N l t WI l l H 1 l I W ‘ 1 — z — _— _..____,, :- — — F, W ‘ ‘ WI HQ; 100 m (0000') STRESWOCQCC! AS (NDECES OF SEWAGE FOLLUTICJN '§”§':esis fear “we? Degree of M. S, MiCHEGAN STATE COLLEGE Reg-her? Mines Buriasws 19:12.9 This is to certify that the thesis entitled Streptococci as Indicators of Sewage Pollution presented by Robert James Drieseno has been accepted towards fulfillment of the requirements for JAMI— degree in May fijor professor . Date "a! 5 9 19,49 - l.— I i-..- ——l _—_- 8;.- .J.!.. ;.;___ .‘J—a. ._l—-___L__.___-s o - --.-—_——.——‘_L_‘~_n .__.. STREPTOCOCCI AS INDICES OF SEWAGE POLLUTiON BY Robert James Driesene A THESIS Submitted to the School of Graduate Studies of Michigan State College of Agriculture and Applied Science in partial fulfilment of the requirements for the degree or LlASTER OF SCIENCE Department of Bacteriology and Public Health. 19k9 TH 5818 ACKNOWLEDGEMENT The invaluable advice of Dr. W. L. Mallmann of the Department of Bacterio- logy and Public Health at Michigan State College and the c00peration of Dr. Harry B. Weiser of the Ohio State University, Bacteriology Department, are gratefully acknowledged. The writer wishes also to express appreciation to the staffs of the Sewage Diaposal Plants of the City of Col- umbus,0hio and East Lansing, Michigan for facilitating the sampling of sewage. 333 #- CD Q C3 ‘1 CONTENTS INTRODUCTION.............................. 1 REVIEW OF LITERATURE...................... 2 EXPERIMENTAL Introduction............................ 10 Results................................. 12 Discussion.............................. 29 Summary................................. 32 REFERENCESOOOOOO0....OOOOOOOOOOOOOOOOOO‘O'OO 33 A number of outbreaks of intestinal diseases has‘ been reported in the southwestern part of the United States where vegetables are irrigated with sewage polluted river water. Inasmuch as the water supplies in these areas are generally free of contamination, vegetables have been sus- pected of transmitting enteric diseases. The method of measuring the degree of contamination in irrigation waters, which are known to be sewage polluted, could conceivably be identical to those used for evaluat- ing the potability of drinking waters. However, these ne- thods may not be satisfactory procedures when it is recalled that the water is channeled into shallow trenches in culti- vated soils. It is possible that the waters may become con- taminated with organisms from the soil. If the soils are fertilized with animal manures it is conceivable that the irrigation water may become contaminated with enteric or- ganisms of manurial pollution. If such is the case, then it would be better to test for human pathogens of enteric origin such as the Salmonella group. .However, it has been demonstrated by repeated test- ing of sewage polluted waters that the isolation of such or- ganisms is difficult and unreliable because their incidence is low and the methods of isolation do not adapt themselves to rapid methods of isolation and identification. Thus it would appear that non-pathogenic enteric organisms would be the most satisfactory indicators of fecal contamination. The purpose of this thesis is to investigate the prob- able utility of streptococci, particularly of the enteric group, as satisfactory indicators of dangerous sewage pol- lution under conditions related to the problem suggested above. Review of Literature Coliforms in water The coliform group is used as an index of stream pol- lution and water potability. It is generally accepted that the incidence of water-borne diseases is low in areas where the coliform index of drinking water is low, high where the index is high. However the literature on the subject shows considerable disagreement with respect to the sanitary inter- pretation of any given index. Savage and Weed (31) studied the longevity of coliforms and streptococci in water inoculated with excreta. At the end of one week, both the coliforms and the streptococci had dimin- ished rapidly. The streptococci persisted for two weeks and the coliforms (mainly finon-fecal' types) continued to persist at very low levels up to six weeks. Rogers (29) inoculated loopfuls of human exreta into sterile water and found that at the end of nine months the ratio of surviving coliforms was approximately 39 aerogengg (non-fecal) to one goli (fecal). The inference is that when aerogenes predominate, the pollution is remote and when the poll predominate pollution is recent and more dangerous. Platt (25) showed that Esch. coli survive longer in sterilized waters than in raw river water and that they disappeared rapidly at 370 C. in both waters. He noted that upon aeration by shaking, their numbers increased mar- kedly. Organisms incubated in the dark persisted longer than those eXposed to daylight. Bigger (2) suggested that carbon dioxide in raw river waters was the reSpnnsible inhibitory agent. He found that coliforms multiplied in autoclaved tap water but died rap— idly in therevawater.. The long persistence of coliforms in tropical waters which are not apparently liable to con- tamination may be explained by the low carbon dioxide con- tent of the waters. Koser (11) proved that after four months sojourn in soil, coliform organisms did not change their biochemical characteristics. Thus, it would be possible to determine the longevity of the Esch. coli in the soil by checking the organisms by biochemical tests (IMViC). Taylor (37) complicates the picture of so-called focal and non—fecal types by showing that the urines of patients having genito—urinary infections contain mostly aerogenes or intermediate types. He suggests that Aerobacter are at least excretory if not typically fecal. Parr (23) inoculated sterilized pieces of string and wood with Eggh;.ggli obtained from feces. These were sus- pended in sterile tap water. The organisms remained viable for over a year. It appears that the presence of either "fecal" or ”non- fecal" coliforms in a water is not necessarily evidence of recent fecal pollutions. The coliform test has proved to be inadequate in at least one instance where no coliforms were detectable in a treated water supply which proved to be the source of an epidemic of diarrhea (Ziegler, #3). Mailmann (l6) concludes that "the presence or absence of coliform organisms in marginal waters is arbitrarily based on the selection of the medium used for their isolation and not their actual presence". Pathogens in Water Prescott, Winslow, and McCrady (27) state that pathogens such as Salmonella typhosa and Clostridium welchii are not con- sidered to be satisfactory indicators of pollution because their numbers are too low to be of practical value. Neverthe- less, it may be well to note their probable longevity in water. Craig (6), citing many observers, reports that Endomoeba histol- ,EEE (vegetative form) survives in feces at room.temperatures up to nine days, in water from.9 to 29 days, the latter only in the presence of few bacteria. Concentrated suspensions of the organisms inoculated into distilled water persisted at room.temperature for ten days and to 211 days at 12°- 22° C. MacConkey in 1906 (lb), noted that cultures of Cholera gibrios were viable in distilled water after several months when inoculated in very large numbers but in concentrations of the order of 10,000 per m1. they disappeared within two hours. Tanner (36), citing the work of Rachel: (28) remarks that concentrated cultures of pathogens survive longer in pure cultures than in mixed. Rochaix is reported to have shown that both Vibrio 22mm§_and Salmonella typhosa sur- vived 3% months in sterilized tap water and nearly seven months in sterile distilled water. Salmonella schottmulleri in sterilized sewage water persisted 85 months. Although virus infections (e.g. poliomyelitis) may re- sult from.sewage pollution, the arduous methods of isolation make their quantitative estimation and evaluation impractical. Since our problem involves waters in intimate contact with soil, it is well to note the activity of coliforms in soils and manure. Coliforms in Soils and Excreta. Rogers, Clark and Evans (30) isolated Eggh:_ggli_fro- grain and grain fields apparently not focally contaminated. None of the coliform.organisms was like the characteristic flora of bovine feces. Young and Greenfield (#2) mixed Eggh:flggli with soil in 27 cu. ft. galvanized tanks and in bottles. Survival was noted in some cases to seven years, others to three years. Opinion expressed was that differentiation between fecal and non-fecal strains was of little practical value to the sani- tarian, since both become saprophytic to the soil. Skinner and Murray (33) inoculated soil with one per cent manure and found that both aerogenes and fecal types of coli disappeared after 150 to 200 days. In manure alone they persisted about 50 more days. Aerogenes types were iso- lated from many soils under "natural conditions", 1.6., - in the absence of known or gross pollution. Tonney and Noble (39) noted a rapid pOpulation decrease under winter conditions. By inoculating decayed stumps with cultures of g; aerogenes and Esch. coli, they noted an in- crease in numbers after 60 days, most of the persisting or- ganisms being aerogenes types. Kulp (l3) inoculated sterilized soil with Eggh; gal; and §;_aerogenes. The soil was kept moist throughout the period of the experiment. The organisms persisted over 3% years. Kline and Fuller (12) in a similar experiment recovered the organisms after 410 days. Taylor (0p. cit.) claims that there is no evidence of coliforms multiplying in grasses, except silage, and insists it is virtually impossible to determine whether a soil is polluted or not under natural conditions. He does not be- lieve evidence warrants the conclusion that coliforms are normal inhabitants of soils, grasses and grains. Tonney and Noble (39) suggest that §;_aerogenes is primarily non-fecal but appears irregularly as a transient organism in feces. They showed (40) s by direct plating that the ratio of Agrppapter to Escherichiakin feces was 1 to 100 and in soil and vegetation 20 to 1. Some explana- -6- tion other than that both are of direct fecal origin must be given, since in feces the aerogenes are in an unfavorable en- vironment. Enteric PathOgens in Soil and Excreta Data are meager with respect to the viability of patho- gens in the soil. The factors of antibiotic substances, des- iccation and temperature fluctuation would suggest a short survival period. Jordan (9) noted that typhoid bacteria did not multiply in stored feces and disappeared within 3 to 52 days. The average number of bacteria in all kinds of feces (i.e., from health and from ill persons) was estimated to be about 75,000,000 per gram. Hurray (19) noted that 333g; 321i in manure piles were rapidly crowded out by other organisms. Tanner (37) citing Rochaix (28) reports the survival of g; typhosa and other Salmonellgp up to 75 months after inocu- lation into sterilized sewage. The writer feels that this is not a valid method of determining response to normal environ- mental conditions. The sterile sewage may be considered to be a prepagation medium. Suckling (35) suggests that manures may not be as in- nocuous as is often supposed. Gulls have transmitted typhoid organisms in their excreta. Brucellosis and some Salmonellas infections are suspected of being transmitted. It seems clear that for purposes of evaluating the pollution of irrigated soils, the coliform test might be grossly misleading especially if made in the warmer sec- tions of our country. Considerable numbers of either types of coliforms may be expected to appear long after pollution by fresh manures or human excreta should have been rendered innocuous. Streptococci and Water Streptococci have long been utilized as supplementary indices to verify the pollution inferred by coliform indices. Prior to the recent advent of Special selective media, these enteric streptococci have been difficult to isolate in num- bers reflecting their actual numbers in water, sewage and feces. Seligmann (32) showed that streptococci pOpulate sewage waters in numbers 10 to 100 times greater than has generally been recognized. For this reason the data collected prior to the last ten years may be only of historical interest. By older me- thods of estimation streptococci appeared to disappear from waters more rapidly than coliforms. Savage and Wood (loc. cit.) using both decimal and intermediate dilution tech- niques showed that streptococci failed to appear usually after two weeks in water, but that in domestic non-indus- trial sewage they persisted from A to 8 weeks, paralleling coliform indices. in the above experiments, an average of 165 ml. of sewage was added to 40 l. of tap water and incu- bated at room temperatures. Houston (8) estimated that the concentration of one part per million of fecal matter in water is represented by 17 streptococci per ml. Hallmann (15) showed that streptococci (presumably oral) failed to multiply in swimming pools whereas under certain conditions coliforms did multiply. Streptococci indices fluctuated with the bathing load and presumably with the de- gree of pollution. Hallmann and Sypien (18) demonstrated similar parallelism.in natural bathing places. I Streptococci in 5011 andExcreta Broadhurst (3) believed that streptococci are neither in- digenous to the soil nor to grains and that the grains are only accidental carriers. She noted that they were most easily obtained from equine and human feces and difficult to obtain from.feces of dogs, cats and cattle. Ostrolenk, Kramer and Cleverdon (22) using soils contam- inated with bacterial suspensions noted that enterococci were detected somewhat longer than ggghL‘ggli but that in soils contaminated with chicken manures, the 3222;.22li were re- covered after 66 days but enterococci only for 21 days. Ostrolenk and Hunter (21) using Pprry and Heine's "8.3. jledimm' (of. Perry and Hajna,'2h) examined 52 specimens of human and animal feces. 26 of 28 fecal samples contained streptococci. 0ppenheim (20) basing his identification on the scheme of Holman (7) found about 7k per cent of the nonhemolytic strepto- cocci in feces to correspond with Str. fecalis (Andrews and Herder). Torrey (kl) noted that the presence of S33; fecalis may depend on the diet of the person from.whom a stool was taken. A low protein, high carbohydrate diet appeared to enhance the number of these bacteria. Suckling (op. cit. p. 50A) reports streptococci to be present in numbers up to 100,000 per g. of feces and that sewage contains about 10,000 per g. It appears that streptococci may occur in sufficient nwm- bers to warrant an investigation of their relation to pollution and sanitation. EXPERIMENTAL One early method for estimating the probable numbers of streptococci from.mixed bacterial sources was to examine mi- croscOpically the sediment from.tubes used for the determin- ation of coliform.popu1ations. Another (Suckling, 35) has been to dilute these broths and heat the same to 60° C. After subculturing on‘NacConkey's agar the small red colonies were examined microscopically.' Prescott and Baker in 1907 (26) observed that when dex- trose peptone broth became acid by the growth of coliforms, the streptococci began to overtake the previously predomin- ant coliforms. They suggested an acid broth medium with an acidity sufficient to inhibit the coliforms and thus allow the early development of the streptococci. -10- Ostrolenk and Hunter (21) grew streptococci on "S.F. broth"and from both "positive" and”negativé‘tubes made streaks on 1.5 per cent agar - S.F. broth plates. In their opinion S.F. broth failed as a presumptive medium because about seven per cent of the tubes were "false positives", 1.6., showed production of acid and sediment without presence of streptococci and five per cent "false negatives" as indicated by microscopic observation. All false negatives were found in the maximal dilutions of 1-100,000 to 1-10,000,000. Chapman (4) recommended two media for the isolation of streptococci. These media were develOped primarily for the separation of enterococci and pathogenic streptococci from mixed cultures. Although these media may be satisfactory for the purpose for which they were designed, they do not fit into the plan of this thesis because they are laborious to make for a repeated routine study of streptococci of fecal origin. Mallmann, Botwright and Churchill (17) noted the se- lective bacteriostatic effect of sodium azide as did Snyder and Lichstein (3h). The fecal streptococci showed marked tolerance of the azide. The general tedhnic of preparing and using the medium were both economical and simple. -11- Preliminary Streptococci may be prepagated on a number of non- selective sugar media. However, the writer found such media to be of no value for enumeration studies because the streptococci were obscured by overgrowth of other or- ganisms. Diagnostic media containing dyes such as those made with 0.1% methylene blue were too deeply colored to facilitate enumeration. Therefore a few exploratory tests were made on "SF" broth and agar (Perry and Hajna, 2h). To check the Specificity of "SF" media, a number of possible conflicting organisms were tested. Stock cul- tures of Str. lactis, Str. citrovorous, Str. pavacitrovorus, Micrococcus pyogenes var. aureus, several hemolytic strep- tococci, Serratia indica and several common molds failed to grow at room temperatures or at 370 0. within 48 hours on SF agar or in SF broth. This indicates that the media has marked selectivity. The SF broth formula is as follows; Glucose...........5.0 g. NaN3......................O.5 g. NaCl..............5.0 g. Tryptone.................20.0 g. KHzPOh............l.5 g. Distilled water........1000 m1. KQHPOA............A.O g. Brom-Cresol Purple (1.6% alcoholic)........2.0 ml. The SF agar was prepared by adding 1.5 g. agar to the SF broth. -12- A series of tests were made by substituting tryptose for tryptone in the 3.1. formulation. It was found that a more rapid appearance of turbidity occurred in 2b hours. Accord- ingly this substitution was made for the medium used in this study. No correlation was found between s.r. broth indices and direct plate counts on S.F. agar. Only a few colonies ap- peared in the latter medium. However, streak plates made from "doubtful positive" broth tubes frequently showed typi- cal streptococci colonies. For this reason only the 5.1. broth medium.was used. Lactose was substituted later for dextrose in the formu- lation because some streptococci (S53; eguinus) from.horse manure do not ferment lactose. Inasmuch as the medium.is to be used for the examination of soils irrigated by sewage laden water the use of lactose would offer a medium.more selective for human pollution and would reduce the confusion from.man- urial sources. When tests were made of horse manure, non- lactose fermenting streptococci were isolated from the 8.1. (dextrose) broth. Eventually Brom.thymol blue was substituted for Brom cresol purple because it manifests a color change at a higher pH than does the former. In view of the fact that the medium is buffered the writer believed that small numbers of strep- tococci mdght not produce:enough acid sufficient to affect color change in the range of pH5 - pH 6. The formula for the altered SP broth used in evaluating river water and manure was as follows: *Lactose............5.0 g. NaN3....................0.5 g. NaCl...............5.0 g. *Tryptose..............20.0 KHQPOh.............l.5 g. Distilled water......1000 m1. KéHPO .............A.0 g. *Brom.thymol blue h (0.5% al°°h0116)eeeeel§eo m1. *Altered ingredients Streptococci Pppulations in Sewage Samples were obtained thrice weekly in January and Febru- ary 19A8 from.the Columbus, Ohio disposal plant. The sewage from.this plant is fairly representative in that there appears to be no overbalancing of industrial or of biological wastes. The raw sewage samples collected at 9 A.Mt were relatively di- lute. This sewage, because of the eight-hour travel period to the disposal plant represents the waste discharged at about 1 A.M. Sterilized salt-mouth bottles were used to collect the sewage. Within two hours after collection, 11 m1. portions were diluted serially in sterile tap water. The samples were planted in 8.3. (tryptose) broth in quadruple decimal dilu- tions. Each tube contained at least 5% m1. of medium to in- sure a minimum concentration of 0.0L per cent sodium.axide. Readings based on presence of acid production and turbidity verified by microscopic examination, were made after incuba- tion at 37.5° C. for 48 hours. MicroscOpic examinations using Gramis stain were made only from the critical series of dilu- tions and from.both positive and negative tubes of these dilu- tions. The substitution of lactose for dextrose in the tryp- tose SF broth was made on February 13. The results of a survey of the Columbus,Ohio sewage treatment plant are presented in Table 1. Samples were col- lected from the various stages of treatment over a period of one month. These figures do not show the degree of puri- fication from a sanitary standpoint as measured by strepto- cocci indices because of fluctuation in the concentration of sewage entering the plant. It is impossible to trace with any degree of accuracy the bacterial changes in a given lot of sewage through the plant. No correlation be- tween the streptococci pOpulation and the total solids was noted. The data show that streptococi persist in large num- bers after prolonged heating at 85° F. in the heterogeneous pepulations of the digesters and that reduction in numbers is not evident. Streptococci and Soil Five wood boxes 20 x 17 x 30 cm. were each filled with approximately 10,000 c.cm. of soil of three types, none of which was known to have been contaminated with either human or animal manure. One liter of raw sewage was added to each boxful. Two samples (muck #2, Sand #1) were allowed to dry gradually at room temperatures indoors and three were set outdoors exposed to natural conditions. -15- manpomq** omonpxom* be www.ma 05m.am mmN.m mom.n .>4 oanuoaoow . m 000.m 000.0 000.0 Had m 0 a 00m.HH 000.0» mam a .ssa sum 000.0m 00m.aa 000.0 00a mm m 00m.m 000.0m 000.0H Ham 0m m 000.0N 00m.m 00m.0H ewe mm mm 000.00 000.0 00m.aa mom 0m 00 000.0m 000.00 00m.m Ham ea 00 *+ 000.maa** 000.mm** 000.0** sum ma 0v .. 000:2... 000.3:. 0023.. a? 3 “Ha 000.HH 000.00 000.HH meg AH 00 000.0 000.0m 00m 0am 0H 000.4 000.0H 000.N 00m.aa 5mm 0 000.0 000.05 000.00H 000.4H mmm 0 am ”a 000.0ma 000.0N 0am m .noa peosammm mmesam owesam mmesom omesom 30m meaaaeem as... damages Ewwmwmm as Mama“ .3 as H0909 pdmam peoapmone aflao menadaoo one Song eoeampno amazom ca mooaecfi HooOOOpmmapw .H sense -16- J to 1‘ FL -[ill 5-4% [.il (I! fit .Hi 1“ < W‘ 1.3\\\/< r uranium 442E moonnw 8.535 m o<3mm w H... u . D Z _ H magmas macaw 32254 Dmkdmmk Edda mma meazmm 34m L .UuOFawakm w0< one: coca... one «o. Oooqon Buplicate surface samples (two inch depths) were col- lected using a flamed spatula. At each sampling one portion was checked for moisture content, the other was used for bac- teriological tests. The latter sample was diluted with ster- ile water and decimal dilutionSprepared. Care was exercised to be sure that suspended solids had not settled during samp- ling. One ml. portions were planted into the lactose S.F. .medium. The cultures were incubated at 37° C. for #8 hours. The data reported are computed on a dry weight basis. The results are presented in Table 2. The results show a very low streptococci population. This was due to the fact that the sewage was chiefly storm water. Because the pepulation was low initially it was dif- ficult to trace accurately the diminution of streptococci during the period of test. At the end of 26 days, zero strep- tococci indices were obtained from several soils. A.second series of tests was made using the same soils. In this case cultures of streptococci were added so that heavy inoculations were obtained. The results are reported in Table 3. lhe data show a marked decrease in population in two weeks (over 90 per cent) and a decrease of more than 99 Per cent in three weeks. Characteristics of the soils do not appear to be important factors in the diminution observed. Because of the low moisture content of the soils, desicca- tion may have been a major factor in this rapid reduction of population. The soils were examined over a period of 18 days. A similar series of tests was made at.Michigan Hews pdmflapsz do uneaseopoa pedoo Hmpoe* *amaw 000.0m0.0 000.0m0.m 000.004.H 000.00e.a 000.0mm.0 s00 masseuse H0009 HsHsHeH me o m m 0 0m 0 m m N m oa mm mm 0 ma ma w «a M052 Hm M052 mm comm Hm edsm 800A meao doapeaeoodH nonoasz eonoasz nouns: aspnoz apeom Hopes when Edam mom Hooooopmonpm ommsom 50m spas oopsaaoqu maflom meoaam> ma Hooooopmonpm mo zpfiaflnma> .m edema -18- 0 000.00 a 050.m a 0mm a 000 a 00m 0H d 000.ma m 00m.ma a 000.00m a 000.maa a 000.00 ea ma 000.00m Hm 000.0ma 0 000.00 a 000.00H.e a 000.00H 0 mm 000.000.m~ Hm 000.0an.e m 000.00m.ea m 000.000.HH e 000.0m 0 0H 0004000.0e 0m 000.mma.m a 000.000.H0H a 000.004.5a a 000.0ea.a N 0mm Sena 0mm ammo omm anew 0mm Seem omm seam deepeaaooeH R Hoe R mom a you a use & Hoe amped when .moapm .monpm .monpm .aonpm .moapm me H% mm H% esoa asao spasm soda emeoasa soda assuage ease asseoa ease nachos mooshnsm Haom QH oHan> HooooOpmmnpm .m edema -19- 4am enema-m «:m shaman f 9. *3 3 $20 \ \/\/ m m .m .. ~ m9 I- ma m o. .1 o 0 a W 3 o. a m m s a _ w m m -m m L n N n n N “W a n. a I? W 3 un mmaah-Du waaa wo<3mm 3(a- I...... a. E... owkds-DUOZ club-W-Duoz. i. wn-n-e-(m > .0 oan0e -2g- VIABILITY 0F STREPTOCOKI IN Two amen warren sans-Les -. \ ' D “I | \ 0 I O LEGEND: A s COL! FORM 3 O = STRt-Prococu - 0 - : VARIAOLE TEMPERATURES --— : REFRIGERATED Loc ,, PROBABLE- NUMBERS O l I l I I I o#¢” , Q0 ‘7 -II 14 1 9 I1. Lt DAYS AFTEPIUOCULATIW i Figure 5 Upon the first disappearance of streptococci, coliforms were found to be present in large numbers, but also gradually diminished by #7 days. Streptococci in manures Twelve samples of animal manures were made by mixing fresh fecal matter from.several animals. These samples were-prepared from.the excreta of horses, cattle and chickens. These differ- ent manures were then mixed with sandy loam soil using 16 parts of manure to 1000 parts of soil, The soil-manure mixes were each placed in tinned steel cans like those described previ- ously. Two samples of each type manure in soil were stored at 516° C. and two each at prevailing autumn temperatures until freezing weather necessitated removal indoors. Duplicate samples were tested in the same manner as de- scribed previously for the pure culture inoculation studies. Soils were wetted periodically with distilled water to reduce the effect of desiccation, if any. One ml. portions of the soil suspension-dilutions were added to each tube containing 6 m1. of tryptose lactose SF broth and incubated AS hours at h5.5° C. only. Microscopic and SF agar plate confirmations were made as previously described. A presumptive coliform index was made at the termination of the study. Data compiled in Tables 6, 7 and 8 and in ac- companying graphs show that streptococci may occur in fairly large numbers in horse manure and chicken manure, but are rel- atively few in cattle manure. maooedH moamaom coeoz * 0H mm KoodH SHOMHHOO , OH mN moch Snowflaoo 00 OH Hm OH mm 00 wa 5004 I I m: I I ma 000.H 00 ma I 0 Na 5m ea 000.0 0H mum.a 00 00 000.: am 000.: 00 I I as I005.0H 0H 00 000.5 I I me I I 50 000.00 0 mm 005.00g 50 000.000 0 onopmaoz R Seam you NoedH onspmfioa R Seam Hoe MoeeH doapoadoqu Hooooopmospm Hooooopmoapm nopud when “Hoapeooq .o o0I0 p0 eoaoem monoponomaoa neop54.po nonopm oedema omnom R0.H spas consasoodH Haom o0 Hooooopmoapm mo mpfl>omdoq .0 manna Loo“, mosseu: numsess STREPTO(O(( I IN HORSE MANURE ‘ CEOME-TRIC MEANS \ PER GRAM mcussvec AT 45°C. " “I LEGEND : An (Old FORM S $ 3 O: sveepvococu ~= Resume RATE-D '- " ’ NOT RE- FRICIERATE-D DAYS 4 Figure 6 mnoooeH oaaaon 0o>o2* x m mN 0H mN NocdH Showwaoo 00 r 0 00 3 0m 00 0a m0 I I mg I I ma 0N NJ I I 4H 0N 5m 0H 0 5a e on 50 mm 5H me mN I * I ma *0 ma NN om I I ma I I HN 0ma 0 NN mo0 mN 00N o onspmaoz R scum you Hccam onepmaos R .8090 you Noch coapoasoodH HooooOpmonpm «ooooopmoapm scene when, aaonpqoov .o o0Im 00 nonopmn monsoonomaoa massed 00 009090 oedema Boo R0.a n00; oopsaeooeH Haom a0 aooooopmoapm no hpfipomooq .5 canoe I -27- STREPTO(O(( I IN (OW MANURE 'I'f 1w GEOME-TRIC MEANS PER GRAN INcuBA‘re-DM 45°C. LEGEND: O A 2 (cu roams O : STREPTococh ""= REFRIGERATED “ "‘ NOT REFRIGERATED Lee ,, PROOABLE' wuneeas Figure 7 mH000dH oaqaom 0o>05* 000.H smeaH asoeHH0000 5H III 0H 5H 0Hm 0H 0N m0 H0 050.0 ms 004 00 I It“. ma 5mm 5m 5H 000.00 50 000.0 00 0H 0HH.NH 0H 000.0 00 I I.II 0H Isam.0 me am 000.0HH I mg I III 00 000.000 0 00 000.00: 00 000.00 0 cadpmaoa R seam Hon NoeoH oaspmaoz R _a0no Hem NoenH dofipoasoodH Hooooopmoapm Hoooooemonpm sound when Aaonpeoov .o o0Im so nonopm mosspsnmmaoe manned p0 coaopm oedema doxoano R0.a and; oopoaooonH Haom ca HoooOOpmonpm ho hpapomqoq .0 canoe -28- LOCI“, PROBABLE-.- Munoz-Rs psmmmmm II POULTRY MANURI': GEOMETRIC MEANS OF Two SAMPLES EACH. 45.5 ° C. INCUBM’ION REF, \ I \ \ NOTREF. LEGEND'. \ A COLIFORMS' \ o sraeevc <0ch ‘ \ - Rat-moan?!» \ - - wov anemone-rec \ \ DAYS In no so 60 Figure 8 Reduction was slower in the manures than in the soils inoculated with pure streptococci cultures or in the river water. By three weeks time reduction was over 90 per cent. 'At 69 days the coliform indices were generally of the same order as those of the streptococci. The manures represented approximately a concentration of fecal matter,Itwice as great as is customarily employed on farms. The manure was not rotted. Recalling the large numbers in sewage sludges, one notes that reduction of the streptococci was less rapid in the presence of organic matter and very rapid when organic matter was less concentrated. DISCUSSION Published data concerning coliforms though voluminous, are not readily compared. iMany early studies on lonvevity were not quantitative. Studies on sterile soil are not come parable to those made on natural soils because the bacterial environments in each are very dissimilar. Again, studies of sterilized or distilled waters cannot be evaluated on the same basis as waters which contain either living organisms or greater concentrations of possible nutrients, both of which may greatly affect the viability of the organisms. Methods and media used very probably affected the numerical results obtained. However, it appears that streptococci do not increase in the soil or river water and that coliforms have been shown by other workers to increase under certain conditions. Soils and waters which have a low content of fecal matter may be expected to show higher coliform indices than streptococci, but where pollution is greater, the streptococci pepulations closely parallel the coliform numbers. The close parallelism shown in the 37.50 C. and h5.5° C. indices where samples had been stored and pOpulations were low suggests that either of the incubation temperatures may serve to determine populations of marginal environments. The more hardy streptococci may be resistant to both prolonged expo- sure in the soil for water and to high incubation temperatures. Recent studies by Seligmann (32) show marked variations in the streptococci indices of identical samples occasioned apparently by the differences in the composition of the var- ious media. These studies suggest the probability of devel- oping media more suitable for the estimation of streptococci than those used in this study. It is obvious that soils freshly manured within 10 weeks of the sampling time cannot be prOperly evaluated by either coliform or streptococci enumerations. The media used does not aid in differentiating between human and ani- mal sources of pollution. -30- The streptococci may be tentatively said to conform to the requirements for a test organism in the following re- spects. 1. Streptococci more closely resemble the pathogens ‘Iith respect to lonvevity in water and soils than do the coliforms. They remain viable longer than most enteric pathogens but diminish rapidly, percentagewise. 2. Streptococci appear universally in mixed sewage and sewage polluted strams. They may not be present in certain individual stools. The latter may be said also for other organisms. 3. Evidence indicates that streptococci of excreta do not multiply or becmme saprophytic in environments which are not heavily polluted with fecal organic matter. h. The technic of enumeration utilized in these studies are relatively simple, call for a minimum.of special equipment and require only the ordinary technical precautions exercised by bacteriologists. The methods give results within a short enough enterval to permit adaptation to routine work. The relationship of the quantity and recency of pollu- tion to the levels of streptococci pepulations required fur- ther study utilizing various measured amounts of fecal mater- ial in soils and water. Further studies on the effects, if any, of non-fecal organicmatter on the viability of the streptococci may prove valuable. The study completed demonstrates that streptococi may prove useful for sanitary evaluations of soil and water under certain conditions. Data presented show streptococci populations levels to be high in raw and treated sewage, in polluted river water and in soils inoculated with animal wastes. Longevity studies show a streptococi reduction of over 99 per cent in manured soils within 18 days and from 70 to 80 per cent in 13 days even under conditions of refrigerated storage. In polluted river water streptococci are shown to de- crease from between 85 and 99 per cent in two days. They were completely absent as early as 12 days in water stored at early autumn temperatures, and were absent by the h7th day in a refrigerated sample. Studies of the viability of cultures of streptococci placed in soil show reductions of 90 to 99.97 per cent in eight days. A method which may be adapted to routine enumeration of streptococci is described. 1. 2. 3. 5. 6. 7e 8. 9. 10. 11. 12. 13. ll... 15., REFERENCES Breed, R. 8., Murray, E. G. D., and A. P. Hitchens. Bergey's Manual of Determinative Bacteriology, Balti- more, 6th. Ed., Williams a Wilkins 00., 191.8 . Bigger, J. W. The growth of coliform bacilli in water. Icur. Path. and Beet. 22: 315, 1937. Broadhurst, 1. Environmental factors. Studies of Strep- tococci. .Tour. Inf. 1318. 1.1. 277, 1915. Chapman, G. H. The Isolation of Streptococci from mixed cultures. Jour. Bact. g: 113, 19%. Conn, H. 1.; The most abundant bacteria in the soil. Beet. Rev. ;._2_: 257, 19h8. Craig, 0. I. Amebiasis and Amebic Dysentery. Baltimore, Charles C. Thomas, 1931., Holman, W. L. The classification of Streptococci. Jour. lied. Res. 25: 377. 1926. Hougtcn, A. 0. Studies in Water Supply, London/Hacxillsn 191 . . ' Jordan, E. O. The changes in the bacterial content of 13511; storgd normal and typhoid feces. Jour. Inf. 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W013“ "(F ONlY MICHIGAIN STATE UNI VEIRSITYI |L|B RRIA R‘IES I IIII IIIIIIII III IIIIII