIGOROUSLY 50 times.

2. Perform a series of dilutions—1/10, 1/100, 1/1000, 1/10,000, 1/100,000, 1/1,000,000 (see notes on performing a soil dilution below).

3. Add 1.0 cm³ samples of each of the dilutions, 1/100,000 and 1/1,000,000, to each of two petri dishes that have been sterilized previously.

4. To each of the dishes, add 10-15 cm³ of soil-extract agar at approximately 45ºC. (refer to separate instructions for preparation of soil-extract agar). Immediately upon addition of the agar, the dishes are rotated by hand in a broad swirling motion so that the inoculum is uniformly dispersed in the medium.

5. Allow the agar to solidify and then incubate the plates at 28ºC for 7 days.

6. After 7 days of incubation, when there is growth of organisms on the two sets of plates (using soil dilutions of 1/100,000 and 1/1,000,000), examine the petri dishes carefully. Hold them up to the light and look for CLEAR zones or HALOS around actionmycetes colonies. The zone of inhibition may be small or the actinomycete colony may be completely surrounded by an area free of growth by other organisms.

Soil Dilution

1. Adding 1.0 g of soil sample to 9 cm³ to sterile water dilutes the soil to a concentration of 1:10 (soil is assumed to be a volume of 1 cm³).

2. Consecutively taking 1 cm³ of a diluted sample and adding 9 cm³ of sterile water produces the next 1:10 dilution (results would be 1:100, 1:1000, 1:10,000, 1:100,000, 1:1,000,000). To transfer 1 cm³ of any diluted sample, use a STERILE pipette with a transfer bulb. DO NOT USE YOUR MOUTH TO DRAW UP THE FLUID INTO THE PIPETTE.

Procedure 2

1. To isolate those organisms that show zones of inhibition, remove a part of an actinomycete colony with a STERILE transfer loop or needle. Aseptically transfer the material to a test tube that has been prepared with what is known as a slant containing nutrient agar. Incubate the tubes at 28ºC until there is good growth. Mark each tube with a number to designate the organism.

2. To test for a particular organism's ability to produce antibiotics, further isolation is necessary.

3. Using a minimal amount of STERILE distilled water, pour the water over the slant being evaluated. To produce a solution of the organism, GENTLY scrape the agar with a sterile transfer needle (loop) such that the living material is added to the water.

4. Using previously prepared nutrient agar plates, inoculate the plates with a drop of the solution prepared in Step 3, then streaking the solution in a straight line across the center of the medium surface, essentially dividing the plate into two halves with the solution acting as the dividing line.

5. Incubate the plates at 28ºC for 5 days, permitting the actinomycetes to grow AND produce its antibiotic.

6. After 5 days, remove the plates and prepare to test the antibiotic production and effectiveness by adding streaks of various bacteria. To do this, you need to have solutions of various bacteria prepared from stock cultures. For purposes of relating antibiotic effectiveness against particular bacterium, a collection of different bacterial types (Gram-positive, Gram-negative) are suggested. They include Sarcina lutea (+), Serratia marcescens (-) as well as the yeast, Saccharomyces cerevisiae.

7. Water solutions of the various bacteria are made by transferring a sterile loop of the bacteria taken from a stock culture to a sterile test tube containing 5 cm³ of sterile distilled water. From this water solution, a loop of the bacteria is transferred to the nutrient agar plate containing the center streak of the antibiotic-producing actinomycetes. The bacterial streak is made at a right angle to the actinomycetes center streak. Multiple streaks of different bacteria can be placed on the same plate.

8. Incubate the plates at 28ºC for 2 days.

9. Examine the plates for evidence of antibiotic activity against the various bacterial streaks. Is there any correlation between those bacteria that are affected by the antibiotic and their designation, Gram positive, Gram negative? Refer to literature that explains Gram staining results relative to the type of bacterial cell wall composition. How is this related to the activity of antibiotics, including penicillin?

For more information, at other Web sites...

Soil Bacteria and Actinomycetes — from the U.S. Bureau of Land Management National Science and Technology Center.