THE DEVELOPMENT OP METHODS FOR THE ISOLATION OP ENTEROCOCCI PROM WATER AND SEWAGE By Warren Litsky AN ABSTRACT Submitted to the School of Graduate Studies of Michigan State College of Agriculture and Applied Science In partial fulfillment of the requirements for the degree of DOCTOR OP PHILOSOPHY Department of Bacteriology and Public Health 1951 Approved Warren Litsky Ethyl purple azide broth, a new medium, was developed# The formula for this medium is as follows: Ingredient Grams per liter Tryptose 20 Dextrose 15 Sodium chloride 5 KgHPO^ 2#7 KHgP04 2,7 Sodium azide 0*4 Ethyl purple 0*00125 pH - 7#0 Sterilized at 121° C# for 15 minutes When tested with laboratory strains of bacteria, it was found that the enterococci were the only group that would grow in this medium# These organisms also showed a char­ acteristic growth of a purple compact button on the bottom of the tube of medium after 48 hours of incubation at 37° 0# The specificity of this medium has been demonstrated also with samples from river water, sewage, and soil# A new test for pollution of river water, sewage, and soil has been advanced* This test employs dextrose azide broth as a presumptive medium and ethyl purple azide broth for confirmation* A comparison of methods for the detection of enterococci was made and it wasdemonstrated that the newdextrose azide-ethyl purple azidebroth testwas the best and easiest of those in use today* THE DEVELOPMENT OP METHODS FOR THE ISOLATION OP ENTEROCOCCI PROM WATER AND SEWAGE ByWarren LJtsky A THESIS Submitted to the School of Graduate Studies of Michigan State College of Agriculture and Applied Science In partial fulfillment of the requirements for the degree of DOCTOR OP PHILOSOPHY Department of Bacteriology and Public Health 1951 ProQuest Number: 10008367 All rights reserved INFORMATION TO ALL USERS The quality of this reproduction is dependent upon the quality of the copy submitted. In the unlikely event that the author did not send a complete manuscript and there are missing pages, these will be noted. Also, if material had to be removed, a note will indicate the deletion. uest ProQuest 10008367 Published by ProQuest LLC (2016). Copyright of the Dissertation is held by the Author. All rights reserved. This work is protected against unauthorized copying under Title 17, United States Code Microform Edition © ProQuest LLC. ProQuest LLC. 789 East Eisenhower Parkway P.O. Box 1346 Ann Arbor, Ml 48106 - 1346 TABLE OP CONTENTS Page INTRODUCTION .................................. 1 LITERATURE REVIEW.............................. 4 EXPERIMENTAL ...................... . . . . . . DISCUSSION........... SUMMARY. 20 49 .................................... 60 BIBLIOGRAPHY .................................. 62 ACKNOWLEDGEMENTS The author wishes to acknowledge the help of Dr# W# L# Mallmann, both during the progress of the research in this thesis and in the preparation of the manuscript# His criticisms were at all times constructive and tempered with the reason and kindness of a scholar# H© was always more than willing to help in the procuring of materials and in the unraveling of difficult problems# To him, the writer1s heartfelt thanks# Thanks are expressed to Mr# Charles W. Fifield and Miss Marianne Kingsbury for their help throughout the entire work, and to Mr* Irving Delappe for proof-reading this manuscript# The author wishes to take this opportunity to express his gratitude to all the members of the Department of Bacteriology for their kindness and for making his work enjoyable# INTRODUCTION Workers throughout the years have Investigated, the problem of polluted waters and are all In agreement re­ garding the seriousness of the situation# Recently Dunlop, Twedt and Wang (15) have reported that 23 out of 113 samples of water used for Irrigating vegetables in Colorado were positive for Salmonella and one sample yielded Salmonella typhosa# This organism was also Isolated from East Lansing sewage and from the Red Cedar River. Although only a few epidemics have been charged to contaminated vegetables, It Is possible that unrecog­ nized endemic enteric infections or sporadic cases may be caused by eating vegetables grown on contaminated soil Up to now the test organisms employed for pollution are members of the coliform group, because these bacteria are supposedly indicative of sewage contamination and can be easily Isolated and confirmed by a simple bacterio logical test# It was felt, however, that the coliform organisms do not give a true indication of pollution because: (1) these organisms are found in uncontaminated soil and may be of non-fecal origin, (2) these organisms may persist In the soil and water for long periods of time and might not Indicate a recent pollution, and (3) 2 fecal strains of coliform bacteria cannot be distinguished from non-fecal strains# Streptococci are used as an Indicator of pollution on the same grounds as Escherichia coli because (1) they are present in feces and sewage and are found in known polluted waters, (2) they are not found in pure waters, virgin soil, and sites out of contact with animal and human life, and (3) they do not multiply outside the animal body (except in such media as milk, etc#)# With respect to their numbers in feces and sewage, streptococci are subject to great variation# A review of literature reveals that while at times they may be almost as numerous as E#_ coll, at other times they may be consider­ ably less numerous and absent# Mallmann and Lit sky (41) shovel that In soil that was treated with sewage the most probable numbers of streptococci was approximately that of the coliform organisms* It was also demonstrated that the streptococci disappeared from the soil rapidly while the coliform group persisted for long periods of time* Tested against the longevity of the typhoid bacillus, it was found that the virulent typhoid organisms died out much more rapidly than did the streptococci* This indi­ cated that the fecal streptococci are a much more Indica­ tive organism of recent fecal pollution than is the coli­ form group# A new medium, dextrose azide broth, DIfco, was reported by Mallmann and Seligmann (42) to be an excellent 3 enrichment medium for the detection of the enterococci and other streptococcic This medium, however, must he confirmed for streptococci by microscopic examination. With the above data indicating a new and more practical test organism for recent fecal pollution, as well as an excellent enrichment medium, work was started to devise a simple test for the confirmation of the enterococci after the presumptive test-growth in dextrose azide broth# 4 LITERATURE REVIEW Ever since Escherich (16) in 1886 recognized the streptococci as normal inhabitants of the bowel of infants and described their morphology in detail, numerous workers have investigated these organisms but because of the lack of a well defined system of classification, much confusion still exists in the literature. Even today there is still some doubt as to what constitutes a fecal streptococcus, an enterococcus, or whether Streptococcus faecalis is a single species or a group of species# The first important work in the classification of these organisms was carried out by Gordon in 1903-11 (19), who introduced the series of biochemical tests associated with his nameo By means of seven chemical tests, Gordon distinguished 48 varieties among 300 streptococci from normal saliva# Houston (29), working along this line, examined and determined the main features of fecal strepto­ cocci* Andrews and Horder (3), utilizing the results of Gordon and Houston, applied an extended series of tests to a large number of streptococci isolated from disease con­ ditions and undertook a wide statistical study of t he genus# As a result of this study, they were able to arrange the entire series of organisms into seven large groups, each 5 cantering in a definite type demarcated by its biological activity* and connected with the other type by a graded series of intermediates# It was these authors who first described Streptococcus faecalls# The streptococcus-like organisms found in feces have been the subject of numerous descriptions* and a host of loosely described types encumber bacteriological liter­ ature# It is evident that many of these should be entirely disregarded In the light of more modern investigations* since the distinctions made use of In defining them are now recognized as insufficient# There appears, however* to be certain constantly recurring and outstanding types* all considered more or less distinct by those who have worked with them# A survey of a mass of confused liter­ ature suggested the following three groups: 1# Described by European workers (a) The Enterococcus of Thiercelin (Micrococcus ovalls of Escherich)# (b) The 81rep to o6cctls enteritis (Hirsh and LIbman 1897)# 2* Described by English workers (a) The 81reptococ cus faecalis of Andrews and Horder# The enterococci* which were described by Thiercelin (59) In 1899 before the introduction of more recent methods and alleged by him to be the causative agent In certain types of diarrhea* biliary Infections and appendicitis, have received little attention in the literature prior to 6 1925, whereas the Str# faecalls Is well represented# The enterococci wsre not recognized, as such, in the majority of* the English text-books, and the descriptions of i#iis group in French and American works differed in some of the most important details from those of the Str# faecal Is1 , as well as from such descriptions of the enterococci as appeared in more recent literature* Mace (35) considered the enterococcus as identical with the Str# enteritis of Escherich, which appeared to be the chain-forming type described under the same name by Hirsh and Libman (24) • He stressed the pleomorphic morphology of the organism and further stated that it was able to grow at 46° G^; it constantly coagulated milk# never liquefied gelatin, in­ It was killed by an exposure of 15 to 20 minutes at a temperature of 60° C#, and did not act upon any of the sugars# Houston and McCloy (31) described an organism iso­ lated from trench fever patients and from suppurating wounds# Among other characteristics, they laid much stress on the ability of the streptococcus to withstand heat and found it capable of surviving an exposure of one and one-half hours to a temperature of 55° C# This may be comparable to an earlier observation of Logan (34) i who found a coccus in the feces of infants capable of surviving pasteurization for ten minutes at 80° G# Wright (68) isolated a diplococcus from a wound which 7 grew more luxuriantly than Str* pyogenes and was able to grow at room temperature* This non-hemolytic coccus was identified as an enterococcus and also as Str* faecalls# although Gordon1s tests did not appear to have been em­ ployed* This was the first time a connection between the two groups was made* Donaldson (14) in 1917 gave a brief summary of the characters of the enterococci* He noted that they gen­ erally grew in the form of pneumococcus-like diplococci, did not produce hemolysis, and produced acid from glucose, lactose, maltose, saccharose, raffinose, glycerol, mannitol and inositol* Weissenbach (61) in 1918 devised a differential test for distinguishing Str* faecalis from Str* pyogenes by employing a liquid medium containing 10 per cent bile* This medium supports the growth of the enterococci but not other streptococci* Bagger (4) utilized this observation and advocated the use of ox-bile with one per cent of peptone for the classification of the enterococci* Dible (13) in 1921 established the relationship and connection between the enterococci and the Str* faecalis* In a classical piece of work he also reported that the power of withstanding exposure to heat was not a property of all intestinal streptococci* By its use, these are divisible Into two classes: one of which largely consisted of organisms having fermentative reactions corresponding to 8 those of Str* faecalis* The other group consisted of types which frequently occur in saliva and included those raffinose fenaentors of the feces which are not thermoresistant and may be regarded as survivors of the salivary organisms* Alston (1) confirmed the work of Dible and showed that there is a clearly defined group of organisms sufficiently differentiated to be classified together as enterococci* The primary attributes of this group are: (1) cocci tending to be oval in shape and occurring in pairs or short chains, (2) heat resistant up to 60° G* for 10 minutes, and (3) non-hemolytic and capable of fermenting mannitol, as secondary and not invariable characters* Among the 51 strains of streptococci iso­ lated from the alimentary tract of man, dog, and rat, 16 or 31 per cent conformed to the description of entero­ cocci suggested by Dible* Holman (25) based a form of classification on the ability of hemolysin production and the ability to ferment lactose, mannitol and salicin* The streptococci derived by this method from feces were found almost entirely in five of the 16 types, viz*, Str* faecalis* Str* equinus* Str* mltis, Str* pyogenes and Str* infrequens* Almost half were in the Str* faecalis group* Welch (62) in 1929 indicated that there were six strains of streptococci common to human stools* Those 9 fermenting (a) all sugars used: glucose, lactose, sucrose, sallcin, maltose, mannitol and galactose; (b) all but sucrose; (c) all but sucrose and mannitol; (d) all but mannitol; (e) all but mannitol and salicin; and (f) all but lactose* Sherman, Mauer and Stark (56), in an exhaustive study of the enterococci, stated that because fermentation tests are extremely variable with Str* faecalis, there is con­ siderable confusion concerning the boundaries of this group and whether or nut one or more species are involved. Of 434 cultures identified as Str, faecalis all grew at 10° C, and 45° C, Other members of the enterococcus group, as defined by Bergy et ai, (5) grow ao these temperatures also (Str, zymogenes, Str, liquefaciens and Str, durans), In a later paper Sherman (55) also shows that only members of the enterococci grow in the presence of 6,5 per cent NaCl, The fermentation tests are diverse within the species and these characteristics are regarden by Sherman as of minor importance. He stresses the above two tests, the ability to grow at 4b° C, and in the presence of 6,5 per cent NaOl as major tests in the classification of these bacteria. Excellent reviews of the enterococci group, as well as its individual members, have been published by Sherman (55,56), In 1894 Laws and Andrews (32) reported for the first time that streptococci could be isolated from sewage- 10 polluted water# This observation was not emphasized until six years later when Houston (27, 28) stressed the fact that streptococci, as well as staphylococci, were charac­ teristic of sewage waste# The former being a more specific indicator of sewage pollution since they are wreadily demonstrated in waters recently polluted and seemingly altogether absent from waters above suspicion of contamin­ ation11# Streptococci were found in 0#1 to 0*001 ml of water from six rivers that were extensively polluted# On the other hand, eight rivers showed no streptococci in 0#1 ml, although ordinary chemical and bacteriological tests gave results which would condemn the waters# Horrocks (26) in 1901 found these organisms in great abundance in sewage and waters which were known to be sewage-polluted, but which contained no trace of Escherichia coll# Winslow and Hunnewell (63, 64) were the first to study this organism in America in 1902 and later reported the isolation of streptococci from 25 out of 50 samples of polluted water# Prescott and Baker (49) found these organisms present in each of 50 samples of polluted water* On the other hand, Winslow and Nibeeker (65) found strepto­ cocci by the direct plating method in only one of 259 presumably unpolluted water samples# Clemesha (9) in 1912 reported that in India, streptococci were present in 0 o001 to 0*00001 gram of feces, but were rare in waters that were not grossly polluted* Ostrolenk and Hunter (46), In a study on the distri- 11 bution of enteric streptococci, examined feces from the human, cat, mouse, guinea pig, dog, rabbit, chicken, flies, monkey and soil* TJsing Perry’s S*F# broth, 51 fecal and two soil samples were examined* The two soil samples were negative for both the enterococci and E* coll* Forty-nine specimens representing 10 animals contained enterococci while E* coli was present in only 46® Mallmann (36) reported that the streptococci were constant indicators of intestinal pollution and the number found in the swimming pool were parallel to the amount of pollution as indicated by the number of swimmers® It was also reported that the E* coli tended to multiply in the swimming pool while the streptococci did not* In a later paper, Mallmann and Sypien (43) compared the colon and streptococci indices of samples taken five feet from the shore of a bathing beach* It was found that while the colon indices and total plate count did not always respond to changes in the bathing load, the streptococci indices always did* The latter were not found at points free from the bathing pollution while the colon bacteria were# It was also reported that the streptococci disappeared overnight while the colon organisms and the total count sometimes showed an increase, although they were generally lower* Ritter and Treece (52) isolated 79 strains of strepto­ cocci from swimming pools* Fifty-two or 65*8 per cent 12 were classified as Str* faecalis; these were confirmed by the Lancefield technique and were further classified as Type D* Winter and Sandholzer (66) confirmed the work of Mallmann and Sypien in that they reported that coliform organisms persisted for a great distance from the source of pollution in water and the streptococci did not* Horrocks (26) found by experiment that E* coll gradually disappeared from specimens of sewage kept in the dark at the temperature of an outside veranda whereas the streptococci and staphylococci persisted* Prescott (47) showed that the streptococci often overgrew E* coli in a few hours when inoculated into glucose broth* This was contradicted by Prescott and Baker (49) in a later paper* Clemesha (9) found that streptococci disappeared very rapidly in water, within two or three days at the most, when stored in bottles in the laboratory or in an arti­ ficially polluted outdoor tank* Savage and Wood (53), in their study of the viability of streptococci in water, found that they died out in parallel with the coliforms, although a trifle faster* Prescott (48) in 1906 reported streptococci occurring on hay and grain* Moore (44) reported as early as 1893 that streptococci were frequently isolated from garden soils* The gardens, however, had been heavily and fre­ quently manured* Andrews (2) in 1906 stated that the streptococci cannot grow and multiply for any length of 13 time apart from the human body* Eighty-four samples of hay, grass, and leaves from country roadsides, pastures, park, and garden paths were examined# showed streptococci# Only two samples Soil and water from wood edges, moist railroad hanks, brooks, woodland humus, etc*, were examined# Only one of these eighteen samples, from a country roadside overflow, yielded a short-chained coccus organism# Broadhurst (6) in 1915 was of the opinion that strep­ tococci occurred less commonly in soils and water than most of the literature of that time implied* Sherman in 1937 reported, nUnpublished investigations have shown enterococci of the Stir* faecalis and Str# ljquefaciens types to occur rather commonly on plants* This may mean of course that these organisms were merely surviving, rather than growing, under these conditions”# Winter and Sandholzer (64) reported that while strep­ tococci were present in all samples of human and animal feces tested, these organisms were never found in virgin soils or in aoil<§ from wooded areas* Mallmann and Litsky (41), using dextrose azide broth as an enrichment medium, could not isolate enterococci from soils which were not treated with sewage* They also stated that other than the coliform organisms, the entero­ cocci were the only organisms found in sewage that could be used as indicators of fecal pollution# While the coli­ form organisms were found to persist in sewage-treated 14 soil, the enterococci were found to die out rapidly "but not as rapidly as virulent typhoid bacilli* It was also noted that the longevity of these three organisms In sewage-treated soils was prolonged with an increase of the organic content of the soil* Both solid and liquid media have been used to estimate the number of streptococci In water and soil* Prescott, Winslow and McCrady (50) suggest a litmus lactose agar on which the streptococci colonies are generally distinguished from the other acid formers by their size, structure, and the formation of a permanent deep red color* These colonies, however, may be overlooked due to their slow growth or if they are present only in small numbers* Representative colonies must be examined microscopically* In 1906 Prescott and Baker (49) reported that when E* coll and streptococci were grown In mixed cultures, E* coli reached a maximum growth before the streptococci but were gradually displaced by the latter 20 to 60 hours after the start of the experiment* From this time on, the streptococci predominated, on some occasions eliminating B* coli completely* Since similar succession of growth occurs In lactose broth, Mallmann and Gelpi (40) suggested the ordinary lactose broth enrichment tube be employed in a similar manner* After the usual coliform confirmation tests were made, the tubes are reincubated for 48 hours, centrifuged and the sediment examined for streptococci by microscopical 15 examination# Another method suggested by these authors is to allow the tube to stand at room temperature for one to three days after the initial incubation to permit sedi­ mentation* It was reported that a heavy sediment in the bottom of the tube, similar to that of the deposition in a macroscopic agglutination test, is an indication of the presence of streptococci, but this should be confirmed by microscopic examination* Mallmann and Cary (38) in 1933 recommended holding the Incubated tube to the light and looking for a granular precipitate, floes of which may adhere to the wall of the outer tube, or to the surfaces of the inverted vial* They stated that this indication is not always confirmed microscopically but that confirmation is not necessary If the granular precipitate Is absent# Houston (30), using the presumptive lactose tubes also, removed one ml to a nine ml blank, Incubated this dilution in a 60° C# water bath for 15 to 20 minutes, and subcultured on MacConkey agar* After 48 hours incubation at 37° C* the pin-point red colonies were transferred Into lactose broth, from this to the condensation water of a nitrate agar slant, and then streaked on the same slant# Acid production without gas from lactose, with a milky precipitate, usually signifies the presence of strepto­ cocci* Short-chain cocci in the condensation water of the nitrate agar slant, and the aosence of nitrate reduction confirms the presence of streptococci* This method Is still recommended by the Committee of the British Ministry 16 of Health. (10) * In 1918 Weissenbach (61) was one of the first to describe a selective medium for enterococci in contrast to hemolytic and non-hemolytic streptococci# ox-bile was used for the inhibitory agent* Sterile filtered Beggar (4) also used sterile ox-bile with one per cent peptone to grow fecal streptococci* As confirmation he suggested the heat resistance test* Fleming (17) in 1932 reported that fecal streptococci will grow in a concentration of lil5,000 potassium tellur­ ite which Is inhibitory to coliform bacteria as well as most other gram-negative bacteria# It was applied to water analysis by Harold (21) In 1936 when he incorpor­ ated this chemical In a solid tellurite agar* Fecal streptococci appear as bluish-black colonies, about one mm* in diameter, with a peripheral opalescence* These were studied and confirmed by the method described by Houston* In 1937 Harold (22) compared this medium to those used at that time and found that from a series of over 250 positive MacConkey broths, 13*9 per cent contained fecal streptococci by Houstonts heating technique, 30*6 per cent by the direct tellurite method and 44*0 per cent by the enrichment tellurite method# Hartman (23) in 1937 was the first to use sodium azide to suppress the growth of gram-negative bacteria while permitting the streptococci to grow* Since this discovery many Investigators have used this chemical in 17 media for the Isolation of fecal streptococci. Mallmann (37) reported a medium containing sodium azide, which was found useful in estimating the number of strepto­ cocci in sewage as It was found to support the growth of these bacteria while inhibiting the coliform group. Eajna and Perry (20) published another selective streptococci medium which was almost an exact duplicate of the medium suggested by Mallmann (37), but the former workers used an Incubation temperature of 45° C. Growth and the production of acid in this medium were stated to be almost complete evidence of the presence of Str. faecalis. Winter and Sandholzer (67) in 1946 published a pro­ cedure for detecting the presence of enterococci which is used by the U.S.P.H.S* at the present time* This method consists of a sodium azide - presumptive broth and a peni­ cillin -methylene blue-NaCl broth-*slant confirmation medi­ um. This method, however, must be confirmed by a micro­ scopic examination and a catalase test. A study of the Inhibition of gram-negative bacteria by sodium azide has been reported by Snyder and Lichstein (57). Folpmers (18) in 1940 recommended two media for the detection of fecal streptococci. The first contained litmus as the effective agent and should be employed In deep columns. Microscopical examination of the sediment must be made. The second medium contains one per cent caffeine. 18 The author stated that coliform bacteria do not interfere, but aerobic spore formers are sometimes troublesome# Chapman (7) in 1944 published formulae of two media for the isolation of streptococci in mixed cultures# Tellurite streptococcus medium contains crystal violet, trypan blue and sodium tellurite as active agents# On this medium, Str# salivarius' produces pale blue opaque colonies from 2 to 5 mm in diameter, Str# mitis produces blue colonies about 0#2 mm in diameter, while the enterococci give a dark brown or smooth black slightly-raised colony from 0*5 to 1*5 mm in diameter* Ninety-seven per cent of the transplants from feces were pure streptococci; the remainder were staphylococci* Azide violet blood agar medium contains S# T* 37, sodium azide and crystal violet as inhibitory agents* In this medium Str* mitis grows in colonies producing large halos, enterococci produce large blue colonies with halos In some, while Str* salivarius produces no halo# It was reported that with the Increase of sodium azide in this medium, more coliform bacteria were Inhibited but the size and number of streptococci colonies were also reduced# In 1946 Chapman (8) again perfected a medium for the Isolation of fecal streptococci* This was mltis-salivar- ius agar, a modification of his former medium. Entero­ cocci produces a dark blue or black slightly-raised colony about 1 mm in diameter, as contrasted to a blue "gum-drop*1 colony of Str* salivarius or the minute colony of Str* mitis* 19 Schuman and Farrell (54) In 1941 published a synthetic medium for the cultivation of Str* faecalls* This medium, however, is employed in vitamin and amino acid determinations* Mallmann and Seligmann (42), in a comparative study of media for the determination of streptococci in water and sewage, after a screening procedure, studied the 'following media: lactose broth, azide broth (Mallmann), S* F* broth (Hajna and Perry) and azide dextrose broth (Roth)* The results of this study can be summarized by the following: Average streptococci indices of river water were as follows: Lactose broth 930 Azide broth (Mallmann) S* F* broth (incubated 3700 at45° C*) Azide dextrose broth (Roth) 600 9200 The authors stated that the positive azide dextrose tubes should be checked microscopically because some of the gram-positive rods may show turbidity as well as the streptococci* It was concluded that azide dextrose broth offers a new means of testing for and measuring strepto­ cocci In water, sewage, and shellfish as well as other materials suspected of sewage pollution* 20 EXPERIMENTAL In order to formulate a confirmation medium for enterococei, a "base medium first had to he perfected that would not only support the growth of a minimal Inoculum of these organisms, hut would also permit them to demon­ strate a very short lag phase* Many Investigators are of the opinion that the lag phase is the most critical stage in the bacterial growth cycle* The following experiments were carried out on the assumption that the shorter the lag phase of a bacterial growth curve, the better the medium Is for the particular species* This point can be further demonstrated by the fact that, under optimum con­ ditions, bacteria multiply In a geometric progression, and that the more bacteria which are able to survive the critical lag phase, the better the chance for these organisms to begin multiplying, and the sooner the log­ arithmic growth phase will be reached* Using E* coli as the test organism, Mallmann and Darby (39) noted that brilliant green lactose base medium gave a more rapid growth rate when one or two per cent tryptose was added to the base medium# In order to ascertain the affect of various concen­ trations of the Ingredients in the selective media on the 21 growth rate of Stfr* faecalls In small inocula, the culture media listed in Table 1, were prepared# position is given for each ingredient# Percentage com­ Azide dextrose broth (Medium 7) was the only dehydrated medium used; the rest were made in the laboratory from the listed formulae* The procedure for determining the growth rate of the Str» faecalis is the same method which was used by Darby and Mallmann (12) and is as follows: A 24 hour culture, which was transferred each day for four days in brainheart infusion broth, was diluted so that between 20 and 100 organisms per ml were present# This was seeded into flasks that contained 100 ml of the respective media* Initial plate counts were made immediately after the original inoculation and every three hours thereafter, up to and through the ninth hour* These flasks were in­ cubated at 37° C# and were shaken for two minutes prior to plating* Azide dextrose agar, without the sodium azide, was used for the plating medium# All plates were incu­ bated for 24 hours at 37° C# before counting* This procedure was repeated five times in order to determine the medium that would best support the growth of the test organism# Each trial yielded similar results and a typical set of data is shown in Table 2# A close examination of these data revealed three outstanding facts: the toxicity of a large concentration of salt, which will be discussed later in the paper; the toxicity of the dehy- 22 •ri *d t- 10 • H to • O 03 O lO • rH to • H O« 10 © 03 rH to • H IS 03 • O IS 03 ♦ O 03 O • O g •o H •d 10 •P :S w o« to • rl to • O fr03 • O IS 03 • O 03 O • O to • tS 03 ♦ O IS 01 • 03 O • O > E* G © © pq © O © P © d o m O © g rH «k O to o•k to a p O to H* a p •H o to o> •k rH © a g o to 00 •k H to H to 03 o o t- © a g P •H •d H © a o•k to o o o •k rH to o o o «k o 00 to o o o•k 05 rH to "d to p •H *d 02 © a O O O O O •k o 02 H to Q o •k 03 O O o * to to rH 1> H to I> O O o> •V 02 O o o•k {> t- H COUNTS OP STR. FAECALIS ON THE VARIOUS EXPERIMENTAL BASE MEDIA 23 PLATE a o •H •P ctj © £J »H O 43 H O to to O 24 drated medium; and the advantage of buffered media# Medium 3 contained three per cent sodium chloride and no buffering agents# The inhibition of growth was quite evident after three hours of incubation when compared to the other media# Medium 6 contained one and one-half per cent sodium chloride and buffering agents* It did not support the growth of the test organism as well as Medium 5 which was of the same composition but only contained one-half per cent sodium chloride# It was observed that Medium 7 made from prepared dehydrated material, showed the lowest count other than Medium 3 after six hours of Incubation* It should be added that Medium 1 is of the same composition as Medium 7 but was made up from the Ingredients In the laboratory* This medium was not as inhibitory as was Its dehydrated counterpart# As is demonstrated In Table 2, Medium 1 showed a count slightly less than double that of Medium 7 after six hours* After nine hours of incubation Medium 1 contained 177,000 organisms per ml while Medium 7 contained only 54,000 per ml# Of the three media which contained buffering agents, Media 4 and 5 were of the same composition, with the exception that the former contained one-half per cent lactose while the latter contained one and one-half per cent dextrose* Upon examination of the data, It was noted that these two media were the best of the experimental 25 base media for the support of Str# faeoalis growth* Upon further repeated investigations, Medium 6 containing dextrose, was judged the better of the two* It is this medium that was employed as a base for the further experi­ mentation for a selective confirmation medium for the enterococci* In an attempt to Increase the inhibitory action of the medium for the gram-negative bacteria, as well as to determine the limiting concentration for the enterococcl, five media were prepared with a concentration of sodium azide ranging from 0*00 to 0*10 per cent# An actively growing culture of Str* faecalis was seeded into the above media In minimal inocula and growth curves were plotted as previously outlined# The results of this investigation, listed in Table 3, Indicated that a concentration of 0#05 per cent and above demonstrated marked Inhibition* It would seem that sodium azide increased the lag phase so that at the end of 24 hours no visible growth was observed in the medium with a concentration of sodium azide as low as 0*02 per cent* The control, base medium minus sodium azide, demonstrated normal growth and turbidity at the end of 18 hours of In­ cubation# The above results Indicated the necessity of extending the incubation period to 48 hours* Media were made with the concentration of sodium azide ranging from 0*00 to 0.05 per cent* Again these media were tested by growth 26 O HI P P S w w *© © o Hi (S3 -aj a p Hi to a PQ EH p co §s p o o « Hi e> eh c Ho i Eh H i P 02 o H to o o o 02 02 O 0> o 02 to o o o to o o o o 02 o o to •* 02 o o o •k o> 02 H o o o o o to * to o o o * o o o •» o to t0 02 P 8 O *s O o H o o o o o to El © O S o o O O O * rl O at *H O PQ « Eh g COI Hi to •H •H g CO EH to o • © 05 02 O * © Pi « 0 O «tf CO s H33 f Oe 1H to O O o * o o o g to o a M p A Q H O to E3 O O P P HI 0> o to P H < Eh W P EH oo ,©• Ph ^ © <+H tS ° o o o * o o o •v 0 to 01 0 0 •H P © 1El •H H CO H 27 curves as previously described* The results of this investigation, shown in Table 4, indicated again that the growth peak for the control, Medium 1, was 18 hours# The growth peaks for media con­ taining 0#02 to 0#03 per cent sodium azide was 42 hours, while for those containing 0*04 to 0#05 per cent, it was 48 hours or more# The medium with 0#05 per cent was the only one in this group that did not demonstrate visible growth after 48 hours of incubation# It must be stated here that 0#02 per cent sodium azide was found to inhibit the coliform bacteria and this same concentration is used in dextrose azide broth as an inhibitory agent# It was felt that a greater concentra­ tion of sodium azide, which would allow Str# faecalis to grow and yet inhibit the gram-negative organisms, should be used in a confirmatory medium# Therefore, the medium containing 0#04 per cent sodium azide was chosen as a base medium for further investigations# It can be argued that 0#02 and 0.03 per cent sodium azide could be used but the results Indicated that after 36 hours there is no appreciable difference in growth of the test organism using the two concentrations# It was found that the above medium, as is the case with dextrose azide broth, demonstrated Inhibition of the gram-negative bacteria but allowed some spore-formers, such as Bacillus subtilis, to grow. For this reason the 28 o to 02 O iO 03 o to HI Q O H* o o 03 ID o• o o o•4 o o> HI o o o*k o o o•k o rH +5 £ o O o O o*k 02* rH o o to•k 02 • H* •k £> Pi •H U IO 59 O • o o 03 «k to O O O •* IO H o o o*k to CO H o 03 o o o«\ o o o•k o to to o O O o O O o•k O•k O «k o o o o o o o•k o•k o •k o co o to to o IO to to 02 to to d o ra 3 rH 03 • 'rf* • co • rH rH rH • erf 03 +3 COS t o 'rf* 02 • 'rf* •H CO • TO GROWTH .Cl 43 shH © 'rf* 03 PS O •H E4 ■g O O LO rH •rf & o CO 02 31 hibit the enterococcus used as a test organism* To carry out this experiment B* subtilis, Staph* aureus, and Str* faeoalis were used* Twenty-four hour broth cultures of the above-listed bacteria were mixed and 0*1 ml of the resulting mixture was inoculated into 10 ml of base broth* Incor­ porated into this base broth were various concentrations of dyes as shown in Table 6* These were incubated at 37° C* for 24 hours and examined microscopically* The following represent the highest concentration of the dyes which supported the growth of Str* faecalls (but not B* subtilis or Staph* aureus): crystal violet, 1:700,000; ethyl pur­ ple, Is800,000; brilliant green, 1:1,500,000; malachite green, 1:1,000,000; and methyl violet, lsl0,000* Various concentrations of the dyes were incorporated into a base medium containing 1*5 per cent agar* Plates were made and divided into sections, in which were streaked a loopful of the above cultures* incubated at 37° C* for 48 hours* was recorded as positive* These plates were then Growth on the plates The results of this investi­ gation are shown In Table 7* Prom the above table certain facts are evident* B* subtilis Is not completely Inhibited at a 1:600,000 con­ centration of crystal violet, while the same dye Inhib­ ited Staph* aureus at 1:1,000,000 concentration* Str* fae calls exhibited growth at 1:600,000* Ethyl purple inhibited both B* subtilis and Staph* aureus at a concentration of 1:800,000 while no effect TABLE 6 TWENTY-FOUR HOTJR MICROSCOPIC EXAMINATION OF BASE MEDIA CONTAINING DYE INOCULATED WITH A MIXED CULTURE str# Dye and dilution Crystal violet faeoalis B. subtilis Staph* aureus 6002? - - - 700T / - - 800T / - - 1M / - - 1.2M / - mm 800T / - - * 1M / - mm 1*2M - - 1»5M / / - - 2M / mm - Brilliant green 1*5M / - - 2M / - - 2*5M / / - 3M / / - 4M / / - 1M / - mm 1*2M - - 1#5M / / - - 1*7M / 2M / / - 10T / - - 25T / - - SOT / / - 100T / Ethyl purple Malachite green Methyl violet - - 33 TABLE 7 FORTY-EIGHT HOUR PLATE ANALYSIS OF GROWTH ON MEDIA CONTAINING DYES Dye and concentration Crystal violet Str. faecalls B. subtilis Staph* aureus 600T £ £ - 700T / - 800T / £ £ 1M 1.2M £ £ £ / 800T / - - 1M / £ 1.2M / / 1.5M / 2M / £ £ Brilliant green 1.5M / 2M / 2.5M / 3M / 4M / 1M / 1.2M £ 1.5M / 1.7M / 2M / 10T - 25T / 50T / 100T / Ethyl purple Malachite green Methyl violet 9m £ £ £ £ £ £ £ £ £ £ £ £ £ - £ £ £ £ £ £ £ £ £ £ £ £ £ - £ 34 was demonstrated on Str* faecalls* Brilliant green inhibited B. subtilis at lrl,500,000 but did not have a complete inhibitory effect on Staph* aureus at this concentration* Str* fae calls was nou in­ hibited at the above concentration* Malachite green, on ohe other hand, inhibited the growth of Staph* aureus at a concentration of l:l,000,00u but allowed B* subtilis as well as Str* faecalis to grow* Methyl violet inniDitea the Str* fae calls at a con­ centration of 1:10,000 but did not inhibit B* subtilis or Staph* aureus» It must be adaea that the above experi­ ments were used only as rapid screening methods* There was a possibility that some of the light growth, reported as plus-minus, could have been due to the carry-over of media on which the organisms were cultured* All things considered, the results of these experiments indicated that a lr800,000 dilution of either crystal violet or ethyl purple could be used to inhibit the growth of the bacilli or staphylococci without effecting the growth of the streptococci considerably* The investigation thus far suggested that the base medium with 0*04 per cent sodium azide and a 1:800,000 of either crystal violet or ethyl purple might be used as a selective medium for Str* faecalis* To Investigate this possibility further and also to determine which Is the better dye, the above media were prepared; one containing 35 crystal violet, the other ethyl purple* Raw sewage, polluted water from the Red Cedar river, and soil were collected and the streptococci indices were determined using dextrose azide broth in triplicate for each dilution* Readings were made after 48 hours incubation at 37° C* and confirmed by microscopic examination* The following most probable numbers of streptococci were obtained from the dextrose azide broth presumptive tests: Source Visible MPN Microscopic MPN Raw Sewage 9,200,000 9,200,000 430,000 43,000 27,000 0 River Water Soil Comparing the visible MPN with the microscopic con­ firmation, it was noticed here, as well as by Mallmann and Seligmann (42), that dextrose azide broth supports the growth of organisms other than streptococci, especially in soil samples* In order to test the above two media as confirmatory media, three loopfuls of broth from each dextrose azide broth tube above media* were seeded into tubes of the After 48 hours incubation at 37° C* these were read visibly and confirmed by microscopic examination* Results of the visible growth are as follows: Source Ethyl Purple Raw Sewage 9,200,000 4,300,000 43,000 43,000 0 0 River Water Soil Crystal Violet 36 It can bo soon that the confirmation in ethyl purple azide broth was exactly the same as the microscopic readings on the dextrose azide presumptive test* The crystal violet broth displayed some toxicity in the confirmation of the raw sewage sample but was similar to the other medium usually* A compact purple button formed by the sediment seemed to be specific In all cases of positive growth. This was repeated and similar data were obtained* In order to test the specificity of these two media the following experiment was performed* Cultures of various micrococci, Isolated by Mr* Edward Seligmann and Miss Lisa Neu of this department, were seeded into the confirmation media* After 48 hours Incubation at 37° C* observations were made for growth* The results of this experiment, as shown In Table 8, demonstrated the speci­ ficity of the ethyl purple azide broth, in that it only supported growth of the Str* faecalls (Nos* 1, 8, 9, 10, 11, 12, and 40) while the crystal violet azide broth sup­ ported the growth of these organisms plus three strains of Staph* aureus and possibly Str* mitis (Nos* 5, 6, 13, and 17) • Since most of these cultures were only partially Identified the certainty of the species is dubious* Such strains are Indicated in Table 8 by a question mark* Cultures 23-36 were not identified but believed to be members of the buccal group of streptococci* 1 -4 Cultures were obtained from the American Type Culture Collection* 37 TABLE 8 GROWTH OP VARIOUS ORGANISMS IN CONFIRMATION MEDIA (48 Hrs.) No# Organism 1 2 3 4 5 6 7 8 9 10 11 12 13 Str# faecalls Str# salivarius Staph# aureus Str# mitis Staph# aureus Str# mitis ? Staph# aureus Str. faecalls Str# faecalls ? Str# faecalls ? Str# faecalls Str# faecalls Staph# aureus or Str# faecalls Str# mitis ? Staph# aureus ? Staph# aureus Staph# aureus or Str# faecalls Str. salivarius 15 16 17 18 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 Staph# aureus ? Isolation ATCC ATCC ATCC Crystal Violet /// /// River tt n n it % Pool !9 It W It tt It It ft ft If tt ft It tt tt If ft ft ft ft Staph# citreus Staph# epiderleus Staph# aureus Str* faecalls Proteus vulgaris Ethyl Purple Miss Neu tt tt tt it 38 On the basis of the results obtained from the pre­ ceding preliminary investigations, ethyl purple was found to be a better selective agent, for the gram—positive bacteria than was crystal violet# The latter dye was also found to be more toxic when used in a confirmation medium than was ethyl purple# On the basis of these find- ings, a more complete investigation of ethyl purple azide broth was carried out in order to prove the merits and dis­ cover its limitations# The formula for this medium, which will be used in the following experiments is as follows: Ingredient Grams per liter Tryptose 20 Dextrose 15 Sodium chloride 5 KgHP04 2 #7 KH^P04 2#7 Sodium azide 0#4 Ethyl purple 0#00125 pH - 7.0 Sterilized at 121^ C# for 15 minutes To investigate the specificity of ethyl purple azide broth more thoroughly, actively growing cultures of the following organisms were seeded into this medium: Pseudo­ monas aeruginosa, Staphylococcus aureus, Staphylococcus auranuica, Staphylococcus alous, Sarcina citreus, Sarcina lutea, Serratia marcescens, Proteus vulgaris, Escherichia 39 ooli , Es cheriohia communior, Salmonella typhinrurium, Sal­ monella typhosa, Shigella alkalis cum, Micro coccus agilis, Bacillus subtilis, Bacillus cereus, Alkaligenes faecalis, Cfaromobacter violaceum, and Streptococcus faecalis# After incubation at 37° C# for 43 hours, these tubes were examined for visible growth. Str# faecalis was the only species which showed visible growth# Prom the previous data it was quite obvious that 6thyl purple azide broth supports the growth of streptococci but further information was needed to find its specificity limitation within the streptococci group# For this, actively growing cultures of streptococci, acquired from Cornell University and Iowa State College, were seeded into the test medium* Observations for visible growth were made after 48 hours of incubation at 37° C# It was found as shown in Table 9, that only the enterococci, Str# faecalis, Str# durans, Str# liquefaciens, and Str# zymogenes, demon­ strated growth at 48 hours# Str# bovis grew out after five days and the rest did not show any visible sign of growth after a week of Incubation# This set of data was repeated with similar results# It showed that ethyl purple azide broth was not only specific for the growth of the streptococci, but it was specific to the extent of supporting the growth of only the enterococci# Again it was noted that all the enterococci demonstrated a compact purple button on the bottom of the tube after 48 hours# 40 TABLE 9 GROWTH OP VARIOUS STRAINS OP STREPTOCOCCI IN ETH3CL PURPLE AZIDE BROTH G-rowth after 48 hours incubation Species Source Str# bovis Bovine mouth - Str# faecalis Intestine / Str# durans Intestine / Str* liquefaciens Intestine / Str# mitis Mouth - Str# zymogenes Intestine / Str# equisimilis Pig heart valve - Str# laotis Milk - Str# thermophilis Milk - Str# salivarius Milk - Str# agalaetiae Milk - Str# dyagalactiae Milk - Str# uberis Milk - Str# canis Dog - - Str# equi MLB Porcine abscess - 419-9 Pig liver - 41 UnidentIfied streptococci cultures were classified as to whether they were enterococci by their ability to grow at 45° C* and in the presence of 6*5 per cent sodium chlor­ ide* Tryptose phosphate broth was used as the base medium in both cases* These cultures were also seeded into ethyl purple broth, Perry's S# F* broth, Winter and Sandholzer presumptive medium and then positive tubes were confirmed in Sandholzer*s confirmation broth-slant* Perry*s S* P* broth and Winter and Sandholzer*s presumptive broth were incubated at 45° 0* while ethyl purple broth and Winter and Sandholzer*s confirmation medium were incubated at 37° C* All tubes were incubated for 48 hours with the exception of those containing Winter and Sandholzer* s pre­ sumptive medium (directions call for 8-12 hours of incu­ bation) which were incubated only 24 hours* The results of this test indicate, as shown in Table 10, that those strains having the characteristics of fecal streptococci, growth at 45° C* and in the presence of 6*5 per cent sodium chloride, grew in ethyl purple azide broth, with the exception of cultures 17, 18, and possibly 33* Hone of the enterococci showed acid production or visible growth in Perry's S* F* broth, while only Culture No* 25 was confirmed by the Sandholzer method* in dextrose azide broth* All cultures grew These data indicate the selectiv­ ity of ethyl purple azide broth for the enterococci as compared to other methods* The toxicity of these latter 42 3 § Hi Eh H gj § O tt «H P *3 tt O MO rH O A • •Cf Pi P S t tp co tt © Sh PH 1 1 1 i 1 * i t i i i i t '" k i i i i | t i i t ■i i i B o H s £3 H tt fr* P • * i *> i i■ii ititi iiiitiii ii ii i tt co Ph M O O O o o EH Pt O H ES 9 EH Eh CO tt H rH Pi & P -P 3 W pL, (x, O CO (H 3 EH •PH H O tt CO .P +3 e o jco • cSb § ■P tt • H w o PS Ct5 O LO •3° %O rUH3 P h H X X x 'Sk ' V i O k I "X. I I H s ' I O C C k I 'Sfs. I l l V k 1 X 1 I tb s § CO t-i PS <*? o o © § • +3 O r^iZJ O H ' ^ ^ O i O C Q L O £ > 0 0 0 » O H 0 3 i O l > ‘ COC3>OrHOH03tO r HrHi Hr HH rH 02 0J Q2 C2 Cv i 02 03 fc Ol Qt Ql Ot Ot 0r j* r Hr Hr H 43 media la clearly demonstrated* In order to test the effectiveness of ethyl purple azide broth as a confirmatory medium for the enterococci the following Investigation was carried out* Samples of water were taken along the Red Cedar River at bridges and points at which sewage entered the river* samples were also collected* Raw sewage The river water (undiluted in ten-fold dilutions up to 1:1,000,000) and sewage samples (diluted in ten-fold dilutions up to 1:10,000,000) were seeded In dextrose azide broth, incubated at 37° C* for 48 hours* Microscopic examinations were made after 48 hours and used as a control for the positive streptococcus growth* Three loopfuls of the dextrose azide broth were then seeded Into the ethyl purple azide broth, incubated at 37° C* for 48 hours* Microscopic examinations were also made of the 48 hour confirmation tubes* One hundred and sixty-four samples of river water were examined by this method* A representative sample of the data is shown In Table 11* While this table does not include all of the results obtained, those not included tend to show the same trend* Ethyl purple azide broth confirmed the microscopic positive tubes of dextrose azide broth* It was also found that all positive confirmation tubes only contained streptococci when examined microscopically* Again it was noticed that the growth after 48 hours In ethyl purple azide broth 44 TABLE 11 RESULTS OF USING ETHYL PURPLE AZIDE BROTH AS A CONFIRMATION MEDIUM FOR STREPTOCOCCI IN TERMS OF M.P.N. Site of Sampling Sample Dextrose Azide Presumptive Visible Railroad Bridge . 4,500 9,500 4,500 45,000 45,000 45,000 4,500 9,500 950 45,000 20,000 25,000 4,500 9,500 9,500 95,000 110,000 20,000 X* 4* 4,500 9,500 4,500 950 25,000 2,500 4,500 4,500 4,500 950 2,000 2,500 4,500 2,500 140,000 45,000 2,000 2,500 F. L. R. Y. 5. 12. 2,500 25,000 45,000 140,000 2,500 2,500 4,500 95,000 45,000 14,000 2,500 500 4,500 25,000 95,000 20,000 250 1,150 Io 0* U. 4,500 140,000 45,000 25,000 150,000 9,500 140,000 45,000 9,500 25,000 4,500 140,000 95,000 25,000 35,000 C. 3 P. V. 3. 9* Women* s Gym Bridge Hotel Bridge Kalamazoo Bridge Microscopic Ethyl Purple Azide Confirmation Visible D. E. K. z. 8. 45 appeared as a compact purple button on the bottom of the tube® Since some streptococci were found microscopically In the 48 hour dextrose azide tubes, but were not con­ firmed in the confirmation medium, an investigation was carried out to determine whether these were of fecal or non-fecal origin# Tubes which were not confirmed were diluted by loop dilution, and plated on brain-heart infusion agar# After incubation at 37° C# for 24 hours, colonies were fished and purified in tryptose phosphate broth# After purity was established these cultures were grown at 45° C# and also in a 6,5 per cent sodium chloride broth# The results indicated that of the 82 cultures checked only 7 were classified as fecal streptococci by Sherman1s method of classification# Of these 7 cultures, five showed typical enterococci growth when reinoculated in the confirmation broth* These false negatives in ethyl purple azide broth may have been due to faulty laboratory technique when seed­ ing from the presumptive to the confirmatory medium# These results indicated that ethyl purple azide medium inhibited not only bacilli but also non-fecal streptococci while allowing the enterococci to grow readily# To further prove this, tubes which did show typical growth were picked at random and seeded in tryptose phosphate agar by loop dilution# above# Cultures were isolated, purified and tested as Of the 116 cultures tested by this method only 46 three typed out as non—fecal streptococcic One culture grew in the presence of 6*5 per cent sodium chloride and not at 45° C#, while the other two did not grow in either test* It must he reported, however, that these cultures were isolated from plates that demonstrated fecal strepto­ cocci# Also, these three cultures did not produce a typical purple "button in the confirmatory medium "but showed very scanty growth upon reseeding# To compare the dextrose azide-ethyl purple azide broth method, for the detection and confirmation of entero­ cocci with present-day methods, samples of river water and sewage were seeded into these media and confirmation pro­ cedures carried out as previously described* The results of these studies, as shown in Table 12, proved that the ethyl purple azide confirmation method was far better than Perryfs or Winter and Sandholzer's methods# In every case, with the exception of sample 21, ethyl purple azide de­ tected and confirmed from 100 to 1000 as many enterococci as did the above methods# Samples 6 to 12 were ta&en from a relatively unpolluted stream. The results of both Perry1s and Winter and Sandholzer!s methods indicate no fecal streptococci in any of the six samples when the ethyl purple azide method detected two samples, 11 and 12, with strepto­ cocci indices of 73# Samples 1 to 6 and 36 show higher indices in Perry's than in Winter and Sandholzer's method but in the case of the remaining 35 samples Winter and 47 TABLE 12 THE M.P.N. OP ENTEROCOCCI WHICH WERE DETECTED AND CONFIRMED BY VARIOUS METHODS Sample Number Perry1s S. P. Method Winter and Sandholzer1s Method 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 91 91 36 36 240 91 0 9 0 0 0 0 0 0 0 0 0 0 0 0 0 91 240 7.3 0 40 91 0 0 40 240 91 0 0 0 91 0 40 0 0 36 0 0 0 0 0 0 0 0 0 0 0 0 36 0 0 73 91 240 150 1,470 36 90 150 210 70 450 450 1,500 450 3,000 40 0 40 40 40 Dextrose Azide and E.F.A* Broth Method 920 430 430 740 920 150 0 0 0 0 73 73 480 240 920 430 9,200 2,500 140 920 240 2,500 4,300 91 1,500 700 4,500 4,500 2,000 25,000 20,000 25,000 110,000 2,500 15,250 15,000 35,000 20,000 tj 48 Sandholser* s method detected as much or more enterococci than did Perry* s medium* It must be noted here that microscopic readings of the dextrose azide presumptive broth invariably resulted in higher indices than did the readings of the other two presumptive broths* Positive confirmation in the ethyl purple azide broth was noted by the compact purple button In the bottom of the tube# 48 hours of incubation* This was easily noticed after The tubes must not be shaken before this characteristic growth is noted# 49 DISCUSSION Many investigators, especially those In England, have demonstrated that the enterococci can be used as Indicators of fecal pollution* Mallmann and Lit sky (41) have shown that these organisms can be used to test recent pollution in soils where the coliform bacteria are normally found, as well as In water* However, in the light of these reports, results have been published to the effect that the entero­ cocci are not good test organisms for pollution because so few could be isolated as compared to the coliform organisms* This belief has been dominating the Investigations for the past fifty years and consequently the enterococci were never given their proper role as test organisms* The primary reason for not using the enterococci for testing for pollution was that there never has been a satisfactory medium that could be used for the isolation of these organisms* If and when these organisms were detected a complicated identification procedure had to be employed for their confirmation* It was not until 1937 that Sherman proposed the two test methods for the identi­ fication of enterococci* Prom 1930 on, numerous media were reported in the literature fort he detection of fecal streptococci* These, 50 however, were too toxic for the detection and demonstration of the actual number of streptococci in a sample of water, especially if the water was not grossly polluted* Conse­ quently, those that did confirm were so few in number that the test was not practical* These media had to be confirmed by microscopic examination, which only tended to complicate the method as well as to add more work* In 1950, Mallmann and Seligmann published a paper in which they showed that azide dextrose broth was the best test medium for the quantitative determination of strepto­ cocci as compared to the media used at that time* This medium had to be confirmed by microscopic examination also* It was felt that a microscopic determination defeated the purpose of this test because it required a trained tech­ nician and valuable time to prepare the stained slides and to examine them. The average water analysis laboratory unfortunately is poorly equipped and cannot employ a trained bacteriologist for routine analysis* It can also be stated that these workers have little time to examine confirmation slides* Another objection to microscopical confirmation is that streptococci may take any coccus-like form Including that of micrococci, diplococci, sarcina, staphylococci, etc* and conversely* It also must be added that It Is very easy to call a smear negative for streptococci and find them on further examination* It can be stated further 51 that -under the microscope all streptococci look alike and that fecal streptococci can not be differentiated from nonfecal streptococci* Because of these objections, a medium was sought to confirm the dextrose azide positive tubes without the use of the microscopic examination* The preceding work in the development of ethyl purple azide broth is self-explanatory* It should, however, be noted that when 0*04 per cent of sodium azide was used in the base medium, a 48 hour incubation period was necessary for the visible growth determination* When the ethyl purple dye was incorporated into the medium, most of the positive tubes were observed in 24 hours or less* This suggests that the dye is in some way decreasing the toxicity of the sodium azide* The same phenomenon was reported in brilliant green bile medium, where the bile decreases the toxicity of the brilliant green dye concentration used* To test the effect of dyes from various sources on the selectivity and toxicity of the medium in which they are employed, three batches of ethyl purple azide broth were made, each having the same ingredients with the ex­ ception of the dye source* When toxicity tests were carried out, it was found that media made from the dyes from National Aniline, Difco Laboratories, and Haricco showed different toxic effects* The media made with the dye received from Difco Laboratories supported the growth of Str* faecalls as well as dextrose azide broth, which was 52 run as the control* All five tubes supported growth, when one ml of the culture containing approximately 1*56 organ­ isms was seeded in the broth tubes and three out of five tubes showed growth when they were seeded with one-tenth of this number of organisms* The dyes from National Aniline and Haricco demonstrated more toxicity than the above* National Aniline dye* which was used in the devel­ opment of ethyl purple azide broth* showed four of the five tubes positive when one ml of the dilution (containing approximately 1*56 test organisms) was seeded* and only two positives out of five when one-tenth of the above dilution was used* The medium made up with Haricco dye showed four out of five positive tubes in the first dilu­ tion and none in the second* All showed five out of five positive tubes when dilutions were used containing 15*6 organisms per ml or more* Litsky, Mallmann and Fifield (33) showed that these dyes differ in actual dye content very greatly* Using spec topho tome trie analysis* they showed that the dye pre­ pared by Haricco contained the greatest amount of active dye content of those examined* When these results were compared with the biological toxicity results it was dis­ covered that there is a correlation between the toxicity of the ethyl purple azide media and the actual dye content of the dye* Haricco dye* with the largest content of active dye* was found the most toxic while the dye obtained from 53 Difco Laboratories containing the least amount of active dye was found the least toxic. Until further investiga­ tions are carried out as to the dye content, and until this dye is certified by the Certified Dye Commission, a con­ centration of 1:800,000 or 0.00125 gram per liter of ethyl purple dye produced by the National Aniline Company should be used in this confirmation medium# The above results also show that very few organisms are required in the initial inoculum for growth in the medium* This indicates that although there are two inhib­ itory agents in the medium, together they demonstrate very little effect on the growth of enterococci* A word of caution as to sterilization of this medium should be emphasized at this time. It was noticed that when the medium was sterilized for more than 15 minutes at a temperature of 121° C* browning took place and the longer the medium was sterilized the more the browning. Toxicity tests, in the same manner as described above, were made using a lot of medium that was sterilized for 15 minutes as a control. It was found that the longer the sterili­ zation process was prolonged the more toxic the medium proved to be# It must be stressed that this medium should be sterilized only for 15 minutes at 121° C. for its great­ est efficiency* As was reported in the preceding sections, ethyl purple azide broth supports the growth of the enterococci and in- 54 hlblts all the other bacteria that were tested* The specificity of this medium for the fecal streptococci can be put to much use in the field of medical bacteriology* With the use of this medium an enterococcus can be disting­ uished very rapidly and easily from that of an hemolytic or pyogenic streptococcus without employing a long series of media and reagents necessary for the separation at present* Streptococcus faecalia has been named by Dack (11) as the causative agent in many food-poisoning outbreaks* Ethyl purple azide broth may find a major role in this field, thereby, making it possible for the investigator to trace this causative organism very rapidly and without too much effort* It has been philosophized for many years that the fecal streptococci have a common characteristic which separates them from the rest of the group* Sherman found that these bacteria are the only streptococci which will grow at 45° C* and also In the presence of 6*5 per cent sodium chloride* The results of these investigations In­ dicate another characteristic common among the enterococci, the fact that they will grow in the confirmatory medium while the other streptococci will not* This investigation was not made to determine whether this ability is due to tolerance or an enzyme system* It does, however, suggest very strong­ ly, a common characteristic among the enterococci, and the 55 reason for this may he found in a further investigation of this medium# A new method for the detection and confirmation of fecal streptococci in water* sewage and soil has been suggested* This method employs dextrose azide broth as a presumptive medium and ethyl purple azide broth for con­ firmation* Using the microscopical examination data as the control, it was demonstrated in the preceding sections that ethyl purple azide broth satisfactorily confirms the posi­ tive presumptive tubes that contain enterococci* In fact it may confirm a larger number of those than are called positive by the presumptive microscopical examination as can be seen in Table 11# This may be due to mistakes in the smear examination, in that the streptococci are con­ fused with other forms or that they were so few In number that they were not detected* It also may be due to the original condition of the organism when first isolated# Weak strains may take 48 hours In dextrose azide broth to be revived* These few organisms may not be detected by the microscope* When they are transferred to the confirmation medium It Is possible that they may be revived to the extent that they multiply more rapidly and consequently show visible growth at the end of an additional 48 hour period# Dextrose azide broth presumptive tubes, showing streptococci by microscopical examination, but not growth in the ethyl purple azide broth, were plated and cultures isolated and classified# Of these only seven out of 82 56 cultures which, were studied were classified as fecal strep­ tococci. Five of these seven demonstrated typical growth on subsequent inoculation into the confirmation medium# The absence of growth in the primary confirmation medium might be attributed to faulty laboratory technique in the transfer# Of those not confirming, the majority were classified as micrococci; although staphylococci, diplococci and tetrads were frequently encountered# The above method was compared with the former methods used for the detection of enterococci and it was found that it was superior to the Hajna and Perry method and also wThe Winter-Sandholzer Enterococcus Test11 in that it detected indices from 100 to 1000 times as great# One reason for the toxicity of these established tests might be due to either the inhibiting agent in the primary enrichment medium or the 45° C# incubation temperature# It is logical to surmise that the more optimum the growth conditions are, the more organisms will survive the critical lag phase of the growth cycle# The primary medium should be employed only to enhance the growth of the test organism and not to Identify It# When growth occurs and a large number of these organisms are present, then and then only should con­ firmation be attempted# Both Hajna and Perry*s S# F. broth and Winter and Sandholzer*s presumptive broth are incubated at 45° C# It Is true that the enterococci grow at 45° C# but this temperature is not the best temperature to grow a very small number of bacteria as often occur in 57 water and soil samples# Dextrose azide, on the other hand* is incubated at 37^ C# and results in a larger index# Winter and Sandholzerfs confirmation medium was compounded with the idea that if t he enterococci are able to survive 6#5 per cent sodium chloride, 0#1 per cent methylene blue and penicillin, then these organisms should be able to grow in a medium containing all three# This is not the case# As the experimental data show, this medium is far too toxic to grow fecal streptococci when seeded in pure cultures, let alone enterococci found in water# Ethyl purple azide broth, on the other hand, showed visible growth when approximately 1#56 organisms were seeded into it# In ordinary routine analysis this is not important because dextrose azide broth increases the number of organisms and a very large inoculum is transferred for confirmation# The following paragraphs were taken from a preliminary report of a ”Tri-State Survey of Lake Michigan Waters11 by the United States Public Health Service (60) and demon­ strates the attitude at present concerning enterococci as test organisms for pollution;: "The Enterococcus Test of Winters and Sandholzer was made simultaneously with the coliform exam­ ination of each sample# Without evaluation at this time of Its usefulness as a pollution in­ dicator, the following summary describes the results* nl. The enterococcus group was isolated from most sample points at one time or another during the period of the survey, including areas where the sanitary survey showed no evidence of im­ mediate sewage pollution# 58 !,2. In areas where the sanitary survey showed no evidence of immediate sewage pollution, the density of the enterococcus was extremely low. Generally the results were negative to the largest portion examined, In frequent instances the most probable number of organisms exceeded 3*6, but rarely did they exceed 43. * . . w3. The enterococcus density was much higher in areas where fresh sewage or treated sewage effluent entered the waters of the area. The most probable number of organisms exceeded 3.6 more than 50 per cent of the time, fre­ quently exceeded 43, and in some Instances exceeded 240. n4. The highest enterococcus densities occurred at the outlets of Pettibone and Sunderland Creeks in Lake County, Illinois. At these two points the density exceeded 240 per 100 ml in 60.4 per cent and 33.3 per cent of the samples examined* ,f8* No characteristic enterococcus-coliform ratio existed for the entire section of Lake Michigan waters studied. However, it is apparent from the results secured that the enterococcus deter­ mination is a less sensitive measure of bacterial densities of waters than is the coliform deter­ mination. n Compared to the method advocated in this paper, the above results derived by the Winter-Sandholzer entero­ coccus test are very low and result in erroneous conclu­ sions. TJsing the dextrose azide-ethyl purple azide broth method, as is shown in Table 11, samples from areas where no evidence of immediate sewage pollution could be detected (Women’s Gym Bridge) showed enterococcus Indices from 2,000 to 140,000, whereas the above report indicated that they rarely exceeded 43. Where sewage and treated effluent entered the waters the above report recorded indices more than 3.6 and frequently exceeding 43. Samples from the 59 Kalamazoo bridge, a quarter of a mile downstream from the East Lansing Sewage Treatment Plant, yielded indices from 25,000 to 140,000 per 100 ml of water* It is obvious that the figures of the TJ* S. P. H. S* would have been higher and more significant If a more efficient method for de­ tecting enterococci were employed* In conclusion it might be repeated that the method developed and described in this paper is the most accurate and easiest of all the methods used for the detection of fecal streptococci* It eliminates the microscopic examin­ ation and only two tubes of media are required* This test takes little time to carry out and can be employed in the smallest of water laboratories* It does not require any additional tests such as the oatalase test of the Winter and Sandholzer method* It can be incubated In the ordinary 37° C* incubator and does not require 45° G. Incubation* 60 SUMMARY It was demonstrated that a concentration of 0*04 per cent sodium azide inhibited the gram-negative organisms but not the streptococci* Data indicated that the following dye concentrations did not inhibit Str* faecalls' for more than 48 hours; methyl violet, 1:500,000; ethyl purple, 1:800,000; brilliant green, 1:2,000,000; malachite green, 1:1,800,000; and crystal violet, 1:800,000* Ethyl purple azide broth, a new medium, was developed* When tested with laboratory strains of bacteria, it was found that the enterococci were the only group that would grow in this medium* These organisms also showed a charac­ teristic growth of a purple compact button on the bottom of the tube of medium after 48 hours of incubation at 37° C* The specificity of this medium has been demonstrated also with samples from river water, sewage, and soil* A new test for pollution of river water, sewage, and soil has been advanced* This test employs dextrose azide broth as a presumptive medium and ethyl purple azide broth for confirmation* A comparison of methods for the detection of entero­ cocci was made and it was demonstrated that the new dextrose 61 azide-ethyl purple azide "broth test was the best and easiest of those in use today. 62 BIBLIOGRAPHY 1* Alson, J* Mo An investigation of streptococci isolated from the alimentary tract of man and certain animals* Jour* Bact*, 16:397, 1928* 2* Andrews, P* W* The evolution of the streptococci* Lancet, 2:1415, 1906* 3* Andrews, F* W* and T* J* Horder* A study of the streptococci pathogenic to man* Lancet, 2:708, 1906* 4* Bagger, So V* 225, 1926* 5* Bergy, D* H*, R* S* Breed, E* G* D* Murray and H* P* Hitchens* Manual of Determinative Bacteriology* 5 ed* Williams and Wilkins do*, Baltimore, Maryland^ 1939* 6* Broadhurst, J* Environmental factor studies of streptococci* Jour# Inf* Dis*, 17:277, 1915* 7* Chapman, G* H* The isolation of streptococci from mixed cultures* Jour* Bact*, 48:113, 1944* 8* Chapman, G* H* The isolation and testing of fecal streptococci* Am* Jour* Dig* Dis*, 13:105, 1946* 9* Clemesha, W* W* In the Tropics* The enterococcus* Jour* Path#, 29: The Bacteriology of Surface Waters Thacker, Spink and^Co*, Calcutta, 10* Committee, Ministry of Health* The Bacteriological Examination of Water Supplies. 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