1. Preparing Serial Dilutions

2. 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 121°C, 15 min
Vortex
Incubator
(Based on AS )

3. Calculations – what dilution should be plated out?
Don’t 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

4. 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.

5. 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

7. Calculations – working with dilutions
Use the inverse dilution (ID) in calculations
Dilution Inverse (ID)
No dilution (1/1, 100) 1
1/10,
1/100, , 102
1/1000, , 103
etc

8. Calculations
All calculations used here assume that the volume of your inoculum is 1 ml ie POUR PLATES

9. 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

10. Calculations – liquid samples
Example 1
1 ml orange juice is inoculated onto AC plates
55 colonies grow
Concentration of aerobic bacteria in orange juice is 55 cells per ml (or 55 cfu/ml; cfu = colony forming units)

11. Calculations – liquid samples
Example 2
Orange juice is diluted 1:10 (i.e. 1 ml diluted with 9 ml saline)
1 ml of 1/10 dilution is inoculated onto AC plates
87 colonies grow
Concentration of aerobic bacteria in orange juice is 87 x 10 cfu/ml = 870 cfu/ml

12. Calculations – liquid samples
Example 3
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)
1 ml of 1/1000 dilution is inoculated onto AC plates
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

13. Calculations – liquid samples
Example 4
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)
1 ml of 1/500 dilution is inoculated onto AC plates
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

14. Calculations – liquid samples
Example 5
Orange juice is diluted 1:100 (i.e. 1 ml diluted with 99 ml saline)
1 ml of 1/100 dilution is inoculated onto AC plates
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

15. 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

16. Calculations – solid samples
Example 6
8.9g mince meat is blended in 90 ml saline ~ 1/10 dilution w/v
1 ml of ~1/10 dilution is inoculated onto AC plates
38 colonies grow
Concentration of aerobic bacteria in mince meat = 38 x 10 x 10/8.9 cfu/g
= 427 cfu/g

17. Calculations – solid samples
Example 7
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
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