perform gram staining this technique
utilizes a four step standing procedure
with two different dyes and is one of
the first tests used when trying to
identify unknown species of bacteria
using a wax pencil label a microscope
slide with your initials
then draw a circle about one centimeter in diameter
on the slide
using a metal inoculation loop sterilize
it by flaming
using aseptic technique at a drop of
sterile water so that a film appears
across the loop
transfer the water into the circle drawn
by the wax pencil
flame the metal loop again to sterilize
after allowing it to cool use the metal
lube to transfer a colony of bacteria
growing on an auger plate make sure to
lightly touch the bacterial colony to
avoid transferring too many bacteria
when finished flame the loop to
sterilize
you
let the liquid completely evaporate
leaving behind a dry spot on the slide
this will take at least 10 minutes
once the liquid has dried completely
heat fix the bacteria to the slide by
quickly passing the slide through the
flame three times the flame should touch
the slide for about one second during
each pass ensure that all Bunsen burners
are turned off before obtaining the set
of gram stains the decolorizer contains
a high percentage of alcohol and is very
flammable add a few drops of crystal
violet stain to the bacteria until the
wax circle is filled with stain let the
stand for one minute prints the slide by
dipping it in a beaker of water two to
three times until most of the stain has
been washed off
next add a few drops of grams iodine to
cover the bacteria and then let this
stand for one minute
Prinze the slide in a beaker of water
until most of the iodine is gone a
second beaker of water can be used to
ensure most of the iodine has been
rinsed off
you
now add a few drops of decolorizer
let the decolorizer flow over the
bacteria at a 45 degree angle until the
flow is clear
then finish by rinsing the slide in a
beaker of water
counterstain by adding safranin stain to
cover the bacteria and let it stand for
one minute
after one minute completely rinsed the
slide in water
allow the slide to dry completely
once the slide is completely dry observe
the sample under a microscope.
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