Gram Staining

     

     You will learn how to

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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