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Counting cells using a haemocytometer

Counting cells using a haemocytometer. Kate Andrews Lorraine Bruce. Cells and Proteins (Advanced Higher). (f) Aseptic technique and cell culture

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Counting cells using a haemocytometer

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  1. Counting cells using a haemocytometer Kate Andrews Lorraine Bruce

  2. Cells and Proteins (Advanced Higher) (f) Aseptic technique and cell culture Use of inoculum, explants or cells. Use of: haemocytometers to estimate total cell counts; vital staining to estimate viable cell counts. Complex media containing growth factors from serum for animal cell lines. Lifetime of primary cell lines and cancer cell lines in culture. Use of growth regulators in plant tissue culture. Sterilisation of containers, equipment and materials. Disinfection of working area. Culture bacterial, yeast and algal cells using aseptic technique. Use a haemocytometer to make an estimate of cell count.

  3. Yeast cells x100

  4. Saccharomyces cerevisiae as seen using an electron microscope ~ x 500,000 Image: Society for General Microbiology

  5. Blood counts: for patients with abnormal blood cells, where automated counters don't perform well. Sperm counts Cell culture: when subculturing or recording cell population growth over time. Beer brewing: for the preparation of the yeast cultures. Cell processing for downstream analysis: accurate cell numbers are needed in many tests (PCR, flow cytometry) while some others require high cell viability. Measurement of cell size: in a micrograph, the real cell size can be inferred by scaling it to the width of a haemocytometer square, which is known.

  6. www.sserc.org.uk Home / Biology Home / Resources / Microbiological Techniques / Enumerating micro-organisms / Counting cells using a haemocytometer

  7. See SSERC website: http://www.sserc.org.uk/index.php/biology-2/biology-resources/microbiological-techniques265/enumerating-micro-organisms141

  8. C-Chip Haemocytometer • Cheaper! • Disposable • No need for coverslips! • Virtually non-breakable! • Reusable (within limitations)

  9. Image: www.invitrogen.com Image : https://www.labtech.com/disposable-haemocytometers

  10. https://www.labtech.com/disposable-haemocytometers

  11. 1mm 0.2mm The grid has 25 squares, we will sample 5 squares

  12. C-Chip grid view x40

  13. C-Chip grid view x100

  14. Boundary lines Top left square x400

  15. Bottom right square x400

  16. Loading the haemocytometer

  17. Yeast cells in grid x100

  18. Yeast cells in one square of the grid x400

  19. Systematic counting....

  20. What should be counted?

  21. Volume in sample area (5 squares) Length of side of grid = 1 mm Area of grid = 1 mm2 Depth between coverslip and slide = 0.1 mm Volume under grid = 1 mm2 x 0.1 mm (25 squares) = 0.1 mm3 Volume under 5 squares = 0.1 / 5 mm3 = 0.02 mm3 Estimating cell concentration (in 5 squares) Number of cells in sample = n Number of cells in 1 mm3 = n x 50 (because 0.02 mm3 is 1/50 of 1 mm3) Number of cells in 1 cm3 = (n x 50) x 1000

  22. If the concentration of cells is very low, all the cells in the 25 square grid could be counted. Volume in grid (25 squares) Length of side of grid = 1 mm Area of grid = 1 mm2 Depth between coverslip and slide = 0.1 mm Volume under grid = 1 mm2 x 0.1 mm (25 squares) = 0.1 mm3 Estimating cell concentration Number of cells in grid = n Number of cells in 1 mm3 = n x 10 Number of cells in 1 cm3 = (n x 10) x 1000

  23. For counting large cells, some protocols advocate counting the cells in the ‘big’ squares... • Some protocols suggest averaging the number of cells counted prior to doing the calculation of cell concentration

  24. In some situations cell viability is also taken into consideration – • Trypan blue • Methylene blue Yeast cells (x 400) stained with methylene blue Live cells actively pump out the dye. Non-viable cells are dark blue. Image: http://braukaiser.com

  25. Cell counting apps iPhone HemocyTap £1.49 https://itunes.apple.com/us/app/hemocytap/id617767138?mt=8&ign-mpt=uo%3D4

  26. Counting cells

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