Determination of Bacteria in Water 243 



that have been cleaned with great care, rinsed in dean water and 

 sterilized with dry heat for at least one hour and a hah at i7o°C., or 

 in an autoclave at 15 lbs. (i2o°C.) for fifteen minutes or longer after 

 the pressure reaches 15 lbs. Great care should be exercised to have 

 the samples representative of the water to be tested, and to see that 

 no contamination occurs at the time of filling the sample bottles. 



The samples should be examined as promptly as possible after 

 collection as rapid and extensive changes take place in the bottled 

 samples even when stored at temperatures as low as io°C. 



The time allowed for storage and transportation of a bacterial 

 sample between the filling of the bottle and the beginning of the 

 analysis should not be more than six hours for impure waters and 

 not more than twelve hours for relatively pure waters. During the 

 period of storage the temperature should be kept as nearly at io°C., 

 as possible. 



If the number of bacteria per cubic centimeter be small, large 

 quantities may be used; but if there be millions of bacteria in every 

 cubic centimeter, it may be necessary to dilute the water to be 

 examined in the proportion of i : 10 or i : 100 with sterile water, 

 mixing well. From the water sample, with or without dilution as 

 may be appropriate, o.oi; o.i; and i cc. respectively are carefully 

 measured with a sterile pipet into a tube of melted agar-agar cooled 

 to a temperature that can be comfortably held in the hand. 

 After thorough mixing without shaking or forming bubbles, the con- 

 tents are poured into a sterile Petri dish which is inverted when the 

 medium sohdifies, and is then stood in the incubating oven for 

 twenty-four hours. 



It is best to count all the colonies developed upon the culture, if 

 possible; but when hundreds or thousands are scattered over it, an 

 estimate can be made by counting the number of colonies in each 

 of several of the divisions of some counting apparatus, such as have 

 been devised by Wolfhiigel, Esmarch, or Frost, and computing the 

 total number on the plate. In counting the colonies a lens is 

 indispensable. 



In ordinary city hydrant-water the bacteria number from 2 to 

 50 per cubic centimeter; in good pump-water, 100 to 500; in filtered 

 water from rivers, according to Giinther, 50 to 200; in unfiltered 

 river-water, 6000 to 20,000. According to the pollution of the water 

 the number may reach as many as 50,000,000. 



The waters of wells and springs are dependent for their purity 

 upon the character of the earth or rock through which they filter, 

 and the waters of deep wells are much more pure than those of 

 shallow wells, unless contamination take place from the surface of 

 the ground. 



Ice always contains bacteria if the water contained them before 

 it was frozen. In Hudson River ice Prudden found an average of 

 398 colonies in a cubic centimeter. 



