For a laboratory exercise of basic techniques in microbiology: Gram stain and Microscopy.
What are the sources of error in performing Gram staining?
Transcribed Image Text:Materials:
.Bacterial cultures: E. coli & Staphylococcus aureus
Crystal violet (primary stain)
.Iodine solution/Gram's Iodine (mordant that fixes
crystal violet to cell wall)
Decolorizer (e.g. 95% ethanol)
Safranin (secondary stain)
• Water (preferably in a squirt bottle)
Procedure:
Preparation of smear for staining
Smear of bacteria from cultures on solid media are made
upon clean glass slides as follow:
1. With the loop place a small drop of saline on each
section of the slide.
2. Sterilize the loop by holding it vertically in the Bunsen
flame until it is heated to redness along its length.
3. Allow the loop to cool.
4. Holding the loop like a pen, pick off a small portion of
bacteria growth from the colony to be examined.
5. Transfer a little of the growth to the water drop on the
slide and, using the flat-slide of the loop, emulsify the
growth, smearing it out well over the area of the slide.
Aim at thin smears, i.e. almost too thin to be seen
when dry. The individual bacteria will then be well
spaced for examination under the microscope.
6. Sterilize the loop.
7. Leave the slide to dry in the air, then heat-fix by
passing the slide once through the Bunsen flame. This
causes the bacteria to adhere firmly to the slide.
Gram staining method
1. Saturate the smear with crystal violet stain for 1
minute.
2. Rinse the slide gently with water (no more than 2 or 3
seconds).
3. Saturate the smear with iodine for 1 minute.
4. Rinse the slide gently with water, shake off excess (2 to
3 seconds).
5. Decolorize with decolorizer (95% ethanol). Apply
95% alcohol, drop by drop, on the top edge of the smear
until no more purple colour runs out of the lower edge of
it. The decolourization time is about 10-20 seconds.
6. Immediately rinse the slide with water (2 to 3 seconds).
7. Counter stain with safranin for 1 minute.
8. Rinse the slide gently with water, carefully blot the
slide (on paper towel) and dry in air.
9. Observe the slide under the microscope, using proper
microscope technique.
10. Record the colour (and hence Gram reaction) and
shape of the micro-organisms.
Result:
Definition Definition Technique that uses crystal violet or methylene blue as primary staining colors to distinguish gram-positive from gram-negative organisms. Under a microscope, the gram-positive organisms appear purple-brown, retaining the primary color. Gram-negative organisms appear pink or red as they do not acquire the color of the primary stain.
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