Cell counting and Staining

Yama Atri
MSc (P) Biochemistry

Cell Counting
General name for various methods for the quantification of cells.

Requirement?

In medicine, the concentration of various blood cells, can give
crucial information regarding the health situation of a person
The cell concentration needs to be known for many experiments in
molecular biology, in order to adjust accordingly the amount of
reagents and chemicals that are to be applied in the experiment.
Studies that examine the growth rate of microorganisms (in other
words: how fast they divide to create new cells) require cell
counting.
Measurments of cell viability, i.e. measuring and calculating the
fraction of dead and live cells, for example of cells exposed to
poison.

Methods
Counting chamber: Hemocytometer.
Plating on a petri plate with growth medium.
Spectrophotometry: Cell cultures are turbid:
they absorb some of the light and let the rest
of it pass through. The higher the cell
concentration is, the higher the turbidity.

Cell Counting using
Hemocytometer
Hemocytometer
A counting chamber, also known as hemocytometer, is a microscope slide that is
especially designed to enable cell counting
To determining the number of cells per unit volume.
Was originally designed for performing blood cell counts.

The device is carefully crafted so that the area bounded by the lines is known,
and the depth of the chamber is also known. It is therefore possible to count the
number of cells or particles in a specific volume of fluid, and thereby calculate
the concentration of cells in the fluid overall.
Advantage is being cheap and fast;.
Usually the culture examined needs to be diluted, otherwise the high density of
cells would make counting impossible. The need for dilution is a disadvantage, as
every dilution adds inaccuracy to the measurement.
Counting grids of hemocytometer
By Richard Wheeler (Zephyris) 2007.
Hemocytometer grid:
red square = 1.0000 mm
2
,
green square = 0.0625 mm
2
,
yellow square = 0.040 mm
2
,
blue square = 0.0025 mm
2
,

at a depth of 0.1 mm
1
2 3
4
5 6
7
8
9
To count cells/ mL

1. the volume of each large square is 0.1 mm
3
.
1000 mm
3
= 1 mL


0.1 mm
3
= 10
-4
mL


2. The cells in four large (red in figure) squares are counted and the
average of the cells found in the 5 squares (the four corners and
the middle one) is taken.
3. The concentration in cells per ml
= cells in four red, large squares/4 10,000.
4. Get the concentration as

C= Number/ vol. X 10,000cells/ mL X dilution factor
To prepare the counting chamber
The mirror-like polished surface is carefully cleaned with lens
paper. The cover slip is also cleaned.
The cover slip is placed over the counting surface prior to putting
on the cell suspension.
counting chamber is then placed on the microscope stage and
the counting grid is brought into focus at low power.
One entire grid on standard hemocytometer can be seen at 40x
Clean the cover slip and the mirror like surface after use.

Never scratch the mirror like surface of hemocytometer while
cleaning.
The tip of the pipette is placed in the V-
shaped groove on the hemocytometer to load
the sample into the chamber (about 10 l).

Capillary action will draw the fluid into the
chamber.

The sample is allowed to settle for 2 or 3
minutes so that the cells stop drifting around
the chamber and most will be in the same
plane of focus.

Not to allow the sample to settle too long or it
will dry out, concentrating the cells over the
grid.

Load the sample
The example at right shows red lines
where cells on the line would be counted.

If red dots represent cells, one would count
3 cells in the top middle large square.

Cells over or touching the lines on top and
on the left are counted, but cells over or
touching the right or bottom lines are
ignored.
How to count
Cell Viability Assays
Trypan blue dye Exclusion Methods
Live cell membrane is impermeable to trypan blue, but dead cell
membrane absorbs trypan blue.
Hence trypan blue is used in cell viability test.
Dilution by trypan blue
- Viable cells : small, round and refractive
- Non-viable cells : swollen, larger, dark blue


Cell viability: trypan blue staining.
Living cells exclude the dye, whereas dead
cells will take up the blue dye.

Dilute 10 l of the cell suspension (1:5)


Place 10 l on a hemocytometer (between
the counting slide and the glass cover slip).

Count the cells under a microscope.

Count the number of unstained cells on the
hemocytometer under a microscope (Dead
cells will take up the Trypan Blue stain).
Then, count the total number of cells .
Determine the percentage of viable cells:

Cell viability
(Number of unstained cells/Total number of
cells) x 100 = Percent Viable Cells
Other Assays
LDH (lactate dehydrogenase) Leakage
Assumptions of LDH assay
: Intracellular enzymes are only released after damage to the cell membrane
: Rapidly released from damaged cells.
Fluorescent dyes
Ethidium bromide (EtBr) and propidium iodide (PI)
PI binds to nucleic acids upon membrane damage and is impermeable to
intact plasma membrane.
Intercalates with DNA or RNA red
Fluorescein diacetate (FDA) is a nonpolar ester which passes through plasma
membranes and is hydrolyzed by intracellular esterases to produce free
fluorescein, the polar fluorescein is confined within cells which have an
intact plasma membrane and can be observed under appropriate excitation
conditions.
Undamaged cell : highly fluorescent Damaged cell : fluoresce only weakly
greenish-yellow at 450-480 nm



Intact cell
PI and FDA is added
Fluorescein in
intact cells
PI /FDA cell viability assay
FDA (Fluorescein diacetate)
PI (Propidium iodide)
Plasma membrane is damaged
; fluorescein leaks out
PI enters and strains
nucleic acids
Cell Staining
Cell staining is a technique that can be used to better visualize cells
and cell components under a microscope.
By using different stains, one can preferentially stain certain cell
components, such as a nucleus or a cell wall, or the entire cell.
Most stains can be used on fixed, or non-living cells, while only
some can be used on living cells (vital stains); some stains can be
used on either living or non-living cells.
Need
To enhance visualization of the cell or certain cellular components
under a microscope.
To highlight metabolic processes or to differentiate between live and
dead cells in a sample.
Cell staining and Slide preparation
Cell staining techniques and preparation depend on the type of stain
and analysis used.
One or more of the following procedures may be required to prepare
a sample:
Permeabilization - treatment of cells, generally with a mild
surfactant, which dissolves cell membranes in order to allow larger
dye molecules to enter inside the cell.
Fixation - serves to "fix" or preserve cell or tissue morphology
through the preparation process.
Most fixation procedures involve adding a chemical fixative that
creates chemical bonds between proteins to increase their rigidity.
Common fixatives include formaldehyde, ethanol, methanol, and/or
picric acid.

Mounting - involves attaching samples to a glass microscope slide
for observation and analysis. Cells may either be grown directly to
the slide or loose cells can be applied to a slide using a sterile
technique. Thin sections (slices) of material such as tissue may also
be applied to a microscope slide for observation.
Staining - application of stain to a sample to color cells, tissues,
components, or metabolic processes. This process may involve
immersing the sample (before or after fixation or mounting) in a dye
solution and then rinsing and observing the sample under a
microscope. Some dyes require the use of a mordant, which is a
chemical compound that reacts with the stain to form an insoluble,
colored precipitate. The mordanted stain will remain on/in the
sample when excess dye solution is washed away.

Vital Stains
A vital stain in a casual usage may mean a stain that can be
applied on living cells without killing them.
Vital stains have been useful for diagnostic and surgical techniques
in a variety of medical specialties.
in "vital staining" - the most accepted but apparently paradoxical
meaning of this term, the live cells exclude the stain i.e. stain
negatively and only the dead cells stain positively and thus viability
can be assessed by counting the percentage of total cells that stain
negatively.
Eosin dye exclusion.
Propidium iodide, DNA stain that can differentiate necrotic, apoptotic
and normal cells.
Trypan Blue, a living-cell exclusion dye
Erythrosine, which is Red No. 3 in food coloring, can be used as an
exclusion dye.

Crystal violet - stains cell walls purple when combined with a
mordant. This stain is used in Gram staining
DAPI - a fluorescent nuclear stain that is excited by ultraviolet light,
showing blue fluorescence when bound to DNA. DAPI can be used
in living of fixed cells
Eosin - a counterstain to haematoxylin, this stain colors red blood
cells, cytoplasmic material, cell membranes, and extracellular
structures pink or red.
Ethidium bromide - this stain colors unhealthy cells in the final
stages of apoptosis, or deliberate cell death, fluorescent red-orange.
Hematoxylin - a nuclear stain that, with a mordant, stains nuclei
blue-violet or brown.
Hoechst stains - two types of fluorescent stains, 33258 and 33342,
these are used to stain DNA in living cells.

Iodine - used as a starch indicator. When in solution, starch and
iodine turn a dark blue color.
Methylene blue - stains animal cells to make nuclei more visible.
Neutral/Toluylene red - stains nuclei red and may be used on living
cells.
Nile blue - stains nuclei blue and may be used on living cells.
Nile red/Nile blue oxazone - this stain is made by boiling Nile blue
with sulfuric acid, which creates a mix of Nile red and Nile blue. The
red accumulates in intracellular lipid globules, staining them red.
This stain may be used on living cells.
Osmium tetroxide - used in optical microscopy to stain lipids black.
Rhodamine - a protein-specific fluorescent stain used in
fluorescence microscopy.
Safranin - a nuclear stain used as a counterstain or to color
collagen yellow.