Dr.Gatot CiptadiLab. Central of Life Sciences Fac. Of Animal Sci.Brawijaya University Malang

3. ELECTROPHORES4. pH Meter

1. MIKROSCOPE And 2.HEMOCYTOMETER

Counting Micro-organisms , Cells and Sperms

Counting elephants is easy – counting bacteria is not quite as easy!

2. Hemocytometer:Manual, software

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.

SERIAL DILUTION1ml into 9ml = 1:10 dilution

conc. 10-1 10-2 10-3 10-4 10-5 10-6

Counting Micro-organisms or Sperms

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)

Volume : 0.1mm3

1 ml = 1 cm3 = 1000 mm3

1 mm

1 mm

0.1 mm

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)

• Do not count cells touching– Bottom line– Right line

Counting Rule

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

Cell countingCell counting

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.

Example 1 ; Observation of cell death

M e t h o d s

1) Clean hemocytometer & coverslip and wipe with 70% alcohol before use

2) Place coverslip on hemocytometer

3) Mix the cell suspension gently

4) Aliquot 0.1 ml cell suspensions

5) 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

6) Draw a sample into a Pasteur pipette after mixing

7) Draw the cell suspension in to fill the chamber

8) Using a light microscope at low power, count the number of cells

9) Count the viable & non-viable cells in both halves of the chamber

10) 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 B C 10Ⅹ Ⅹ Ⅹ 4

(2) Total dead cell count

A B D 10Ⅹ Ⅹ Ⅹ 4

(3) To give a total cell count

Viable cell count + dead cell count

(4) % viability

(Viable cell count/Total cell count) 100Ⅹ

Example

Dilution factor Vol.

of CSCell count Total viable cells

0.1 ml CS +

0.1 ml TB (2) 20 ml 23 20Ⅹ2 23 10Ⅹ Ⅹ 4 = 9.2 10Ⅹ 6 cells

0.1 ml CS +

0.3 ml TB (4) 15 ml // 15Ⅹ4 23 10Ⅹ Ⅹ 4 = 1.38 10Ⅹ 7 cells

0.1 ml CS +

0.9 ml TB (10)10 ml // 10Ⅹ10 23 10Ⅹ Ⅹ 4 = 2.3 10Ⅹ 7 cells

1) Vol. : Volume

2) CS : Cell Solution

3) TB : Trypan blue

• 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)

RBC Example…

Automated trypan blue method for optimal cell viability determination

www.innovatis.com

Run results

Electric Cell Counting using a COULTER® COUNTER

Electric Cell Counting using a COULTER® COUNTER