Hemocytometer Instructions

 

Materials

Cell suspension at ~10⁶ cells/mL

Hemocytometer and coverslip

Micropipette

Tally counter (optional)

 

Safety

1.      Wear disposable latex gloves at all times

2.      Wear safety glasses at all times.

 

Procedure

1.      Clean hemocytometer and coverslip with ethanol and kimwipes.  (NOTE:  The coverslip is very fragile.)

2.      After both pieces have dried, place coverslip on hemocytometer.

3.      Gently mix cells. 

4.      Aliquot 10 mL of cells.  Insert pipet tip into the grove between the hemocytometer and the coverslip at the edge of the chamber and eject the cell suspension under the coverslip (see Freshney p. 310).  Hold the micropipette at approximately 45° angle to the table.  You may have to try this several times before you successfully transfer the cell suspension into the chamber.

5.      Let the cell suspension be drawn under the coverslip by capillarity.  Add liquid only until full.  Do not overfill or underfill the chamber. 

6.      Remix the tube of cells.  Reload the pipet with cells and fill the second chamber.

7.      Carefully blot off any excess liquid.

8.      Transfer the hemocytometer to the microscope stage.  The 3.5” x 5” black plate needs to be in place to secure the hemocytometer.

9.      Count the cells in the center 1 mm² area (includes 25 square areas, each bordered by triple lines).  Use the 10X objective.  Look through your left eye or through the left eyepiece if you are seeing double grids.

10.  When counting, count cells that lie on the top and left-hand lines of each square but not those on the bottom or right-hand lines.  This method should help you avoid counting the same cell twice.  Be slow and deliberate when you begin counting cells so that you can establish a routine that ensures that each cell is counted once.

11.  If the cell number is very high (>300 cells/mm²), count only 5 of the 25 squares.  If the cell number is very low (<50 cells/mm²), count an additional 1 mm² square that borders the center square.  Make appropriate adjustments in your calculations.

12.  Count the cells in the other chamber of the hemocytometer.

13.  Clean hemocytometer and coverslip with ethanol and kimwipes.

14.  Dispose of biohazardous material properly.