150 BACTERIOLOGY OF WATER, AIR, MILK, ETC. 



i. The bottles, which should have a capacity of from 25 to 100 c.c., should be 

 sterile. Sterilization may be effected by heat or by rinsing with a little sulphuric 

 acid and subsequently washing out thoroughly with the suspected water before col- 

 lection. The utmost care must be exercised that the fingers do not come in contact 

 with the glass stopper of the neck of the bottle while filling it. If the specimen is 

 to be sent some distance, it should be packed in ice to prevent bacterial development. 

 Frankland states that a count of 1000 became 6000 in six hours and 48,000 in forty- 

 eight hours. In water packed in ice for a considerable time, however, the bacterial 

 count may diminish. 



2. If collecting from city water supplies, secure the sample direct from the mains 

 and let the water run from the tap a.few minutes before collection. If the water be 

 taken from a pond, stream, or cistern, be sure that the specimen comes from at least 

 10 inches below the surface. As sedimentation is the most important method in 

 self-purification of rivers and ponds, it will be understood that any stirring up of the 

 mud on the bottom will enormously increase a bacterial count. 



Quantitative Bacteriological Examination 



i. Deliver definite quantities of the water to be examined into tubes of liquefied 

 gelatin or agar and plate out the same in a series of Petri dishes. 



A more practical method is to deliver the water from the graduated pipette into 

 the empty sterile dish. The water should be deposited in the center of the plate and 

 the melted gelatin or agar poured directly on the water and then, carefully tilting to 

 and fro, mix the water and the media. One set of plates should be of gelatin and 

 incubated at room temperature; a similar set should be of lactose litmus agar and 

 incubated at 38C. If the water is highly contaminated, it is necessary to dilute 

 it; thus, with river water, which may contain from 2000 to 10,000 bacteria per c.c., 

 a dilution of i to 100 would be desirable. 



Ordinarily it will be sufficient to deliver from a sterile graduated pipette 0.2, 0.3, 

 and 0.5 c.c. of the water in each of two sets of plates: one set for gelatin, the other 

 for agar. 



When gelatin is not at hand or convenient to work with, the gelatin plates may 

 be replaced by others of lactose litmus agar for incubation at room temperature. 

 After twenty-four hours at 38C. or forty-eight hours at 2oC., the count should be 

 made. 



Example. Forty colonies were counted on the gelatin plate containing 0.2 c.c. 

 (^5) of the water. The number of organisms would be 200 per c.c. Ten colonies 

 were counted on the agar plate containing 0.2 c.c. and incubated at 38C. Number 

 of bacteria developing at body temperature equals 50 per c.c. 



There is no strict standard as to the number of bacteria a water should contain per 

 c.c. Koch's standard of 100 colonies per c.c. is generally given. It is by the 

 qualitative rather than the quantitative analysis that one should judge a water. 



If there should be very many colonies on a plate, the surface can be marked off 

 into segments with a -blue pencil. If very numerous, cut out of a piece of paper a 

 space equal to i sq. cm. By counting the number of colonies inclosed in this 

 space at different parts of the plate, we can strike an average for each space of i 

 sq. cm. To find the number of such spaces contained in the plate, multiply the 



