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An investigation of the water supply of the city of Vancouver MacLean, Olive Edmonson 1921

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£(k j ^ e ?±i£f*^X°XK j <rt AH INVESTIGATION OP THE WATER SUPPLY OP THE CITY ©F VANCOUVER by OLIVE EDMONDSON MACLEAN. A Thesis submitted for the Degree of The MASTER OF ARTS in the Department of Bacteriology. University of British Columbia. April, 1921, AN INVESTIGATION OF THE WATER SUPPLY OF THE CITY OF VANCOUVER by OLIVE B. MACLEAN, B.A. For some years, the water supply of the City of Vancouver has been under supervision by the Medical Health Officer* Bacterial counts are made weekly of three distinct samples; one sample from Seymour water supply, one from Gapilano water supply, and one taken from a faucet in the city constituting a mixture of the two. Occasionally in this weekly routine examination, organisms giving the presumptive test for Bact, coll have been found. During the past four years however, samples which were positive to the presumptive test have been subjected to a more detailed examination and in no oase has it been possible to identify the organisms isolated as typical strains of Bact. coli. The present investigation was undertaken with four objects in views 1, To determine if possible the frequency of occurrence of organisms giving the presumptive ooli test, 2. To determine if possible the source of suoh organisms. 3. To record what daily variations, if any, occur in the bacterial content of the water supply, 4, To determine the regular Bacterial Flora of the water supply. The writer is indebted to the Medical Health Officer, Dr. F, T, Underbill, for much assistance, and for the privil-ege of consulting the information regarding the data in his - 2 -o f f i c e j and Is indebted a l so to Mr. B. M, LePluffy of the City Engineer's o f f i c e for considerable data regarding the phys i ca l condi t ion of the water supply . 8 8 1 8 » A t P A P A . BgSORIPTIOK OF THB WATBB SUPPLY. Seymour and Capllano Creeks are the sources of the water supply for the City Of Vancouver, They are separate and independent of each o ther , Capllano watershed has a drainage area of about 55 square m i l e s , whi le that of Seymour i s about 80 square m i l e s , They are both rapid, c l e a r streams, mainly g l a c i a l water, with a f a l l of about 70 f e e t per mi l e , She d i s -charge v a r i e s wi th in wide l i m i t s , that of Capllano for ins tance varying from about 15,000 cu, f t , per second when in f l o o d , down to about 40 cu, f t , per second in very dry weather (August With the except ion of a few days in each year during f lood con-d i t i o n s the water i s c l e a r and free from sediment and does not require f i l t r a t i o n , IHTAKES. TASKS AHD SORBBKB-Capllano intake i s s i t u a t e d 7 mi les from the mouth of the Creek at an e l e r a t i o n of about 480 f e e t above sea l e v e l and 10 mi les from the centre of the City of Vancouver, The intake i s constructed of re inforced concrete with head racks of oak to keep out l eaves and s t i c k s . An open conduit or flume 570 f e e t long connects the intake wi th the tanks , the water being - 3 -admitted through two sluice valves. The tanks are in duplicate, each being 100 feet long by 20 feet wide. At low water they are filled to a height of 6 feet. Both are provided with a double row of screens at the mouth of the supply pipe to pre-vent from entering any gross suspended or floating matter, She Seymour supply has two intakes, situated about 7 and 7^ miles respectively from the creek's mouth at elevations of 465 and 485 feet above sea level, The lower intake is similar in general design to the Oapilano intake described above. The upper one is constructed of hewn cedar with rook filling. Oak racks are provided at the entrance and a conduit extends 300 feet to the settling tanks. These are in duplicate, measure 10Q*X20*x6f, and are lined with concrete. Double rows of screens are provided at the lower end of each tank, DISTRIBUTION SYSTEM. The water from both intakes flows into 36 inch mains, the Oapilano supply crossing the Inlet at the First Harrows and the Seymour supply at the Second narrows. Just before crossing, the 36 inch pipes are divided into four 16 inch pipes, one of which goes directly to Burnaby, while the others supply the re-maining Municipalities, The supply from Seymour and Capilano is not distributed as such to different parts of the city, but in some portions, especially in the West End, there are cross connections between these two supplies in order to ensure water in case any one pipe under the Harrows should be destroyed. - 4 -UfllUJUSft. The water flowa by grarity dlraotly iato the maine but la addltloa thare ara two reaervolrs known respectively aa the "Stanley Park" and "Little Mountain" reserroire, Tha Stanley Park reservoir, with a oapaoity of about tea million gallona. Is situated 240 feet above the aea level. It la now uaed only at a balaaoing aad emergency reservoir alnoe Its elevation Is not aufflolent to give adequate preaaure throughout the whole olty, the Little Mountain reaervoir, diataat about one mile aouth of the olty boundary, haa an elevation of approx-imately 400 feet above sea level, with a oapaoity of twenty-five million gallons. This reservoir is filled and maintained from the Seymour Creek aupply main. It la oonatruoted of con-crete and oontrola the preaaure ever the whole olty, mum. The approximate number of aervieea for houae, o f f i c e , and factory oonneotions in the Olty of Vancouver la 25 ,000 . Thia doea not include the other municipal i t lea aerved. The plpea vary in e l s e from •§•" to 6" in diameter, tha majority of the houae connections or domestic aervieea being -fr" in diameter, OOfigVMmpM. The average amount of water admitted at the lntakea t o t a l a t h i r t y m i l l i o n gal lona per day. This does not represent the consumption ef Tanoouver proper, as at the present time the - 5 -Munic ipa l i t i e s of Point Grey, South Vancouver and Burnaby are a l so suppl ied with water from the o i t y mains. She 1920 p a r t i c u l a r s as to population and consumption are as f o l l o w s : -Locat ion. FQPftlflUPfl. Consumption. ( i n m i l l i o n gals* a d a y . / Oity- of Vancouver,-— 125,000 25 Po int Sroy,—•——-»— 17,000 1.5 South Vancouver,——p- 30,000 1.9 Burnaby, —— 17.QPQ 1.6 189,000 30.0 - 6 -L A B O B A T O B Y S T U D I E S . 1 . QUANTITATIVE DETERMINATIONS. Samples of tap water were examined d a i l y , Sundays and one or two o t h e r days excep ted , from November 23rd, 1920 to May 6 t h , 1921, i n c l u s i v e . D i r e c t coun t s and the p resumpt ive t e s t fo r Bae t . c o l i were made as a r o u t i n e p r e l i m i n a r y p rocedure each day . Media Bnraloyeft. Beef a g a r , g e l a t i n and l a c t o s e b r o t h made accord ing to S tandard Methods (1920) (1) were u s e d . The r e a c t i o n was ad jus t ed so t h a t the f i n a l r e a c t i o n was PH 7 . Brom-thymol b lue was used as the i n d i c a t o r in a d j u s t i n g t h i s r e a c t i o n and as i n d i c a t o r i n the l a c t o s e b r o t h . Technique . The mouth of the t a p was s t e r i l i z e d by f laming fo r 3 minutes a f t e r which t h e wa te r was al lowed to run f o r 5 m i n u t e s . About 50 cc were then c o l l e c t e d i n to a s t e r i l e f l a s k , 1 co and 0 .1 ce amounts were p l a t e d on agar and g e l a t i n , and the p l a t e s incubated a t 37*C and room tempera tu re r e s p e c t i v e l y . For the presumpt ive t e s t 1 cc of the wa te r was i n o c u l a t e d i n t o l a c t o s e b r o t h f e rmen ta t i on t u b e s . The agar p l a t e s were counted a f t e r 24 hours i ncuba t ion at 37°C and the g e l a t i n a f t e r 3 days a t room t e m p e r a t u r e . The r e s u l t s of the p resumpt ive t e s t s were r ecorded - 7 -after incubation at 37°C for 48 hours. The production of acid and 10$ or more gas at the end of this period was regarded as a positive test. The counts and the correlation of the same with heights of water at both Oapilano and Seymour intakes are graphed in Table I. The points in each curve are joined by a continuous line. The substitution of a dotted line indicates the occurr-ence of "spreaders" on the plates preventing counts. ./ The bacterial count at 37°C varies from 0 per cc to 12 per ee. The range at 22°Q is from 29 per cc to 105 per cc. It is interesting to note that there appears to be a correla-tion between both the 37°C counts and the 22°G counts, and the height of the water at the intakes. This is close enough to be significant. Undoubtedly, a higher count is to be expected after a period of high water at the intake when a certain num-ber of soil bacteria must have been washed into the supplies by rains or melted snow. On three days only were presumptive tests positive,^ namely, December 2nd, December 10th, and December 11th. There does not seem to be correlation between the occurrence of these presumptive tests and the height of the water at the intakes, but there is an apparent relation between the occurrence of the presumptive test and the rainfall. At least it may be stated that on the three occasions upon which the presumptive tests were positive there had been an immediately preceding heavy rainfall, as is shown by Table II, TABLE I ... No. OFBft£fEf?lA> P E K c c iftT ^ ^ 0 C .... HETi&HTof WKTER flTCAPlLftfJO INTAKE ljO IfOCHES Ht£l<^LSiL WATEW Ar sev ^QUisaMCflKc. iM IHCHE^ . f laorencre-Rif t PER cc ftT ^"["c I a g a. < r © TABLE I I . Date Capilano Seymour Presumptive Test Showing Deoeir 1 .07 1.32 0 iter 2 1.66 .96 + relate 3 • 85 .96 0 onshi 4 .70 .05 0 p between 5 .03 .56 0 6 .34 .97 0 rain 7 1.54 .68 0 fall and positive 8 1.03 .45 0 9 .17 1.33 0 10 1.86 2.40 + pres 11 3.6 .oe • ump t i v 12 .10 1.63 0 e test &> - 9 -2, QUALITATIVE DBTBRMIKATIOHS. Madia employed and methods adopted. The following media made according to the Standard Methods of Water Analysis (1920) (l) were used* Agar, broth, gelatin, pej>tone, phosphate broth for Methyl Bed test, sugar broths and endo-agar. Potato medium was made in the ordinary way, The milk media are described later (see pages 15 & 16), Heutral red bile salt lactose agar (4) was used in search for lactose-fermenting organisms, Srhlich's Indol test, (3) was used. The morphology is described on 24 hour agar cultures incubated at 37*0, unless otherwise stated, Stirling's modi-fication of Gram stain was used; Staphylococcus aureus and Bact. coli being employed as positive and negative controls in each oase, In determining liquefaction of gelatin, cultures were kept under observation for eight weeks before a negative liquefier was recorded, . Types of organisms found in Daily Counts. Colonies for investigation were fished from both agar and gelatin plates. In selecting bacteria for study it soon became a matter of comparative ease to pick off typieal colon-ies of various types. On the three occasions on which the presumptive test was positive, lactose plates were made from the fermentation tubes and from them, lactose fermenting colon ies picked. The following five distinct types of organisms - 10 -were obtained: 1. Sarcinae and Rhodococci. 2. Spore formers* 3. Rapid lactose-fermenters. 4. Slow lactose-fermenters. 5. Fluorescent non-lactose fermenters, 5JYPB 1, SAROIHAE ASP BH0POOOC0I. Several strains of the genus sarcinae were commonly found on the agar and gelatin plates, general Characters. These bacteria are spherical in shape, and divide under favorable conditions in three planes producing packet groups of eight. In these cultural studies growth was abundant on artificial media, pigment being formed on agar, gelatin and potato* Gelatin was usually liquefied, 0R6AHI3M SO. (l). The morphology was determined on beef peptone agar after 48 hours incubated at 22*C, The organism occurs as large spherical cocoi 1,5 to 2 mierons in diameter, arranged in packets of eight, non motile. Optimum temperature 22°C, Slightly decolourized by Gram stain, QttiUttral Characters. Agar slant;- growth abundant, lemon yellow, echinulate, raised, Brotht- heavy clouding, yellow sediment. Gelatin;- abundant growth along line of puncture. Slow napi-form liquefaction* - 11 -Carbohydratest- dextrose broth faintly acid, alkaline in lac-tose and sucrose broths. This organism is classified after Winslow ; as Sarcina flava (De Bary) (5). ORGANISM HO. (2) Similar to S. flava except that the pigment produced is a brownish yellow. This organism is classified after Winslow 1 j as Sarcina canescene (Stubenrath) (5). OB&AHISM HO. (5) Morphologically identical with S. flava except that it is Gram positive* Cultural Characters. Agar;- abundant golden yellow growth. Gelatin;- rapidly liquefied, a heavy orange precipitate. Organism Ho. 3 is classified as Sarcina anrantiaca (Flugge) after Winslow (5). 0RGASI5M HO.(4) Morphology. Large spherical cells occurring in packets. Gram negative. Optimum temperature 22°C. Cultural Characters. Agar*- abundant surface growth. Gelatin;- pini growth along the line of puncture, very slowly liquefied. Carbohydratess- dextrose broth faintly acid, lactose and suc-rose broths alkaline. Classified as Bhodococeus rosana (Plugge) afrter Winslow (*).(6). - 12 -TYPE 11. 8P0BB F08MBR3. Several strains of the myeoides, cereus, subtllls and megatherium groups were commonly found on the room temperature plates, as follows (using Ford's Classification) (7). PBMI3M ffQ. (5). Morphology, In young cultures on plain agar the organisms are long and thin and tend to arrange themselves In short chains. Motile, Gram positive. The spores appear after about 48 hours; at first they are situated at one end of the eell, but soon the cytoplasm disintegrates, leaving an oval spore. These spores often remain attached in long chains. Cultural Characters. On solid media the growth is glistening and rhizoid. Agar colonies are characteristics from the central nuclei! filamentous strands of growth extend on all sides forming the typical "myeeloid" colony. Gelatin:- is slowly liquefied, Carb0hydrates;- slight acidity is produced in dextrose broth and alkali in lactose and sucrose. Classified as Bacillus myooidag (Flugge) (Ford) (J?), OfiGAifSSM HO, U ) Shis b a c i l l u s i s a rapid "spreader" and was u s u a l l y found af ter a heavy ra in , Morphology. Spal l rods with rounded ends, often growing in short - 13 -chains and in old c u l t u r e s , shoving a tendency to thread. Ac t ive ly m o t i l e . Gram p o s i t i v e , sometimes s l i g h t l y decolour-ised* The spores appear ear ly , and have the cytoplasm attached to e i t h e r end. Cultural Characters . Agar s l a n t ; - the growth i s abundant, spreading and g l i s t e n i n g . • c h a r a c t e r i s t i c green pigment i s d i f fused throughout the medium. Agar c o l a n l f l s i - spreading and amoeboid with s l i g h t l y ra i sed edges and the vernal greem co lourat ion , G e l a t l n i - rapid saccate l i q u e f a c t i o n . The medium soon acquired the gX9*n. f luorescence , Lltama Mlllci- peptonised without aoagulat ion . A c l e a r greenish r ing s t a r t s at the top and rapidly proceeds dowaard, CarbghyAgataai- dextrose i s fermented to ac id , l a c t o s e and suc -rose unchanged. C l a s s i f i e d as faoUJUfl OflfQftS fluoreafisag (Yard)(7) . 2MAMWLMU21 Morphology. Small square ended rods. Motility doubtful. Gram positive. Cultural Characters. Agar atab:- thin, glassy, spreading growth. Agar plates 1- colonies spread ooncentrioally, dry and glassy, and usually firmly attached to the medium. - 14 -Gelat int- slow napiform liquef action. Carbohydrates;- dextrose and sucrose are fermented to aoldj lactose is unchanged. Classified as Bacillus snbtilis (Sternberg) (Ford) (?). Q&gjffig* gO> U)« MofEhPtOKy* Long th ick rods with s l i g h t l y rounded ends. Motile, Gran Pos i t iTe , Spores appear e a r l y ; they are usua l ly s i tua ted la the centre of the organism. In old cu l tu res the cytoplasm is e n t i r e l y d i s i n t e g r a t e d , leaving o r a l , r e f r a o t i l e spores . Qui U r a l ChftraptnTB. Agar g i a n t ; - th ick, creamy, cretaceous growth, s l i g h t l y rh lzoid at edges, Amg e a l n a l a i i " th ick and round with regular edges. Young colonies are white and change to a lemon yellow or brown when older* So l a t i a* - saccate l i que fac t i on . Carbohydrates!- glucose i s s l i g h t l y fermented, lac tose and suc-rose unchanged. Class i f ied as Baci l lus megatherium (DeBary) (Ford) ( « ) . £YJ£_U1. RAPIff hAOEQSB gEBMBHTEfla, The charac ters of t h i s cu l tu re are common to those of 23 s t r a i n s each of which had been studied in dd ta i l as de-fined bolow: - 15 -MORPHOLO&Y^, Twenty-four hour plain agar cultures incubated at 37°C were used to determine the morphology. All strains are Gram negative and distinctly rod shaped, with rounded ends; some are slightly thicker than others, the average size being 1-3 microns,long and 0,3 - 0,7 wide. Stain the organism readily with both aqueous fuchsin and methylene blue. Ho spore formation could be demonstrated on ordinary media. Motility tests were made in semi-solid media, and by the "hanging drop" method from the water of condensation of 24 hoar agar cultures. Ho true motility could be detected. Capsules could be readily seen in forty-eight hour milk cul-tures, stained by Hiss' copper sulphate method (10), Cultural Characters -The cultures characters were studied from 24 hour old cultures at 37°C unless variations are specially mentioned), ffutrient Broth;- abundant growth giving a pellicle, clouding and sediment, but no oolour or distinct odour. Agar slants- growth abundant, echinulate, glistening, and slimy; this latter characteristic being especially evident in older cultures. Agar platest- the colonies appear after 12 hours. The grwwth is heavy and greyish, usually heavier in the centret the edges are undulated and the consistency granular, gelatin plataat- 3 days at 22°C, The cultures grow rapidly, - 16 -oausing no liquefaction, the colonies are round and glistening and of the same general character as on agar, Selatin stab;- 3 days at 22°C, growth filiform and slightly beaded, best at the top and brownish in colour, no liquefaction of gelatin up to 2 months. Potatot~ the growth is luxuriant, echinulate and browiiish, BIOCHEMICAL CHABACTBBS-(a} Ljtmus M U L This was fresh separated milk with sufficient azo-litmin as a 2% solution added to give the required tint and sterilized for three successive days in flowing steam. The cultures were examined at the end of 24 hours, 2, 5 and 7 days. At the end of 24 hours the cultures gave acid but no coagulation, the degree of acid varying. After 48 hours about half of the strains coagulate the casein, the other; effecting practically no change. At 6 days all are coagulated, in most cases the coagulum is bleached.at the bottom and broken with gas bubbles and whey is beginning to be extruded at the top or laterally. After a week's incubation the coagulum con-tracts horizontally to about a third of its former diameter in the majority of the cultures, and is surrounded by clear whey, (b) Plain Milk. This medium was made from Klim - a skim milk powder » 100 gms of Klim mixed with 1000 co. of water and sterilized for 55 successive days in flowing steam in 75 co amounts in flasks. - 17 -After being incubated for 24 hours, there is no apparent change. At 48 hours the milk adhers slightly to the bottom and sides, and seems thicker. There is a soft curd and the whey is beginning to be extruded at the sides after 3 days(l At § days the curd is cracked and digested to about one half of its former size. After ten days the curd is very much shrunken is of a pale burnt colour and is broken into small pieces which tend to float submerged in clear whey. Carbohydrates. (a) Gaa Production. To estimate the amount of gas produced dextrose broth fermentation tubes (Smith's) were used. When the column of gas in the closed area became stationary, usually at the end of 48 hours, the amounts of COg and H were measured. The ratio of 002 to H was found to be 1,5 - 1, Gas was produced in the following carbohydrates:-dextrose, maltose, mannit, xylose, arabinose, duloite, lactose, salacin, sucrose, raffinose, mosite, dextrin, glycerose, stare}., and inulin, (b) Acid Production. Acid was produced in the following carbohydra tes : -(Brom-thymol-blue was used as i n d i c a t o r ) . Dextrose, maltose, mannit, xylose, arabinose, dulcite, lactose, salacin, sucrose, raffinose, inosite, dextrin, glycerose, starch, inulin. - 18 -(o) Action on Starch. Production of Diastase. Allen*s method (11) was used - Z% soluble starch solution was added to plain agar, then sterilized and poured into petri dishes. The cultures were streaked on these plates, incubated for 2 days at 37°C and 5 days at room temperature, after which they were flooded with a saturated solution of Iodine in 50$ alcohol, A large clear area about the stroke indicates diastatlc action. All cultures were positive to this reaction, Methyl Bad and Toees froskauer Reactions. (1) (12).(15). All cultures gave a negative methyl red reaction, and a positive Voges Proskauer reaction, Indol graduation. (2). Indol was not produced. This organism is classified as Bact. aerogenes (Escherich) (91.(9), TYPE lYt SLOW LACTOSE PERCENTERS. The characters of this culture are common to those of 11 strains each of which has been studied in detail as defined below, ORSAUISM HO. (10). These organisms when first isolated from the water required 3 to 4 days to ferment lactose to acid and gas, only a small amount of gas being formed. After half a dozen trans-fers cultures split the lactose to acid within 48 hours, in - 19 -this respect resembling the slow fermenting types of oolon described by Bronfenbrenner (13) v" !T and Sadler (14) & llounoe. In no case was it found possible to materially in-crease the amount of gas produced either by repeated subcul-tures or prolonged incubation at 37°C or 22*C. Morphology, Twenty-four old agar cultures showed short rods averaging 1-2 microns long by 0,3 -0,7 wide. They were Gram negative and stained readily with the ordinary alcohol and aqueous stains. Motility tests were made on semi solid media, and by the "hanging drop" method from the water of condensation of 24 hour old agar cultures, True motility could not be dem-onstrated. Ho capsules were found. All strains were aerobes and facultative anaerobes, Cultural Characters. Agar slantt- growth good, spreading, thin, transparent, glassy in old cultures, Hutrient broths- heavy viscid sediment, slight clouding, no pelicle. Agar plates*- the colonies appear early. They are white con-vex with regular edges, and have a granular consistency. Gelatin stab a^ fl plates:- growth is of the same general charac-ter as on the agar. Ho liquefaction of the gelatin at the end of 2 months. Batata:- growth good, medium rendered brownish. - 20 -BIOCHEMICAL CHARACTERS. Action on Milk. Litmus Milk. This type turns the milk slightly aoid after 24 hours growth, hut not enough to cause coagulation. Bo other change is noticed* The cultures were examined for 2 months. Carbohydrates. (a) Gas Production. To estimate the amount of gas produced Smithfs fer-mentation tubes with dextrose broth were used. The column of gas in the closed area was too small to accurately measure the ratio of C©« to H, Approximately the amounts of C02 and X were equal, A small amount of gas was produced in the fermenta-tion of dextrose, maltose, mannit, xylose, arabinose, dulcite, lactose, salacin, sucrose, raffinose, inosite, glycerose, (b) Acid Production. Acid was produced in the following sugars using Brom-thymol-blue as indicator;-Dextrose, maltose, mannit, xylose, arabinose, dulcite salacin, sucrose, raffinose, inosite, glycerose, Ho acid was produced in starch, inulin or dextrin, Methyl Had and Togas Proskauer Reactions. (1) (12) (15). All cultures gave a positive methyl red reaction and a negative Voges Proskauer reaction. - 21-inapi ?roAEotion» The Zndol t e s t gave a f a i n t l y p o s i t i v e reac t ion . This organism i s t e n t a t i v e l y c l a s s i f i e d as a va r i an t of B. (16) neapol i tanus d i f fe r ing from i t by m o t i l i t y ac t ion on milk and on i n o s i t e . TYPE v. ipBgTiFiOATm OF gog-frAOTQgg FfifiMMnflft BACTEBU. This group of b a c t e r i a gave an a lka l ine reac t ion in l ac tose b ro th . All are members of the f luorescent group of b a c t e r i a , (17) , Morphology, The vege ta t ive c e l l s twenty-four hours old and grown on p l a in agar are both long and short rods, often forming short cha ins . They are uniformly Gram negative and s t a i n equally well with the ordinary aqueous and aloohol s t a i n s . Mot i l i ty was observed in a l l cu l tu re s by the "hanging drop" method, and in semi-sol id media t h i s l a t t e r being incubated for 24 hours before being inocula ted . Capsules were found in about 20$ of the cu l tu re s , by means of "Hiss" copper sulphate method, (10) milk being used as the medium. No spores could be demonstrated, All s t r a i n s were aerobes and f acu l t a t i ve anaerobes. Cultural Characters . All cu l tu res were incubated for twenty-four hours »t 57°C, unless va r i a t i ons are mentioned. Agar s l a n t i - a l l s t r a in s produced a d i s t i n c t f luorescencej the growth was abundant, g l i s t e n i n g , ech inu la te . - 22 -Agar plates;- the growth of the colonies is good, the edge ir-regular and elevation flat with raised edges. The colour a greyish white at first and later ohanges to the characteristic fluorescent green. At 22°C and 72 hours old the colonies are distinctly opalescent. Gelatin plates;- 72 hours old incubated at room temperature. The gelatin liquefiers give abundant -growth, forming large saucer shaped colonies, which are filled with a flocculent growth of noticeable fluorescence. These colonies rapidly spread, soon liquefying the whole plate. The non-liquefying strains form colonies similar to those on the agar plates* Gelatin stab;- observations were made for two monght; 95 per cent of the strains liquefied the gelatin, the rate and form of liquefaction varying with the different strains. The com-mon forms of liquefaction are saccate, stratiform and crater-iform. Growth is abundant and fluorescent, Kutrient broth;- growth abundant, the medium being clouded and having agreenish pellicle and heavy sediment. The broth is distinctly greenish and the odour putrid. In the cultures which form capsules the medium is viscid. Potato;- growth good and after a few days incubation spreads throughout the medium, changing it either to a dark brown or green colour, £lbCH2MI0AL CHARACTERS. Carbohydrates. Glucose gives a small amount of acid but no gas-- 23 -other sugars not fermented. Methyl Red and Vogea Proskauer Reactions, (l) (12). (15). The methyl red test is negative. In the Voges Pros~ kauer test all strains give a bright green gluoreseence. the in tensity of which varies with different strains* India! Production. (2). Inall cases a red fluorescent pigment is secured, which varies from a pale pink to a deep red. It is interesting to note that the production of this red pigment correlates with the production of the green pigment noticed in the Yoges Pros-kauer test. Cultures which give an intense green fluorescence with the phosphate broth and the XOH give a deep red colour in the indol tests. This red pigment is not indol, as it is not soluble in chloroform also the cultures lack that character-istic odour of putrefaction associated with the production of indol. Action on milk. The action on milk divides this large group of bac-teria into three classes, (a) Those which have no action on milk, (b) Those which peptonize without coagulation, (c) Those which coagulate the milk and then peptonize . . the medium. Litmus Milk. Fresh separated milk with pure azo-litmifl as an indicator was used. The milk was inoculated from 24 hour old agar cultures and incubated at 37°0 for two weeks. - 24 -(a) In about 4% of the cultures the proteolytic enzyme is absent and the milk remains unchanged. [ . (bl In 85$ of the cultures the milk becomes alkaline and peptonization begins at the top, leaving a ring of green whey which extends downward until the whole tube is digested. In a few instances the whey is yellow, finally changing to a distinct orange. (e) In the remaining 11$ of the cultures, the milk remains alkaline or slightly acid, and is coagulated by a renn-in-enzyme and then peptonized. Plain, mite. This medium was made by mixing 100 gms of Klim with 1000 cc of water, and sterilized in 75 ce amounts in flowing steam. Twenty-four hour old cultures were inoculated into the milk and the flasks incubated at 37°0. (a) A slight acrid odour is the only noticeable chang< in the milk. Observations were carried on for two months. (b) The majority of these fluorescent bacteria belong to this gfoup, digesting the casein without coagulation. In most cases the action is rapid, being completed often within 48 hours. A dark green, transparent layer is formed which ex-tends downward rapidly, leaving a wrinkled pelliole in the sur-face and a greenish yellow whey; later this changes to a golden yellow in the most cultures; while a few retain their green colouration. All strains have the characteristic putrid odour. - 25 -(o) The milk is first coagulated and then digested. The curd is invariably soft; digestion starts usually after 48 hours, beginning at the top and along the sides. When com-pleted a greenish yellow fluorescent whey remains. In the absence of more detailed studies the strains of type are classified as belonging to the fluorescent group of bacteria. Efforts were made to discover the possible source of the various types of organisms found in the daily counts, especially as concerns the occasional occurrence of slow and rapid lactose fermenting types. Previous data in the hands of the Medical Officer of Health seemed to indicate that the probable source of these organisms was in Seymour and not in Capilano Creeks, With him, an investigation was undertaken of the conditions immediately above the Seymour intakes, A series of samples of soil and surface water on the west bank of the Seymour Creek above the upper intake were taken as high up as Iron Creek. The exact sampling points are approximately indicated on the annexed map. In taking samples of the soil, sterile wooden tongue depressors were employed to remove the soil to sterile test tubes. Surface water samples were collected in sterile test tubes. Smples were taken also from the intake, from the sett-ling tanks and from between the screens. After collection, the samples were transported as rapidly as possible to the labor-atory so that plating was accomplished within two hours of the - 26 -taking of the last sample. Seymour Creek above the upper intake is a comparat-ively wide and fairly rapid stream. Just above the uppor in-take there is a clay bank over whioh surface water trickles. This extends for a distance of approximately a quarte* of a mile above the upper intake close to the edge of the stream. It is succeeded by a series of flats., the mountains receding from the stream for some distance. The banks become con-siderably higher and at one point, known locally as the "Large Slide", there has been a considerable excavation and erosion of the banks, this being two to three hundred feet at its greatest width. Between the large slide and the clay banks a very much smaller slide has occurred. The large slide is with-in five hundred feet of Iron Creek. In the slide the various strata can be seen. Nearest the surface there is a looseloam occupying a depth of about a foot; beneath this is a layer of sandstone down to a clay layer about six feet below the surface> The clay layer at this point is about eight inches thick. Be-low the clay there is a layer of sandstone. The sampling point i may be described as follows, their approximate position being indicated on the blue print, (Description of Sampling points). I, Surface soil from first clay bank, taken about ten feet above the leven of the stream, II, Surface water which trickles down the clay bank of Sample - 27 -I, but fifty feet further lip stream. III. Clay from this bank - see Sample I - taken about six inches below the surface, (deep sample) IY4 Water from a small spring which flows over this bank ~ see Sample I - entering the stream about one hundred feet north of Sample II, 7. Surface water collected from clay bank mentioned in Sample I. TI, Surface water which was trickling over a large decayed log Til, Water from a spring which flows through a dense under-brush. This sample was taken inland about one hundred yards from creek, between the clay bank and big slide. Till, Sand at a small washout. IX, Sandstone between slides. X, Clay Oollteted about three feet above the mouth of the largest washout, XI, Water at the big washout taken near the outlet, XII, Soil above the clay layer taken from a surface which had been recently exposed by a big washout. This layer is about a hundred feet higher than the surface of the stream. XIII, Sandstone taken directly beneath the day layer at this washout, XIT, Clay from the layer above the sandstone at big washout, XT, Surface sand on the north bank of Iron Creek where it enters Seymour Creek, > - 28 -XVI. Water from Iron Greek. This stream is about ten feet wide where it enters Seymour Creek, but fairly shallow. It is called "Iron" Greek because of the peculiar red silt deposited in its bed. XVII. Subsurface sand on the opposite bank of Iron Greek, taken twelve inches below the surface. RESULTS OBTAINED. The organisms especially sought for were the lactose fermenters and the fluorescent organisms. No specific atten-tion was paid to the cocci forms. The aeyogenes type of organ-ism was found in the following situations - Samples Nos, 1, 2, 3, 4, 5, 7, 8 and 15. The fluorescent type of organism was found in all samples except Nos, 12 and 13. The slow lactose fermenting type was found in Samples 2, 3, 6, 9, 10, 11, 12, 14, and 17. Sample No. 13 showed no growth of any kind. This sample is from the sandstone beneath the clay layer at the "Large Slide", which would indicate that the clay layer was acting as a filter against any organisms in the soil above the clay. From the studies of these samples it seems that the fluorescent type of organism is found almost constantly in all samples. The aerogenes type of organism is found in samples taken from surface water or surface soil. Where deeper soil samples were taken the alow lactose fermenting typei and not the aeroeenes type of organism is found; and this slow lactose - 29 -f a rata n ting typa is found in all samples of water. It la inter-esting to find that at tha intake and in both the east and west tanks of tha upper intake all three types of organisms ware found, but that tha sample between the screens showed only tha alow laotaaa farmanting types and the flftprflBOaB* types of organlama, QQJfgfcUBIPgg. From the data collected in this investigation it would seem that the fluorescent type of organism found la a water form constantly present In tha water supply; that the alaw lactose fermenting type is a soil organism which obtains fairly easy access to the supply, and that the ftflrogsaai type la probably a grain or grass form whloh obtains antranoe to tha aupply only after the watershed has been drenched with a con-siderable rainfall. fl(L+S ?'% - \ ^ r J * •J v\{\\ miffs. 1 * ' '«* t, /7 / ^ -5: IjSSK on atv - 30 • HEgERBHOBS. (1) Standard Methods of Water Analysis 1920. American Public Health Association, Boston, (2) Bohme. Centrall fur Bakt. Abl. I. Orig. XL. pp. 129-133. (3) Standard Methods of Water Analysis. American Public Health Association, Boston, p.107, (4) Savage, 1906* Bacterologioal Examination of Water supplies. London, Pg. 221 , (5) Wiuslow ; 190 8, Sys temat ic R e l a t i o n s h i p s of the Coccaoeae. Hew York, Wiley & Sons . p g . 2 2 6 -248 (6) Flugge, 0. 1886, Die Mioroorganismen. Leipzig, (7( Ford W, W, and others, 1918. Studies on Aerobic Spore bearing non-pathogenic Bacteria. Journal of Bacteriology. I , 3 , 273-320 and I I , 5, 493-532 (8) Migula, 1886. System der Bakt . pg . 396 . Bscher ich 1886. T)armbiafc: des S a n g l i n g s . S t u t t g a r t , - 31 -(9) Jackson, 1911. Classification of the B. ooll group. Journal of Infectious Diseases 8, 241, (10) Hiss, 1905, Copper Sulphate Method of Capsule Staining, Journal of Experimental Medicine 71, 1905, (11) Allen, P, W. 1918, A Simple Method of the Classification of Bacteria as to Diastase Production, Journal of Bacteriology III, I, 15-17, (12) Voges and Proskauer, 1898. Zeit, fur Hyg. 28, 20, (12) Clark and Lubs, 1915, Differentiation of Bacteria of the colon Aerogenes Croup, Journal of Infectious Diseases, 17, 160-173, (12) Max Levine, 1916. The S ign i f i cance of the Voges-Proskauer Reaction, Journal of Bacter io logy , I , 2 , 153-164, (13) Bronfenbrenner and Davis, 1918. I s o l a t i o n and I d e n t i f i c a t i o n of the Members of the Colon Typhoid Group, Journal of Medical Besearch, XXXIX, 1, 33-37. (14) Sadler and Mounoe, 1920. The Bacteriology of Swelled Canned Sardines. Proc. Royal Society of Canada. Ser. Ill, Vol, XIII, 135-141, - 32 |15) Harden and Walpole, 1905-6 Chemical Action of B. lactia Aarogenes (Escherich) on Glucose. Proc. Hoyal Society of London (B) 77, 399. (16) Emmerich, 1885* (17) Tanner, F. W, 1918« A Study of Green Fluorescent Bacteria from water. Journal of Bacteriology, III, I, 63-101, 32 -| 15) Harden and Walpole, 1905-6 Chemical Act ion of B. l a c t i s Aerogenes (Esche r i ch ) on Glucose . P roc , Soyal Soc ie ty of London (B) 77, 399. (16) Emmerich, 1885* (17) Tanner, F. W, 1918. A Study of Green Fluorescent Bacteria from water. Journal of Bacteriology, III, I, 63-101. 

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