ZoBell 



46 



Marine Microbiology 



bated at the higher temperatures are larger than those found on plates 

 incubated at lower temperatures, thereby giving the former plates the 

 superficial appearance of having more colonies. 



The decrease in the counts recorded for plates after prolonged incu- 

 bation is due to the merging of colonies and the liquefaction of the agar 

 by some of the bacteria which obliterate surrounding colonies. 



Very few colonies develop on plates incubated at 37° C. In fact, many 

 marine bacteria are killed by ten minutes' exposure to temperatures no 

 higher than 30° C. The thermal sensitivity of marine bacteria is indi- 

 cated by the fact that some perish by being exposed for a few seconds to 

 the temperature of agar near its congealing point, as is shown by the data 

 in Table X. While too few bacteria are killed when the medium is cooled 

 to near 42° C. to invalidate the use of agar plates for enumerating bac- 

 terial populations, it is emphasized that prolonged exposure at 42° C, or 

 short exposure at temperatures a few degrees higher, is lethal for an ap- 

 preciable number of bacteria occurring in marine materials. 



Table X. — Relative numbers of colonies developing on nutrient agar inoculated with sea 

 water or marine mud when the agar was poured at different temperatures, the plate counts being 

 expressed as percentages of the average plate count on media poured at 42°C. (from- data 0/ ZoBell 

 and Conn, 1940): — 



Dilution water blanks for marine bacteria : — It is always necessary 

 to dilute mud samples before they can be used to inoculate plating or ex- 

 tinction-dilution method media, and it is often necessary to dilute sea 

 water or other marine materials such as surface slime, the intestinal con- 

 tents of organisms, etc. A dilution water used for this purpose should 

 provide for the suspension and prolonged survival of the microorganisms 

 in the samples being analyzed. 



Sterilized sea water might be considered to be the best dilution water 

 for marine microorganisms but it is surprising to note the number of work- 

 ers who have employed distilled water, tap water, physiological saline 

 solution, or other solutions for dilution purposes. Some workers merely 

 specify that "sterile water" was employed and others fail to mention the 

 dilution water at all. The effect of the composition of the dilution water 

 blank upon the plate count of marine mud is illustrated by the data sum- 

 marized in Table XI on page 47. 



In this experiment, samples of marine mud were diluted with raw sea 

 water until it was estimated that i.o ml. of the mixture should contain 

 around 20,000 bacteria which would develop into colonies on Medium 

 2216. Then with two people working together, i.o-ml. quantities of the 

 mixture were transferred to 99-ml. sterile dilution water blanks. The 

 latter were shaken uniformly for one minute, after which i.o-ml. quanti- 

 ties in duplicate were used to inoculate plates to which Medium 2216, 

 cooled to 42° C, was added. The data in Table XI summarizes the aver- 

 age of five such experiments, the plate counts from the different dilution 

 water blanks being expressed as percentages of the number of colonies 



