Hi everyone and welcome to Biology Professor. Today we're going to be talking
about the Gram Stain procedure. The Gram Stain is a very commonly used method in
laboratory and medical settings. It is called a differential stain, so that
means that it helps someone to differentiate or distinguish between two
different classes of bacteria, specifically gram positive bacteria and
gram-negative bacteria. For more information on the differences between
those two types of bacteria, see my video on gram positive and gram negative
bacteria. Today we're going to talk about the steps of the Gram Stain.
It starts with heat fixing a sample. This is when you have a biological sample
that has been heat fixed to a slide simply by passing it over a flame. This
helps the sample containing all of the bacteria to stick to the slide through
all the subsequent steps. The second step is the use of crystal
violet dye. This is a purple dye that stains all of the bacteria. So you can
see here that after we use this crystal violet dye, all of the bacteria in the
sample are going to stain purple. This is because crystal violet dye associates
with peptidoglycan and all bacteria have at least some peptidoglycan in their
cell walls. Now crystal violet is a positively (+) charged molecule and iodine
is a negatively (-) charged molecule. This means that together they form a complex.
Iodine for this reason is called the mordant.
It helps the crystal violet to link to the peptidoglycan in gram positive cell
walls because they have a much thicker layer of peptidoglycan. Then these cells,
after they've been treated with the crystal violet primary stain and the
iodine mordant, they are treated with a decolorizing
solution but the decolorizing solution, it is usually some form of alcohol
that helps the crystal violet and iodine to be washed away from cells where they
aren't very strongly adhering. These would be the gram-negative cell walls
that only have a very, very thin layer of peptidoglycan, leaving the
crystal violet dye and the iodine in the gram-positive cell walls, which have that
much thicker layer of peptidoglycan. So after using this decolorizing solution,
you have the gram positive cells still retaining that purple crystal violet dye
and then you have gram-negative cells that are suddenly colorless again. So
that is from the decolorizing solution. At this point, you can visualize which
cells are gram positive because they have retained this crystal violet dye
after using the decolorizing solution, but then the final step is the use of a
counter stain, safranin, which has a pink color just to help distinguish further
between those two cell types. So after the use of safranin, crystal violet is
still in all of the gram-positive cell walls so that these are all still purple
stained and you can distinguish that they are gram positive, whereas the
gram-negative cell walls have taken up this safranin counterstain making them pink.
Now at the end of a procedure like this, you can see how it's fast, it's
inexpensive, it's relatively simple and one of the reasons it's so useful in a
laboratory or hospital setting is it helps to give what's called a
presumptive diagnosis. We see that the majority of cells in the
sample are gram positive rods, so purple gram positive rod-shaped bacteria.
The most common, or one of the most common causes of hospital-acquired
diarrhea in adults is called Clostridium difficile, and it is a gram
positive rod-shaped bacterium. It is the most likely cause of the infection that
was take the sample was taken from. And then knowing this information not only
do we have a presumptive diagnosis that can then be confirmed through later
studies, we also know what kind of bacteria we're working with, which helps
doctors to prescribe the correct antibiotic regimen to treat this
infection and so this is why a Gram Stain is so important and
this is how it works.