How to count cells with a hemocytometer.

An improved Neubauer hemocytometer with its cover glass, 0.4% trypan blue, a pipette that delivers 10 microlitres, a microscope with a 10x objective, a tally counter or a pen, and a written protocol that says which squares are counted and at what dilution. The chamber is glass and reusable; the only thing you use up is the dye. The word is also spelled haemocytometer, in British English, or hemacytometer, and the device is also called a counting chamber or a Neubauer chamber.

A hemocytometer is a thick glass slide with a grid ruled into a polished floor and a raised support on either side of it. A cover glass laid across the supports sits exactly 0.1 mm above the ruled floor, and that fixed depth is the whole principle: each square of the grid marks off a known volume of sample, so the cells counted over a square are the cells in that volume. Labs use it to count cultured cells before seeding, passaging or freezing, to read viability with trypan blue, and to count blood cells, yeast and other cells wherever a concentration is needed and a microscope is at hand.

An improved Neubauer chamber has two counting areas, one on each side of the moat, each ruled with the same 3 by 3 mm grid of nine large squares. The corner squares are divided into 16 squares of 0.25 mm, the side squares into 20 rectangles, and the centre square into 25 squares of 0.2 mm, bounded by triple lines and each divided again into 16 of 0.05 mm. All of them sit under the same 0.1 mm of sample, so the size of a square sets the volume over it and the number its average count is multiplied by.

1. 1

   Mix the sample with trypan blue

   Resuspend the cells well and mix one part sample with one part 0.4% trypan blue. Count within a few minutes, because the dye eventually enters live cells too. The one-to-one mix is a dilution factor of 2; multiply in any dilution you made before it.

2. 2

   Load the chamber

   Seat the thick cover glass made for the chamber on a clean improved Neubauer hemocytometer: press it down onto the two raised supports and rub it gently back and forth until bands of colour, Newton’s rings, show on both. Then touch about 10 microlitres of the mix to the edge of the cover glass and let the chamber fill by capillary action without overflowing into the moat. Give the cells a moment to settle.

3. 3

   Count the large squares

   Under the 10x objective, count one 1 mm square at a time; the four corner squares, or the four corners and the centre, is the usual pattern. Clear cells are live, blue cells are dead. Pick one rule for cells on a line, for example count those touching the top and left lines and not the bottom and right, and keep it.

4. 4

   Turn the count into concentration and viability

   Cells per mL = the average number of cells in one 1 mm square × 10,000 × the dilution factor, because a square over the chamber’s 0.1 mm depth holds 0.1 microlitre. Viability = live ÷ (live + dead). Aim for roughly 100 cells per square, and dilute or concentrate the sample if you are far from it.

5. 5

   Write down how it was counted

   Record the dilution, which squares, who counted and when, next to the number. Two people who follow the same written protocol get comparable counts; two blinded experts with no shared protocol have been measured to differ by about 20% on the same cultures.

The 10,000 in the formula holds only if the cover glass lies on the supports with nothing between glass and glass. When it does, bands of colour called Newton’s rings show on both supports: light interfering in the very thin gap where the two surfaces touch. No rings on a support means the glass is not down on it, the chamber is deeper than 0.1 mm, and the count comes out high.

A trace of moisture helps the rings form, which is why some protocols suggest breathing on the supports before the cover glass goes on. It has a cost: a film of water can hold the glass up, and any that reaches the counting area dilutes the sample. The same seal without it: press the cover glass down onto the supports and rub it gently back and forth until the rings show on both. Use the thick, flat cover glass made for counting chambers; an ordinary coverslip bends, and the depth bends with it.

Four corner squares, sample mixed one to one with trypan blue, so the dilution factor is 2.

Average per square

411 ÷ 4 = 102.75

Cells per mL

102.75 × 10,000 × 2 = 2,055,000

Viability

376 ÷ 411 = 91.5%

About 2.06 million cells per mL, of which 1.88 million are live. A seeding calculation uses the live concentration, so keep both numbers. The cell count formula, with a calculator for your own count.

The method is simple and the spread is not. On latex beads of a known concentration, the cleanest possible case, one lab re-counting the same sample spreads by ~5%, and different labs by 14.6%. Two blinded experts counting the same cell cultures, with a stain and clumps and a live-or-dead call to make, have been measured to differ by roughly 20%. The differences come from the loading, the choice of squares, the rule for cells on a line, the dilution arithmetic and the judgement of what is a cell, and a written protocol removes most of them. The studies are cited on the validation page and the terms are defined in the glossary.

Clean the chamber and its cover glass straight after counting, before the sample dries on them. Rinse both with distilled water, then with 70% ethanol, which also disinfects, and dry them with lens tissue. Wipe the ruled floor gently and never with anything abrasive: a scratched ruling is harder to read, by eye and in a photo. Handle both by the edges, keep them in their case, and follow your lab’s rules for anything infectious.

CellCount does this count from a photo of the chamber: a microscope camera on the port, a phone held to the eyepiece, or a file you already have. It measures the rulings in the image to get the scale (if they cannot be found, you place the crossings by hand), counts every cell inside the square, calls each one live or dead, and applies the dilution and the counting pattern from a protocol the whole lab shares, so the arithmetic above happens the same way every time. Every detected cell is marked on your image; you add, delete or flip the ones you disagree with and accept the count, and the record keeps who accepted it and when.