198 PRINCIPLES OF SOIL MICROBIOLOGY 



sufficient for one liter of cellulose agar, is moistened with water and added to the 

 acid which is then vigorously agitated till the cellulose is dissolved. The flask is 

 then quickly filled with cold tap water. The whole process of dissolving the 

 paper and filling the flask requires about one minute. The precipitate is now 

 filtered through paper and washed until the filtrate no longer gives any test for 

 sulfuric acid. When the volume of the suspension is drained to about 200 cc, 

 a hole is punched in the bottom of the filter and the whole precipitate washed out 

 and made up to 500 cc. 



The cellulose may also be obtained from plant tissues by extracting well ground 

 material with 2 per cent KOH solution, washing, chlorinating, extracting again 

 with boiling 2 per cent NaOH for 30 minutes and washing. 



Cellulose suspension (500 cc.) prepared by any of the above methods is now 

 added to 500 cc. of a solution containing 0.5 gram K 2 HP0 4 , 0.5 gram MgS0 4 , 

 0.5 gram NaCl, 1 gram (NH 4 ) 2 S0 4) 1 gram CaC0 3 and 500 cc. of tap water. Ten 

 grams of agar are dissolved in the mixed solutions and medium tubed and ster- 

 ilized as usual. 



When soil suspensions are directly inoculated upon poured plates 

 with cellulose agar, filamentous fungi will develop and tend to overgrow 

 the plate, making difficult the isolation of the bacteria. The sample of 

 soil or manure is added to sterile flasks containing the cellulose broth or 

 peptone cellulose broth and, as soon as the cellulose shows signs of dis- 

 integration, transfers are made upon fresh sterile flasks. This avoids 

 the development of fungi. After several preliminary cultivations upon 

 the liquid enrichment media, the cultures are plated out on the cellu- 

 lose agar. 



The colonies of cellulose-decomposing organisms developing on the plates 

 show a translucent area due to the decomposition of the cellulose in the agar, 

 as well as neutralization of the CaC0 3 by the acids formed from the ammonium 

 sulfate and cellulose decomposition. Starch agar and finally nutrient agar 

 may be used for the final cultivation of the organisms. The cellulose-destroy- 

 ing bacteria studied by Kellerman and associates were found to grow more 

 rapidly under aerobic conditions, although some anaerobic development has also 

 been observed. They show a more vigorous growth on media containing organic 

 nitrogen (peptone) than inorganic nitrogen. They usually reduce nitrates to 

 nitrites, attack various carbohydrates, and do not form any gaseous products 

 from cellulose or the lower carbohydrates. The paper is disintegrated into fine 

 fibres and a small amount of organic acids is formed. 



The following liquid medium was used for demonstrating the dissolution of the 

 filter paper: 



Soil extract 500 cc. Peptone 2.0 grams 



Distilled water 500 cc. CaC0 3 excess 



K 2 HP0 4 0.5 gram Reaction pH 7.0 



(NH 4 ) 2 S0 4 1.0 gram 



All the forms studied were rod-shaped organisms varying in length from 0.8 

 to 3.5ju. Only in three species were involution forms observed. Five species 

 produced spores. Twenty-seven of the thirty-six species isolated were motile. 



