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 121°C, 15 min Vortex Incubator (Based on AS5013.11.1)

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

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 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)

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

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

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

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

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 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

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