TOTAL VIABLE COUNT (TVC) Media Plate Count Agar( CM463B-Oxoid) Peptone Water(CM9-Oxoid) Media Preparation Add 235gm plate count agar to 1 litre of distilled water, dispense 9ml into universal bottles and sterilize for 15 minutes at 15psi When cooled the agar will solidify and can be stored in this state for several months For use the agar should be melted in a boiling waterbath and allowed to cool to 45-50C Method Take the 0. 1% dilution of the sample as prepared under sample preparation and pipette 1.0ml via a sterile petri dish, add gmls of cooled melted agar and mix by gently swirling the petri dish(or plate). Allow the mixture to set, place upside down in the incubator at 30'C and leave for 2 days After this time the number of colonies are counted and multiplied by 1,000 to give a count per gn If counts in excess of 300,000 per gm i.e. 300 per plate are expected then the 0.01% dilution should be used. The dilution should be increased so that the count does not exceed 300 per plate. Initially it may be necessary to carry out E(ESCHERICHIA) COLI Media MacConkey No 3(Cml15-Oxoid) Media Preparation Dissolve 515gm of agar in 1 litre distilled water, by bringing to the boil, dispense 10mls into universal bottles, sterilize for 15 minutes at 15ps When cooled this media will set, prior to use melt in a boiling water bath and allow to cool to 45-50C Method Pipette Iml of the 10% homogenate onto a sterile petri dish, add 10ml of cooled, melted agar, swirl to mix and allow to set. Incubate upside down at 44°Cfor24 hours Count only the red/ purple colonies and multiply by 10 to give the lumber of coloniesTOTAL VIABLE COUNT (TVC) Media Plate Count Agar (CM463B - Oxoid) Peptone Water (CM9 - Oxoid) Media Preparation Add 23.5gm plate count agar to 1 litre of distilled water, dispense 9ml into universal bottles and sterilize for 15 minutes at 15psi. When cooled the agar will solidify and can be stored in this state for several months. For use the agar should be melted in a boiling waterbath and allowed to COO^ to 45-50°C. Method Take the 0.1% dilution of the sample as prepared under sample preparation and pipette 1.0ml via a sterile petri dish, add 9mls of cooled melted agar and mix by gently swirling the petri dish (or plate). Allow the mixture to set, place upside down in the incubator at 30°C and leave for 2 days. After this time the number of colonies are counted and multiplied by 1,000 to give a count per gm. If counts in excess of 300,000 per gm i.e. 300 per plate are expected then the 0.01% dilution should be used. The dilution should be increased so that the count does not exceed 300 per plate. Initially it may be necessary to carry out several dilutions in order to ascertain the range of results. E. (ESCHERICHIA) COLI Media Media Preparation Dissolve 51.5gm of agar in 1 litre distilled water, by bringing to the boil, dispense 10mls into universal bottles, sterilize for 15 minutes at 15psi. When cooled this media will set, prior to use melt in a boiling water bath and allow to cool to 45-50°C. Method Pipette lml of the 10% homogenate onto a sterile petri dish, add lOml of cooled, melted agar, swirl to mix and allow to set. Incubate upside down at 44°C for 24 hours. Count only the red/purple colonies and multiply by 10 to give the number of colonies per gm. MacConkey No 3 (Cm115 - Oxoid) 247