260 



SCIENTIFIC AGRICULTURE. 



June. 1921. 



tion is by no means solely to be found with Radio- 

 bacter: l>ut we feel siure that thase pictures, as shown 

 on Plate 68, G-L, will be found most valuable for 

 diagnostical purposes. 



The flagellation is the same with Radiobaeter 

 (Plate 68, C) and Ra.dicicola rPlate 68, B), w^hile the 

 bacteria of the cowpea-soybean grroop are characterized 

 by one coarse, fairly straight polar flagellum (PI. 68, 

 A). Jusb before fission one eilium may be seen at 

 each end ; as a rare exception one tnft of polar 

 f lamella w-as observed occa.sionally. Fretpiently a 

 darkly stained body becomes visible within the rod 

 just at that point wfhere the flagellum springs forth, 

 w.hich may be considered to be a flagellated, not yet 

 liberated, gonidinm, such as can be seen occasionally 

 with many other bactKjria, especially with Bacillus 

 radicicoU, too. When liberated thus becomes the mono- 

 trichic small "swarming body" described by Bei- 

 jerinck in 1888 (4). 



The growth on mannite-nitrate agar, as well as on 

 l)eef agar slants, as described in Table I, is quite 

 characteristic, and after tihe eyes have been .sufficiently 

 trained, one seldom makes a mistake in guessing the 

 group to wjiich a culture pre.sented for inspection may 

 belong. But it must be admitted that occa.sionally 

 and temporarily a strain of the cowijea-soybean group 

 can show the flat, transparent gro-wth characteristic 

 of Radicicola, whereas it is a very rare occurrence 

 tliat a member of the last-named group simulates the 

 fonner one. The growth of Radiobaeter is always 

 ver>' typical, except when a \-ery weak straia is en- 

 countered, whic.ii does, not frequently occur within 

 this group. Plate 6!1, A, demonstratftsi the characteris- 

 tic differences noticeable on mannite-nitrate a^rar as 

 clearly as they can be .siho^vn in a photographic re- 

 production.' 



Cviltures on beef gelatine and in beef brotii dif- 

 ferentiate clearly Radiobaeter and nodule bacteria, 

 while, as stated in Table I, the two groups of no<lule 

 organisms gi-ow very muclli< alike on these .substrate^!. 

 Jlicroscopic tests, however, made from gelatine and 

 brotji furnish, in mo.sti cases, especially characteristic 

 pictures, provided that the growth has not been al- 

 together too poor to get a .satisfactory preparate. 



The growth in milk and on potato, as described in 

 Table I and illustrated on Plate 69, is very charac- 

 feris'tic and can be iLsed to great advantage for 

 diagnosis. It is not to be denied that with old stock 

 cultures atypical residts may sometimes be obtained 

 in tHiis direction also. Espwially cultures rich in or 

 entirely made up of the globular regenerative bodies, 

 whicji are produced by these as w^ell as by all other 

 bacteria, furnish whitish, yellowish, or only slightly 

 browui.sh growth on potato in the case of Bacillus 

 radiohacte-r and B. radicicola. But we have never 

 seen sucih atypical growth with new isolations. Here 

 the eoli-browai color of t|lie potato cultures sepai'ates 

 Radiobaeter .sharply from the nodule bacteria, and 

 these in turn are equally sharply to be distinguished 



I As was the case with AzoLobacter. for which the 

 mannite-nitrate agar was first used (13. p. 686). so also 

 the nodule bacteria and Bacillus radiobaeter grew very 

 rapidly^on this substrate. Allen (1, p. 33) asserted recently 

 that he could not get any growth of Aaotobacter on a 

 dextrose agar, which he erroneously called "Lohnis and 

 Smith's medium." But not even the formula 'used by u.s 

 has been quoted correctly by Allen, and it is, of course 

 quite obvious that on account of the alterations made by 

 Allen his agar must indeed have been quite un.suitable. 



by the behavior of their milk cultures. It is true 

 that sometimes milk cultures of the B. radicicola 

 group also leave the milk undhanged. but t.he micro- 

 scopic test of such almormal eases probably will 

 always show, as it did in the ca.ses .studied by us, 

 that tlie abnormality w-as simply caused by the fact 

 that the bacteria w'hich were inoculated did not 

 multiply at all. Furthermore, no alteration may be 

 .seen if milk is used whic.h has been kept for a long 

 time and has been concentrated by evaporation of 

 part of its water. 



To determine on a larger scale whether this dif- 

 ferent behavior of the two gi-oups of nodule bacteria, 

 when grown in milk, can be correctly accepted as of 

 real diagnostic value, all cultures of nodule bacteria 

 at our dispasal were tested simultaneously with the 

 following results: 



Milk u-as chaufjed as typi- 

 cal for Bacillus Radici- 

 cola bfi the foUowiuf/ 

 cultures : 



Millx- was left unchanged 

 hjl the following cultu- 

 res : 



10 from cow-pea. 

 S from soybean. 

 5 from peanut. 

 4 from Japan clover. 

 2 from beggar weed. 

 2 from Cassia chaniaecrista. 



5 from red clover. 

 4 from sweet clover. 



6 from navy bean 



1 from vetch. 



2 from, lupine. 

 ■i from black locust. 

 ■^ from Amorpiha. 



2 from Stropjiostyles. 



If kept for longer than four weeks milk cultures 

 of the cow[:)ea-soybean organi.sms usually become more 

 or less transparent on account of partial decomposi- 

 tion of the ca;=iein; but they never mIiow the perfectly 

 clear zone dharaeteristic of tfle other group. 



The bacteria were also tested on other media be- 

 sides the standard substrates, of w-hicli sterilized soil, 

 moisteued with 0.5 per cent mannite solution, mannite- 

 nitrate solution as used for i^tudying the life cycle 

 of Aziit()l)acter. tap w^ater plus 0.") per cent beef broth, 

 and 2 per cent salt agar furni.shed the most satis- 

 factory results, especially with regard to a more 

 complete knowledge of the cell morpholep- of t.he 

 organisms. For diagnostic pui"poses, Ihowever, these 

 sub.strates are of minor importance, as they do not 

 bring out anything which is not already to be seen 

 on the .standai-d media. Nevertheless, it should be 

 pointed out that cultures of the nodule bacteria in 

 soil are to be recommended for tw-o reasons. First, 

 they are useful in keeping t,he organisms in a noi'mal 

 state of virility for a lx>ng time, and, in the .second 

 place, they demonstrate very clearly, when .studied 

 microscopically, that it Ls erroneous to believe — 

 though numerous authoi"s have promoted sauvi h\-po- 

 theses — that the nodule bacteria beiliave very differ- 

 ently in soil and could, therefore, not be isolated in 

 their typical form from tiheir natural habitat. Our 

 results are in complete ag-reement with those recently 

 obtained by Bart.iiel (3) concerning the grovviii of 

 bacteria in sterilized soil. 



Tap water containing 0.5 per cent beef broth gave 

 also very good development and proved repeatedly 

 helpful in re%'iving old, weakened strains wHhich re- 

 fused to grow on solid substrates. 

 DISCUSSION 



Our rxpcrimcntal results leave no doubt that the 

 nodule bacteria of the legiuninous plants are to be 

 divided at least into two distinct groups, differing 



