We most often use basic stains to examine bacteria. Basic
stains, due to their positive charge will bind electrostatically to negatively
charged molecules such as many polysaccharides, proteins and nucleic acids.
Acid stains bind to positively charged molecules which are much less common,
meaning acidic stains are used only for special purposes. Some commonly encountered
basic stains are crystal violet, safranin (a red dye) and methylene blue. Basic
stains may be used alone (a simple stain) or in combination (differential stain)
depending on the experiment involved.
Slightly different techniques must be used for preparing
bacteria for staining depending on whether the bacteria are growing on agar
or in broth. The concentration of bacteria in a colony on an agar plate is extremely
high, thus the major problem is having so many bacteria that they form a dense
mass in which individual cells stain poorly and whose morphology is hard to
distinguish. In broth, the bacteria are relatively dispersed so we have to ensure
that we get a sufficient number onto the microscope slide so they're not too
hard to find.
Step 1. Bacteria from colonies. Clean a glass
slide and place a small mark slightly off center using a grease pencil. Using
your loop, transfer one small drop of water to the center the slide, being careful
to be close to but not overlapping the grease pencil mark. Do not transfer too
much water because these drops will have to air dry. Sterilize your loop and
touch a single colony and transfer the bacteria to the water droplet on the
slide and mix well. DO NOT scoop up a whole colony; you'll have vastly too much
bacteria. If while mixing you see an opaque slurry of bacteria on the slide
you have too many bacteria for effective staining.
Step 1. Bacteria from broth. Clean
a glass slide and place a small mark slightly off center using
a grease pencil. Mix the broth containing the bacteria well because
the bacteria may sediment to the bottom of the container. Use
a sterile loop and transfer one or two droplets of bacteria to
the center of a cleaned glass slide, close to but not overlapping
the grease pencil mark.
Step 2. Drying. Allow the bacterial slurry
(called the smear) to air dry. You CAN NOT heat the sample nor blow on it to
hasten drying time because that could force bacteria into the air leading to
contamination and possible infection.
Step 3. Heat fixation. Holding the slide
by one edge, pass it slowly through a bunsen burner flame. Do not move so slowly
that the edge of the slide you're holding heats up to uncomfortable levels.
This heat fixation step denatures bacterial proteins causing the cells to stick
to the slide while also killing the bacteria making them safe for the following
Step 4. Staining. Place the stain in a
staining rack and cover the smear with the stain of choice. Allow the stain
to work for 30 seconds (some stains may have different staining times but this
time will work well for simple stains). Remove the stain by rinsing with water
from the squeeze bottle and gently blot (do not rub) the stain dry using bibulous
paper. The slide is now ready to look at under the microscope. Because the bacteria
were heat fixed, it will not be necessary to use a cover slip.
The Gram Stain
The Gram stain is classifed as a differential stain because it allows us to
distinguish between different types of bacteria. Bacteria can
be quickly divided into two distinct morphological and functional
groups on the basis of the Gram stain. By this technique, Gram
positive bacteria stain purple and gram negative stain red. The
bacteria are first stained with crystal violet followed by a brief
treament with Gram's iodine. The iodine functions as a mordant
to help the crystal violet bind more firmly. The bacteria are
then rinsed with ethanol. Gram positive bacteria, which have multiple
layers of peptidoglycan, retain the crystal violet while it is
quickly rinsed out of Gram negative bacteria because their peptidoglycan
is a single layer thick. The bacteria are stained a second time
(counter stained) with the dye safranin which will not show up
on the already purple Gram positive but will stain the decolorized
Gram negative bacteria red.
Step 1. Preparation. Smear
and heat fix the bacteria as described above (steps 1 through 3) for the simple
Step 2. Primary
stain. Cover the smear with crystal violet and incubate for
30 seconds. Rinse the dye off with distilled water (dH2O)
from the squeeze bottle.
Step 3. Mordant. Cover the
smear with grams iodine. After 20 seconds, rinse the slide with dH2O.
Step 4. Decolorization Rinse the stain
with 95% ethanol. This step must be done very carefully. Hold the slide
at a 45o angle over the staining rack and rinse with ethanol one
drop at a time. Watch the ethanol as it runs off the slide looking for blue
color. Stop dropping ethanol as soon as no more color is releases and rinse
the slide immediately with water. A few drops of ethanol too many and the gram
positive bacteria will also lose their crystal violet.
Step 5. Counterstain. Cover the bacteria
with safrinin for 30 seconds. Rinse with dH2O
and and blot the slide dry with bibulous paper.