Chapter IV —^51 — Methods of Enumeration 



velop, they can be readily counted in reflected light against a dark back- 

 ground. 



The oval tube technic is applicable not only to the enumeration of 

 anaerobes in general, but by employing appropriate differential media, 

 the abundance of various physiological ty]ies can be estimated. If, how- 

 ever, the anaerobes produce gas, the medium in the oval tubes will be 

 shattered, thereby rendering it useless for counts. 



Colonies of anaerobes can be removed from the medium for further 

 study either by breaking the oval tube (after scoring it with a file) near 

 the colony and then aseptically dissecting out the colony with a sterile 

 inoculating needle; or the entire plug of agar can be expelled from the oval 

 tube into a sterile Petri dish. Then, by dissecting away the surrounding 

 agar with a hot needle, the desired colony can be isolated. To remove the 

 agar, the oval tube is immersed for a second in boiling water to loosen the 

 agar from the walls of the tube, after which heat from boiling water or a 

 Bunsen flame is applied to the bottom of the tube until the increasing 

 vapor pressure expels the intact plug of agar. 



When using the successive dilution method to enumerate marine 

 anaerobes, oxygen must be excluded from the differential media to pre- 

 vent the multiplication of aerobes. Wide-mouth Mason jars, which are 

 inexpensive and rugged, have proved to be useful for this purpose in the 

 field. One-quart bottles accommodate 15 5/8-inch test tubes or 21 

 1/2-inch tubes. Immediately before placing the inoculated tubes of 

 media in the jar, 5 grams of powdered chromium metal and 100 ml. of 

 15 per cent sulfuric acid are introduced (Rosenthal, 1937). After the 

 initial vigorous evolution of hydrogen has subsided, the jar is sealed. 



If it will not interfere with the specific functions of the medium, the 

 enrichment of differential media with o.i per cent of either ascorbic acid 

 or sodium thiogly collate is recommended to maintain reducing conditions, 

 and 0.0002 per cent methylene blue helps to poise the medium while 

 serving as an indicator of anaerobiosis. 



Media which are rendered semi-solid by the addition of o.i to 0.3 

 per cent agar are better than either solid or liquid media for the cultivation 

 of anaerobes. Semi-solid media for the cultivation of anaerobes have been 

 recommended by Spray (1936), Reed and Orr (1943), and many others. 

 Kitchens (1921) has reviewed the literature on the advantages of semi- 

 solid media for the cultivation of microorganisms. 



Direct microscopic counts : — Wherever careful comparisons have 

 been made, direct microscopic counts have detected the presence of many 

 more bacteria in water and mud than have plate counts. According to 

 Waksman et al. (1933c), direct counts on sea water are 1000 times as large 

 as plate counts. Direct counts made by Bere (1933) on water from Lake 

 Mendota were from 20 to 335 times as high as plate counts, while water in 

 Lake Wingra gave direct counts which were from 8 to 125 times as high as 

 plate counts. 



Direct counts are higher partly because of the failure of any one nutri- 

 ent medium to satisfy the cultural requirements of all bacteria and pri- 

 marily because of the tendency of bacteria to occur associated together 

 either as zoogleal masses, chains, sheets, or clumps, or tenaciously at- 

 tached to particulate material. The studies of ZoBell (1943^) show that 

 most bacteria appear to be associated with particulate material, both 

 living and inanimate. This is in agreement with the observations of 



