5’71“" A COMPARISON OF VARIOUS MUEATION TEMPERATURES FOR THE PRIMARY ISOLATION Of COLIFORM ORGANISMS FROM WATER Thesis Io: the Dogm of M. S. MICHIGAN STATE COLLEGE Joanna R. Bonioco I949 . . cfl. I,.I.Inu1u§ . I . « HI .»,dw$ virufllkfilvhlrI nu- .n Ik JIII ,llcf3111: cal. w-MIL fir. s..v..|! ‘1 I I}; A COMPARISOH 01' VARIOUS INCUBATION TEMPERATURES FOR m PKWY ISOLATION 01' COLIFOBI ORGANISIS FROM WATER By JOANNA a. 333mm A 553813 Submitted to the School of Graduate Studiee of Michigan State College of Agriculture and Applied Science in partial fulfillment of the requirements for the degree of MASTER 01' fl mo: Department of Bacteriology and Public Health 1949 IIHESIS ACKNOWLED @MER‘I' The writer wishes to express her sincere appreciation for the guidance and assistance in conducting this investigation to Dr. I. L. Mallnann of the Department of Bacteriology and Public Health. 2.16866 TABLE OF CONTENTS IMBODUCTIONOOOO0.00.00.00.00...0.0.0.000000000000000001 EXPERIMENTIL PROCEDURE.................................§ 1. Growth Curves..............................u...) B. Water Samples..................................ll DISOUSSIONL...........................................16 CONCLUSIONS.........................................a.l9 WC...OOOOOOOOOOOOOOOOOOOOOOOO0.0.0.COO...0.0.20 INTRODUCT ION In the routine bacteriological examination of water for the pre- sence of organisms of the colifonn group. the accepted presunptive fen- mentation test involves the inoculation of lactose broth with suitable aliquots of the water semis. followed by incubation at 37° 0. The op- timum temperature for the growth of 1: 99}; (l) and other organisms of intestinal origin has been considered to be 37° 0.. and hence primary isolation is attempted at this "optimum temperature". The question arises whether the optima temperature for the growth of coliform on- gsnisms in their normal habitat is also the optimum growth and fermen- tation temperature after those organisms have existed in water for some time at temperatures normally much lower than 37° C. A review of the literature reveals no instances where presqutive fermentation tests were attempted at taperatures lower than 37° 0.. however a few investigators have tried primary isolation at temperatures as high as 16" O. For exasple. Hinhevich (2) in applying the principle of Bijkman's (3. u. 5) fermentation test for determining the “coli titre” of water. found that at temperatures of I46". ’45". MP, andlh3° maximum dilutions of sewage polluted water and suspensions of coli cultures are not always able to ferment mannitol. being inhibited more by the higher temperatures. nevertheless. Hinhevich and his workers concluded that 143° 0. is the highest temperature harmless for coli in “maximum dilu- tions“ of water samples. although higher fermentation titres were ob- tained when 37° 0. was used as the incubation temperature. Other in- vestigators. unlike Minknvich. employed pure cultures of intestinal on- ganisms isolated from various sources in testing the efficacy of ten- -1. peratures higher than 37° 0. for the primary isolation and detection of organisms of fecal origin. merwood and Olegg (6. 7) contended that the most reliable test for the detection of “Bacterium coli" was incubation in neoconlney's broth at MI" 6. Experimental results obtained with pure cultures of organisms isolated from shellfish. sewage and feces were offered in support of their argument. However. conclusions drawn from experimental results obtained with pure cultures grown at the normally optimum tauperatures are not necessarily valid for the primary isolation or detection of those same organisms after they have existed for some time in a sub-optimum temperature environment. Intestinal organism that survive for extended periods in water at temperatures that are usually much lower than 37° 0. conceivably might become so adapted to these lower taperamres that a relatively sudden return of the organisms to the temperature of their normal environment could. in many cases. seriously impair their ability to survive and mul— tiply. Therefore. this investigation was undertaken to determine the optimum tauperature for the primary isolation of colifom organisms from water. l'our specially constructed incubators. each with a capacity of one cubic foot. were used. throughout these studies. These incubators were carefully controlled to maintain the desired '10 O. 3 0.50. and were regulated to 32°. 35°. 37° and 39° 0.. respectively. The first phase of the experimental work involved growth curve de- terminations at the above four temperatures for important representatives of the colifon group. All of the organisms (13 in number) thus studied had been isolated in pure culture from various water sources. The pur- pose of these growth curve runs was to ascertain whether there were sig- nificant differences in the growth rates of the various coliform types at the temperatures under consideration. It should be emphasised that since their initial isolation from water. all sub-cultures of these test organisms had been incubated at 37° 0. The second and perhaps most important phase of the experimental procedure involved actual presumptive fermentation tests of a series of water samples obtained from a call lake near Lansing. lichigan during the months of August and September. 19,48. In order to obtain as great a variety of coliform organisms as possible. the seaples were collected in the customary manner from all parts of the labs. to more than twenty samples were taken at any one time. since that figure represented the limited capacity of the test incubators. Within 24 hours after col- lection. the presumptive tests were performed concurrently at 32°. 35°. 37°. and 390 0. upon each sample of water. In every case where the pre- sumptive fermentation test was positive. an attempt was made to isolate the organim or organisms responsible for the gas production. EWTAELPEOOINRI A. Growth Ourves The following organisms were used in the growth rate studies: lecherichia 392:; J-I cultures. lloso 2. 5. 7 and 8- Aerobacter aeroggeg - b cultures roe. 25. 26. 27 and 29. Intermediate 1 (- + - 4-) .65 cultures. los. 29. 31. 32. 33 and 35. Oultures of the above organisms were carried on plain agar slants. Uniform saline suspensions of the organismswere prepared from 18 to 211 hour single strength lactose broth cultures that had been incubated at 37° 0. The saline suspensions were so diluted that when 1 ml. of a -3- suspension was added to 99 mls. of single strength lactose broth in an Erlenmeyer flash. the number of organisms present per ml. in the flask did not exceed 1CD. The flasks were machine-shaken for five minutes to insure uniform distribution of the organises. 15 ml. aliquots from each broth culture thus prepared were pipetted into each of four sterile test tubes. After plating out 1 ml. from each tube to determine the initial counts. the tubes were distributed among the four incubators set at 32°. 35°. 37° and 39°. respectively. At the end of 2. II. 6. 8. 12 and an hours 1 ml. aliquots from each tube were plated with tryptone-glucose- extract agar. All plates were counted after ‘48 hours incubation at 37°. At the same time the 15 ml. portions of the broth cultures were pipetted into sterile tubes. 1 ml. aliquots were added to each of four Durham single strength lactose fermentation tubes which were then incu- bated with the other cultures. one at each tmsperature. Gas production in percent was read and recorded for each incubation temperature after 12. 2n and 148 hours. A total of 29 “ch determinations were made. Three of the _l_._ gall cultures were run twice. and one was run three times. The four _A_._ 2.9.22: mg; cultures were handled similarily. Of the five atemediate ;. cul- tures. four were run twice and one three times. renewing the accepted procedure for handling such data. the logs-- rithmic average counts were determined for each coliform type. the data for which are given in Tables I. II. and III. The logarithmic growth curves were plotted from those data. and are represented by Gsmphs I. II and III. -14- TABLET A Comparison of Various Incubation Temperatures for £3 11‘ Log Average Counts 2:3? __3_2_° lambs; n T 37 O W o 1.78 1.79 1.81 1.80 2 2.1a. 2.6} 2.77 2.65 ‘1; 3.73 14.30 n.63 11.71; 6 5-36 5.85 6.33 6.31. s 6.55 ms 7.72 7.32 12 8.31 8.63 8.57 8.61 "2!: 8.80 8.72 8.57 8.51 Average Gas Production Time in ° Hours 32° 35° 37° 39° 12 us . 1M 8s 1% 211 an 33 31 32 us 32 36 3h 3” “I cultures (HOBO 2s 59 7 and 8) .. 9 MB warn A Comparison of Various Incubation Temperatures for A; aeroggnes’ Log Average Counts 9:11:33 32° WWW"— o 1.77 1.7!. 1.75 1.76 2 2.33 2.53 2.63 2.9!. h 3.98 11.35 Inns M534 6 5.36 6.35 6.31 6.32 ' 8 6.93 7.67 7.6!; 7.62 12 8.117 8.51 8.13 8.31 2h 8.89 8.71 7.152 7.20 Average Gas Production Iran... in 0 Hours 32° 3 37° 39° 12 6f 8% 10$ 7‘ 2h 38 no 32 26 ’48 51 51 fl 29 '- u cultures (1m. 25. 26. 27 and 29) .- 9 runs. -6. TABLE III A. Comparison of Various Incubation Temperatures for Intermediate I t .103. Average Counts Time in Incubation Temperature °c. near: 32° 35° 37' 395 0 1.70 1.71 1.70 1.69 2 2.12 2.21 2.23 2.15 n 3.51 3.52 3.15 3.02 6 ‘u.71 n.92 u.u1 u.o7 8 6.07 5.1.3 5.5!. 5.33 12 8.02 8.17 6.61 6.55 2h 8.71 8.61 7.17 6.24 Average Gas l’rodnction Time in 7 'IncubatioLTe m° 0. Hours ‘ 35° 37° 39°” 12 0i 0% 0% 05$" 2!. 5 11 5 3 us 23 27 11 7 *5 enema (r... 29. 31. 32. 33 and 35) - 11 m... GRAPHI. Growth Curves for E. coli at Various Incubation Temperatures 10- Logo 5 A - 57° and 59°C a - 55°C 0 - 32°c Time in Hours ~80 GRAPH II. Growth Curves for A. aerOgenes at Various Incubation Temperatures 10}- Log 0 A - 55°. 57°. and 5900 B - 3200 L l l l J O 2 4 6 8 12 Time in Hours -9- GRAPH III. Growth Curves for Intermediate I. at Various Incubation Temperatures 10 - 9 .. A B C D LogC 1 L ‘ c 4 l— l L 0 2 4 6 8 12 Time in Hours 4:0- 3. Water Samples Of the l58water samples which produced gas in lactose broth within 118 hours. 1147 of them were successfully confirmed by isolation of the coliforms responsible for the fermentation. A modified. 1: - tube presumptive fermentation test wee performed on each water sainple at each of the four incubation temperatures. In every case. 10 mls. were added to each of two double strength lactose fermentation tubes. and 1.0 ml. and 0.1 ml. were added. respectively. to two single strength lactose fermentation tubes. All tubes were ex- emined for gas production. which was recorded in percent for each pos- itive tube. at the end of 12. 2h. and 1&8 hours. Isolation of the colifoms was attempted by streaking Levine cosine methylene blue agar plates from those tubes showing the maximum gas pro- duction after 1&8 hours for each sample. After 2h to ’48 hours incubation at 37° 0.. morphologically distinct. suspect colonies were piched fium each 1.11.3. plate and transferred to plain agar slants. The lactose fermenting ability of each culture thus isolated was verified before carrying out the Imvic reactions. As recommended by Standard Methods. Kevacs' (8) modification of the indol test. Barrit's (9) modification of the Voges-Proslmuer reaction. and Koser's (10) procedure for the citrate test were employed. Ihenever the Imvic reactions tallied a. + + + or + + '0' + the purity of the cultures responsible for them was checked. Al- together only three types of intermediates were isolated. i.e.. those giving -'+ - +. a 45 + +. and + + + + Imvic reactions. respectively. .11.- can .eew madness AsapummoMraaawnea on» ma my eons» no sense: on» concedes“ access assay one .aneouea ma newcomuenn new Bananas on» ouceuehaea nepssu.cn000m any 91... RA 0 mun m4 m4 8.... are me 84.. mum m4 0 o 0 RA 0 end 93 o 91 o o om; 97m n... 84 SA 0 07m name. 094 o mun Sum 07H 0 o oeua om-H o om-a mum o o omna 91m 84 o omum omnm omum 0 8A w: am ma we em 095mm, macaw. ; .0 can .0 cpm -. .c o saspsaopaoa coauepsonH measpmaepess cowampnocm mdoaam> on» we copeaoen sneaks: .mepsa mowpnecmomm ebuuamom mo panama measonm >Hn: (W 08 .mesoaaom you same .enmvememmea noes as summon .Hem. sens» : no alumnae ‘ Rm mmH was Sm 2. m: Rn one am unn m: an SH .38 a. n N a i. n a. m m m n H m 685...: e .monoe .38 m n o w o o m m H a. m o .n .eofloosH e .mfloo. m m H w o o a m H n m H m 6.8.35 s 38 mm on m mm mH n on 3 m um AH m 3 .eeHeoflooS on on 3 mm . Hm HH as an S R R m no eoaomaos e :3 an mm on H: 2. H HmH SH R 3: 2. mm mm 3388 em 5 mH am «a l. 8 mm 3 mm m: a an HH.. 3 tum: «H 3 .uwem NH 3 .mwem NH 2 one NH Juan“. eooeHSH 2.1.838 .e an 6 .5 .o em” .0 ensues canon. sounded—emu cede-cu mower and so» use nausea $33. no aspen: Hence one no «suspense-ca scavenge” no post" ea > manna -13.. RH 8H m: an mm on SH nn H 9. EH mHH ton SH 38 n m H m m m m H H n m H n 6.!33 e .688 .39.. m m o H o o m m H n m o n 6.!33 e .monos m m H H o o m H H m H H N 625th e :8 nH. MH m 3 HH n S :H m nH «H m S 3362:35 um ma. m we . 5 2 mm om m mm am m mm . nonsmoaee. a «use mm Hm mm mm .3 mH mm mm mm mm E 9 mm nosemonoo mm mm HH an R m mm mm NH an R m mm :8 ea .eumohf NH, 3 :nwom NH 3 .MMoFNH 3 .mwmm NH .stuum eoeeHSH . £833. 6 omm .o ok 6 eon .o own suspense-ea 3333mm .saeea cage-«mesh 05303 new»; season no .HepmHfi one no manganeaaea scavenge” no «new»! can ab 53 -114- II: |.lll|‘u mu VII Average Maximum"I Gas fmduotion for All Positive Seaplee at the Various Incubation Temperatures Tine 32° 0. 35° 0° 37° C. 39° G- 2“ hours 533$ 236$ 294$ 286% he hours hofifi 56.7% 39.1% h7-9fi *In calculating the average maximum gas percentages. a bubble of gas was arbiH trarily considered as 2%. -15.. A complete tabulation of the data for a number of the water searples thus tested is given in Table IV. A compilation of all the pertinent data obtained is given in Tables V. VI and VII. DIS'JUSSION from a study of the logarithmic growth curves obtained for both 1:. 391.1 and £1 aerogeiies. it is evident that their Optimum growth temper- ature must lie in the 37° to 39° 0. range. since the growth curves plotted for either organism at those two temperatures are practically coincident. However. there appears to be no marked decrease in the growth rates of _l__._ 22;; and A; aerogenes when they are incubated at 35° 0. Also. it is apparent that the optima growth temperature for Qty: mediate I. is quite definitely close to 35° 0.. even though that organism is only slightly inhibited by a temperature of 32° 0. On the other hand incubation at 37° or 39° 0. appreciably decreases its rate of growth. as is obvious from the much lower bacterial pepulations at the stationary phases for those two incubation temperatures. The data tabulated in Tables V and VI are most illuminating. Since the total number of tubes showing gas for all samples proved to contain coliforms is appreciably greater at both 32° and 35° 0.. than at 37° or 39° 0., it is evident that the optimum or critical temperature for the primary isolation of coliform organisms from water is less than the tem- perature of their normal environment. Further, it appears that the best incubation temperature for the primary isolation of coliforms from water is closest to 35° (3.. since the greatest number of tubes showed gas after 24 hours incubation at that temperature. However. longer incuba- tion at 32° 0. produced equally good results. These observations are further substantiated by a consideration of the number of confirmed nos- -16- itives obtained for the various incubation temperatures. which data are given in Table 71. Although 32° and 35° 0. produced the same number of confirmed positive presumptive tests after 1L8 hours. again 35° 0. seems to be the best. since the most positives after 21; hours incubation are recorded for that temperature. A further study of the data with regard to a comparison of the type or types of coliforms isolated with the results obtained at the four incubation temperatures is also very interesting. The data clearly testify to the undesirability of subjecting both 1; _c_o_l;_i_ and g; aerogges to a relatively sudden return to the temperature of their normal envir- onment. after these organisms have soJourned for some time in water at temperatures far below the normal optimum. There is a striking corre- lation in each case between the total number of tubes showing gas. the total number of positive presumptives. and the incubation temperature: which heavily favors the two lower temperatures. especially 35° C. One can only conclude that the normal optimum temperature; often has 9. def.- initely harmful influence upon the ability of certain coliforms to sun-- vive and multiply. when it is applied for their primary isolation from water. The data obtained for these water samples from which only inten- mediates were isolated are also suggestive of this viewpoint. although not so conclusively. since relatively few such samples were encountered in the series tested. . An examination of the percentage gas production data (Tables I. II and III) accumlated during the pure culture studies reveals some ten- dency (least remarkable for _l;_._ £934) for greater gas production at the lower temperatures. especially 35° 0.. in the case of each coliform -17.. type. This is true even though that temperature can be correlated with the growth curve optimum temperature only in the case of Intermediate 1: Although a study of the average maximum percent gas production data at the four temperatures. given in Table VII. for all 1‘47 positive water sainples is equally inconclusive. there again is some suggestion that 35° 0. may be the optimum fermentation temperature. The results of the pure culture studies in no way hint as to the probable outcome of the more practical water sample tests which followed. mm. the importance of using ”water-adapted" organisms. and hence actual water samples. rather than pure cultures for a study of this nature is 6715.“ t 0 ~18- CONCLUS IONS l. The normal optimum growth temperature for Escherichia 21;; and Aerobacte; aegggenes is in the 37° to 39° 0. range. 2. The normal optimum growth temperature for Intermediate h is approximately 35° Co 3. The most favorable incubation temperature for the primary iso- lation of coliforms from water is closest to 35° 0. h. Incubation at 32° 0. for primary isolation is also superior to 37° 0.. although a longer incubation period is necessary at that tem- perature than at 35° 0. 5. Data are presented which suggest that 35° 0. may be the opti- mum fermentation temperature for coliform organisms. It is recommended that 35° 0. be accepted as the incubation temper- ature for presumptive tests for colifoms in water. inasmuch as that tem- perature is now officially recomended (11) for determining plate counts in milk. -19. 1. 2. 8. 9. 10. ll. @RENOES Barber. ii. A. Rate of Multiplication of .31.: coli at Different Tem- peratures. J0 Inf. D180 '53 379. (19035 Iinkevich. J. 1.. et al. The Application of the Principle of Iikaan's Fermentation Test for Determining the 0011 Titre of later. Jo Hygo £250 (195) 113m. 0. m. Gamingsprobe bei h6° a1. Hilfsmittel bei der Trink- Yagsle‘runtersuchung. Oentr. Bath, Abth. I. Orig. 51: 71k? 1 ) Thomann. J. Zum Nachweis des Bacterium coli comune in lesser vermittels der Eijkmannschen Methods. Hyg. Bundschau 3.1: 857 (1907) In... I. fiber die Mrtellmg des Colibaltterienbefundes in Trink- wasser nebst Bemerlmngen fiber den lachweis und das Vorbmmen der celibasillen. z. Hyg. Q5: '25). (1910) Olegg. L. r. and Sherwood. E. P. Incubation at Rh" 0. as a Test for rascal Ooli. J. Hyg. 32: 361. (1939) Sherwood. 13. P. and Glegg. In I. l‘urther Studies of Incubation at c. d. a Test for rascal Goli. J. Eye 33: “5 (19h?!)- Kovacs. I. line vereinfachte lethods sum lachweis der Indol-bildung durch Batterien. . Z. 1mm unmfsforsch . £33” (IQ-:5) Barrit. M. I. The Intensification of the Vogee-Prosbuer Reaction 139%.). Addition of alpha-Naphtholo J’- Path. Bact- _1_¢_2_: mu. 1 . loser. S. A. Utilisation of the Salts of Organic Acids by the Oolon-aerogenes Group. J. Sect. g: 1493. (1923) Standard Methods for the Examination of Dairy Products. American public Health Association” 9th Ed. (19%). {a an; an ha a E a; , 3. '1 92;" .34 i ’5’! ' Q. Lilli SIPTE UNIV ERSIT: II I III IIIIIIII II 479 3196 IFIB HNF E'S