Chapter IV — 49 — Methods of Enumeration 



tions, plate counts differ very little from dilution method counts of water 

 bacteria. Butkevich (1932&) reported that the inoculation of tubes of 

 liquid media with successive dilutions of material from the Barents Sea 

 demonstrated the presence of from ten to a hundred times as many bac- 

 teria as plate counts on solid media. It is possible that his solid media 

 were poured too hot, or the plates may not have been incubated suffi- 

 ciently long. 



Since it requires much more time and material for dilution method 

 counts in order to approach the accuracy of plate counts, the use of the 

 former for estimating the general bacterial population is not recom- 

 mended. However, if one lacks the facilities for plating samples of 

 marine materials in the field, valuable information can often be obtained 

 by inoculating a few tubes of nutrient media with various dilutions. It is 

 far better to know the order of magnitude of the bacterial population than 

 to have no information at all, and from the tubes in which growth occurs, 

 cultures for further study can be isolated after returning to the shore 

 laboratory. 



The successive dilution method finds its widest application for esti- 

 mating the abundance of different physiological types of bacteria by the 

 use of selective or differential media. For example, the number of bac- 

 teria in samples which reduce nitrate can best be estimated by inoculating 

 nitrate broth with different dilutions of the sample and then determining 

 the highest dilution which caused the reduction of nitrate. 



The enumeration of marine anaerobes: — Anaerobic bacteria are 

 found in nearly all samples of marine bottom deposits and in most sam- 

 ples of sea water. Many of them prove to have the faculty of growing 

 either in the absence or in the presence of free oxygen although some are 

 strict anaerobes. 



There is no sharp demarcation between aerobes and anaerobes. For 

 purposes of discussion, bacteria which grow in the absence of free oxygen 

 or in media having an oxidation-reduction potential of Eh to —0.4 volt 

 or lower are regarded as anaerobes while those which grow in the presence 

 of free oxygen or in media having an oxidation-reduction potential of 

 Eh o to -I-0.4 volt are regarded as aerobes. Fifteen species of strict an- 

 aerobes studied by Reed and Orr (1943) were found to grow best at 

 Eh —0.2 volt. Microaerophilic bacteria are those which grow best in the 

 presence of traces of free oxygen or at Eh +0.1 to — o.i volt. 



There is no relationship between the oxidation-reduction potential of 

 the medium and the oxygen tension except in so far as the oxygen tends 

 to influence the Eh. According to Kligler and Guggenheim (1938), 

 anaerobes require a low Eh only if oxygen is present. It has been amply 

 demonstrated that certain so-called strict or obligate anaerobes will grow 

 in the presence of free oxygen if the Eh is sufficiently low (Hewitt, 1936). 



Considerable difficulty has been experienced in estimating the abun- 

 dance of marine anaerobes. Most of the conventional procedures (Hall, 

 1929) have failed to yield reproducible results, and they require the use of 

 complicated, bulky and time-consuming apparatus which is impractical 

 for field studies at sea. No one nutrient medium has proved to be satis- 

 factory for estimating the general anaerobic population, although good 

 results have been obtained with Medium 2216 enriched with i.o per cent 

 glucose, 0.1 per cent sodium thioglycollate and 0.0002 per cent methylene 

 blue. If protected from atmospheric oxygen following autoclave steriliza- 



