Preparing

Serial
Dilutions
 Materials
Diluents
Sterile 0.1% peptone
0.85% saline 8.5g salt in 1 litre water
Pipette
Pipettors and sterile tips
Stomacher bags a tricky one
Sterile bags commercially available
Bottles for dilutions
Lots commercially available
Sterilised 121C, 15 min
Vortex
Incubator
(Based on AS5013.11.1)
 Calculations what dilution should be
plated out?
Dont know depends on number of
bacteria in the sample
Solution plate out several dilutions and
hope for the best
Experience will allow you to better estimate
the need for dilution
 Calculations working with dilutions

To make a 1 in 10 (1:9, 1/10, 10-1) dilution

add 1 ml of sample to 9 ml of diluent or 10
ml of sample to 90 mls of diluent etc.
 Calculations working with dilutions

To make a 1 in 100 (1:99, 1/100, 10-2)

dilution add 1 ml of sample to 99 ml of
diluent or 10 ml of sample to 990 mls of
diluent etc.
OR
Make a 1/10 dilution and then dilute this
1/10. This is a serial dilution.
1/10 x 1/10 = 1/100
 Calculations working with dilutions

Use the inverse dilution (ID) in calculations

Dilution Inverse (ID)

No dilution (1/1, 100) 1
1/10, 10-1 10
1/100, 10-2 100, 102
1/1000, 10-3 1000, 103
etc
 Calculations

All calculations used here assume

that the volume of your inoculum
is 1 ml ie POUR PLATES
 Calculations liquid samples
Milk, juice, water, beer etc

The concentration of cells or colony forming

units (cfu) per ml of liquid
= colony number x ID
 Calculations liquid samples
Example 1
1. 1 ml orange juice is inoculated onto AC
plates
2. 55 colonies grow

Concentration of aerobic bacteria in orange

juice is 55 cells per ml (or 55 cfu/ml; cfu =
colony forming units)
 Calculations liquid samples
Example 2
1. Orange juice is diluted 1:10 (i.e. 1 ml diluted
with 9 ml saline)
2. 1 ml of 1/10 dilution is inoculated onto AC
plates
3. 87 colonies grow

Concentration of aerobic bacteria in orange juice is

87 x 10 cfu/ml = 870 cfu/ml
 Calculations liquid samples
Example 3
1. Orange juice is diluted 1:1000 (i.e. 1 ml diluted
with 99 ml saline, then 1 ml of 1/100 dilution is
diluted with 9 ml saline)
2. 1 ml of 1/1000 dilution is inoculated onto AC
plates
3. 216 colonies grow

Concentration of aerobic bacteria in orange juice is

216 x 1000 cfu/ml = 216,000 cfu/ml = 2.16 x
105 cfu/ml
 Calculations liquid samples
Example 4
1. Orange juice is diluted 1:500 (i.e. 1 ml diluted
with 99 ml saline, then 1 ml of 1/100 dilution is
diluted with 4 ml saline)
2. 1 ml of 1/500 dilution is inoculated onto AC
plates
3. 17 colonies grow

Concentration of aerobic bacteria in orange juice is

17 x 500 cfu/ml = 8,500 cfu/ml = 8.5 x 103
cfu/ml
 Calculations liquid samples
Example 5
1. Orange juice is diluted 1:100 (i.e. 1 ml diluted
with 99 ml saline)
2. 1 ml of 1/100 dilution is inoculated onto AC
plates
3. Colonies grow but too numerous to count (tntc) -
assume max countable no. = 300

Concentration of aerobic bacteria in orange juice >

300 x 100 cfu/ml > 30,000 cfu/ml > 3.0 x 104
cfu/ml
 Calculations solid samples
Solid & semi-solid food

Try to make the initial dilution about 1/10

The initial dilution is assumed to be 1/10 even if
this is not so. A correction factor is used.

The concentration of cells or colony forming

units (cfu) per ml of liquid
= colony number x ID x 10/wt of food
 Calculations solid samples
Example 6
1. 8.9g mince meat is blended in 90 ml saline ~
1/10 dilution w/v
2. 1 ml of ~1/10 dilution is inoculated onto AC
plates
3. 38 colonies grow

Concentration of aerobic bacteria in mince meat =

38 x 10 x 10/8.9 cfu/g
= 427 cfu/g
 Calculations solid samples
Example 7
1. 11.1 g mince meat is blended in 90 ml saline
(~1/10 dilution), 1 ml of ~1/10 dilution is diluted
in 9 ml saline (~1/100 dilution
2. 1 ml of ~1/100 dilution is inoculated onto AC
plates 196 colonies grow

Concentration of aerobic bacteria in mince meat =

196 x 100 x 10/11.1 cfu/g
= 17,658 cfu/g