1. MIKROSCOPE And 2.HEMOCYTOMETER 3. ELECTROPHORES 4. pH Meter Dr.Gatot Ciptadi Lab. Central of Life Sciences Fac. Of Animal Sci. Brawijaya University Malang

Counting Micro-organisms , Cells and Sperms 2. Hemocytometer: Manual, software Counting Micro-organisms , Cells and Sperms Counting elephants is easy – counting bacteria is not quite as easy!

Counting Micro-organisms A single tiny drop of nutrient broth incubated overnight may contain 5 000 000 cells – this is a lot to count. 1cm3 may contain 108 cells. In order to estimate numbers it is necessary to dilute the sample.

Hemocytometer Chamber Depth = 0.1 mm Grids RBC use 5 small squares in the center large square WBC use 4 corner large squares

Thoma Pipet RBC Aspirate to 0.5 or 1.0 Dilute to 101 DF = 200 or 100 WBC Aspirate to 0.5 or 1.0 Dilute to 11 DF = 20 or 10

The most common routine method for cell counting which is efficient and accurate is with the use of a hemocytometer.

Counting Micro-organisms or Sperms SERIAL DILUTION 1ml into 9ml = 1:10 dilution conc. 10-1 10-2 10-3 10-4 10-5 10-6

Counting Micro-organisms It is possible to dilute a cell suspension in known steps in order to plate a specific range of cells. This set of plates contain a 1 to 10 dilution series.

Hemocytometer Originally used in counting RBCs Consists of a thick glass microscope slide with a rectangular indentation that creates a counting chamber Counting chamber - engraved with a laser-etched grid of perpendicular lines Raised edges hold cover slip above these marked grids

Cell quantification using hemocytometer See microscopes on the table.

Cell Counting - haemocytometer

TOTAL COUNTS Direct counts The haemocytometer is a specialized microscope slide used to count cells. The centre portion of the slide has etched grids with precisely spaced lines.

Hemocytometer Counting chamber is a 3 x 3 mm square and divided into nine large squares Each resulting square measures 1 mm2, which are used for white blood cell counting (Square A) The center square is divided into 25 smaller squares. (1/25 mm2) Each smaller square is subdivided into 16 even smaller squares, which are used for counting red blood cells. (Square B = 1/400 mm2)

1 mm 0.1 mm Volume : 0.1mm3 1 ml = 1 cm3 = 1000 mm3

TOTAL COUNTS To get an accurate count there should be between 40 and 70 cells in a 1 mm square. If not you dilute or concentrate the cell suspension as necessary .

Determination of Number of Cells per mL Coverslip sits 0.1 mm over the chamber 1mm 3 = 10-3 cubic centimeters (cc) or ml Volume of a square = 1 mm x 1 mm x 0.1 mm = 0.1 mm3 = 10-4 cc = 10-4 mL Take the average of the cells found in the 5 squares (the four corners and the middle one). Avoid counting the cells twice by disregarding the cells found at the upper and left borders Multiply this by 104 to get the # cells/ml in the suspension. If, however, the suspension was diluted prior to counting, then one would also multiply by the dilution factor to get the # cells/ml in the culture flask. Avg. # cells counted x 104 = # cells/ml (x DF if any)

Counting Rule Do not count cells touching Bottom line Right line

Cell counting Measurement Characteristics Methods Direct Distinguish viable and non-viable cell - Simple, quick, cheap - A small fraction of the total cells from a cell suspension - Microscopic counting(Hemocytometer) : Trypan blue, Crystal violet - Electronic counters : Coulter counter Indirect - Monolayer - Immobilized in matrix - Lactate dehydrogenase activity - Functional assay - DNA labeling assay

Example 1 ; Observation of cell death A group of hepatoma cells exposed to a diffusing wave of digitonin. Intact cells (green) are damaged by digitonin, loose the green fluorescence and acquire red fluorescence of PI.

M e t h o d s Clean hemocytometer & coverslip and wipe with 70% alcohol before use Place coverslip on hemocytometer Mix the cell suspension gently Aliquot 0.1 ml cell suspensions Add 0.1 ml (2-fold dilution), 0.3 ml (4-fold dilution) or 0.9 ml (10-fold dilution) trypan blue : appropriate range of cells to be counted Draw a sample into a Pasteur pipette after mixing Draw the cell suspension in to fill the chamber Using a light microscope at low power, count the number of cells Count the viable & non-viable cells in both halves of the chamber

Calculations A = Vol. Of cell solution (ml) B = Dilution factor in trypan blue C = Mean number of unstained cells D = Mean number of dead/stained cells 104 = Conversion of 0.1 mm3 to ml (1) Total number of viable cells A Ⅹ B Ⅹ C Ⅹ 104 (2) Total dead cell count A Ⅹ B Ⅹ D Ⅹ 104 (3) To give a total cell count Viable cell count + dead cell count (4) % viability (Viable cell count/Total cell count) Ⅹ 100

Example Dilution factor Vol. of CS Cell count Total viable cells Vol. : Volume CS : Cell Solution TB : Trypan blue Dilution factor Vol. of CS Cell count Total viable cells 0.1 ml CS + 0.1 ml TB (2) 20 ml 23 20Ⅹ2Ⅹ23Ⅹ104 = 9.2 Ⅹ106 cells 0.3 ml TB (4) 15 ml // 15Ⅹ4Ⅹ23Ⅹ104 = 1.38 Ⅹ107 cells 0.9 ml TB (10) 10 ml 10Ⅹ10Ⅹ23Ⅹ104 = 2.3 Ⅹ107 cells

RBC Example… Aspirate blood to 0.5, then dilute to 101 DF = 200 Counted 500 erythrocytes in 5 small squares RBC = 500 rbc x 25 Sm Sq x DF (200) 5 Sm Sq 0.1 mm3 = 5.00 x 106/mm3 (L)

Automated trypan blue method for optimal cell viability determination www.innovatis.com

Electric Cell Counting using a COULTER® COUNTER Run results

Electric Cell Counting using a COULTER® COUNTER