(REFLECTION)

USING A HEMOCYMOMETER
Based from the video I watched hemocytometer was originally developed
for counting blood cells today it is used to count many other cell types as
well. A hemocytometer consists of a special glass microscope slide that
contains two separate counting chambers, each chamber is etched with a
carefully grafted grid with exact dimensions, there are two supports on
either side of the counting chamber when a cover glass is placed on the
supports the depth of the chamber will be exactly point one millimeters.
Make sure the hemocytometer and the coverslip is clean 70% ethanol can
be used for the cleaning carefully place the coverslip on the supports using
gentle pressure and small circular motions, moisten the shoulder of the
hemocytometer before you fix the coverslip make sure your diluted cell
sample is mixed well and load 10 microliters by gently resting the end of
the pipette tip at the edge of the chamber or the edge of the coverslip do
not overfill the chamber let the liquid you’ve drawn out of the tip fill the
counting chamber by capillary action with a microscope and a ten times
magnification you will be able to see approximately one square per viewing
window use phase contrast to distinguish the cells, live cells will not be
stained by trypan blue and will appear white while any dead cells will be
faint or dark blue. Based on the number of viable and dead cells you can
now calculate the cell viability, to calculate the density of viable cells you
need to determine the number of live cells in a particular volume this
volume is defined by the number of squares that you counted and the
volume of one such square we also multiple by the dilution factor in order
to determine the cell density of your original cell solution prior to diluting
with trypan blue solution. The hemocytometer is designed so that the
volume at each of the larger squares is constant.

COUNTING CELLS WITH A HEMOCYMOMETER

Based from the video I watched cell counting is an important step in many
cell-based assays for determining cell number and viability. In general, the
goal of counting is to understand how much a sample of an unknown cell
concentration should be diluted for further use. The most common
laboratory tool for counting cells is the hemacytometer. A hemocytometer
is a device that is used for counting cells. It’s a modified microscope slide,
containing two identical wells, or chambers, into which a small volume of a
cell suspension is pipetted. We have already removed 100 uL of our cell
suspension and placed it in a micro-centrifuge tube. Dilute the suspension
by adding 100 uL of trypan blue. Trypan blue is a dye that helps us
distinguish between living and dead cells. The dye passes through the
membranes of dead cells so they will appear blue under a microscope.
Living cells exclude and will appear mostly clear. So based from the video I
watched each chamber is divided into a grid pattern, consisting of 9 large
squares. Each square has the same dimensions and contains 10 to the
negative-fourth power mL of suspension. The rules for counting cells
sometimes differ from lab to lab. In other laboratory they count cells in the
4 large corner squares and the center square. Proper storage, cleaning, and
handling of the hemocytometer will minimize the number of artifacts. In
the video there 10 viable cells and 1 non-viable cell in the first square next
there are 9 viable cells and no non-viable cells in the top right square. In the
bottom right square there are 11 viable cells and no non-viable cells after
that the bottom left square there are 10 viable cells and 2 non-viable cells.
Sometimes cells will appear as clumps or small groups. It may be difficult to
determine exactly how many cells are in a group.