Use sterile techniques to prepare your slide! Use an inoculating needle to
add a drop of saline to a clean slide. Take a sample of a single bacterial colony
and mix it into the saline. Let air dry until a white film appears. Heat fix
by passing the slide through a flame a few times.
Follow the above procedure but try to get a portion of the
colony on the slide intact. You can try lifting it with a sterile
scalpel. It will be too crowded to observe on most of the slide,
but at the edges of the colony you will be able to see the pattern
that the filaments form.
Lift a portion of the colony intact onto a clean slide (it will still be attached to the agar), add a cover slip and observe without staining. Look at the edges of the colony where the sample will be thinner and there will be enough light to observe.
Gram staining can be used to make slides of bacteria and actinomycetes:
Preparation of Gram Stains
Dissolve 2 g of crystal violet in 20 ml of 95% ethanol. Add
this solution to 80 ml of a 1% Ammonium Oxalate solution. Let
stand for 24 hours and filter.
Add 1 g Iodine and 3 g Potassium Iodide to 300 ml distilled
water. Store in an amber bottle.
Decolorizer: 95% Ethyl Alcohol
Add 2.5 g safranin to 10 ml 95% ethanol. Add this solution
to 100 ml distilled water
Procedure for Gram Staining
1. Flood slide with crystal violet - 20 sec.
2. Wash with distilled water - 2 sec.
3. Flood slide with Gram iodine - 1 min.
4. Decolorize by tilting slide and drop by drop rinsing with 95% ethanol until
ethanol runs clear - about 10 to 20 sec.
5. Wash with distilled water - 2 sec.
6. Flood with safranin - 20 sec.
7. Wash with distilled water - 2 sec.
8. Blot dry.
These protocols were written by Elaina Olynciw, biology teacher at A. Philip Randolph High School, New York City, while working in the laboratory of Dr. Eric Nelson at Cornell University as part of the Teacher Institute of Environmental Sciences.
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Cornell Waste Management Institute ©1996
Ithaca, NY 14853-5601