I .1. 'K4 a :U H 3> :r" t“ aalmadgy; ! U I | L WIN 1 ! III MINIMUM ‘ HI ”WI I I W l I I IN .4 I ‘ .b I I 1 It“ :E‘ES {HE RHATiON CfiF FUbAREA C VAREC US [‘3‘ LLLI E924 L‘RE MEDIA ?viinzwyj(:. (jarp)b31 Crystal violet lethyl violet He found gentian violet more toxic than malachite green for the bacteria he studied. These results suggest group reactions of the dyes toward the growth of the organisms. Tests were made using various concentrations of the dyes to find the most suitable concentrations for use in plate cultures. The presence of the agar seemed to exer~ cise a protective action, a greater concentration of the toxic dye being tolerated in a solid medium than in the broth cultures. Tables ill and TV show the results of such tests for malachite green and gentian violet. The tests were set up in duplicate on two different occasions. 1‘T'Iote- These leauits may alerherese it the true relation- shios,since they are based on per cent comparisons and not on moi eculer comparisons. 10 name .o.o oom mom soaps IHom ass gm.o mo .o.o m.H heme .o.o oom you define IHom aha fim.o mo .o.o H + @ I I + I I I + l I + I assassin...“ + I I I + I .I l I | IT I ._I nogogouoaoné I I I + I T I I I a. I I + EnemaunQHSdem I I I + I + I I I + . I I + annommhaoso.m I I I + I I I I t + I .T ® gauge; + I I I + I I I I t I AHV +I «whooonpuo.m a. I II II .+ I I. I. .I I. +. .7 I. ufiuwpmn.m 4. II .+ II +. .I I. .I .I II a. +. II 39.33 mnafiuusqoofi S m .e . an e .d m m m .u S 6 ea s I s s I e u s as m c vino .t u S e s c o c Emanwmno R 0 O "was "J 11 O w S O 0 Va 2. e m mo mama r. au+u n s n ea mwaa o u .1.t n r «in; on . e C n 0 e O o r O C n 0 e o o m .1 e +tri +U m n mmnu gr .1 .1 e .t m m n m n .0 r m +tfia +u Dana a a 11 r m .t .D a a a a u e 0 none 0 {lhu ilk“ O .1 e O O u .1.n .l.n ca aa+u c a c Au+t aa+u n v s.t c S .d.t .d.t qdfipnom no unofiudppnoonoo mzoauwp mo nomanamsoo deHuwoa hue b .poaofib HHH mdmde 11 5:8? no :3 25 _. I I I I + I I I I ..,.I + I I + I + I I eyepmafluna I I I I I I I I I + + I I + + + Q + 3.303933» .m ® I ® I T I IT t I + I | I + I I + I 5.333959% kUIsSUQ II +. .I I. II II I. II II .I I . + ... + + + + + Eamohaouofi imamc I I I I + I I I I I + Q I I I I I + ganged i I I I I + I I I I I + I I I + I + I neaoomnaao.m I I I I I I + I I + I I + + I I +. .+ unpwudn.m I I I I I? I.. I + L. I I I I I + I? I I ungpaamnoofi .e. . e. s.e. Emandmho e S 0 none +u e S c 0 c .t u s 8 gene 0 c at y E e m w S V. g e m w 0 O S v. E, l m W n r l 2 2 0 u n r l 2 2 0 e t u n r 2 2 0 o as . . r o o e . r c n .u o e . . r t m m n m n R 1 t m m n m n R .1 e l t m m n m n 2:. +U .0 a a nana 11 .t .D a a a a r m 11 +U .0 naaa a a 0 u .1h .1h 0 .1 O u 1h .1h 0 60.1 0 u ihih O c S d t d t n v c S d t d t n S t v c S d t d t n mama .o.o oom mom Heaps .ndmd .o.o com mom scans mama .o.o com non Iaou one Rm.o no .o.o n . IHom aha mn.o no .o.o m.a noduuaom aha Rm.o no .o.o m.o was»: and N. 53% bu. as 33032” no unogdnunoodoo 2.3.3» no nomaadmaoo 1a The readings marked in circles represent cases where the first and second trials did not give the same results. These contradictions may have been due to varying amounts of inoculum. FINAL EYTWR MTITS WITH PLAT? CULTURES The media selected for the final tests were made up as follows:- Number of c.c. of 0.5» dye solution per 200 c.c. of cheap synthetic agar (page 7) malachite green* 1 Gentian violet l methylene blue for hact. l. Eosin (yellowd 4 The method described above was used and all experiments were done in duplicate. All the Fusaria available were grown on these colored media and on uncolored cheap syn- thetic medium as checks. All the organisms named in the tables were grown in duplicate at three different times, and many of them were grown four times. The results of all the trials were consistent with but slight differ- ences. The organisms tested showed constant characteristics in their behavior toward the dyes used. The source and age of the culture used appeared to be an unimprdfiant factor in the growth on these media as plantings from corn meal and from potato dextrose cultures, and from 2 weeks old and 2 months old cultures of the same organism showed no uifTerences in growth reactions. The following tables are typical of the results obtained. The readings given are the results of the fin l duplicaJe FGTlGS. ~ ‘ . 7-- - «I “- JXI- -,.I incae txmms were adltlel s. r" I "*3 A;‘1..'.Jv4 Showing growth on uncolored synthetic medium, 10 day reading Size 0 Type of Type 0 colon colon m cel. Tame of organism Diam. more than 2 cm. submerged Culture number aerial mycel. villous produced V.culmorum o 9. ma n F. oeru 14‘ Ill) IJI. aura tie a '1 7‘. .L‘n casts VI Showing growth on gentian violet medium, 10 day reading Type of mycelium flame of organism Culture submerged aerial mycel. abundant sericeous tomentoee number villous H . " var ca . F.redolens t . .8110)? S orum .VhS H.190 um 0 LI Ii S o 1" 00 so . o 800 01‘ o ViiI‘.SU . o a n Thbulbigenum .auran iacum .ma li H " 174 rsici l7 l5 decolorized deep purple zone below i5 TABLT VII Showing growth on malachite green medium, 10 day reading Type of Type of colony mycelium Name of organism less Culture no growth Die-mo more than 2 cm. than 2 cm. aerial mycel. abundant villous sericeous tomentose D1831. uox soorum e t F.batatis T tricothecio des F.discolor F. var.su p . .cu morum .eumar H .I‘I‘d C CO 8. H 1' .so an F.coeru eum .zona um . u genum .auran acum .ma um 10 TABLE VIII Showing growth on eosin medium, lO day reading Size of Type of Type of colon colon m celium Name of organism aerial mycel. Culture number abundant villous oottony appressed sericeous tomentose zonation 7' F. " var F.redolens . .GIIOX soorum .vas 1’1 0 um Ii a d H H H 1' F.solani .coeru eum .zona um F.bulbi enum .auran acum o m“- H n F.l on Showing growth on methylene blue medium, 10 day reading Type of Type of colony mycelium Name of organism aerial mycel. medium beoomming green below medium becomming Culture abundant villous eericeous tomentose number 4. J. 4 4. 4. euox s orum V.vasinfectum H H H F.solani at bulbi‘inum F.aurantiacum ersici lb ‘ _ - . . .- .., .1 ,, ,, .. '., . , ‘ . The accompanyinr gnotoaiaohr shot 1L0 tyges Oi ° r" 1’- V onies described. inere are three distinct tyges. The F_J co term cottony is used to describe the most common type. The mycelial threads are fine and s ft, not matted to- gether but interminsled like the fibers of cotton. Cottony appressed designates a similar tyge of mycelium , but less dense, and closer to the surface of the medium. The villous colony is covered with fine, soft, short hair, not matted toeether, and not very dense. in the colony described as sericious tomentose, the mycelial threads are long, soft,and silky, several threads being stuck together to form the long silky tufts as shown in the photoeragh of P. solani . F.0ulmorum and F.trico- thecioides are classed as cottony because their mycelium is of that type, but it is loose and spreading like a weft of cotton pulled out very thin. The growth of E; culmorum usually spreads to the to; of the Betri dish and is torn in removing the cover. Ho good pictures were obtained for this reason. F. culmorum produced its typical red color even when grown in the dark. None of the other Fusaria tested produced any color on un- treated synthetic medium under these conditions. In many cases only the center portion of the colony produced any aerial mycelium. Such colonies are classified in the tables under the heading "center aerial? while the head- ing "aerial mycelium abundant" is used to designate those colonies which were covered over the entire sur- face with earial mycelium. F.sclerotioides differed from all the other colonied on eosin and gentian violet in havine a colony entirely submerged save for a zone 19 of sericious tomentose aerial mycelium near the edge of the colony. The color changes in medium recorded appear on the under side of the plate, and are very marked. On gentian violet, F.malli, F.solani, and two of the cultures of F.radicicola decolorize the medium so that not the slightest trace of color remains, and the medium appears white against the white mycelium above. The col- onies of the other organisms appear distinctly lavender below. Certain organisms as F.03ysporum show a deep purple ring near the edge of the colony. This ring is much deeper in color than any of the rest of the medium. The colored zone recorded for F.euoxyspprum on eosin appears in the mycelium which is white save for a narrow pink zone half way to the edge of the colony, where the dye has been absorbed by the mycelium. F.aurantiacum changes eosin from its normal yellowish pink to a pure pink. This is observed on the lower side of the plate. The lavender spotting on the methylene blue plates is due to the alkalinity produced by the fungi. The characteristics of growth selected and recorded in tables 5 to 9, are definite and constant under the con- ditions of the experiment. The four cultures of F. radicicola tested were dint/aren‘t from entirelyAisolations, and identified by different authorities; the growth on the media used was identi- cal in every respect save one. Although the appearance of the mycelium was the same on gentian violet medium, two of the cultures decolorized the dye, while the 20 other two did not. F. lycoonrsici 170 and 155 were from different isolations, and grew identically alike in every respect. The histories of cultures 171 and 206 of F.eumartii Show them to be sub-cultures of the same isolation, al- though they were obtained from different laboratories. Their growth was identical. Three separate isolations of F. asclerotium produced cultures which grew alike on these media. Of the six different cultures of F.0xysoorum tried, two strains appear on the media used. Cultures 209 and 211 are alike but quite distinct from the other four cultures. The two cultures of F.conglutinans are from separate isolations, and produce the same type of colony. The results show that the cultural characteristics of these Fusaria are constant under a given set of con- ditions. As pointed out by Apoel and Wollenweber, the type of colony whether submerged or aerial, depends somewhat upon moisture. Moore (15) also shows that the production of aerial mycelium depends upon the H ion contration as well as the sugar concentration of the medium. However, under constant conditions, the cultur- al characteristics seem to remain constant. The following ébematic outline was devised to test the usability of these cultural characters as a basis for identification. No work was done in this investiga- tion on identifying the organisms according to spore measurements. Only such cultures were used as had been identified previously by an authority on Fusaria, but all of the cultures had been transfered several times, L1 and no responsibility is assumed for the correct naming of the cultures used. It was not found necessary to use the characteristics on all the media to distinguish between these fungi. Other classifications could be devised using other characters. The following outline is given to illustrate the possibilities in this regard. UNCOLORED . MALACHITE GEETIAH EOSIN tans JED‘*M GREEK VIOLET MEDIUH HEDIEi MEDIEI submerged F.coeru- no growth/’/' leum \\\ sericious . ” , tomentose F.discolor , submerged F.zonatum center ,. cottony mycleium cottony subm rged center sericious , tomentose F.conglu- tinans sericious tomentose zoned F.sclero- tioides center submerged sericious F.brthade- tomentose as villous F.or§§por- sericious < um tomentose cottony FF.batatis small F.eumartii cottony large no growth cottony F.culmorum sericious F.bulbige- . . tomentose hum mycelium ———' flarlal cottony medium - . F.s a .,. decolorized ———El—£l sericious tomentose sericious tomentose F.ascler- 221.191 CC dun UTCOLOPYU MALACHITE GTHTIAN SOS N HAM? MEDIUM GRETN VIOLET MEDIUK KB UM MED UJ small regular F.redolens small irregular F.trocothe- cioides large sericious tomentose zoned F.1y00perr sici not zoned F.congl.var gal.- mycelium large aerial cotton cottony cont. zoned F.0xyspor- .Efl zoned . F.euoxyspor- um pink not below F.aurantia- zoned cum not pink below F.vasin- fectum The behavior recorded in this paper is based on the react- ions of cultures merely transfered from laboratory media wdthout any attempt to produce the condition refered to as "hoch kulture”. EKZYAE EXPTRIHENTS Studies were undertaken to discover the cause of the color changes and decolorizationbf the dyes used. Since the triphenylmethane dyes had already been found to be toxic to Fusaria, the plan was adopted of gwowing the organism several weeks in uncolored synthetic broth, and then adding a high concentration of dye after abundant growth was obtained. Flasks containing sterile Coons'synthetic broth were inoculated with F.radicicola and F.conglutinans 25 and grown at room temperature in diffused light for three weeks. A thick mat of mycelium had formed at that time. 1 c.c. of malachite green was added to certain of the flasks, the others being held as checks. The flasks were incubated at room temperature over night. All the flasks treated with dye were completely decolorized. 0.5 c.c. of the dye was added to each flask previously treated. The dye was decolorized in three hours. One of these flasks containing a 25 day old culture of F.radici- £313 which had decolorized malachite green was used in the following experiment: The mat of mycelium was filtered off on a Buchner funnel. The clear culture fluid left was designated as the"filtrate? The mycelium was washed to remove traces of the filtrate,and ground up in a mortar with sterile distilled water and quartz sand. The liquid was filtered off and refiltered through sterile filters until clear. This liquid was designated as the "extract?. Both filtate and extract were divided into two portions, one part being boiled for 5 minutes and cooled. A slight precipi- tate formed on boiling. Sterile tubes containing 10 c.c. lots of filtrate and extract, boiled and unboiled, were set up, and 1 drop of 0.5 % solution of malachite green added to each tube. Tubes containing the same amounts of sterile distilled water and dye were used as checks. The tubes were incubated at 25°”. for 12 hours and read. The results of this experiment are given in table X. Showing the effect of heat on mycelial extract and culture fluid in their re- actions toward malachite'green filtrate boiled -- color unchanged filtrate unboiled -~ decolorized extract boiled -- color slightly paler extract unboiled -- decolorized checks -- color unchanged This result pointed toward an enzyme reaction. There was a possibility that the decolorization might be due to the presence of ammonia which could decolorize malachite green by precipitating the carbinol-form of the dye, or might produce sufficient alkalinity to allow the aldehyde sugar to reduce the dye as Gates and Olitsky (7) demonstrated. The NHé would be removed by boiling which would explain the results above. It was therefore necessary to determine if NH3 was the factor. A two weeks old culture of F.conglutinans on Coons synthetic broth in which the asparagin was replaced by K1105 to avoid the SHE groups, was tested for its ability to decolorize malachite green. It was found to decolor- ize rapidly. The pH value was determined by the colori- metric method described by Clark (3) to be 6.5.A stand- ard buffer solution with a pH value of 6.5 was made up and 10 c.c. portions tested with 1 drop each of malachite green. The tubes were incubated at 250C. for 24 hours. Results: buffer solution pa 6.5 color unchanged culture fluid pH 5.5 decolorized buffer solution pH 6.5 with dextrose unchanged distilled H20 checks unchanged These results indicate that the M ion concentration is not the significant factor and that some other agency than NH3 was responsible for the decolorization. Tests were then started to see if the decolorization was due to enzymic action. PRECIPITATION TEST A ten day old culture of F.conglutinans var.cal. grown on cheap synthetic broth was tested for ability to decolorize malachite green. It decolorized 10 drops of 0.5fi malachite green in one hour. The mycelial mat was filtered off, and 103 c.c. of the clear culture fluid treated with enough 97% alcohol to make an 80% alcohol- ic solution.A flocculent precipitate was formed. This was allowed to settle, and the supernatent liquid was siphoned off. The precipitate was dried at room temp- erature, and ground to a powder. A yield of 250 mg. was obtained. 100 mg. of the powder were redissolved in 100 c.c. of sterile distilled water. All the material ‘did not dissolve on 15 minutes standing. The solution was divided into two equal lots, one half being filter- ed through sterile filter paper. Both the filtered and unfiltered solutions were further divided into equal portions , one half being boiled for 10 minutes. A precipitate was formed on boiling. 5 c.c. portions of the solutions thus prepared were placed in sterile tubes by means of sterile pipettes, and 1 drop of a 0.5% dye solution was added to each tube. The test was set up in duplicate. After 12 hours incubation at 25°C. the results were read as follows: unfiltered unboiled unfiltered boiled filtered unboiled filtered boiled distilled water checks (3" a0 Malachite green decolorized unchanged decolorized unchanged unchanged Gentian violet partly decolorized unchanged partly decolorized unchanged unchanged Hethylene blue partly decolorized unchanged partly decolorized unchanged unchanged The agent responsible for the decolorization was found to be precipitated by 80% alcohol, not removed by filtration, but was destroyed by heating. It is well known that the triphenylmethane dyes may be decolorized by oxidation or reduction, while methylene blue is decolorized by reduction. Flask cul- tures of these organisms treated with methylene blue had several times been observed to have a colerless layer at the bottom, while the ton layer remained blue. This suggested the influence of atmosphereic oxygen. Several flasks of modified Coons synthetic solution were inoculated with F.radicicola and F. conglut growth had progressed for 10 days, dye solu inans. After tions were added and part of the cultures were sealed with parafin melted and cooled to about 50° C. These cultures were incubated at 25°C. After one hour all the sealed cultures had completely decolorized while the unsealed cultures were only slightly paler. The seal was broken on the cultures and the contents of the flasks shaken up. 27 Methylene blue regained its color almost immediately on contact with the air, but malachite green, gentian violet, ponceau, and chrysoidine remained pale. 100 mg. of the alcoholic precipitate described above were dissolved in 100 c.c. of sterile distilled water and a test with dyes was set up as before. The tubes were sealed with parafin and incubated as before. All three colors were completely decolorized in the un- boiled tubes. These results indicate that decolorization is due to enzymic action,and is probably a reduction. It is a matter of contention whether living tissues se— crete reductases capable of reducing dyes to their leuco- bases as claimed by Ricketts (l5) and Harris (8) or whether such decolorization is due to the SH groups in proteins and amino acids as cystein. Heffter, Strassner (l7), and others maintain the latter View. As the evi- dence against the existence of such reductases is not generally accepted, and since so little is known of the nature of fungus proteins, it seems that the results of these tests are most simply explained by attributing the decolorization to a reducing enzym of the types already found in organic material. STHHARY The growth of various Fusaria was tested on twenty different dyes. In their effects upon these organisms, groups or classes of dyes seemed to show reactions. The triphenylmethane dyes are the most toxic to the fungi tested. Some of these organisms are able to decolorize the triphenylmethane, monazo, and quinone-imide dyes -_=._l__ 42 _ _ (3" 60 tested. The agent of decolorization is believed to be enzymic in nature, and the reaction is probably a re- duction since it takes best in the absence of air. Under the conditions of the experiments, the fungi tested showed definite and consistant.characteristics of growth on synthetic media both colored and uncolored. The previous cultural conditions of the fungus do not seem an important factor in the growth on synthetic media. The results indicate that synthetic media and synthetic media colored by dyes may furnish valuable means for quick and accurate identification of certain groups of fungi difficult of identification by the ordinary morphological methods. l. 9. 10. ll. 15. L9 LITERATURE CITED Appel 0.,and Tollenweber, H.V. Grundlagen einer Monographie der Galtung Eusarium (Link) arb. K. Biol.Anst.Land.u.Forstw. §, 1-207 Bernstein, E.P. and Lowe, K. organism 1919 A medium for the isolation of influenzaA qur. Inf. Dis. fig, 78 Clark, -3". III. 1920 The determination of hydrogen ions. Churchman, J.T. 1912 The selective bacteriocidal action of gentian violet. Jour. Exp. Med. lg, 221 Coons, G.H. 1916 Factors involved in the growth and pycnidia formation of Plenodomus fuscomaculans. Jour.Ag. Res. V 1e Endo, 8. 1905-4 Uber ein Verfahren zum Nachweis der Typhus Bacillen. Cent. f. Bakt. l Abt. pg, 109 Gates,F.L.and Olitzky, P.K. Factors influencing anaerobiosis with special reference to the use of fresh tissue. Jour. Exp. Med. pg, 51 Harris, D.F. 1910 Endo-enzyme in liver and kidney. Biochem. Jour. 5, 145 Holt-Harris, J.E. and Teague, Oscar 1916 A new medium for the isolation of p; typhosus from stools. Jour.Inf.Dis. lg, 596 Klieger, J.J. 1918 A study of the antiseptic prouerties of certain organic compounds. Jour. Exp. Med. g1, 453 Krumwiede, C.Jr.,Pratt,J.S. and Mc Williams,H.I. 1916 Brilliant green for the isolation of typhoid bacilli. Jour. Inf. Dis. lg, l Wilcox,E.M.,Link,G.K.,and Pool,V. A dry rot of the Irish potato tuber. Res. Bu1.}l U. of Heb.Ag. Bxpt.Sta. Moore, 3.8. 192 Physiology of F.00eru1eum.‘ Ann. Bot. pg, 137 14. 15. 16. 17. 18. Petrof, s.e. 1915 Ricketts, H.T 1904 50 A new and rapid method for the isolation and cultivation of tubercle bacilli directly from sputum and feces. Jour. Exp. Med. 31, 38 Reduction of methylene blue by nervous tissue. Jour. Inf. Dis. 1, 590 Sherbakoff, C.D. 1915 Strassner, W 1910 ”78.111, A. 1918 Fusaria of potatoes. Mem. 5 Cornell U. Ag.Expt.Sta. Die Reduzierenden Wirkungen des Gewebes. Biochem. Zeit. 29, 295 The manufacture of organic dyestuffs. Translation by F.fl.atack Fig.1. A- Sericeous tomentose type of colony (T.solani) B— Cottony type of colony (F.vasinfectumi C- Cottony type of colony (F.zonatumf D- Villous type of colony (F.31scoIor sulph.) on malachite green mediwm Fig.2. a F.00nglutinans on gentian violet medium B- F.tricothecioides on same medium ' my T BLIiL-‘ .LI Fig.1. A- F.0rthaceras longius on gentian violet B- F.1ycopersici on same medium Fig.2. A- F.radicicola on malachite green 8- F.0xysporum 211 on the same medium 0- F.0xysporum 204 ‘ PLATW III Fig.1 e- F.radicicola on gentian violet B- F.0xysporum 211 on same medium 0- F.0xysporum 204 Fig.2 F.cong1utinans on malachite green 1 ...(uiurL... ..... , -.. ., .z'.‘.;(.:: a .. ...... .... ......,.~,....Lur. ~-|.l5.... .. 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I. . . .. . . .. . u .. ... . . . . u .. 4.. .. w . . . .. . . . A .. _ .I.