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SCIENTIFIC AGRICULTURE. 



June, 1921. 



A Bacteriological Analysis and Cultural Test of 

 **Nitro-Bacter Soil Vaccine" 



DAN. H JONES, Professor of Bacteriology, O.A.C., 

 Guelph. 



"Nitro-Bacter Soil Vaec-ine" i.s a liquid preparation 

 recently put upon the market and widely advertised 

 a-' giving wonderfully increased crop returns when ap- 

 plied to the soil on whicli the crops are grown. It is 

 claimed by its manufacturers to be a culture of bac- 

 teria, which, on addition to the soil, "attach themselves 

 ti> the plant roots and draw nitrates and phosphates from 

 the air and soil and act in place of fertilizers." 



As we have received many letters of enquiry regarding 

 this preparation we obtained from the Toronto branch 

 of the firm that manufactures it a free gallon sample 

 for analysis and test. The sample was from a lot manu- 

 factured May 10th and the culture tests here reported 

 w-ere commenced June 2nd. A portion of this sample 

 lias been applied to various crops and a portion used for 

 bacterial analysis. The crop test will not be complete 

 until the end of the growing season but the bacterial 

 analysis is sufficiently far advanced to warrant a re- 

 ]H)rt being made. 



The analysis included bacterial counts made on var- 

 ious culture media and cultural tests for production of 

 ammonia, nitrites and nitrates. Standard metliods were 

 used throughout. 



Coincident with the "soil vaccine" test we ran for 

 jiurpcses of comparison similar te.sts with a soil sus- 

 pension made by adding (me part of garden soil to two 

 parts of sterile water, shaking and allowing the soil to 

 settle and then using the supernatant liquid for cul- 

 tural purposes. 



The results of these comparative te.sts are as follows : 

 Number of Decomposition Bacterial Colonies Growing 



on Beef-Agiir-Phiies. 

 ' ' Soil . Vaccine ' ' (hirchn Soil. 



50,000 per c.e. 16,000,000 per gram. 



Azotohacter Colonies (nitioijen-fi.ring bacteria) (flow- 

 ing on Ashby's-Agur-Plates. 

 "Soil Vaccine". Garden Soil. 



1 in 3 c.e. 900 per gram. 



Other Types of- Bacterial Colonies on Ashby'.t-Agar- 

 Plates. 

 "Soil Vaccine" Garden Soil. 



Numerous streptothrix, Few streptothrix. 



A few acid producers. Some legume bacteria. 



Few others luidetermined. Many others uiuletermined. 



From the above it will be .seen that when comparing 

 the bacterial content of 1 c.e. of the vaccine with that 

 of 1 gram of ordinary garden soil, the decomposition 

 bacteria were thirty-two times as manj' in the garden 

 soil as were found in the "soil vaccine", and tliat the 

 azotobacter, or nitrogen-fixers, were twenty-seven-hun- 

 dred times more numerous in the garden soil than in the 

 "soil vaccine". 



Ammonificaiion Test. 



In this test a comparison was made in triplicate be- 

 tween the "soil vaccine" and the water suspension of 

 jrarden soil for their power to ammonify gelatin solu- 

 tion. To each of three flasks containing 100 c.e. of 

 gelatin solution 5 c.e. of the "soil vaccine" was added. 



and to each of a similar set of flasks 5 c.e. of the garden 

 soil (equivalent to 'l.ii grams soil) was added. 



After four days a cuemical test for ammonia was 

 applied and this showed in tiie "soil vaccine" culture a 

 slight amount of ammonia, wliile the garden soil culture 

 sl'.owed iieavj' ammonia production. Two days later a 

 similar test was applied which showed moderate amount 

 of ammonia for tlie vaccine culture and very heavj- am- 

 monia for the garden soil culture. These results tally 

 with the bacterial counts matle on the beef agar plate 

 cultures, the slow production of amnKuiia in tlie "vac- 

 cine" flask cultures agreeing with the small number of 

 decomposition bacteria found in the "vaccine" plate 

 cultures, while the rapid ammonia production by the 

 garden soil flask cultures corresponds to the large num- 

 ber of decomi)osition bacteria found on the garden soil 

 plate cultures. 



Nitrification Tests. 



A. Formation of nit)ites (This is a necessary stage in 

 the Iniilding up of nitrates). 



Comparison tests were made in triplicate between the 

 "soil vaccine" and the water suspension of garden soil 

 to form nitrites in a solution made for nitrite produc- 

 tion. 



To each of three fla.sks containing .^0 c.e. of the solu- 

 tion 0.5 c.e. of "vaccine" was added and to each of a 

 similar set of three flasks was added ().') c.e. of garden 

 scil suspension (equivalent to a quarter of a gram of 

 garden soil). 



After six days a chemical test for nitrites was applied 

 with the following results : 



"Soil vaccine" cultures showed no nitrite produc- 

 tion ; garden soil cultures sliowed moderate amount of 

 nitrites present. After twelve days the same test was 

 again applied when it was found that a trace of nitri- 

 tes was present in the "soil vaccine" cultures while a 

 lieavy nitrite content was found in the garden soil 

 cultures. 

 B. — Production of Nitrates. 



For this test 0.2 c.e. of the "soil vaccine" was added 

 to each of three flasks containing 50 c.e. of a solution 

 ]u-epared for the formation of nitrates and tlie same 

 t|iiaiitity of garden soil suspension, eciuivalent to 0.1 

 gram of .soil, was added to a similar set of flasks. 



In this test for nitrate production it is desirable to 

 incubate the cultures for fi-om four to six weeks before 

 making the chemical test for detei-mining presence of 

 nitrates. At time of w^riting the cultures for this test 

 liave lieen incubated only two weeks. However, we have 

 just made a preliminary test and we find no nitrates 

 j.roduced in the "soil vaccine" cultures whilst there is 

 a slight amount of nitrate found in the garden soil 

 cultures. 



Conclusions. 



A consideration of the above findings shows that for 

 increasing the denitrifying, ammonifying, nitrifying 

 and nitrogen-fixing bacterial content of the soil the 

 "soil vaccine", if the sample forwarded to us is a re- 

 presentative sample, is of little, if any, value. Further, 



