ZoBell — 54 — Marine Microbiology 



microscope method makes it possible to observe the organisms which 

 settle out of rather deep counting chambers, provided the latter have 

 thin bottoms made of optical glass. Utermohl (1931) gives detailed 

 directions for staining microorganisms with complimentary colors, and 

 the use of light filters in order to provide contrast for the microor- 

 ganisms. 



Like most of the direct microscopic methods, Utermohl's in- 

 verted microscope method cannot be recommended for counting bacte- 

 ria at sea due to the effect of vibration of the boat on the focus when 

 using the necessary high magnifications. However, the samples can be 

 preserved with formalin or otherwise for observation in the shore labora- 

 tory. 



Benecke (1933) has proposed filtration methods for concentrating the 

 bacteria in sea water prior to making direct counts, as suggested by the 

 work of KoLKWiTZ who concentrated microplankton with membrane 

 filters. However, such filters do not retain the free bacteria which are 

 smaller than 5 m in diameter. 



After treating samples of fresh water with formalin in preservative 

 concentrations, Cholodny (1929) filtered 100 ml. through a membrane 

 filter having a porosity of only 2 ju. When all except 4 or 5 ml. had passed 

 through the membrane, the exact volume was determined and 0.05 ml. 

 was transferred to a measured area of glass slide. This smear was air 

 dried, fixed, and stained with erythrosin preparatory to making micro- 

 scopic counts from which the bacterial population of the original material 

 could be estimated. Cholodny found from a hundred to a thousand 

 times as many bacteria in water samples by this direct method as by plate 

 counts although his fine filter membrane may not have retained all of the 

 bacteria. Cholodny recognized that one big disadvantage of the direct 

 method is its failure to differentiate between dead and living cells. The 

 filters employed by Cholodny were manufactured by P. Altmann, 

 Berlin. Such membranes may be dissolved in acetone to facilitate the 

 separation of bacteria. 



Gee (193 2 J) reported indifferent success in his attempts to enumerate 

 marine bacteria, employing the aluminum hydroxide adsorption method 

 of Snow and Fred (1926) and Cholodny's ultrafiltration procedure. The 

 primary difficulty was caused by agglutination of the organisms, giving a 

 preparation in which the bacteria were collected in bunches. Gee pro- 

 posed a method of concentrating bacteria in sea water by their adsorption 

 to CaCOs precipitated by the addition of ammonia. He believed that the 

 bacteria entrained with the precipitated CaCOs could be concentrated by 

 filtration. Then weak acids or carbon dioxide could be used to dissolve 

 the carbonate, thus releasing the entrained bacteria. We have not found 

 the method to be promising. 



It is difficult to recover all of the bacteria from filters of very fine 

 porosity. Furthermore water passes very slowly through such filters, 

 thus limiting the volume that can be filtered. Certain workers have 

 undertaken to obviate the former difficulty by the use of filter mem- 

 branes which may be dissolved with chemical reagents. For example, 

 collodion membranes are soluble in ether, and filters made of powdered 

 CaCOs or MgCOs dissolve readily in dilute HCl. Carbonate filters have 

 yielded the best results for concentrating bacteria in sea water, but many 

 technical difficulties must be overcome before this method of enumerating 

 bacteria can be recommended for general use. 



