Counting trypanosomes:

Using the inverted scope, estimate the cell density of the culture to determine the appropriate dilution for counting (this takes some practice to get the eye for it – remember being in the ball park is usually sufficient). To start with, a mid-log culture can usually be diluted 5 – 10 fold prior to counting.

Dilute a small aliquot of your culture with the correct amount of PBS to achieve your desired dilution. ( e.g. 100 ul of culture + 900 ul of PBS = a 1:10 dilution).

Place 10 ul of your diluted culture on each side a hemocytometer.

Using the 40X objective, start in the top left box of the 5 x 5 grid and count the number of tryps in each box of the grid. Be consistent when counting cells on the edges of a box so as not to double count them.

To determine the concentration of your cells, use the following equation:

(# of counted cells)(dilution)(1 ee4 cells/ml) = culture density in cells / ml

The equation to determine to what concentration to dilute your cells in order for them to grow to a desired concentration in a specific time is:

A₀ = A_i / 10^{T (k)}

Where A₀ = the new “dilute to” concentration

A_i = ideal or desired final concentration

T = time of growth in hours

k = constant = 0.043

e.g. If you want a culture to be at 3 x 10⁶ cells/ml in 24 hours, then:

A₀ = 3 ee6 / 10 ^{(24) (0.043)}, = 3 ee6 / 10^1.032, = 3 ee6 / 10.76, = 2.78 x 10⁵ cells / ml

Therefore, you can determine the necessary dilution factor of your current culture to be at the density of 2.78 x 10⁵ cells/ml in the final volume of your new culture.