Experimental Procedure

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

Materials

4 10-mL beakers 3 20-mL beakers 10 screw cap test tubes 20 sterile Petri dishes, 60 x 15 mm (if McConkey agar is used) Chronometer 10 sterilized 1-mL pipets Commercial ozonizer UV lamp Incubator Test tube rack

Reagents wastewater sample 4.5 ppm NaOCl Ti02

coliform counting plates or McConkey agar medium Saline isotonic solution, (NaCl 0.9%)

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.

After fixed time intervals of exposure to a given disinfection agent, 1-mL wastewater aliquots are spread on coliform plaques (e.g., 3M Petrifilm™). The use of these plaques avoids the preparation of a sterilized culture medium specific for coliform microorganisms. (Instead of the plaques, McConkey agar medium can be used and sterilized according to the manufacturer instructions, and Petri dishes prepared). McConkey agar contains crystal violet, which is inhibitory to Gram-positive bacteria, and allows the isolation of Gram-negative bacteria. Incorporation of the carbohydrate lactose, bile salts, and the pH indicator neutral red permits differentiation of enteric bacteria on the basis of their ability to ferment lactose. Coliform bacilli produce acid as a result of lactose fermentation and exhibit a red coloration).

The plaques are incubated for 24 hours at 37°C, and the resulting red-colored colonies are counted. There should be fewer than 50 colonies per plaque; if not, dilute the samples before spreading them onto the plaques. If dilution is necessary prepare a dilution series as follows:

1. Take 1 mL of the sample and mix with 9 mL of saline solution in a sterile screw cap test tube; mix thoroughly to have a 10_1 dilution.

2. Transfer 1 mL of the 10_1 dilution to another test tube containing 9 mL of the saline solution, using a sterile pipet and repeat the step in order to have 10 2 and 10-3 dilutions. Mix thoroughly.

Hypochlorite Treatment

Take five 5-mL wastewater samples in 10-mL beakers. Add 1 mL of a 4.5 ppm NaOCl solution to beakers 2, 3, 4, and 5. Make sure that the final concentration of NaOCl is less than 2 ppm. Stir at constant speed. Take a 1-mL aliquot from beaker 1 at time 0 (this is the control), a similar aliquot from beaker 2 after 5 min, another aliquot from beaker 3 after 10 min, and so on up to 20 min. Place each aliquot on a plaque, incubate, and count the red colonies as described above. If Petri dishes with McConkey agar were used, a 0.1 mL aliquot of each dilution can be used.

Ozone Treatment

Take a 10-mL wastewater sample in a 20-mL beaker. A commercial ozonizer with a porous filter can be used to deliver small ozone bubbles to each sample. (For instance, one can use a BIOZON FAGOR ozonizer that produces 4 mg of 03/min). Take a 1mL aliquot from the beaker after 0, 30, 90 and 180 s, and place each aliquot on a plaque. Incubate and count as described above.

UV Treatment

Take a 10-mL wastewater sample in a 20-mL beaker and place it in a dark compartment. Irradiate the sample with a 254-nm, 9-W lamp placed at 12 cm above it. Stir at constant speed. Take a 1-mL aliquot from the beaker after 0, 60, 150, and 240 s, and place each aliquot on a plaque. Incubate and count as described above.

T1O2 + UV Treatment

Take a 10-mL wastewater sample in a 20-mL beaker and add 20 mg of Ti02 (the anatase phase) to form a suspension. Stir at a constant speed. Place the experimental set-up in a dark compartment. Irradiate the sample with a 254-nm, 9-W lamp placed at 12 cm above it. Take a 1-mL aliquot from the beaker after 0, 120, 300, and 480 s, and place each aliquot on a plaque. Incubate and count as described above.

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

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

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