Experimental Procedure

Estimated time to complete the experiment: 3 h the first day and 30 min after 24-48 h

When doing microbiological laboratory work, the following rules must be followed:

2. Never eat or drink in the laboratory, and avoid placing objects in your mouth

3. Always wash your hands before leaving the lab

4. If you spill living organisms, cover the spilled material with paper towels and pour a laboratory disinfectant over the towels and the entire contaminated area. Wait 15 min before you clean it up

6. Using a sterile pipet, transfer 0.1 mL of the 10-2 dilution to another test tube containing 9.9 mL of the saline solution, and repeat the step in order to have 10"4 and 10-6 dilutions. Mix thoroughly.

Use an aseptic technique when making serial dilutions and plating. Always use a clean, sterile pipet for all transfers.

Materials

Reagents

20 sterile Petri dishes,

60 x 15 mm 10 1-mL pipets 3 500-mL Erlenmeyer flasks 1 mixing plate

1 Bunsen burner

8 screw cap 13 x 100 mm test tubes Test tube rack Autoclave

2 Incubators (37°C and 30°C) Inoculating loop

Glass slides and cover slips

Optical microscope

Immersion oil

Soil and water samples

Sterile spatula

Sterile mortar and pestle

Dextrose potato agar Nutrient agar Sabourad agar Saline isotonic solution

(NaCl 0.9%) Gram reagents, solutions of:

- crystal violet stain

- Gram's iodine solution

- acetone-alcohol mixture 70:30

- safranine stain

7. Label each Petri dish (as 1-7) and add aliquots of the dilutions and media according to the following table:

Dilutions and ^^ media

^^ Petri dishes

1 2 3

4 5

6 7

ItT1 mL

1

1 —

10"4 mL

— 1 —

1

1

10"* mL

— — 1

— 1

PDA agar mL

10 10 10

10 10

10 10

Nutrient agar mL

Sabourad agar mL

1. Prepare 100 mL of an isotonic saline solution. Add 9.9 mL to each of six tubes and cap them. Sterilize for 15 minutes at 121°C.

2. Sterilize six 1-mL pipets.

3. Prepare the necessary amount of nutrient agar, of Sabourad agar, and of potato dextrose agar, as indicated by the manufacturers (each Petri dish requires 10 mL of the medium). Mix thoroughly. Heat gently and bring the mixture to boil. Autoclave for 15 min at 15 psi and 121°C. Maintain at 45°C until they are poured in sterile Petri dishes.

Soil sample

4. If necessary, place some soil in a sterile mortar and break up the lumps with a pestle.

5. Weigh 0.1 g of the sample soil. Suspend in the 9.9 mL of saline solution and mix thoroughly. (This means a 1:100 dilution, or 10~2).

8. Slowly, move each Petri dish so that the samples become mixed with the culture medium.

9. Let the agar solidify and incubate (at 37°C for nutrient agar and 30°C for PDA and Sabourad media).

Water sample

10. Measure 0.1 mL of the liquid sample, dilute in 9.9 mL of saline solution and mix thoroughly. (This means a 1:100 dilution, or 10~2).

11. Repeat steps 6-9. Air sample

12. Prepare Petri dishes with each agar medium.

13. When the agar has solidified, open the dish for 20 min to allow airborne microorganisms contact the medium.

14. Close and incubate, as in step 9.

15. Observe and count the cultures after 24 and 48 h for bacterial growth, and 48-72 h for yeast and fungi growth.

Bacteria

1. Observe the macroscopic characteristics of the colonies of microorganisms that grew on each plate.

2. Prepare separate smears of the unknown bacteria in the following way:

• Place a drop of tap water at the center of a glass slide.

• Using the inoculating loop, aseptically remove half a loopful of bacteria from the culture.

• Mix the large amount of organisms on the loop into the drop of water on the slide.

• Flame the loop to prevent contamination of the worktable, your culture and yourself.

• Heat-fix by passing the slide back and forth through the flame of your Bunsen burner.

3. Perform the Gram stain as follows:

• Flood the slide with crystal violet and allow to react for 1 min.

• Handle the slide with slide forceps. Tilt it to an angle of approximately 45° and drain the dye off the slide into a pan or staining sink.

• Continue to hold the slide at 45° and immediately rinse it thoroughly with a gentle stream of water from the wash bottle.

• Hush the slide with iodine solution. Allow the iodine to react for 1 minute.

• Tilt the slide and allow it to drain.

• Immediately rinse the slide thoroughly with water.

• With the slide still held at a 45° angle, decolorize it by allowing the acetone-alcohol mixture to run over and off the smear.

• Rinse immediately with water.

• Rood the slide with the safranine counterstain. Allow the countestain to react for 1 minute.

• Rinse the slide thoroughly with water.

• Carefully blot your stained slide and examine each smear in the microscope under high power and oil immersion objectives.

The Gram stain separates bacteria into two groups, depending on whether the original stain (violet crystal) is retained or lost when the stained smear is treated with an iodine solution and then washed with the acetone-alcohol mixture.

Organisms that retain the stain when washed with alcohol are termed Gram positive; those that fail to retain the original stain but take the counterstain (safranine) are called Gram negative. The Gram stain is of considerable value in identifying and classifying bacteria.

Yeast and fungi

Yeast and fungi are observed directly under the microscope by placing a loopful of each colony in a drop of water between a slide and a cover slip. Observe the form, size, and number of different colonies under the microscope (at high power and oil immersion objectives).

Autoclave all Petri dishes when the experiment is concluded to kill the isolated microorganisms

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