ture of MPA and yeast-agar 

 at ion. 



The number of colonies is counted after incub- 



Treatment of Samples with 96 Percent Ethyl Alcohol 



Romanenko and Daukshta (1975) have shown that the vegetative cells of 

 microorganisms die almost instantly under the action of strong ethyl alco- 

 hol, but the spores are preserved for a considerable period of time. This 

 is the basis for the second method of determining the quantity of spores in 

 a sample of water or mud. 



The spores can be separated from the vegetative cell by several methods. 

 The tested water may be mixed in a test tube with alcohol and then inocu- 

 lated. To 5 ml of test water, 10 ml of ethyl alcohol is added (ratio 1:3 in 

 any volume), and 0.5 ml of the mixture is inoculated 

 MPA by the depth technique (with or without dilution 

 Another method is to pass a known volume of the test 

 brane filter, then 3-5 ml of ethyl alcohol is passed 

 lowed by 3 ml of sterile distilled water to wash the 

 The filters are then placed upon a layer of agar in 



into Petri dishes with 

 using sterile water) . 

 water through a mem- 

 through the filter fol- 

 alcohol from the filter. 

 Petri dishes. The number 



of spores is estimated after incubation by the number of colonies present. 

 This method is particularly good in the case of clean water, where the number 

 of spores is small, because of the concentrating effect of filtering. 



The number of spores in mud deposits or sediments, can be determined by 

 yet another method. A row of test tubes with sterile water for dilution is 

 used. One test tube, for example N 3 or 4, is filled with 9 ml alcohol (see 

 Figure 3). Thus, the water is passed through the alcohol in the process of 



xJJ v^ 



s > 



1 



Figure 3. Position of the test-tube (N 2) with ethyl alcohol in 

 determining the spores of bacteria in mud deposits. 



61 



the row when 



