The haemocytometer Flashcards
How can the population of a yeast culture be investigated?
Adding a sample of yeast suspension to a flask of nutrient medium which has been previously sterilised. e.g. 2% glucose solution and mineral salts (e.g. ammonium and phosphate). Plugged and incubated at 25ºC. Samples then taken and counted using a haemocytometer.
How do you calculate cell density?
A suspension containing the cells to be counted should be placed on the grid. Then decide what type of square A, B or C to be used. Always use the North-West rule when counting the cells.
What is the usual pattern of growth that is observed?
It should show a phase of exponential growth, followed by saturation and may even include a decline phase.
Exponential - Plentiful resources, no restriction to growth.
Plateau - When a factor becomes limiting e.g. space, nutrients or accumulation of waste.
Decline - Death rate exceeds growth rate.
What are some important points about using the haemocytometer?
1) Mix the yeast suspension thoroughly before taking a sample
2) Sample from the same depth in the flask
3) If sampling from a natural environment e.g. phytoplankton - samples taken from the same depth and at the same time of day
4) Avoid getting the yeast suspension on top of the coverslip or in the grooves - this changes the distance from the coverslip to the grid from 0.1mm.
5) Carry out an appropriate number of replicates.
What do you do when there are too many cells to be able to count them as discrete entities?
A serial dilution. Dilute the suspension by a factor of 10. 1cm³ of of the original suspension is diluted with 9cm³ of an isotonic buffer. And the multiply by the dilution factor at the end.
How can you distinguish between living and dead cells?
Stain with methylene blue. Dead cells are blue while living cells are colourless.
How can a colorimeter be used to estimate bacterial numbers?
1) Explanation of sampling technique from bacterial culture
2) Initial calibration of bacterial culture with no bacteria in the sample
3) % transmission will be lower for a more dense bacterial population
4) The population becomes more dense over time as the population grows
5) Use a calibration curve to compare % transmission with bacterial numbers.
What is an advantage of using the colorimeter?
Faster / Less time consuming
