Dilution and plate counts

A broth culture holds far too many cells to count. Dilute it until the colonies on a plate can be counted by eye, then multiply back — that number is the answer.

Labs 3 and 4. The arithmetic is small; the marks are lost on choosing the wrong plate and on forgetting to divide by the volume plated.

Work it out

Sample transferred

What you pipette from one tube to the next.

Diluent in each tube

1 mL into 9 mL is the standard 1:10 step.

Number of tubes

Between 1 and 12.

Dilutions multiply

Three 1:10 tubes give 1:1000, not 1:30. Add the exponents if you prefer: 10⁻¹ × 10⁻¹ × 10⁻¹ = 10⁻³.

Each step

1:10

Sample ÷ total volume — 1 mL in 10 mL.

Final tube

10⁻⁶

1:1000000

Working

01Each tube takes 1 mL of sample into 9 mL of diluent, giving 1 mL in 10 mL total.
02That is a 1:10 dilution at every step.factor 10
03Dilutions multiply down the series — they never add.

Fig. 1The series drawn out, with a plate poured from each tube. Colony density falls with dilution, and only one plate lands in the countable window.

Tube	Dilution	As a power of ten	Cells per mL from a 2.4 × 10⁷ sample	Colonies on 0.1 mL	Usable
1	1:10	10⁻¹	2.40e+6	240,000	Too many
2	1:100	10⁻²	2.40e+5	24,000	Too many
3	1:1000	10⁻³	2.40e+4	2,400	Too many
4	1:10000	10⁻⁴	2.40e+3	240	Countable
5	1:100000	10⁻⁵	2.40e+2	24	Too few
6	1:1000000	10⁻⁶	2.40e+1	2	Too few

The cells-per-mL column assumes an original sample of 2.4 × 10⁷ per mL, which is why one plate lands between 25 and 250. In a real experiment you do not know that number — which is exactly why you plate several dilutions and count whichever one lands in the window.

The rules behind the numbers

25–250 colonies

Below 25 the count is statistically unreliable — a handful of colonies is mostly noise. Above 250 the colonies crowd each other, merge, and compete for nutrients, so the count reads low.

Spread vs pour

A spread plate takes 0.1 mL on the surface. A pour plate takes 1.0 mL mixed into molten agar, so colonies grow inside as well as on top. The volume goes in the denominator either way.

TNTC and TFTC

Too Numerous To Count and Too Few To Count. Both are reported as such rather than guessed at — a made-up number is worse than no number.

Viable count only

A plate count sees only cells that grow on that medium under those conditions. Dead cells, injured cells and anything fastidious are all invisible to it.

The formula, and where it goes wrong

Three ways to lose the mark, all of them arithmetic rather than biology.

The formula

CFU/mL = colonies ÷ (volume plated × dilution)

The dilution is written as a fraction — 10⁻⁵, not 10⁵ — so dividing by it multiplies the answer back up. If your answer comes out smaller than the number of colonies, the dilution went in the wrong way round.

Slip	What it does
Forgetting the volume	Plating 0.1 mL and dividing by 1 makes the answer ten times too small.
Using the tube dilution, not the plate	The dilution that matters is the one on the plate you counted, not the last tube in the rack.
Counting an out-of-range plate	A plate with 8 or 600 colonies produces a number, and the number is wrong.
Adding dilutions	Four 1:10 tubes give 10⁻⁴, not 1:40.