MEDIA. 293 



Allow the agar to solidify so that a slanting surface is 

 obtained. 



Peptone Water. This contains 1 per cent. Witte's peptone 

 and 0*5 per cent, salt ; the solution is heated till clear, filtered, 

 and quantities of about 1 c.c. placed in Durham tubes, which are 

 plugged with cotton wool, and sterilised. 



Sugar Media. The sugars used are glucose, lactose, and 

 cane sugar ; other carbohydrates, such as dulcitol, adonitol, 

 and inulin, may also be used, but the three sugars give a fairly 

 good distinction between the organisms of intestinal origin. 



These media are made up, containing 7-5 per cent, gelatine, 

 2 per cent, peptone, 1 per cent. Lemco, and 1 per cent, of the 

 sugar ; they are heated till clear, filtered, 1 c.c. of 5 per cent, 

 potash solution added to each 100 c.c., and the media tinted 

 blue with litmus. Quantities of about 1 c.c. are placed in Durham 

 tubes, five of the small tubes being held together by an india- 

 rubber band, and contained in a 3-inch X 1-inch plugged test 

 tube. They are sterilised as usual. 



Milk Tubes. Plugged test-tubes, each containing 10 c.c. of 

 separated milk, are sterilised. 



It is a convenience to plug tubes containing the various media 

 with different coloured cotton wool ; this saves labelling, and 

 minimises the chance of error. The use of different coloured 

 cotton wool also serves to distinguish different quantities of 

 water taken. 



Prepare also a number of test-tubes, each containing 9 c.c. of 

 distilled water ; plug these with cotton wool ; and sterilise. 

 The tubes containing nutrient media and sterilised water must 

 be covered with a rubber cap to prevent evaporation. 



Procedure. Sterilise a pipette delivering 1 c.c. by heating to 

 150 C. (350 F.) ; the pipette is best sterilised in a test-tube 

 plugged with cotton wool. As soon as this is cool, open the 

 bottle containing the sample, and take out 1 c.c. Add this to 

 one of the tubes containing sterilised water and replace the 

 plug immediately. Take out another 1 c.c. and add this to a 

 tube of nutrient gelatine, which should have been previously 

 liquefied and allowed to cool to 27 C. (80 F.). Pour into a 

 sterilised Petri dish. 



With another sterilised 1 c.c. pipette add 1 c.c. of the mixture 

 of water with sterilised water to a tube of nutrient gelatine, 

 which has been previously liquefied by heating and cooled to 

 about 27 C. (86 F.), and pour into a Petri dish. 



The Petri dishes should be placed in an ice chest to solidify 

 the gelatine. Place the gelatine cultivations in an incubator 

 kept at about 22 C. (or 72 F.) ; after two and a-half days the 

 number of colonies that have developed are counted. 



