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 mm2, green square = 0.0625 mm2, yellow square = 0.040 mm2, blue square = 0.0025 mm2,

at a depth of 0.1 mm

To count cells/ mL 1. the volume of each large square is 0.1 mm3. 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

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.

Load the sample

The tip of the pipette is placed in the Vshaped 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.

How to count
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.

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

(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

PI/FDA cell viability assay

Intact cell PI and FDA is added FDA (Fluorescein diacetate) PI (Propidium iodide) Fluorescein in intact cells

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.