Download Serial Dilutions and Basic Calculations

Preparing
Serial
Dilutions
Materials
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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
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Lots commercially available
Sterilised 121°C, 15 min
• Vortex
• Incubator
(Based on AS5013.11.1)
Calculations – what dilution should be
plated out?
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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
Calculations – working with dilutions
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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
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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
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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.
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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