Chapter IV 



— 45 



Methods of Enumeration 



this material has certain applications, it is not suitable for plate count 

 procedures. 



When prepared with fresh water by the method outlined by Hanks 

 and Weintraub (1936), silica gel contains about 3.5 per cent of sodium 

 chloride, which is formed when the sodium silicate is neutralized with 

 hydrochloric acid, thereby giving without dialysis a solid medium which 

 is isotonic with sea water. Using a procedure similar to that described by 

 Moore (1Q40), it has been possible to prepare and sterilize a solution of 

 sodium silicate which sets slowly into a suitable gel when mixed with a 

 dilute solution of hydrochloric acid containing peptone and other nutri- 

 ents. About half as many colonies developed on this silica gel medium as 

 on Medium 2216 solidified with agar. Although its use is not advocated 

 for general plate-count purposes, silica gel prepared in this way has 

 proved to be useful for plating autotrophs and other special physiological 

 types of bacteria. 



The threatened shortage of agar, imported from Japan prior to the 

 War, has encouraged an extensive search for agar substitutes. To date 

 neither Irish moss extract (Walker and Day, 1943), fibrous sodium 

 pectate (McCready et al., 1943), nor alginates have proved to be satis- 

 factory agar substitutes for the cultivation of marine microorganisms. 



Incubation temperature for plate counts: — The numbers and kinds 

 of bacteria which develop into countable colonies on plates of nutrient 

 agar are influenced by the time and temperature of incubation. This is 

 illustrated by the data in Table IX compiled by ZoBell and Conn (1940) 

 who counted the number of colonies on replicate plates incubated at dif- 

 ferent temperatures and for different periods of time. The media were 

 inoculated with identical quantities of appropriately diluted sea water or 

 marine mud. Results with water and mud samples were almost the same 

 regardless of whether collected from great depth where the ocean temper- 

 ature was less than 5° C. or from shallow water of higher temperatures. 



Table IX. — Relative number of bacterial colonies appearing on nutrient sea-water agar after 

 dijfferent periods of incubation at different temperatures, the average plate counts being expressed 

 as percentages of the plate count at 18 days at 18° C: — 



During the first few days of incubation the most colonies appear on 

 plates incubated at 25° or 30° C, but after seven to ten days greater 

 numbers of colonies appear on the plates incubated at 12° to 22° C. The 

 bacteria which multiply at the higher temperatures develop into visible 

 colonies more rapidly than those incubated at lower temperatures, but 

 there are relatively few bacteria in the sea which grow at temperatures 

 higher than 25° C. Most of the colonies which develop on plates incu- 



