was then dropped on the slide until no more crystal violet was washed off.
Afterwards, the slide was rinsed then drained. Safranin was then dropped on the
slide, and after a minute, the slide was rinsed with tap water. After the
staining was done, excess moisture was blotted off with tissue paper. The slide
was then air-dried. The slide was next studied under OIO (immersion oil was
used) of the microscope (the slide was placed under LPO first, where a good area
to examine was located). Gram-positive will retain the violet color,
gram-negative bacteria will be stained red. II. Endospore Staining This process
required at least 36-hr. cultures of the organisms in the NA slant enumerated
earlier that were fixed on a slide (like the smears on Gram staining). 5%
malachite green and 0.5% safranin (see Appendix) were the stains used for this
staining method. A disposable plastic, forceps, a microscope and an alcohol
burner were used in this method. First, the working area was covered with the
plastic because the stains might splatter out. Then the slide was flooded with
malachite green. This was passed over low flame several times for five minutes,
allowing the stain to steam but not to boil. The stain was replenished from time
to time and after five minutes, the slide was rinsed. The slide was then stained
with safranin and was allowed to stand for a minute. The slide was then rinsed
with tap water and air-dried. The dried slide was then examined under LPO, to
locate a good area, then placed under OIO (immersion oil as used) for a more
detailed study. The presence of green bodies the presence of endospores.
684
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