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Cell counting and Staining

 Yama Atri

MSc (P) Biochemistry

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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 cellcounting.

Measurments of cell viability, i.e. measuring and calculating the

fraction of dead and live cells, for example of cells exposed to

poison.

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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. 

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Cell Counting using

Hemocytometer 

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 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.

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 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.

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Counting grids of hemocytometer

By Richard Wheeler (Zephyris) 2007.

Hemocytometer grid:

red square = 1.0000 mm2,

green square = 0.0625 mm2,

yellow square = 0.040 mm2,blue square = 0.0025 mm2,

at a depth of 0.1 mm 

1 2 3

4 5 6

7 8 9

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To count cells/ mL

1. the volume of each large square is 0.1 mm

3

.1000 mm3 = 1 mL 

0.1 mm3 = 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

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 To prepare the counting chamber

The mirror-like polished surface is carefully cleaned with lenspaper. The cover slip is also cleaned.

The cover slip is placed over the counting surface prior to puttingon the cell suspension.

counting chamber is then placed on the microscope stage andthe 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 whilecleaning.

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The tip of the pipette is placed in the V-

shaped groove on the hemocytometer to load

the sample into the chamber (about 10 μl).

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 

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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 

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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

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Cell viability: trypan blue staining.

Living cells exclude the dye, whereas dead

cells will take up the blue dye.

• Dilute 10 μl of the cell suspension (1:5)

• Place 10 μl 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

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Other Assays LDH (lactate dehydro genase) 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 tointact 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

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Intact cell – 

 PI and FDA is added

Fluorescein inintact cells

PI /FDA cell viabi l i ty assay  

● FDA (Fluorescein diacetate)

● PI (Propidium iodide)

Plasma membrane is damaged

; fluorescein leaks out

PI enters and strains

nucleic acids

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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 onlysome 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.

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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 mildsurfactant, 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 thatcreates chemical bonds between proteins to increase their rigidity.

Common fixatives include formaldehyde, ethanol, methanol, and/or

picric acid.

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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.

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 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.

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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.

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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.