SAMPLING AND SAMPLE PREPARATION Reagents Peptone Water Powder(CM9-Oxoid 0. 1% Peptone Water 180ml in 200 ml medical flat bottle 0. 1% Peptone Water 9ml in Universal media bottles Reagent Preparation Dissolve 1gm of peptone water(CM9)in 1 litre distilled water and dispense either 180ml or 9ml per bottle as given above. Autoclave for 15 minutes at Samplin g Samples should be taken using clean equipment and cross contamination must be avoided. This is most easily effected by using a polythene bag which is inverted over the hand, the product is grasped by the gloved hand and enclosed by re-inverting the bag Sample Preparation Place part of sample into grinder and mill, reject, repeat operation twice, the third part is retained. Transfer 20gm into 180ml of sterilized peptone water using a flamed spoon/spatula, mix well, allow to stand for at least 20 minutes to resuscitate the bacteria A 10% homogenate has been produced i. e 1ml contains 0.1gm Further dilutions are obtained by taking lml and adding to 9ml sterilized peptone water as shown below: .Ogm+180ml = 10% lml contains 0. 1gm or x 10 1ml10%+9ml=1% lml contains 0.01 gm or x 100 lml1%+9ml=0.1% lml contains 0.001gm or x 1,000 1ml0.1%+9ml=0.01% 1ml contains 0.0001gm or x 10 and (1)10% homogenates can be obtained by using 10gm+90ml, 25gm+ 225gm or 5gm+ 45ml if desired (2)When moving from dilution to dilution a fresh clean sterilized pipette must be usedSAMPLING AND SAMPLE PREPARATION Reagents Peptone Water Powder (CM9 - Oxoid) 0.1% Peptone Water 180ml in 200 ml medical flat bottles 0.1% Peptone Water 9ml in Universal media bottles Reagent Preparation Dissolve lgm of peptone water (CM9) in 1 litre distilled water and dispense either 180ml or 9ml per bottle as given above. Autoclave for 15 minutes at 15psi. Sampling Samples should be taken using clean equipment and cross contamination must be avoided. This is most easily effected by using a polythene bag which is inverted over the hand, the product is grasped by the ‘gloved’ hand and enclosed by re-inverting the bag. Sample Preparation Place part of sample into grinder and mill, reject, repeat operation twice, the third part is retained. Transfer 20gm into 180ml of sterilized peptone water using a flamed spoon/spatula, mix well, allow to stand for at least 20 minutes to resuscitate the bacteria. A 10% homogenate has been produced i.e. lml contains 0.lgm. Further dilutions are obtained by taking lml and adding to 9ml sterilized peptone water as shown below: 20gm + 180ml= 10% - lmllO% + 9ml= 1% lml1% + 9ml= 0.1% - lml 0.1% + 9ml= 0.01% - lml contains O.lgm or x 10 lml contains 0.01 gm or x 100 lml contains 0.001gm or x 1,000 1ml contains 0.0001gm or x io4 and so as may be required NOTE (1) 10% homogenates can be obtained by using lOgm + 90ml,25gm + 225gm (2) When moving from dilution to dilution a fresh clean sterilized pipette or 5gm + 45ml if desired. must be used. 246