Experimental Procedure

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

Materials

Reagents

3 500-mL Erlenmeyer flasks 6 1-mL pipets 20 sterile Petri dishes,

60 x 15 mm 10 1-mL pipets 10 10-mL pipets 10 screw cap 13 x 100 mm test tubes Cotton caps

Incubators (37°C, 30°C) Autoclave 1 mixing plate 1 Bunsen burner Test tube rack Inoculating loop Glass slides and cover slips Sterile spatula

Wastewater sample Saline isotonic solution (0.9% NaCl) Nutrient agar

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.

Preparation of the agar medium

Nutrient agar is prepared in the concentration indicated by the manufacturer. Mix thoroughly. Gently heat and bring the mixture to boil. Autoclave for

15 min at 15 psi and 121°C. Maintain the agar at 45°C until it is poured in sterile Petri dishes.

Autoclave ten 10-mL screw cap tubes for each toxicant tested, 100 mL of saline solution (0.9% NaCl) and 1- and 10-mL pipets.

Preparation of the control plates A small sample of spent water can be used (e.g., the effluent of a wastewater treatment plant). Dilute 1 mL of this sample to 1/10, 1/100, and 1/1000 by using the screw cap tubes in the following way:

• Add 9 mL of sterile saline solution to each tube.

• Add 1 mL of the sample to the first tube. Mix thoroughly. This is a 10-1 dilution. Transfer 1 mL of this solution to the second tube and repeat the procedure to prepare two additional dilutions (10~2 and 10"3).

Use an aseptic technique when making serial dilutions and plating.

Always use a clean, sterile pipet for all transfers.

-—. ... Petri dish no. Toxicant (conc.), Vol ~~ -——

1

2

3

4

5

6

7

8

9

Sodium azide (2 mg/mL)

(PL)

0

25

50

75

100

25

50

75

100

Aseptic saline solution

m

100

75

50

25

0

75

50

25

0

Diluted sample, 10~2

OIL)

100

100

100

100

100

-

-

-

-

Diluted sample, 10~3

(ML)

-

-

-

-

-

100

100

100

100

Nutrient agar medium

(mL)

10

10

10

10

10

10

10

10

10

Preparation of the samples

Prepare small Petri dishes in the following way for each of the proposed toxicants.

Petri dish no. Toxicant (conc.), VoL

123456789 10 11

K2Cr207 (29.4 mg/10 mL = 0.02 mmol Cr6+/mL) (pL)

0

25

50

75

100

200

25

50

75

100

200

Aseptic saline solution (fxL)

200

175

150

125

100

0

175

150

125

100

0

Diluted sample, 10-2 (pL)

100

100

100

100

100

100

-

-

-

-

-

Diluted sample, 10~3 (|iL)

-

-

-

-

-

-

100

100

100

100

100

Nutrient agar medium (mL)

10

10

10

10

10

10

10

10

10

10

10

Petri dish no. Toxicant (conc.), VolT~~~—

1

2

3

4

5

6

7

8

9

10

11

Q2O3 (15.2 mg/10 mL = 0.02 mmol C^/mL) (pL)

0

25

50

75

100

200

25

50

75

100

200

Aseptic saline solution (pL)

200

175

150

125

100

0

175

150

125

100

0

Diluted sample, 10~2 (pL)

100

100

100

100

100

100

-

-

-

-

-

Diluted sample, 10~3 (pL)

-

-

-

-

-

-

100

100

100

100

100

Nutrient agar medium (mL)

10

10

10

10

10

10

10

10

10

10

10

Prepare each Petri dish in duplicate.

• Reagents are added first and the agar is added at 45°C, before it solidifies. Move with slow horizontal movements to homogenize. Let the mixture stand still for 20 minutes.

• Count the number of colonies in each plate. Determine the number of colony forming units

(CFU)/mL in the control plates and in each dilution (take the average of two or three determinations).

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

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