Hemocytometer

A hemocytometer has a calibrated grid and depth. ATCC describes $1{\mathrm{mm}}^2$ squares $0.1\mathrm{mm}$ deep, each sampling $10^{-4}\mathrm{mL}$.^[ref]

An original constructed hemocytometer count uses four such squares after mixing the biological cell preparation with the same volume of stain. The dye-excluding counts are $36,44,40,40$; the dye-stained counts are $4,6,5,5$. The dilution factor is $D=2$. With $v$ the sampled volume per square and $\bar{n}$ the arithmetic mean count per square,Hence the undiluted preparation has dye-excluding number density $2(40)/10^{-4}=800000{\mathrm{mL}}^{-1}$ and total number density $2(45)/10^{-4}=900000{\mathrm{mL}}^{-1}$. Its dye-excluding fraction is $160/180=8/9\approx 88.9\%$. If the well-mixed preparation contains $3\mathrm{mL}$, it contains an estimated $2400000$ dye-excluding biological cells. The chamber sample itself contains only $160$ counted dye-excluding biological cells.

The relation $c=D\bar{n}/v$ follows from counting material in a known volume, then undoing dilution. It assumes a representative mixed sample and correctly identified single biological cells. Clumps, inconsistent decisions at grid edges, wrong chamber depth and nonrepresentative sampling introduce measurement error. A fixed convention such as including top and left edges while excluding bottom and right edges assigns shared-edge centers to just one square. Adding ten times as much suspension to a correctly filled chamber does not make each square's calibrated volume ten times larger.