STUDIES ON THERMOPHILIC BACTERIA By LETHE ELEANORA MORRISON A.B. University of Illinois, 1919 M.S. University of Illinois, 1921 THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY IN BACTERIOLOGY IN THE GRADUATE SCHOOL OF THE UNIVERSITY OF ILLINOIS, 1922 URBANA, ILLINOIS d»r t z I 322 M 8 3 UNIVERSITY OF ILLINOIS THE GRADUATE SCHOOL August 15, i 9 2 2 1 HEREBY RECOMMEND THAI' THE THESIS PREPARED UNDER MY supervision by Lethe Eleano rs Morris on ENTITLED Studies on Thermophilic Bacteria BE ACCEPTED AS FULFILLING THIS PART OF THE REQUIREMENTS FOR THE degree of Doctor of Philosophy Recommendation concurred in* Committee on Final Examination* 500412 Digitized by the Internet Archive in 2016 https://archive.org/details/studiesonthermopOOmorr Acknowledgement It is with pleasure that the author takes this opportunity to express her appr eolation for much kindly advice and assistance received during the super- vision of this work from Professor P# W# fanner# She also wishes to thank him for encouragement received in all graduate study and teaching done under his supervision# Thanks are also extended to those others, who by various means, aided in the completion of this in- vestigation# 4 . . . LIST OF TABLES * Table I Table II 12 Table III 20 Table IV 27 ■Table V 29 Table VI 29 * m. Tables follow the page numbers given above* 1 I, Introduction A new interest in thermophilic bacteria is rapidly- developing since the investigations of Barlow (1912), Weinzirl (1919), Cheyney (1919), Bonk (1920), and Bigelow and Estey (1920), have suggested their significance in the spoilage of canned foods. The wide distribution of these organisms in nature is demonstrated beyond doubt by a review of the literature which has been publish- ed describing strains isolated from soil, water, foods, manure, ensilage, hay, cotton, etc. The infection of foods with ther- mophiles results, probably, from their contact with soil and water. The object of this investigation was to study the charac- teristics, particularly the temperature relations including ther- mal death point determinations, of some thermophilic bacteria isolated from different samples of water and soil. i 2 II, Historical In order to have the present dissertation more complete in regard to the historical review and bibliography, a table summarizing the literature on thermophilic bacteria used in the Master’s Thesis by this author is also included, (Table I.) Since the experimental work of this investigation deals principally with the temperature relations and thermal death points of thermophilic bacteria isolated from various sources, it was deemed advisable to include here a brief review of that literature on thermophilic organisms regarding these points only. a • . ' ... - mm J - . . - ■ * - — e • - Investigator Organism Described Source •Temperature Remarks Mi quel (1879-99) Bacillus therisophilue eine River water; sewage xcreta; dust; nir 20 -72° C. 15° - 70° G. jptimum Attributes the property of t ic organism to grow at such high temperature to particular character of protoplasm. Isol - *d first in 1879 ; characteristics described in 1388 van Tiornem (1381) (1) Streptococcus (2) Bacillus ;ter in which beano had ear. cooked Jp to 7 4° C. jp to 77° C- A certain umouht of acidity produced by these organisms a.en renderod the media uninhabitable by them Certoa or.d Garrigou (1986) (1) Small rods f (2) Filaments ot epring at Luchon ,5° . 54° c. "Further experiments are necessary to determine the chemical and biological a*ti of these rods, and this knowledge will throw a light on the therapeutics of mineral waters" Globl g (1?93) Many bacilli (30 kinds) arden soil j0° - 70° C. Thought the fact that ros of them were isolated from the superfi:ial layers of tho noil explains that they get heat for high temperature at which they grow from the rays of the sun. source not intestinal t of man or animals, tap or river -v.iber Eurrlli (1339) Two bacilli ilage ; manure 60° - 70° C. Toe initial hig. temperature which these bacteria induce is probably most 6orviceuble by causing the closer packing of the sil»ro and the exclusion of the air, rather than by killing the germs of other ferments Schloesing (1989) table manure Jp to 79 .5° C- At temperature of 60° - 66° C . these organ! -.3 produce 17 times as much carbonic acid as that in sterilized raanure. Cohn (1893) Attributed to thermogenic bacteria a role i., so-called spontaneous heating of r.alt, tobacco leaves, cotton, hay, and manure- Made no attempt to isolate any of these cultures by the plate method. FI Or *9 (1394) Many bacteria Sterile milk 4° - 44° C« or 27° - 34° C. All strongly peptonizing in character; some ere toxic; all formed spores which would withstand heating in water or steam for two hourr.. Griffiths (1994) Bacillus valericus Bacillus therrdcus Silage 50° - 66° C- 56° C* optimum 68° - 680 c. 55° C* opt i um Functioned in the production of the so-called sweet silage. Leichraan (1894) A bacillus Slimy milk 45° - 50’ C. Produced acid UacFadyen and : (1894) Many bacilli Earth, river and sea water river mud, air dust, straw and feces of men, mice and. chickens 60° - 6SP "Their most marked property appears to be the decomposition o- proteid codier. • ich they are able to effect." Most of them possess active fermentation properties. Gorihi (1395) Milk 37° - Ambres (1910) suggested that this organism was ther, ^tolerant since it grew at 37<* G • also - Karlinskl (199 5) (1) Bacillus IUidzensis capsulatue (2) Bacterium Ludwigi Hot springs of Illidze in Bosnia 50° - 58° c. 55° - 57° C. Has no explanation to offer a3 to significance of the presence of these two organisms in hot springs. Su^rests that the examination of other hot springs for similar bacteria would greatly increase the knowledge of biology of water bacteria Rabinowitsch (1895) Eirkt specier. of thermophilic bacteria Many sources; widely distributed in nature 3 4° -75° C. Concluded that peculiar ability of so-called thermophilic bacteria to grow at temperatures so much higher than the optimum temperature for common bacteria is a property of adaptation to environment. Weter (189 5) Bacillus I Bacillu II Bacillus III "Sterile" milk 22° - 50° C. 220 _ 50° C. 3 0° - 650 c. Found thermophilic bacteria in eight out of eleven samples of so-called "sterile" milk . T«o of the three bacilli formed sporec; -.one liquefied gelatin; all 1 - T.-o or three k i -.ds of thermophilic ncteria 50« . 60 o c . Mo attempts made to characterize or iden'ify the • orme fo- - Kedzoir (1396) Cladothrix form River spree 35° - 65° C. 55° C. optimum Facultative anaerobe grows better without oxygen. Spores very resistant to heat p_nd to disinfectants such as 5 per cent phenol Teich (1895) Bacillus form Hot springe of Illidze 54° - 58° G. Large oval spores formed in one end of tie rod* make then pear club-shaped Davis (1897) Probably a bacillus Hot springs of Yellow- stem* Park Up to 85° c. Functions in the formation of -miner 1 deposits in hot springs. Harshbarger (189 7) \Thite filamentous bacteria Hotsprihge of Yellow- stone Park 35.4° C. Eecomes of a sulphur yellow color at 175° F. Yellow '-.rowth due to species of Beggiatoa, a plant which is classed with the Bacteriaceae , and which, during life, deposits sulphur .granules- Koning (1897) B. tabaci III B- tabaci IV B- tabaci V Tobacco Ambro* (1910) speaks of these organisms ns real thermophilic bacVeria Uiyoshl (1397) Many bacillus forms; n zooglian mass of bacteria; iron bacteriui Hot springs in Japan 41-69.8° C. Hot springs of Japan furnish a good medium for thermophilic bacteria Wittlin (1397) Hot springs in Switzer- land Ambroz (1910) explains the negative results of littlir. on the grounds of inadequate methods of research. Laxa (1398) Clostridium gelatinosum F'Jllmass* in sugar manufacture :5° - 58° C. Tnis organism is a facultative anaerobe whose spores are not killed by exposure to steam at 10CP C. for 15 minutes. Oprescu (1898) Bacillus thermophilus lique- facien6 aerophilus Bacillus thermbphilus aerobius Bacillus thermophilus aquatilis Bacillus thermophilus reducens Eacillus thermophilus liquefac- iens tyro genus Soil, Berlin zoological r ardens Canal water Spring water, ice Blood serum test gli-rs Roquefort cheese 21° - 70° C. 36° - 60° C- 36° - 60° C. 36° - 620 C. Room tempera- ture to 60° C. Oprescu gave rather a detailed description of the five forms tudied by him Poupo (1898) An organism simijiar to Clostri- dium gel at in os urn Laxa (1898) Syrup 15° C. Schillinger (1898) Four types Soil 66° C. max. After experiments on bacteria from soil carried on t different temperature , he came to the conclusion that thermophilic bacteria ore not properly so-called; the term thermotolerant should be applied to those organisms which can adapt themselves to high temperatures. Teiklinsky (1898 and 1399) Theraoactinomyces vulgaris Thermomyce3 lanuginocus Soil 48° - 68° C. Also isolated 6 varieties of bacteria from the ot sprines of Island of Ischia hich she called strict thermophiles; 0 timura temperatiuee 60° C- Michael la .(1839) B. thermophilus aquatilic liquefaciens B. thermophilus aquatilis liquefacie s aerobius B. thermophilus aquatilis chromogenes V B. Thermophilus aquatilis anguinosus Spring water, Berlin 50° - 60° C. optimum "ichaelis said of thee, organi 9 that they '-ere not only thensotolerant, 1 ut also thermophilic Vernhout (1899) .cillus tabaci fermentationis Fermenting tobacco at 4 i° - 50° 0. 50° C« optimum Mot a true ther :iophile cince its optimum temperature was IS C. xt vVjJVOpf) >>* , . , , ' c, «>■( . ' "■* • nr,- .01 f ! w -mn r m - iiiuit Ift J ■U. , .01. ' ■ ?} '■ : 7 bio l continued Invest! gator Organisms Described Sources 1 '^re Remarks ?K.T,es ( 10CO) Hino err nl »i not named 7uter, feces, coil, pus, dlk, etc. 309 - 70° 0. c ptimum Suites eu f roots that a distinction be made between "thermophilic" and "thormotolerant" bacteria. I Bacillus thormophilus Gripooni onure 30° - "0" C- ••6° C- optiaUB - - - 1 • • The organism was proteolytic in character. Russell Hastings (1902) A micrococcus Pasteurized milk 200 . 25- C. optimum. 76° C* maximum ' ut a true thermophile : ut probably a tnersotolor&rt organism. Schardinger (1303) Group I 6 aerobic and 1 anaerobic orgar.ioms. Group II 1 aerobic and 5 ana- erobic organisms. ~ooda, etc . i*oods, etc. Rc 001-55® C- 37® - 66° Grouped with h- -rn otato bacilli, produce : Hj fr, r. nitratee. Found that these lacterir "doxtri ire" starch. Setchell (1903) Filamentous schizomcete o- Springs of Yellowstone Park 70° - 89° C. Such bacteria grow at a higher temperature in silicious than in calcareous waters. Tsiklir.sky (1903) :.cilli. TWO streptc- thrix forms Human alimentary tract 57° C- relieved Ve . ppearar.ee of tb.or. their wide distribution in nature and their f£eat resistance; t ermophili-s are probably merely variations of common non-thermophilic organisms* C.tterlna (1904) Bacterium thermophilus radiat Y/ater 60® - 70° c. optimum Also isolated an organism apparently identical with B. thermophilus IS (Rab'nowitsch, 189 5) A CilUrt (1904) Actinomyces therraop: ilue Soil 50® - 55° C- optimum Strict aerobe; liquefied gelatin slowly; coagulated milk. Kehler (1904) Tvr forms , 6 5® C. c; *.i aa . etti ( 1905) A bacterium Hot spring CO® - 76° C. o ptinum An anaerobic ram positive organism v i h produced large central spores. i rulni (1905) Thirteen bacilli, five streptothrix forms Adult and infant stools. F ur bacilli an< one streptothrix aboolute thermophiles ; all strict aerobes; all gram positive; all but one 6pore formers. kiiehe (1905) Bacillus thermophilus alcha. Hay 40° - 70° C. Hay in which spontaneous combi^i ns had occurred. Anitschkow (1906) 59® - 60® C. Only occasionally found thermophiles in alimentary tract (1906) Bacillus thermophilus alpha Bacillus thormophilus beta Bacillus thermophilus gamma Eacillus thermophilus delta Setfage 52° - 60° C. optimum Ato'. roc says Bardou's work on the chenical reactions of thermophiles is fundamental and far surpassed in detail *nd accuracy other work that had been done. Three of organisms described were denjtrifiers; all of them were strongly proteolytic. Blau (1906) B* cylindricus B. robustus B* tostus B. calidus Soil to® c. cptimuo Thermal death point 100° C. fofl' 20 hours Thermal death point 100° C. for 7 l/2 - 8 hours. Extremely resistant spores. Thermal death point 100° C. for 19-20 hours Thermal death point 100° C. for 3 hours Brazolla (1906) Regarded therr.opb.il ic i cleric cf s. unit ary significance in water. Falcior.i (1907) B* thermophilus I B- thermophilus II Hot springs 60° C. optimum Concluded tha* hot springs for vorable medi . for thermophilic bacteria Miehe (1907) E. calfactor Hay 50® - 60° C. optimum Claimed t > i : : organism was responsible for he ti ng c i h^. • 5 1 ;i H - r - » .. ♦ . 1 9 above the coagulation for albumin (60° - 67® C.) and (2) thermo- tolerants with a maximum temperature of 50° - 55® C. f but which also grow well at ordinary temperatures* The organisms which grew best at higher temperatures but also grew, though feebly, at lower temperatures, he termed psychro tolerant* In accordance with the findings of Miquel, Russell and Hastings, and Setchell, Tirelli (1907), from the results of his work, concluded that the ability of thermophiles to withstand such high temperatures must be attributed to the particular nature of their protoplasm, rather than to their adjustment to the circumstances of environment* In work on thermophilic bacteria in the tropics de Kruyff (1910) tested the resistance of the spores of ten forms isolated from soil and found that they were killed in boiling water in 4 1/2 to 8 hours* He also found that thermophilic bacteria grew better aerobically at the higher temperatures and anaerobically at the lower temperatures* The same results were obtained by Rabinowitsch and Tsiklinsky. Ambroz (1910) claimed that if the high temperatures at which thermophiles would grow was not the optimum, as stated by Schillinger, then thermophilic bacteria should grow better at ordinary temperatures than they do at the high temperatures. This is not the case* The conclusions drawn by Oprescu, Sames, and Blau were confirmed by Koch and Hoffman (1911). The organism which they isolated from soil would grow on artificial media at 52® C., but r , f , r , 10 not at 25® - 28° C.; in soil they would grow at 28® - SO® C. but not so well as at 52® C. i'rom these results they also concluded that the nature of the medium used had a great influence on the temperature demanded for the growth of thermophiles.. Some spores of the thermophiles found in soil by Krou- lik (1912) were quite resistant to heat. The spores of two of these organisms withstood sterilization with flowing steam for 2 hours, while those of two others were a little less resistant. Noack (1912) emphasized the importance of the fact that the resistant forms of thermophilic bacteria would stand many changes of temperature below as well as above their optimum temperature. He believed that this was the reason they can be isolated from soil almost any time, whereas, except in the trop- ics, the temperature of soil seldom rises to the optimum temper- ature for these organisms. This might also account for their occurrence in decomposing organic matter in which the temperature, due to spontaneous heating seldom reaches the optimum temperature for their growth. Although the thermophiles studied by Uegre (1912) were isolated from the sands of the Sahara, they were less resistant than many of the forms which have been described. The spores of these forms resisted heating at 100® C. for 15 to 20 minutes. Bergey (1919) divided thermophilic bacteria into two groups, namely, the true thermophiles, and the facultative thermo- philes. In his studies on many organisms in these two groups he found that the spores of true thermophiles resisted a temperature . ♦ t . e of 100° C. for periods of time varying from 5 to 400 minutes, while those of the facultative thermophiles were killed in 15 to 60 minutes at 100° C. In the light of this it appeared pro- | bable to Bergey that the optimum temperature for growth was I related to the heat resisting powers of the spores. He oonsid- J ered that the phenomenal oharaoteristic of thriving at temper- . atures above those at which egg and serum albumin coagulate, ex- hibited by thermophilic bacteria, might be due to the reaction of the medium in which the bacteria were grown, or to the miner- al content of their own protoplasm. While investigating the use of a biorizator for pas- teurizing milk Patzschke (1919) discovered an organism which he named Streptococcus laoticus thermophilus . This organism resist- | ed a temperature of 75® C. for 3 minutes but was killed at 65° C. in 10 seconds. The organism isolated by Bonk (1920) from canned corn was very resistant because it was found in corn that had been processed at 118* G. for 75 minutes. The experiments of Bigelow and Estey (1920) constitute the first very extensive work on the thermal death points of thermophilic bacteria. They described a new method of determin- ing the thermal death points of spores, at a temperature of 100° C. and above, under definite and well controlled conditions. The longest length of time that any of the spores of the organisms which they studied were found to resist the action of heat under the described conditions was 1320 minutes at 100® G. This is a r ' f - « . - ■ . - * ' , - , 12 greater degree of resistance than has been reported by any other investigator* The spores of an organism found to cause the fermenta- tion of spent wattle-bark in the corrosion of white lead by Grieg-Smith (1920) were very difficult to destroy* This was especially true when the spores were contained in the pores of the bark* They lived after an exposure to 186® to 205® C* for 2 1/2 hours* Although Buchanan (1922) gives no authority for his remarks , he makes the statement that thermophilic bacteria "are so resistant that they will withstand boiling water literally for days before being destroyed” • Thermal death point determinations and temperature relations reported by the investigators included in this survey are summarized in the following table* * ■I * , r ■■ . « \ 1 < . > > * ^ w - ' r: t • t Tab^e II Invostiyatcr Referer.ee Organisms Described Source Temperature for Growth Thermal Death Points (1) FlStfe (1894) Ztschr. f . Hyg* u. in- Menv bacteria Sterilized milk 24° - 44© C. or 27° - 54° C • Spores Withstood 3/4 to 5 hours heating at 100° c. fectionskrnnkh. , 17, 272 (not named) ( 2) RaUno«d‘.»ch (1895) Ztschr. f- Hyg-, 20, 154. Bacillus thermephilue I Soil; snow ; various excreta; rrains; milk. 34° - 75° c. Spores survived steam at 100° C. for 5 to 6 hours " II Spil; snow; excreta; -rair.s; " ..ilk . III Soil; excreta; " f rair.s; milk " « IV Soil; excreta " " •• V Excreta; grain " " " VI Excreta " " VII Cow excreta " " " " VIII Excreta; grains (3) Karliaeki (189 5) Hyp. Rundsbau, 5,, 605. Bacillus Illidzer.sis Hot springs of 50° - 50° C. Spores withstood flowing steam at 100° C. for 4 Capsulatus Illid e ir. Bosnia minutes. ( 4) Ked:oir (189 6) Arch. f. Hyg., 27, 328 A thermophilic cladothrix River Spree 35° - 65° C.; 55° C. optimum Spore6 killed by flowing steam at 100° C. in form 3 l/2 7. 4 1/2 hours. Ti) Laxa (1899) Centralbl. f. Eekteriol. Clostridium gelatinosua "FQllmasse" in 25° - 58° C. Spores not killed by exposure to dry heat at 150° Att. 11., 4, 362 sugar manufacture C- for 15 minutes or moist heat i\ lffitf C. for 75 minutes. (6) T.Uin.ky, lilH.P. ( 1899 ) Ann. de l'lr.st. Pasteur, Thermoactinomyces vulgaris Soil 48° to 60° C.; 57° C- optinuir Spores not killed after 20 minute at 100° C. 13, 500 Thermomyces lanurinosus » 42° to 60° C. in an autoclave. 54° to 55° C. optimum Spores killed in one minute at 100° C.; with- stood dry heat at 80° for 3 hourB. (7) SUMS (19 0) Ztschr. f. Hyg., 33, 313 Bacillus I Earth 56 c - 70° C. optimum. At 750.2 ran. pressure spores resisted live 70° - 74° C * maximum steam for 3 hours and 10 minutes. Spores were formed at 62° C . Eacillus II Pus from mouse in 56° - 70° C. optimum Sporeo resisted steam at 743.7 mm. pressure for j ected with eorth 75° C* maximum 2 hours and 50 minutes. Spores were formed at and immunized a- ainst tetanus 56° - 70° C. optimum 62° C. Bacillus III Vaginal mucous Resistance of 6pores formed at 62° C. at 740." during pregnancy Raw milk 66° - 70° C. maximum mm. pressure = 25 minutes. Bacillus IV 50° - 60° C. optimum 66° - 70° C. ma: " " •' 5 hours (13) Kreulik (1912) Centralbl. f. Bacteriol. Bacillus I 1 Soil 60° C. optimum Spores withstood 2 hours sterilization *ith Art. II., 36, 339 flowing steam Bacillus I 2 60° ’ . optimum Spores withstood sterilitation with flowing stearii for 2 hours Bacillus II 1 " 30° - 68° C. 55° - 60° C. optimum Resistance of spores greater than that of E* II 2 Bacillus II 2 " Same as for B« II 1 Spores did not with6tund the action of flowing Bteam for 2 hours. (14) H.jr. (1913) Corapt. renc . Soc. de Bacillus 3 Sands of the 70° C. max. 50° C. o - J i,'. - . -j . >.•* . 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PATHOGENICITY) 15 for incubation at 55® C# than the 1 or 1 l/2 per cent agar as » ordinarily used* i Microscopic features i j The microscopic features were determined by use of carbol fuohsin and Oram stains# ior the ) staining of flagella the nev/ method proposed by Plimmer and Paine (1921) was used, with certain modifications, and proved to be a very quick and efficient method# Since it was found that the thermophilic bacteria under observation grew very rap- idly at 55® C., 8 - 12 hour agar slant cultures were used# Some of the growth was removed carefully and put into tubes contain- ing sterile water which had been held at a temperature of 55® G# for about an hour# These water suspensions were kept at 55® G# for 30 - 60 minutes and then 2 or 3 small drops of the suspen- sions were placed on slides that had been prepared according to the description of Plimmer and Paine and maintained at 55® C# for 15 - 60 minutes# It was found that by keeping the suspen- sions and slides at 55® G#, thus avoiding any change in temper- ature during the preparation of the smears, that the motility was not interfered with and the flagella were then easily demon- strated by staining# The smears were allowed to dry at 55® G. over night and then fixed and stained by the method as described by the above mentioned authors. Miscellaneous Biochemical Reactions Just one of the cultures (iio. 4) was used to test its pathogenicity for guinea 16 pigs* This culture was grown on blood agar slants for 18 hours, the growth removed and a suspension of it in physiological salt solution made* Guinea pigs were given injections of this sus- pension intraveneously and interperitoneally with no effect* Bruini (1905) is the only investigator who attributed a patho- genic effect to a thermophile* A zero is used for this charac- teristic in all of the index numbers simply to indicate not that it was not pathogenic, but that this characteristic was not studied on all of the cultures. For the determination of gelatin liquefaction the provisional method was used* It is designed to distinguish "true liquefiers" (organisms producing ecto-enzymes ) from organ- isms that produce endo-enzymes of proteolytic action that are released from the cell after death and cause liquefaction of the gelatin if incubated for a long period* The cultures were given a preliminary cultivation for 24 to 48 hours (according to the rapidity of growth) in a 1 per cent solution of gelatin at 55° C*; then the surface of gelatin in test tubes was inoc- ulated and incubated at 55° C* for 4 days* By this method all the cultures except 78, 83, and 84 were found to be gelatin liquefiers. To test for the production of nitrites and gas in nitrate media both nitrate broth and nitrate agar slants were used. f . 4 * r - « . ' > : . , » - v : 17 Carbohydrate Reactions The method of Baker (1922) was used, with certain modifications, to determine the production of acid and gas in dextrose, laotose, and sucrose broths. Brom thymol blue was added to these carbohydrate broths before the media was tubed and sterilized. It was found that the addition of 15 co. of a 0.04 per cent alcoholic solution of the indicator to every liter of carbohydrate broth made a good concentration for the detection of acid production, and was not strong enough to inhibit growth. J’or the determination of diastatio action, starch agar plates were used and dot inoculations were made in the center on the surface of the agar in the plates instead of the usual streak inoculations. Other Characteristics Tests for indol were made on nutrient broth and Dunham's peptone solution cultures; Vsfitte's peptone was used in these media since it seemed to be better suited for the production of indol by bacteria. Both the nitroso-indol test and Ehrlich's test were used. Ehrlich's reagent was prepared according to the method described by Horton and Sawyer (1921). It was found that this test gave more satis- factory results when the tubes were heated slightly. All the cultures studied, except Ho. 81, produced indol* In general the conclusions of Horton and Sawyer were confirmed. 18 For the determination of the production of hydrogen sulfide, nutrient broth made with Witte's peptone, over which a strip of lead acetate paper was suspended by means of the cotton plug, was used* The blackening of the paper indicated hydrogen sulfide formation* Streak cultures on ”Bacto Lead Acetate Agar” plates were also used* All but cultures No. 83 and 84 formed hydrogen sulfide* Litmus milk and sterile milk to which brom cresol purple had been added, were used to determine the reactions of the thermophiles in milk. These milk cultures were incubated 7-10 days. Most of the cultures grew well in milk; only two cultures, N 03 . 54 and 59 produced no apparent change in the milk. Cultures No. 83 and 84 produced alkali with no other apparent change. The majority of the rest of the cultures showed coag- ulation and peptonization with an alkaline reaction, some of them having shown a slight preliminary acidity. A few cultures showed slight acidity in milk and a few showed distinct acidity with coagulation but no digestion of the casein. Temperature Relations Of the 89 cultures used in this investigation 18 cultures were chosen for use in a more intensive study of temperature relations and thermal death point deter- minations. The choice of these 18 cultures was made to include organisms from as many sources as possible* These cultures were grown on agar slants, in gelatin tubes, and on agar plates, at , 4 .. . : . ‘ . .> , 4 ' 19 the five different temperatures available for incubation in these laboratories* The agar slants, 5 for each culture, were inoculated by means of a small wire loop from a 24 hour broth culture of the organism to be tested, and these agar slants incubated simultaneously at the five given temperatures* The gelatin was inoculated by placing a loopful of 24 hour gelatin solution cultures on the surface of gelatin in tubes* Five tubes were prepared for each organism, and these tubes incubated at the five given temperatures* Large petri dishes were used for the agar plate cultures, dot inoculations being made in the cen- ter of the plate on the surfaoe of the hardened agar. A small wire loop was used to make these inoculations from 24 hour broth cultures of the organisms. All of these inoculated media were incubated at the five given temperatures for 24 hours in order to have a definite period of incubation; a 24 hour incubation period was used be- cause the agar streak cultures and plate cultures seemed to reach their maximum growth within such a period of incubation* The comparative amount of growth attained in gelatin and on agar slants at the different temperatures was judged as carefully and as accurately as possible with the naked eye; the diameter in mm* of the giant colonies produced on the agar plates at these same temperatures was measured. The data secured are given in the following table {Table III), i’igures are used to indicate the amount of growth, - ■ ' * , . . . . , ■ . ; . ' . » 20 1 indicating the greatest amount of growth at the stated tem- peratures, 2 the next best, etc. This method is used for the lack of a better one* The method, proposed by Bigelow and Estey (1920), for the determination of thermal death points of typical thermophilic organisms, with a few modifications, was followed in this study. Nutrient agar slants were inoculated with pure cultures of the organism to be tested and grown at 55° 0. for 48 hours. The growth from two agar slants of each culture was brought into suspension by pouring 10 co. of sterile nutrient broth onto the slants and emulsifying. These suspensions were then trans- ferred to flasks containing 100 cc. of sterile broth and incu- bated for 7 days at 55° G. At the end of the incubation period the flasks were placed in a refrigerator for 24 to 48 hours, when they were heated to 85° 0. for 15 minutes to kill all vegetative forms, cooled immediately, and placed again in the refrigerator to prevent the germination of spores. These were the stock suspensions used for the determination of the thermal death points of the spores. The concentration of spores in the suspensions was determined by plating in different dilutions. The relative ability of the 18 cultures to form spores under these conditions is shown by the counts. The number of spores per cc. of suspension for the 18 cultures varied from 590 to . ■ ' . . . * . „ . , . Table XII GROWTH OF THERMOPHILES ON 3 DIFFERENT MEDIA AT 5 TEMPERATURES (CENTIGRADE) Agar Plate 0 o to COtO'tf'^tOtOtOr— ltOtOOiO)O0tOO3tOO3rH o LO m HOJIONHHHOJNNHHHHHHHOJ o m i o 03 03rH.HiHC3 03O3tOrHiHO3rHtOO3tOO3tOtO o J> to ■^•^OSlO^^’^'^'^^tOtOtOtO^tO 1 1 0 o to 1 0 m 03 ^mioin-tfio^^T^in^^Tit^LO^ • 1 Gelatin O o to 1 | ^ |03t0t0^'^^ l H^^-^rHrH 0 m m t0r-ltOt0r-l03iH0lr-ltOtQtO03 03r—lrHO3O3 0 m i 0 03 H03HH03HtOH01rHHHtOHN03tOtO o O' to 03t00303t0t0^^^030303'^t0t0t0^'^ o o to 1 o m 03 to ^ in n i iininminm i i i im Agar Slant © o to 03 1 t003tOt003t003rHrHrHrH03t0^rH03 0 m in HrlrlHHHHHH03NN03HHrl03H 0 m a 0 03 t00303t00303t003t0t0t0t0t0t00303t0t0 0 O' to 0 0 1 0 m 03 m i tP i i in m m i i i nn i i Culture Number OOiHtOtOOWlOOHWtOO^OOOi rHrHrHO3O3tOtOtO'^*'^iniOinintOtO00C0 ' ' . • I I I I 1 I I 1 I lilt J . . I . J r r i [ill i i M 73,800,0^0 21 The tubes used for the determinations were hard glass tubes 5 mm. in diameter and 250 mm. in length; they were pre- pared for use by soaking over night in weak hydrochloric acid solution, rinsing thoroughly with distilled water, draining, wrapping in heavy brown paper in packages of 15 each and ster- ilizing. These tubes were inoculated with 1 cc. of the sus- pensions of spores, sealed off to within 40 to 50 mm. of the surface of the liquid, and held in the refrigerator until ready to be heated. The thermal death points of the spores at 100° C., 105° C., 110° C., 115° C., and 120° C. were determined by im- mersing the sealed tubes in an oil bath adjusted to the desired temperature. A DeKhotinsky electric bath containing "Crisco" was used to maintain a constant temperature, and a Wassernan test-tube rack for suspending the sealed tubes in the bath. Before immersing the sealed tubes in the oil bath the tempera- ture was increased one degree in order to compensate for the loss in temperature due to the immersion of the tubes in the oil and 30 seconds were allowed for the heat to reach the center of the tubes and for the temperature to drop to that at which it was previously adjusted, before recording the time. A series of tubes was exposed to the desired temper- ature for definite periods of time, a tube being removed at the end of each period and immediately placed in a bath of ice water r zz in order to prevent further action of the heat on the spores of the bacteria. When cold, the tubes were placed in the refrig- erator and held until the sterility of the medium could be determined. Sterility was determined by inoculating agar plates with the contents of the heated tubes and incubating for 2 to 4 days. In most oases, when spores had survived the heating, growth occurred within 24 hours, and in no case did growth occur after 48 hours. The hydrogen-ion concentration of the suspensions was determined at the beginning and end of each period of heating by the colorimetric method. The color chart of indicators show- ing the colors of Clark and Lube* indicators in solutions of known pH was used to determine the pH of the different suspensions. Charts were prepared to show the thermal death points of these 18 thermophiles at the five given temperatures. These follow. Table IV, following the charts, summarizes the work of the thermal death point determinations on these organisms and also indicates the change in pH of the broth suspensions due to the heating. * 4 , ' r « 23 24 26 Table IV. Tim© Required to Destroy the Spores of Thermophiles at Stated Temperatures. Time Required to Destroy and Changes in pH at • © o o CM r-l w Q« cocooocDcococoyD^QOcoooocoor)co f o c^r-i>L*-c<-i>ot-!>oi>aocococ^c^£>j> i • i i i i i i i i i i i i i i i i C0OC0cOr0(O(0t.0^(0 03OOOQDC0cnt0 ••♦••••••••• • • • • o O OO£>C'-OC'-OC-C-I>C-C0C0Q0t>Ol>£> Min. ooooomomoifloifloooiooii) oOu)ioiowu)>ot>ir)No^ioo3int> W ^ H rH rH rH rH rH rH iH rH rH rH rH rH rH rH rH • O 0 o H H a OOOQOOCOCOOOiiCOCOOOOCOQDcncO 1 1 1 t 1 1 1 1 1 1 1 1 1 1 1 1 1 1 OOcOCOcOCOcc-cD'd^OOCOOOOCOCOCOcO Min. OOOOOOOOOOOOOOOOQO lOOOOOOOOOOiOOOOiOOOO .ft... CMCMCMCMCMCMCMCMCMCMCMCMCMtOCMCMtOtO ♦ o o o H H w p. COOCOOOOOOO^COCOOOOCOCQOOcO C-OO-£>C>r>J>-L''^i>l>C000C0t>£>t>C> 1 1 1 t 1 1 i 1 1 1 1 1 1 1 1 1 1 1 OTOCOCCOCDOcOt^QCOOOOCOCOCOcO C-C'-t>t~£>I>C'-t>!>I>l>COCOCDI>-C'-t>I> Min. ^toioiocon^^inioint't-C'toinacR • o o lO o rH w Ot OOtOCOcOQDcO^OCO^COOOOOCOOCOCncO 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 oo'vDcococx)«o^oco^cocoooococooa«o !>[>C^t>l>t'OC^>>C'COCOCOt>t'C v -f' » C3 •H 2 lOOOOOOOOOOOOtOOOOOO ^-HCMtOtQiOtO'tfiOtQCMtOCM^LOcDt-tO •OoOOT pH ooQoooao o*o> cn coc^t~i'~k N -t'C^L'>t~i>L'~ooa5aDr~c>f>!> f 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 J cDcoooy5coto^QaoDooooocoooco t>£>£>£>-C^>£>C , -l>l>>OOo6o5l>l>l>C> Min. oiooooooooooo m o o o o o HH^maoc\)t>t>o>cMoo rH r-l pH rH CM rH Spores per cc. oooooooooooooooooo OOJOOOOOOOOOOOOOOOO OtOOOOOOOOOOOOOOOOO O lOOOf'OlQOOiOO^^O^OrH CO t*rHtOiO£>OtOC'-.HCM IQ 0> ^ t> O O* rHrHCMCMtOtOtOH<^iOiOlOlO'OtOOOCO i i i I i i i I i i r i t I l ! i t 1 I I I I I I I ! I 1 I I 1 I I I I 1 I I I I I I [ I | I I I I 1 I 28 Index Numbers Table V. includes the source and "Index dumber" for each organism studied. On page 29 is found a key to be used to determine what characteristics of these thermo- philes are indicated by the figures in the given index numbers. Another table (Table YI ) shows the separation of the thermophiles studied into classes according to the index numbers. . ■ 29 Index No.* BRIEF CHARACTERIZATION As each of the following characteristics is determined, indicate in proper marginal square by means of figure, as designated below: C/5 O o *S. w o ! Chromogenesis: 1, flourescent; 2. violet; 3. blue; 4. green; 5. yellow; 6, orange; 7, red; 8. brown; 9, pink; 0, none 2 a +3 CO ctf Length: 1. less than 2 diameters; 2, more than 2 diameters > Chains (1 or more cells): 1, present; 2, absent C/3 M Capsules: 1, present; 2, absent 2 w CO a> u Shape: I, round; 2, oval to cylindrical r* o Cfl Diameter: 1, less than diameter of rod; 2, greater than diameter of rod W o u 2 Surface: 1. smooth; 2, contoured; 3. rugose