ZoBell — 50 — Marine Microbiology 



tion, the oxidation-reduction potential of this medium is generally lower 

 than Eh — o.i volt. The reduced methylene blue helps to maintain a low 

 Eh and it serves as an indicator of the Eh, the Eo of methylene blue being 

 +O.OII volt at pa 7.0. (Eo = Eh when the oxidant is 50 per cent re- 

 duced) . For certain purposes, such as the cultivation of sulf ate-reducing 

 bacteria, o.i per cent ascorbic acid is better than sodium thioglycollate. 

 The influence of ascorbic acid on the growth of anaerobes has been studied 

 by Kligler and Guggenheim (1938). Reed and Orr (1943) found as- 

 corbic acid to give about the same results as sodium thioglycollate. 



Alkaline pyrogallol cannot be relied upon to remove enough oxygen 

 from anaerobe jars to prevent the growth of microaerophiles and certain 

 aerobes. Stone (1936) has found chromous sulfate to be more than forty 

 times as efhcient as pryogallate as an oxygen absorbent. Both chromous 

 sulfate and yellow phosphorus have been employed for depleting the 

 oxygen from anaerobe jars at sea. Burning phosphorus leaves an unde- 

 sirable film on the culture receptacles. Though useful in the shore labo- 

 ratory, Mclntosh-Fildes jars requiring hydrogen are somewhat cumber- 

 some on a research vessel. Moreover, the maritime underwriters look 

 with disfavor- upon the use of potentially explosive mixtures such as hy- 

 drogen and oxygen. 



A 20-quart pressure cooker has proved to be a good anaerobe jar. It 

 is sturdy and has a capacity of 80 to 100 Petri dishes. A glass receptacle 

 must be used in the pressure cooker to contain the mixture of chromous 

 sulfate and sulfuric acid or other oxygen-absorbing agents in order to 

 prevent the corrosion of the metal. A pressure cooker has also been wired 

 with a Mclntosh-Fildes palladinized asbestos heating element to provide 

 for the ignition of hydrogen. The principal disadvantage of these im- 

 provised anaerobe jars is their lack of transparency; it is necessary to open 

 them in order to observe the cultures. 



For field work, extensive use has been made of circular glass disks, 

 slightly smaller in diameter than Petri plates, for maintaining anaerobic 

 conditions in single dishes. After inoculating and pouring the plate in the 

 conventional manner, a sterile glass disk is placed on top of the medium 

 to exclude atmospheric oxygen. The failure of the blue color of methylene 

 blue to return to the medium except around the periphery of the plate 

 indicates reducing conditions under the glass disk. Colonies can be ob- 

 served and counted as they develop. After carefully removing the glass 

 disk, colonies can be transferred to other media for further study. When 

 employing this method of anaerobiosis, the glucose content of the medium 

 should be reduced to 0.1 per cent or less. Otherwise gas production from 

 the fermentation of glucose by anaerobes often causes fragmentation of 

 solid media. 



RiTTENBERG ct al. (1937) employed oval tubes about 380 mm. long 

 and 6 by 14 mm. in cross-section for the enumeration of marine anaerobic 

 bacteria. One end of the tube is closed and the other end is flared to 

 facihtate the introduction of the medium. It is easier to observe colonies 

 in the flat-walled oval tubes than in ordinary round glass tubes. 



The oval tubes are sterilized in a pipette can. Then 10 ml. of nutrient 

 agar, recently heated to 100° C. to expel oxygen and cooled to 42° C, is 

 inoculated with i.o ml. of the appropriately diluted sample, and without 

 undue agitation, the inoculated medium is poured into the oval tube. 

 The medium is covered with a 40 mm. layer of reduced methylene-blue 

 agar to serve as a seal or plug for excluding oxygen. When colonies de- 



