Gram stain

Heat-fix a smear of bacterium as follows: 1. Using the dropper to place1/2 of a normal sized drop of water on a clean slide by touching the dropper to the slide 2. Aseptically remove a small amount of the E. coli from the agar surface and gently touch it 2 - 3 times to the drop of water until the water just turns cloudy Flame the loop and let it cool. 3. After the loop cools, spread the bacteria/water mixture over the entire slide to form a thin film. 4. Allow this thin suspension to completely air dry. 5. To heat-fix the bacteria to the slide, pass about one inch of the slide (film-side up) through the flame of the bunsen burner several times until the slide just becomes uncomfortable to hold. If the slide is not heated enough, all of the bacteria will wash off. If it is overheated, the bacteria structural integrity can be damaged. b. Stain with crystal violet for one minute. Gently wash with water. Shake off the excess water but do not blot dry between steps. c. Stain with gram's iodine solution for one minute and gently wash with water. d. Decolorize by picking up the slide and letting the gram's decolorizer run down the slide until the purple just stops flowing at the bottom of the slide. Wash immediately with water. e. Stain with neutral red for one minute. When you wash off the excess neutral red, be very careful to wash gently and briefly as it is possible to wash out some of the neutral red in the bacterium. f. Blot dry and observe using oil immersion microscopy.

Sensitivity Tests
A. KIRBY-BAUER METHOD Materials supplied: (work in pairs)

1 BAP of a Gram-negative organism 1 BAP of a Gram-positive organism 2 Prompt- Inoculation wands 2 Prompt- Dilution tubes 2 sterile cotton swabs 2 Mueller-Hinton agar plates 1 multi-welled plate containing antibiotic discs

Procedure: 1. One student in each pair will test the Gram-positive organism while the other student will test the Gram-negative organism. 2. Prepare the standardized inoculum for the Kirby-Bauer method as follows: o a.Hold the Prompt Inoculation wand by the handle and touch the bottom of the wand to three colonies on the BAP. o b.Snap off the cap of the Prompt Dilution tube, insert the wand and make a tight seal between the wand and the tube. o c. Vortex on a mixer (one for each side of the room) for at least 5 seconds. o d. Discard wand in Biohazard bag provided. Replace cap onto tube. 3. Moisten a cotton swab in the dilution tube and allow the excess liquid to flow back into the tube. The swab should be wet but not dripping. 4. "Paint" the swab once over the entire surface of the MH agar, as evenly as possible. Turn the plate 90 and without rewetting the swab, paint the agar again. Discard both swab and dilution tube in the Biohazard bag. Allow the plate to dry for 3-5 minutes. Flame your forceps. Apply antibiotic discs to the agar surface in a rectangular pattern, keeping them as far apart as possible, but at least 3/4 inch from the edges of the plate. The chart on page 41 indicates which antibiotic discs should be placed on which bacterial lawn. Gently tap on each disc with the forceps to ensure contact. Incubate the plates inverted at 37C for 18-24 hours.

NEXT LAB PERIOD 1. Compare growth inhibition zones on the Mueller-Hinton plate with the appropriate inhibition patterns on the next page. 2. Record the extent of growth inhibition as R (resistant), I (intermediate), or S (sensitive) for each antibiotic. 3. Sample results 4. Measuring the tetracycline inhibition zone

Kirby-Bauer Sensitivity Results GRAM POSITIVE GRAM NEGATIVE Antibiotic Results Antibiotic Results Ampicillin Ampicillin Erythromycin Nalidixic Acid Penicillin Streptomycin Tetracycline Tetracycline

Demonstration of protozoa, algae, and fungi Materials: Pond water sample Coverslip Microscope slide
1. Use the pond water samples to look for

example of living microscopic algae. 2. Prepare a wet mount of sample. 3. Examine under microscope.