Chapter IV 



47 — 



Methods of Enumeration 



which developed from autoclaved sea-water dilution water blanks after 

 one minute exposure. After 15 minutes the inoculated blanks were again 

 shaken and i.o-ml. quantities were transferred to Petri dishes. The pro- 

 cedure was repeated after a total elapsed time of 30 minutes and 60 min- 

 utes. **Aged" or "rotted" sea water (see page 58) was used. 



Table XI. — Relative numbers of bacteria demonstrated by plate counts in identical samples 

 of marine mud after different periods of time in different kinds of dilution water blanks, expressed 

 as pcrccnfat^es of the number of bacteria found in autoclaved sea 'water after one minute: — 



All of the dilution water blanks except one which was passed through a 

 Berkefeld-N filter were sterilized by heating in the autoclave for 20 min- 

 utes at 121° C. Since Kiribayashi and Aida (1934) have reported that 

 certain bacteria survive longer in boiled than in raw sea water, it seemed 

 desirable to compare boiled or autoclaved sea water with Berkefeld- 

 filtered sea water. Actually the latter was not as good as autoclaved 

 sea water. Apparently in passing through Berkefeld or Handler filters, 

 contact with the brass and nickel-plated connections renders sea water 

 slightly bactericidal. This conclusion was reached after experimenting 

 with Berkefeld-filtered sea water which had been autoclaved and placing 

 sterile Berkefeld filters in autoclaved sea water. However, part of the 

 bactericidal effect of sea water sterilized by filtration is ascribable to a 

 thermolabile, filter-passing, toxic agent which occurs in raw sea water, as 

 has been demonstrated by the use of Coors and Chamberland all-por- 

 celain filters and sintered glass filters having no metal parts. 



From the data in Table XI it will be observed that marine bacteria 

 rapidly perish when suspended in tap water, 0.85 per cent sodium chloride 

 solution, distilled water, or Formula C. The latter is a balanced salt solu- 

 tion having the following composition: 



Calcium chloride 



Magnesium sulfate 



Ferric chloride 



Dibasic potassium phosphate 



Sodium hydroxide to />H 7 . 2 



o. 250 millimols 

 o. 100 millimols 

 0.0005 millimols 

 0.312 millimols 



Formula C, which is based upon the average composition of river water, 

 has been recommended by Butterfield (1932) and others as a dilution 

 water for "water" bacteria. However, even when rendered isotonic with 

 sea water by the addition of 3 per cent sodium chloride, Formula C is not 

 satisfactory for marine bacteria. 



