Populations - Haemocytometer Flashcards
(16 cards)
Investigating growth of yeast populations
Add sample of yeast suspension to a flask of nutrient medium which has been previously sterilised
Suitable nutrient medium for yeast is 2% glucose solution (carbon source) and mineral salts (ammonium - N)
Flask plugged and incubated 25°C over several days - samples taken at intervals (twice a day)
Yeast cells counted with haemocytometer - cell density (mm-3) plotted against time (hours)
Haemocytometer
Special glass slide with accurately ruled, etched grid of precise dimensions
Area Type-C Square
0.0025mm2
Area Type-B square
0.04mm2
Volume Type-C square
0.00025mm3
1/4000mm3
To obtain Cell Count
Representative sample of yeast culture is placed beside coverslip so that capillarity draws it under
Number of cells in 20 type-C squares counted and totalled
40 facilitated calculation of cell density
Formula for Calculation of Cell Density
Graph of Haemocytometer
Graph show exponential increase followed by saturation (curve plateaus)
May even include decline phase (depends on duration of experiment)
Explanation of Graph
Exponential - Resources plentiful and not limiting yeast cell division
Plateau - when some factor become limiting (lack of resources or accumulation of waste)
Decline - many cells are dying (same as above) and not being replaced
Why is culture shaken
Ensure homogenous suspension before sampling
When will procedure need to be repeated
If sample on haem enters the grooves
Counting of Type-C Squares
North West Rule
Include cells touching top and left boundaries - exclude those touching right and bottom
Ensures no cell counted twice
Counting if very Few Cells
Early stage of growth
Type-A or Type-B squares counted as many C will be empty
Area Type A Square
1mm2
If Samples Dense
May be too many cells to count
Accurate dilution (1 in 10) made and final count should then be multiplied by dilution factor
Distinguish Living and Dead Cells
Haem gives total viable and non-viable cells
Distinguished by staining with methylene blue
•Dead=Blue
•Living=Colourless
