:5 I :, mm [[HEb‘5 A NEW MEDIUM FOR THE DETECTION OF EKTERIC ORGANISPS IUDICATIVE OF OF SEWAGE POLLUTION IN WATER by moan D . M cur-DY, JR. AN ABSTRACT Submitted in partial fulfillment of the requirements for the degree of LESTER OF SCIENCE In Michigan State University Department of Microbiology and Public Health 1955 Approval) thjlflfikm (NNV \\ A new medium was deveIOped, the formula of which was as follows: Tryptose 2.0% Dextrose 0.5% Potassium dihydrogen phosphate 0.275% Potassium hydrogen phosphate 0.275% Sodium lauryl sulfate 0.01% Bile salts (Difco) 1.0% Sodium chloride 0.5% Promcresol purple 0.0015% When tested with laboratory strains of bacteria it was found that this medium permitted maximum growth and detection of typical coliform bacteria and other enteric bacteria of sanitary significance in water while inhibiting non enteric forms. The specificity of this medium was also demonstrated in comparisons with Standard Method's procedure. It had a higher percentage confirmation for the presence of typical coliforms than lactose broth. Enteric bacteria were found to be responsible for almost all of the positive tubes obtained. Acid production from dextrose was found to be an adequate indication of the presence of coliform bacteria when used in a selective medium. The use of an indicator (Brom cresol purple) permits the omission of gas vials, thus making the new enteric medium easily adaptable to field work. -.-~.'im‘-nnm ' ' l Inf. A :7: .IJDITIII Ede. T773 “and” 0F :fr‘ilc 0E>.G-~.TiIS 2“ 13320111le ~—. . * ,~ -\ "H 11"”1-‘7 ‘7 v7 "7‘7“ Or sang: : wing-7m I: i h. by Howard Douglas KcCurdy, Jr. ”w" mIYT—‘(llfl I —v ‘I —. a. J....LJ>J c.) Submitted in partial fulfillment of the requirements for the derree of J. “J T ’,A_Or‘1~w~r‘, O"- QC ‘s" ’\""| A 4 .2-) A A". .I;.' -. I "1.”!de In Kichijan State University Departhent of Iicrobiology and Public Health 1955 THEbm .".CKI-I"IILITOCI SITES The author wishes to express his thanks and appreciation to Dr. W. L. Hallmann, Professor of Microbiology and Public Health for his able guidance and advice. He wishes to thank Hr. 0. E. McGuire of the Michigan Department of Health for his assistance in making the field studies. The author is also indebted to Mr. I. L. Dahljelm for his assis- tance and to the personnel of the stockroom of the Department of Piorobiology and Public Health who never hesitated in giving their cooperation. Page I I . IIEIYRCJDUCTIOIE. I O O O O O O O O O O 0 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 0 O C C II . EEj-IIFLIIE:JT-ALL T"! CE—iK O O O O O O O O O O O O O O O 0 O O O O O O O O O Q I O O O O O O O O O O O O O O 0 10 III .COI‘:CLUSIOI\D O O O O O O O O O O O O O O O O O O O O C O O O O O O O O O O O I O O O O O O O O O O O O O O O 27 Iv. LIST OF REED--31;CFJSOOO.0.OO00......0.0.0000...OOOOOOOOOOOOOO 29 LIST OF TABLE. TABLE PAGE I. The growth of E. coli on media containing various COIilbinationS of the SGleCtiVe a.geIII»S........o............o... 1-6 II. The growth of coliforms planted directly from polluted sowces on tlle variOIIS meC-iiaO0.0..00....OOOOOOOOOOOOOOOOOOOOO 17 III. The growth of E. coli on a basal medium containing sodium lauryl sulfate and various concentrations of bile salts.................................................. 18 IV. The growth of various enterics on an enteric medium......... 19 v. The selectivity of bile-sodium lauryl sulfate............... 20 VI. Comparison of the enteric medium with lactose broth (A)..... 24 VII. Comparison of the enteric medium with lactose broth (B)..... 25 INTPODUCTIOI Escherichi (1) first isolated Pactorium coli commune from the feces of a cholera patient. Subsequently, this and related organisms, collect- ively known as the coliform group, were found to be natural inhabitants of the intestinal tract of both man and animals. The significance of this was quickly realized and these organisms became a valuable yardstick of sewage pollution in water. The litmus lactose afar plate introduced by Sedgwick and Hathews in 1893 (2) was the first device developed for their detection and enumeration. On this medium coliform organisms formed red colonies because of their ability to ferment lactose. However, since no inhibi- tory agent was used, the plates were frequently overgrown by other forms,' some of which also fermented lactose; thus colonies had to be picked for further examination. Smith (2), in 1893, showed the value of a preliminary enrichment medium and introduced the use of the glucose fermentation tube. This method was quickly accepted. It was simple and permitted the examination of larger quantities of water. In addition, it could be made quantita- tive by planting a series of tubes with measured quantities of the sample. However, it was found that many organisms, not belonging to the coliform group, could produce gas from glucose. is a result, the more specific lactose broth was substituted. Al- though many attempts were made to include inhibitory agents in that medium to prevent growth of non-coliform types, none was successful and lactose broth is still in use in essentially the some form. The standard procedure of the American Public Health Association (3) is substantially the same now as it was in 1920. It consists in planting a series of standard lactose broth fermentation tubes with equal quantities _ 2 - of water to be tested. The tubes are then incubated at 3500 and exam- ined for the production of gas withi ’8 hours. Tubes showing gas at the end of that time are confirmed for the presence of coliforrns by transferring to a inhibitory diagnostic medium such as eosin methy- lene blue agar or brilliant green bile lactose broth. In some cases, this is not sufficient and the "completed test" is necessary. This involves transferring the organisms to an near slant and a lactose fer- mentation tube. If microscopic examination of the organisms from the slant reveals the pres nee of non—sporinq gram negative bacilli, and there is rras fermentation in lactose broth, then it is a positive, completed test. The method has its limitntions. It is costly and requires much time and materials. Four days are usually required before the results can be co o9i‘ered sisnificant sometim ,3 longer. This difficulty has been recognized for many years as evidenced by the many attempts 1 5:) that have been 1% de to evis e a one tube procedure. However, none of the host presumptive and enrichment media, which have been proposed to replace lactose broth, has proved satisfactory as a presumptive medium w thout co1£ mr mtio on. Only one, lauryl tryptose broth (4), has been included in Standard Kethods as an alternative to lactose broth. Standard Methods is almos t holly unadaptable to use in the field. 1 en if a one tune presumptive medium could be found in which a positive test is based on gas production the inclusion of fermentation vials would cancel much of its practicability in that area. Another limitation which has been given increasing attention in recent years is the failure of Standard hethods to detect many atgpical 1 coliform organisms. These include many slow and even non lactose fermentinn -3- bacteria some of which do not produce gas from any sugar. This group is a heterogeneous one that defies classification and is usually referred to as the paracolon group or as aberrant coliforms. Gilbert and Lion (5) appear to have been the first to describe para— colon bacilli. Ledingham encountered such types in human feces and reported them as numerous in cases of diarrhea but regarded them as of little pathogenic importance. On the other hand, Gyorgy suggested that they played a considerable part in diarrlea in both man and animals. Stuart, Nickle and Eorman (e) prOposed the separation of atypical coliforms into four groups based on a study of more than 10,000 strains isolated from water, soil, milk and other sources. They prooosed the term "aberrant coliforms" for all gram negative non-spore-forminq rods which ferment lactose slowly or weakly at 3700 and these were subdivided as follows: I Microaeroqenic Coliforms - aberrant coliforms producing gas from lac— tose slowly or in small amounts at 3700 or 2000. II Pseudomicroaerogenic Coliforms aberrant coliforms having the charac- teristics of the true microrerogenic strains at 37°C but showing normal lactose splitting activity at 2000. III Papillae—formina coliforms: aberrant coliforms showing the type of dissociation evidenced by Escherichia coli mutabile but not restricted to the genus Escherichia. IV Anaerogenic coliforus aberrant coliforms producing acid but no gas from lactose with or without gas on other sugar. r“ inese authors also recognize the existence of non—lactose fernehting coliforms. In a later paper Stuart, wheeler and Zimmerman (7) presented a study _ A _ of the biochemical and antigenic relationships of paracolon bacteria. They sugfested a sinrle, coliform genus which would include three species: freunlii, aeroeenes and coli. Within this Croup would be included all aberrant coliforms and paracolons. Non-gas producing cultures fermenting lactose rapidly, slowly or not at all would be prouped as anaeroaenic aeroyenes, freundii or coli or collectively as anaerogenic paracolons. Stuart, Nickle and EOrman (6) stated that the microaerogenic coliform probably have a significance in water analysis similar to that of Aerobactor and Escherichia strains showing typical lactose fermentation. They noted that the papillae forming coliforms are only infrequently detected in water analysis in which gas production is used as a criterion' for their presence and the anaerogenic coliforns would not be detected at all. However, they thought that these two eroups were highly important because of their frequent association with gastroenteritis and genitour— inary infections. Reports of isolation of these organisms from such diseases are many. Plass (8) investigated an outbreak of diarrhea which had its source in fricasseed chicken. He was able to isolate paracolon bacteria both from the chicken container and the largest prOportion of the cases. Farnes and Cherry (9) reported paracolon bacteria as the cause of an epidemic of diarrhea in a United States IrVal Hospital. In an area highly endemic for enteric infections, Christensen (10) studied the comparative distribution and possible pathogenicity of £233" colobactrum species. These Organisms were isolated from 60 percent of the gastroenteritis cases as compared to 20 percent of the healthy indi- viduals examined. These findings were considered to be, at least, indi- cative of pathogenicity. Parecolobactrum species were found much more - 5 _ frequently in cases of gastroenteritis than either Salnonella or Shisella types. Ziegler (ll) studied the bacteriology of an epidemic of diarrnea. he isolated late lactose fermenting and non-lactose fermentins organisms from polluted water su plies and from diarrhea patients. The agfilutination of at least two strains of these occurred in high dilutions of patients' serum, indicatinn that they were the probable cause of the infections. During the later part of the epidemic no typical Escherichia coli was isolated but these disease organisms were. Kriebel (12) isolated many nonlactose fermenters which she was able typical EIB colonies by rapid transfer. She also obtained non-lactose fermenters from typical :. ggli, thus establishing a relation- ship. Such late lactose fermenters or ion-lactose fermenters must there- 1) fore be regarded as atypical coliforms as sue ested by Kriebel and cer- tainly are entitled to equal consideration with typical forms in deter- mininq the potability of water. host of the information which has been obtained concerning aberrant coliforns in water has been obtained by the use of procedures supple;enting those used in routine analysis, and frequently, these are quite time con- suming and tedious. In certain cases many of these organisms can be detected by prolonged incubation of the standard methods media but such a procedure aggravates the inherent faults of Strndard Kethods, and, even then, not all coliforms are detected. It would seem, from these considerations, that some new, more practicable procedure is needed which will permit the detection of all coliforms cuickly and dependably whether typical or otherwise. Such a A' procedure should, as much as possible, correct the diificulties inherent in old methods. The purpose of this thesis 1s to present the results of studies which were made on a new er teric medium which, it is hOped, will prove a valuable tool in water analysis. Its formulation is bored on the need for a practi- cable single step procedure for bacteriological analysis of water and for a sensitive and specific medium for all coliform oreanisms. In formulating any bacterial diagnostic medium the first requirement is that it must stimulate the earliest possible development of all dormant and viable cells. After growth and development have begun, the bacteria should reproduce in such numbers that their presence will be re dily recognized by Mh ir phm iolocicr 1 action on the medium. Nallmann and Darby (4) presented a new medium, lauryl tryptose broth, for the isolation of coliform organisms from water supplies. They reported that the use of a medium containing tryptose, sodium chloride and phosphate buffers grew out more coliforns present in water as indicated by the higher colon indices obtain ed bv this medi 1. They also found that the addition of sodium lauryl sulfate in l:l0,000 dilution inhibited the growth of pram pos1Ht1ve organisms while permitting the mm.) mum development of coli1orm bacteria. The formula of lauryl tryptose broth is as follows: Trvptos 2.03 Lactom 0.5% Diootassium phosp m1 te 0.27dfi lknopotassium phosphate 0.275% Sodium chloride 0.51 Sodium lauryl sulf ate 0. 01:“: \ \ McCrady (13), Levine (14) and Perry and Hajna (15) all reported the superiority of this medium over lactos se broth 88 a presumptive enrichment medium. Fewer 1alse presumptives were obtained and more slow lactose fermenters were detected than with the older medium. -7- Lauryl tryptose broth, thouph permitting the rapid develOpment of coliform types, is limited in its use for the detection of those organisms that give acid and visible gas from lactose in 48 hrs. The most constant characteristic of enteric organisms indicative of pollution is their ability to ferment dextrose with the formation of acid or acid and ass. If dextrose rather than lactose was used and acid rather than gas production was used as the criterion of fermentation, more enteric organisms would be deticted, since bacteria attacking lactose slowly or not at all will attack dextrose with comparative ease. Such a procedure would have the additional advantage of not requiring inserts in the fermentation tubes. For this reason, in devising a tentative formula for the enteric :1) medium, dextrose is used 3 the fermentable sugar in a medium essentially the same as lauryl tryptose broth. In addition, brom cresol purple is used as an indicator of acid production. Because of the wider spectrum of bacteria capable of utilizing dex- trose as compared to lactose, an additional selective agent was thounht necessary to surplement the action of the sodium lauryl sulfate. is a result of their habitat in the intestinal tract, enteric bacteria are capable of withstanding relatively hich concentrationflcf bile. For this reason, in seeking an event that will curb the growth of non enteric gram negative bacteria, the use of bile salts was considered. The use of bile as an inhibitory agent in media for water analysis has had a long and, heretofore, somewhat dubious history. N) Jackson (16) first proposed the use 0 ten percent bile as a presump- tive medium with lactose. He found that it inhibited the growth of non coliform, lactose fermenting stra1ns. The original medium consisted of undiluted ox bile to which was added one percent lactose and one percent peptone. Lactose bile broth was enthusiastically received and in 1912 it was adopted by the Committee on Standard Kethods of Water Anal sis The new medium supposedly did not permit the growth of weakened or attenuated forms. The 1912 edition of Standard Methods considered this to be an advantage for it stated that, "attenuated Faeterium coli does not represent recent contamination and all Factsriuu coli not attenuated grow readily on lactose bile.” Jordan (17), however, found that the bile inhibited from one third to fifty percent of the coliforms. He also concluded that attenuation did not have any befring on the results obtained, so that it could not be said that bile eliminates attenuated or weakened forms. Obst (18) and Cumming (19) obtained similar results in comparisons of lactose bouillon and lactose bile. Because of these and other similar results the 1917 Standard Iethods (36) adopted lactose broth for preliminary enrichment and the presumptive test. Somewhat different results were obtained by some workers. Hale (20) found that by the use of five percent bile in lactose broth, gas production was obtained much more quickly and fewer clostridia were able to develop. Levine (22) cc°ried out investigations using Difco evaporated bile and sodium taurochalate in tests with E. coli and g. aerorenes. In a basal medium containing one percent peptone and various concentrations of bile and sodium taurocholate he found growth of these bacteria was actually accelerated by concentrations of from one to two percent. In ado he found that few anaerobic soore formers develOped and sporing lactose fermenters growing aerogical"y did not grow on bile. In 1920 Muer and Harris (93) proposed the use of a brilliant green - 9 _ lactose bile medium for water analysis. They found that the bile empedred to inhibit completely the aerobic and the brilliant green bile to inhibit almost completely the anaerobic soore formers. Although the new medium was found to inhibit coliforms to some degree results okt intd in other labor- atories were cons ioered fever able. Dunham and Schoenlein (2) made a careful study to determine tlie optimum bile: brilliant green ratio and recommer nded the forg' ula of brilliant en bile as it is used now. Jordan (2A) Butterfield (fl) and Parr and Caldwell (25) found that brillient green bile broth inhibited from ten percent to one third of the coliform orgsnisrls end was, therefore, not 5 tisfactory, as a presumptive or enrichment mediimh Little more was done with media containing bile for water analy81s until Perry and Heine 26 developed their 5. C. medium which is essen- idlly the same as lauryl tryptose broth excebt that they used bile instead of sodium lauryl sulfate. They found that their medium was much superior to standard lactose broth. It inhibited almost completely fecel strepto- cocci end other Pram positive organisms with no nnperent inhibition of oliform bacteria. Levine used a more controlled medium, then employed by eerlier authors, to which peptone had been edfed and obtained good results with lower bile concentrations. Perry and Hejna (26) used a bcse medium which had been proved to be more conducive to bacterial groxth and obt m1 very fr -vor°ble esults. It is our thesis that much of thee ifL' icultv encountered throuéh the use of bile was due to the use of base media which were not of themselves as favorable as possible for the develoa ent of bacteria. -10- Early investigators used crude undiluted fresh ox bile. Hence, the con- centration of bile wes extremely high (about 10%) and variable and the nutritive value of the medium ouestionable. - 11 - EI‘TPTTTI‘. BITTZL TREK The experimental work of this thesis was divided in two parts. First, an attempt was made to determine the eliect of adding bile salts to a modified lauryl tryptose broth on the growth of enteric organisms and to determine its selective preperties in such a medium. Secondly, the results obtained with the new medium were compared to those obtained usinr Standard hethods in testing routine water samples. Procedure Piaf-b A. o 9-1 — The rate of growth of escherichia coli was studied on a base medium to which various combinations of bile salts and sodium la ryl sulfate had. been ndied. The base medium had the following formula: Tryptose 2.0% Dextrose 0.5% Monopotassium phosphate .275fi Dipotassium phosphate .2753 Sodium chloride 0.5% Particular attention was given to the lag and early logarithmic phases of growth when minimal inocula were used. Huntington and Winslow (27) showed that during these stages the young cells exhibit all the yo'JfllI'ri . . characteristics of phy51ologiCsl grewth comparable to those exhibited by multicellular organisms. It is, therefore, considered more important to net to new environment to the extent that D CL C...) 0 know whether an organism can figion will occur, and after fission that the two physiologically young cells will survive, than to know the total number at the end of the c m- plete growth cycle. In the first set of experiments a pure culture of E. coli was used. Before seeding in the Various media it was transferred from nutrient aear slant every twenty—four hours for three days to insure uniformity. -12- In order that minimal numbers would To used in the inocula, the organisms were transferred from a slant to a tube of sterile saline (28). They were added until the first turbidity visible to the naked eye appe red. At that density the number of organisms in suspension approximated 50,000, 000 per millilitfir (26). This suspension was diluted in sterile saline until such concentration was obtained that the test medium would contain one organism or less per milliliter when one milliliter was used as the quantity for inoculation. Two hundred and fifty milliliter portions of the medium were used and incubated at 3500. Growth of the pure culture of E. 22;; was studied on the following media. I. The base medium II. The has medium to which was added 0.01% sodium lauryl sulfate. III. The base medium plus lfl bile salts (Difco) IV. The base medium plus 0.01” sodium lauryl sulfate and lfi bile salts. The numbers of organisms at (l. 2, 4, 6 and 2a hours were determined by plating portions of the test medium. Tryptone glucose extract agar was used as the plating medium and the plates were incubated at 350C for 48 hours before counting. In order to supple.ent the findings obtained in the first experiment, a somewhat different procedure was tried. Uhile minimal numbers were used in the firs: set of determinations to ascertain the effect of the two in- hibitory agents, it may be argued that this does not indicate that the more weakened or attenuated forms, found in water will react in the same way. Hallmann and Darby (A), in their original studies on laury tryptose broth, used organisms which had been subjected to refrigeration in ordgr to simulate the condition of attenuated forms. Such a process cannot duplicate -13.. the many factors which affect the organisms in water. It is our idea that in order to ohtsin information concerning the growth of such bacteria in a medium which is to be used for their detection, no more practical way of attenntina to solve this problem can be found, than to study the behavior of these organisms under conditions in which they will actually be encountered. For this ree son, a water sample was ohtqined from a source known to be heavily polluted. The sample was then diluted to such an extent that the final concentration of the coliforns in the sa rle wouli be 250 per nilliliter. This sprocedure was based, by a ppro: :ination, on the estimated coliform dens sit, of the polluted water. One milliliter of the susnenslon was then inoculated into 250 milliliter Quantities of the ane media used in the first set of experiments. Growth was then determined by making plate counts using tryptone "lucose extract agar at 0 and 6 hours. In 0 der ’\ to check the identity 0: the cultures obtained several colOLies were icked . ’ from each plate and inoculated in tunes of lauryl tryntose broth. In the third set of experixents the effect of ilterinq the concentration of bile was determined on a base medium to which sodium lauryl sulfate had been added. The procedure was the sane as in the first set of experiments except that growth was determined only at 0 and 6 hours. Concentrations of 0.57, 1.0%, 2.05, and 5.01 bile were used. Since there are many enteric organis s which have sanitary sienificance it was considered desirable to obtain some information concerning the behav- ior of other enteric organisms in the new mediit. There being so much in— terrelationship among menbers of the Enterohacteriaceae and so much diversi- fication in the coliforn group, it was thOU‘ht that a study of the behavior .L.‘ of nany related species misht indicate the reaction of many of the coliforns that may be encountered. For this purpose sinvle growth r t, xptrrminations were made using stock cultures of Ferocolob>ctrum arizono, Salmonella tynhifiurium, gero- boctor kerosene", Chisella sonnei, Salmonelb.tynhosd, Salmonella enteri- tfdis, folfionollo r--ra+*"ohi and Falmorolla schotlruellcri. Linimal numbers were ursr in the inocula again and plate counts were made at 0, end 6 hours. Three T'edia were tested fihd their composition was as follows: I The base medium containirg .2751 each of diootassium and monOpo— tassium phosphate, .51 glucose and .5? sodium chloride. II. The brse medium pl‘s .013 sodium lauryl sulfate. Ill The base medium plus .011 sodium lauryl sulfate and l5 bile salts. Having determined the effect of the vwrious afents in tne enteric medium on the Crowth of enteric organisms, of its selective ability. For this purpose a number of known stock cultures of dram negative and Pram positive oraonisms were used as well as several unknown eram negative organisms not belonrins to the coliform group which had been isolated from water examined in this loboratory. All cultures were transferred every 74 hours for three days before use. The inhibitive properties of Various percentoses of bile in the enteric medium with sodium leuryl sulfate 0.01% concentrstion was compared to a non inhibitory bese medium containinfi tryptosc. Ten milliliter amounts of the Various media were seeded with standard loopfuls of broth culture and Crowth recorded on the basis of turbidity. R sults In the firs series of exncrinents, it which growth on the bone median containiie tryptose, sodium chloride, slucose and phosphate buffers was compared to that in the Various combinations of the hose medium and the selective events, favorable results WFTG obtained. The results 0: tnese runs are presented in Table I. A critical examination of the data reveals — l \J‘x very little difference between the various media at the end of 51x hours. In most Ceses the counts en the 21—hour interval gave the same picture but the 6-hour counts were cone 1 dered more sirnificant. The se eries of experiuents in which a diluted water semrle was used as an inoculum gnve results hich are considered evcellent. All the colonies which were picked produced yes when seededi to le1r3rl tryntose broth, an indicetion that x st, if not all, of the colonies were those of coliform organisms. In terms of growth, these runs gave a picture somewhat the same as the first experiment. The data ejtained are presented in Table II. Consistently hi ner counts w*re obtained in media III and IV which might :.nd.iCDte a slifb t stimulatory effect on the part of the lauryl sulfate either alone or in co binet ion with the bile salts. In the next series of experiments (Table III) the effect of various concentrations of bile salts in a basal medium of sodium lauryl sulfate glucose broth was noted. The bile salt exerted a very Slijht, if any, U) .‘ inhibitive action in concentration up to Qfl. However, the counts obtained from the of concentr tion was consistently lower in all cases and ir one I case nuite sienificently so, ize dwic.ting that th as bile die inhiiit h. coli in that concentration. The results of the fourth group of exoerinents added further support to the results already obtained, showing that the use of bile salts n the medium had no effect on the growth of any of Ho sodium laur3 1 sm 1fete the ente ric organisms stunied. The selective preperties of the medium was studied next u31n§ pure laboratory cultures of Various bacteria. Table V gives the results of this study. Most of the non enteric gre m neg.:ti_ve org: nisms were completely inhibited by all concentr tions of bile salts with sodium laurvl sulfa -15- Table I o Growth of E. coli on Hedia Containing'Various Combinations of the Selective Agents. Trial Time (hours) I II III IV A O 1* O O O 2 l O l l A 15 10 l l 6 196 254 111 2A2 24 517M' 63 W 483I 5?SH B O O O O O 2 2 2 O O 4 10 10 170 10 6 260 29 254 261 24 447K 798M 474M 506M C O l l l O 2 l O l l 4 ll 0 100 O 6 314 212 240 211 24 563M 425M 4335M 302M D O O l O O 2 2 O O O 4 20 o 20 A0 6 580 637 4/4 619 24 620% 1120M BOAR 960K a. d *ESSHE = Million = Ease medium = Ease medium and sodium lnuryl sulfate I = Ease medium 9nd bile salts 2 Ease medium with sodium leuryl sulfate and bile salts Jumber of beateria per milliliter. -17.. Table II. Growth of Coliforms Planted Directly fr m Polluted Sources on Various Xedia. I* II III IV ' '3 l—lo “I D Trial O 2 " 115 150 179 158 a A 3‘0 L7 '1 U] o H \3 0‘ O H X) t" 1.. |-—’ O“ J l-‘ \ \1 C 0 hrs. 0 O O l D 0 hrs. 0 O O O 6 hrs. - 120 149 163 I* = Pose medium II 2 Ease medium with III = Face medium and - Fase medium with bile udlts emu sodium lduryl sulfete. 2 ‘T‘el'tle III. Growth of E. Coli on a 31331 .ium Contdining Sodium laurvl sulfate and Verious Ceueantretio s Lt of File Salts. Trial Time 0? File 0.53 Kile 1.0” File 77 File 5“ A 0 hrs. 0* l 6 " ‘06 5°? /16 356 257 0“ 0 hrs. 0 O 2 l " 513 3/“ 39‘ hrs. 1 O O l " 312 267 575 0‘0 ’3" {.3 L) o O O O O O\C) )J :\ Io .3 \o J J.) \3 I "l * No. of bacteria per ml. £19.. Table IV. Grovth of Various interics on on Enteric Heoium. Ordanism Time Fedium I Hedium II radium III Paracolobactrum 0 hrs. 0 l O firizona 6 " 215* 172 188 Salmonella O " O 2 O tymhimurium 6 ” 1563 122 1470 fierobacter O " O O 1 aeronenes 6 " 3551 4859 5320 Shigella O " O O O sonnei 6 " 402 536 A30 Salmonella O" O O O tyuhosa 6 " 51 52 41 Salmonella O " l O enteritidis 6 " 38A [87 368 Salmonella O " O 5 O _:jrntyrhi 6 " 453 526 671 Salmonella O " l O 2 schottmuelleri 6 " 113 152 171 * All counts are the average of two plates (per 1 ml.) Medium I - Base medium of 2.75 gm of monopotassium phosphate, 2.75 gm dinotassium phosphate, 20 gm tryptose (Difco) jns. sodium chloride, 5 {ms dextrose, water-one liter. Ledium II - Ease medium + 0.1 pm sodium lauryl sulfate Medium III - Ease medium + 0.1 cm sodium lauryl sulfate + 10 pm bile salts. T able V. Selectivitg of File - Sodium Lauryl Sulfate. Ornanisms Control 1” Tile Pil 3f bile 5? File Paracolobactrum (firizona) 4 4 3 3 2 Peracolobactrum II A 4 4 3 2 Peracolobactrum sp. IV A 4 4 3 2 lneerogenic paracolon III 4 4 4 3 2 Anaerogenic paracolon'VIII 4 4 A 3 2 Salmonella tyehimurium 4 4 4 3 2 Salmonella paratrohi 4 A 4 3 2 Salmonella tyuhosa A A 3 3 l Salmonella enteritidis A 4 A 3 2 Eerobacter aeroeenes A 4 A A 3 Shigella sourei 4 A A 3 2 Salmonella choleresuis 4 A A 3 2 Bacillus subtilis A O O O O Pseudomonas aerufinosa 4* 1 1 l l Eacillus cereus 4 O O O O Streptococcus fecalis 4 4 3 3 2 Ilcrococcus agilis A O O O O Hicrococcus aureus 4 O O O O Flavobacterium sp. (1) 4 O O O O Flavobacterium sp. (2) 4 O O O O OF-‘wa >6! HO acid - same amount of prowth as control - less growth than control medium amount of prowth little Frowth no growth. - 21 - The only exception was Pseudomonas aeruginosa which gave limited growth on all of the bile concentrations. Of the gram pesitive organisms tried only Streptococcus fecalis grew on any of the inhibitory media. Since it is an indicator of fecal pollution itself and would be outnumbered by enteric medium. mm The results of the experiments carried out in part one, indicates that a bile concentration of one percent in an enteric medium containing sodium lauryl sulfate, permits maximum growth of enteric organism while inhibiting non in- testinal forms. In order to check the value of the enteric medium under actual laboratory conditions a study was initiated in which it could be compared to standard procedures. The formula for the enteric medium which was used was as follows: Tryptose 2.0% Dextrose 0.5% Kfimbo 0.275% SOgPO 0.275% um lauryl sulfate 0.01% Bile salts (Difco) 1.0% Sodium chloride 0.5% Brom cresol purple 0.00l5% Water samples which had been brought to the Michigan.Department of Health from private wells for routine testing were used. There, they were tested according to Standard Methods procedure using Standard lactose broth as the presumptive medium with confirmation on brilliant green bile broth. The remainder of the samples were then sent to our laboratory where parallel tests were run using the enteric medium. All positive tubes were con- firmed by streaking on eosin methylene blue agar plates and transfer to lauryl tryptose and brilliant green broths. During the later part of the study Tergitol-7 and tryptone glucose extract agars were added in order to insure _ 22- early isolation of organisms giving acid reactions on the enteric medium. Tubes which gave positive results on all three or any two of the con- firmation media were recorded as confirmed. In all cases when confirmation was obtained on less than two, an attempt was made to isolate the organisms responsible for the positive result and subject them to identification pro- cedures. The method used was adapted from that of Cope gt g; (29) and was as follows: 1. ColOnies were picked from plates showing growth and transferred to dextrose and lactose fermentation tubes containing brom cresol purple as an indicator and nutrient agar slants. Care was taken to pick only from the surfaces of the colonies and to use only one fishing to avoid transferring a mixed culture. The tubes were then incubated at 35°C. At the end of 48 hours a gram stain preparation was made from the slant culture and examined microscopically. The sugars swere incubated for ten days until acid and gas were produced. 2. If the slant culture was found to be a pure culture of gram negative non- sporing rods and no fermentation of lactose was obtained, transfers were made directly to other diagnostic media. If a mixed culture was found, the organisms were transferred to nutrient broth and pure culture obtained by streaking on either MacConkey agar plates or Tryptone glucose extract agar plates. In such cases the identification procedure was begun again as in (l). 3. Other diagnostic tests were run as follows: a. Fermentation of sucrose b. Growth on Simmon's citrate agar c. Motility, hydrogen sulfide production and indole using S.I.M; medium d.'éggggggroskauer test for acetyl-methyl carbinol production using M.R.-V.P. medium (Difco). e. Decomposition of urea broth. Organisms fermenting dextrose and lactose with the production of acid - 23 - and gas in 48 hours that gave positive results on any of the three confirmation media were classed as typical coliforms and the tubes from which they came were considered as confirmed. Those that did not confirm on any of those media were classed as atypical. Those fermenting lactose slowly with the production of gas were also considered atypical coliforms. Strains producing acid and gas on dextrose but not lactose in ten days and which decomposed urea were classed as Eggtggs. Those fermenting dextrose with no gas, were non motile and did not utilize citrate or urea were con- sidered as Shigella-like. Organisms fermenting lactose of sucrose without gas or producing indole were classified as anaerogenic paracolons and were considered as typical coliforms. If gram positive bacterial or non-dextrose fermenters were isolated they were tested for growth on the enteric medium and if no growth was obtained in 48 hovrs they were discarded. Results The enteric medium was compared with those recommended in Standard Methods on the basis of the number of positive samples and the number of positive tubes. The results are given in Tables VI and VII. Referring to Table VI, it will be noted that, although there were fewer presumptive positive samples with the enteric medium, a higher percentage of these showed typical coliforms than did the lactose broth. All the samples positive in bile broth medium, contained coliform organisms which were either typical or of the paracolon group. Seventy-three samples were presumptively positive by both methods. Sixty seven of these confirmed in both. The fact that all 73 were found to contain enteric organisms indicates that all of the samples probably contained gas Table VI. Comparison of the Enteric Kedium with Lactose Broth (A). Enteric Medium Standard Lactose Number of samples tested 324 324 Number of positive presumptive samples 86 97 Number of positive samples confirmed 71 73 Number of positive samples containing enterics 86 - Ihmmer of samples positive on both a) Presumptive 73 73 b) Confirmed 67 67 c) Enterics 73 ? Hunters of samples positive on one, not the other a) Presumptive 13 24 b) Confirmed 4 (l7 tubes) 6 (6 tubes) 0) Enterics 13 - Percent Confirmed 63% 75$ Percent with lactose 94$ - fermenters (acid or acid gas) Table VII. Comparison of the Enteric Medium with Lactose Broth (B). Enteric Hedium Standard Lactose r *to a 'n‘- is t a 1 "£1 1 5L5 iHIIthI' OJ. BHUGS pflrul‘l 8% ,DJ... , -1 Number of presumptive positive tubes 361 361 Ihmmer of presumptive pdsitive tubes confirmed ??4 204 Linker of tubes from which enteric organisms were isolated 35? - Percent confirmed £2 73 limker of tuhes not confirmed 67 - Inmier of tubes having lactose fermenting enterics present 48 lhmmer of tubes having sucrose fer enting enterics present 9 Others-mostly citrate and indole positive organisms 8 Percent of positive tubes having lactos fermenters present (acid or acid and gas) 95% -26.. producing coliforms, some of which were either eliminated or altered by passage through. the media used in the analysis. Thirteen samples were positive on the enteric medium and negative by Standard Methods. Four of these samples, represented by 17 positive tubes, confirmed. All contained enteric organisms. On the other hand, 24 samples gave initial positive reactions on lactose broth and six of these confirmed. None of these samples had more than one positive tube. In terms of rapidity the enteric medium was found to give on the whole, an earlier indication of the presence of pollution than lactose broth . Considering the latter two thirds of the sampling program, 68 percent of the enteric medium positives were pesitive in 24 hours while 42 percent of the lactose broth positives were so. There was no correlation between whether the organisms were typical or not and the speed of detection. During the study, it was found that there was a great deal of variation in the results obtained by the various methods of confirmation used for the enteric medium positives. It was found that lauryl tryptose broth gave a much higher number of confirmed tubes when it was used as the secondary medium. Brilliant green bile broth frequently showed growth but gas production seemed to have been suppressed and in many cases no growth at all was obtained on E.M;B. For this reason, in tabulating the results in Table VI, the lauryl tryptose broth confirmaticrs were used since these were found more dependable. Brilliant green bile did show at least one positive tube in all except one of the confirmed samples. Table VI gives a picture similar to that of Table V. Mere enteric tubes confirmed than did lactose broth positives., All except two of the enteric positive tubes showed the presence of typical coliforms or paracolon bacteria. Failure - 27 - to isolate enterics from these tubes was attributed to technique since the other tubes of the same sample yielded typical coliforms. All the paracolon organisms that were isolated were of the microaerogenic or anaerogenic type. These organisms, as noted earlier, are approximately as significant as typical coliforms. {\J 77 -~ - C(‘T‘T’T-I ”'33:"? 33 The enteric medium is spec ific; it does not permit the detection of any but enteric organisms indica‘ _uive of pollution. Vere coliform organisms are detected neing the bile medium than s poss-ble by standard methods. This includes both typical and atypical Acid production is an adequate indication of presence of enteric bacteria when used in a selective medium. 1'1"" "”1 "—7 ‘ N b S If ‘IV‘RLJI' Cl.“ 1. Halcolm, J. F. ., Classification of Coliform Bacteria. . Jour. H3 giene 38: 395—!23, 193g. 2. Prescott, C. 8., IHnslow, C. E.ii and cCrrdy, H. 3., Hater Bacteriology. 6th Ed. John Hiley and Sons., Inc., Hew'York 1946. 3. (a) American Pub 1 r 10. ( ( ( ( ( ( . "azu nation t4 ic Health Association. Standard Methods for the of Water a.” Sewage 2nd. Ed. 77—110, 1912. b) fimerican Public Health Association. Standard Hethods for the Examination of Water nnd Sewage 3rd. Ed. 93-111, 1917. s l ‘ c) American Public Health Association. Standard {ethods for the Examination of Water and Sewage. 4th Ed. 92-110, 1920. d) American Public Health fissociation. 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Pub. Health. 23: 343, 1933. Levine, Max. Bacteria Fermenting L9ctose and their Significance in Water Analysis. Iowa St79 te Colleg e of Agric. and Mech. Arts. Off. Pub. 20, LEO. 31 blfl-l. 62. Huer, T. C. and Harris, H. L. Value of Brilliant Green in Eliminating *9 Errors Due to anf robes in the Presumptive Test for E. coli. Am. Jour. Pub. Health 10: Sit—o 9, 1920. Jordan, H. H. Brilliant Green Pile for the Determin9_tion of the Colon- Lerogenes Group. Jour. Lm. Water Works Assoc. 18: 337-346,102 . Caldwell, E. L. and Parr, L. H. The Pr sent State of} andling Hater “Hrvlies gm. Jour. Pub. Health. 23: 467-470. 193 qu '1 Perry, 0. A.and Hajna, A. A. A Further Hvalnvtion of h. C. medium for the Isolation of coliform Bacteria and Tscherichia coli. 1m. Jour. Pub. Hcvlth. 34: 735-738, 1944. IX) 03- o I J . 1') Huntington, E. and Winslow, C. E. A. _ 3o _ Cell Si ze and hetabolic Activity at'Vdrious Phases of the Focteria Culture Cycle. Jour. Eact. 33: 123, 1927. Gilmore, Eleenor L. 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