EXPERIMENTS OF A UTHORS 1 3 1 



Notes, — (i) The experiments began in April 1900 at Denham. 



{2) The milking was carried out in the usual way, without notice or warning to the 

 milkmen. The cowshed was an ordinary country byre, and no precautions of any kind 

 were taken, nor was the milk when drawn refrigerated or treated in any way. The 

 temperature of the milk at the time of milking was 34° C. Samples of milk were taken 

 from the pail containing the mixed milk of three cows during the process of milking. 

 The samples were received into sterilised flasks, and conveyed to the laboratory and 

 incubated at 5° C, at 15° C, and at 37° C. These temperatmres were chosen as represent- 

 ing in a general way a cold storage cellar, an ordinary dairy room temperature, and Mood 

 heat. The three temperatiu-es were uniformly maintained throughout the experiment. 



(3) Foiu" Petri gelatine plates were made at once from the common stock of milk in 

 the milk pail from which the samples had been taken. The average number of organisms 

 found, which furnished the initial bacterial standard of the milk imder observation, was 

 812,000 per c.c. 



(4) The succeeding plates were made from a dilution in sterile water from each of the 

 three samples (at 5°, 15°, and 37°) of one in five hundred Criir)- These dilutions were 

 made with great care and absolute accuracy (jee p. 48). Of the dilution, we took -OS of 

 a cjc. (=1 drop from the specially calibrated pipette), •! cc. ( = 2 drops), and -2 cc. 

 ( =4 drops), and added these amounts to tubes of melted gelatine, which were thoroughly 

 mixed and poured into Petri dishes. Thus, for example, at eight o'clock one of the 

 samples of milk which had been incubated for exactly four hours at 5° C. was taken, a 

 portion was diluted to i in 500, and three gelatine plates were made (-05 c.c, -l c.c, -2 cc.) 

 and incubated at 22° C. The same was done with samples of milk at each of the three 

 temperatures every four hours, as the table indicates. Upwards of 200 Petri plates were 

 made during this experiment. 



(5) The gelatine plates were in all cases incubated at 22° C, and counted on the third 

 and fourth days. All countings were checked several times. 



(6) On the fifth and sixth day of the experiment it was found necessary to increase the 

 dilutions from I in 500 to i in looo for the samples at 15° C. and 3° C. respectively. On 

 the seventh day the dilution was increased for both samples to i in 5000. In all respects 

 the technique remained precisely the same throughout the experiment. 



(7) On the thirteenth day the fall in the number of organisms in the sample 

 maintained at 5° C. was so marked that an interim analysis was made twenty-four hours 

 afterwards (tj. after fourteen days) as a check, and this analysis yielded 84,800,000 

 bacteria per c.c, which showed at once that the fall was correctly estimated, and was but 

 one of the steps in a uniformly declining gradation. 



(8) The nutrient gelatine used throughout the experiment was made as follows : one 

 pound and a quarter of lean beef was minced and macerated in one litre of water for 

 twelve hours, and strained through sieve and muslin. The resultant fluid was made up 

 to one litre with distilled water, put into a saucepan, and slowly brought up to boiling 



. point. It was then filtered through damp filter paper. Five grammes of common salt 



, (NaCl) and 10 grammes of dry peptone were added. The peptone was rubbed down in 



' a mortar with a small quantity of bouillon before being added to the bulk. The broth 



was then placed on the stove and stirred until the peptone had completely dissolved. 



iThe gelatine (120 grammes) was then added slowly and stirred constantly imtil fully 



j dissolved. The medium was alkalinised with normal caustic soda, and made fciintly alkaline 



to litmus, and cooled down to 60° C. The white of one egg was well mixed with 60 cc 



of distilled water, and added. The gelatine was then made up to one litre with distilled 



, water, and steamed at 110° C. in Koch's steam steriliser for one hour. It was then filtered 



' Ihot and run into sterilised test tubes. The tubes as finished were sterilised on three 



'raccessive days at 100° C. for thirty minutes each day.^ 



We have discarded the above method of preparing nutrient gelatine since 1900 

 «« p. 37). 



