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Pagination at followi : [19] -28 p. This item is filmed at tha reduction ratio checked below/ Ce document est film6 au taux de reduction indiquA ci-dessous. 10X 14X 18X 22X 26X 30X y 12X 16X 20X 24X 28X 32X Ttw copy fllmad h«r« has b««n rtproducMI thanks to th« ganarosity o1: MMlical Library Mc&ill Univanity Montraal Tha Imagaa appaaring hara ara tha baat quality poaaibia eonaidaring tha condition and lagibiiity of tha originai copy and in icaaping with tlia Aiming contract apacificationa. L'axampiaira fiim* fut raproduit grica A ia gAnAroait* da: IMadical Library McQiil Univariity Montreal Laa imagaa auh/antaa ont 4t* raproduitaa avac la plua grand aoin. eompta tanu da ia condition at da ia nattati da l'axampiaira flimA, at an conformiti avac iaa conditiona du contrat da filmaga. Originai copiaa in printad papar covers ara fiimad baginning with tlta front cover and anding on tha laat paga with a printad or illuatratad impraa- sion, or tha bacic covar whan appropriata. All othar original copiaa ara fiimad baginning on tha first paga with a printad or illuatratad impraa- aion. and anding on tha laat paga with a printad or illuatratad Impraaaion. Laa aKamplairaa originaux dont la couvartura an papiar aat imprimto aont filmte an commandant par la pramiar plat at an tarminant aolt par ta damiira paga qui comporta una amprainta dimpraaaion ou d'llluatration. soit par la sacond plat, aaion la caa. Toua laa autraa axamplalraa originaux aont filmte an commandant par ia pramMra paga qui comporta una amprainta dimpraaaion ou d'iliuatration at an tarminant par ia damMra paga qui comporta una talia amprainta. Tha laat racordad frama on aach microficha shall contain tha symbol ^-^ (moaning "CON- TINUED"), or tha symbol ▼ (moaning "END"), whichavar appiiaa. Un daa aymbolaa auhranta apparaltra sur la damlAra imaga da chaqua microficha, salon la caa: la aymboia —^aignlfia "A 8UIVRE", la aymbola y aignifla "FIN". IMapa, piataa, charta, ate., may ba fiimad at different reduction ratioa. Thoee too large to be entirely included in one expoaure are filmed beginning in the upper left hand comer, left to right and top to bottom, aa many framea aa required. The following diagrama iiiuatrata the method: Lea cartae. pianchae, tableeux, etc., peuvent Atre fiimia A daa taux da rAduction diffArents. lArsque le document est trop grand pour Atre reprodult en un soul clichA, 11 est flimA A partir da i'Migle aupAriaur gauche, do gauche A droite, et do Iwut an bee, en prenent le nombre d'imegea nAcaaaalra, Les diagrammae suivanta illuatrent ia mAthode. 1 2 3 1 3 4 5 6 ''^•^ M •A*,. 'ifU&^i »V»; ter?'-'' 'W^---'V->- ', Jjy'' i-^*^^ On the Collection of Samples of Water for Bacteriological Analysis. By WYATT JOHNSTON. J, M ■"■'i '^.fXiw^ ^t m Record of Science. ^ O Dr. Wyatt Johnston's apparatus for collecting Sampl Reduced one-half (li w^mfnf' .» '" Plate HI. Plan. €tt A. Ptan eti .S -.^jentrt y/iftnystU C. C' tus for collecting Samples of Water for Bacteriological Analysis. Reduced one-half (linear). ikJ.^ ■' / mmm i^,. i.^..*^' "JUprinttdfrom ttu Cd/nadian Beeord ofSeUnee, Janmry, 1882.* On the Collection of Samples of Water for Bacteriological Analysis. By Wyatt Johnston, M.D., Montreal. (One PlaU.) I have been prompted to deaoribe mj method of eoUeot* ing samples of water for baoteriologioal examination, in tiM hope of its being of service to those who are anzioos to do field work in this department of bacteriology. Certain principles govern this work which cannot be neg- lected without introdacing serious sources of errw. First, the bottles in which the samples are to be taken must be sterilized by a dry heat of 150° 0. and afterwards kept oat of the reach of contamination from outside sources (espe- cially from dust) until the moment when the water is ooU lected. To this end the mouths of the bottles must be kept from contact with the fingers, and the stopper is only to be removed in the water. Second, the manipulations must be rapid enough to permit of a large number of . separate samples being collected, and finally, these should be taken from such points as will ensure their afibixling a fair index of the body of water under examination, as the number of bacteria in samples taken at different places from the aaioe water often varies considerably. The method usually adopted, that of immersing the bottle at arm's length and removing the stopper under water, though fraught with much personal discomfort in oold weather, is tolerably secure from contamination at the mouth of the bottle, but it has the disadvantage of only giving ^^^^^W^'^^&^^^'^^S'S^'MiM^Sir - Mmjmmf ' "^i -v^W- .Wii» '. TV '1. Canadian Record ofScUnee. the number of bacteria at the surface of the water. In the case of a rapidly flowing stream this is of little moment as the water is sure to be thoroughly mixed and the bacteria pretty eyenly distribnted. In standing bodies of water, such as IttkoH, ponds, reservoirs and wells, the bacteria for the most part sink to the bottom, so that the number of bacteria found at the surface affords no indication of their number in the deeper part. fVora which usually supply pipes are fed in the case of drinking waters. In the course of a recent biological examination of the waters of the Ottawa and St. Lawrence rivers it was found necessary to take samples at some distance beneath the surface. In winter, when samples were obtained through a hole cut in ice, often from one to two feet in thickness, the water which welled up into the hole was found to be contaminated by the instruments used in cutting it. On one occasion the water in the ice hole yielded 8,000 colonies per c. cm., while a sample obtained from the running stream beneath the ice only gave 30. Lying beneath the solid ice running water there is often found a stratum of " frazil " ice. THIh consists of a dense mass of small, shai'p ice fragments which have at one time been in contact with the bed of the stream and have then become contaminated fiom the soil. That water obtained from the midst of a bed of ^* frazil " ice is unsuit- able for bacteriological examination was shown by one examination of St. Lawrence water made in mid-winter, when two samples from a bed of frazil yielded respectively 473 and 480 colonies per o. cm., while clear water from an adjacent spot gave only 77 and 39. In endeavoring to obtain some apparatus suitable for obtaining deeper samples, I was surprised to find no men- tion of anything of the kind in any dealers' catalogues ; their poverty in this particular contrasting strangely with the wealth of appliances available for other purposes. * It thus became necessary to procure some simple form of apparatus, secure from sooroes of extraneous contamination mmmmimtmmmmmmmmmmmmimm 'P^wm*^' Water for Bacteriological AnalytU. n he as ria ler, for r of heir ipes 'the was teath ftinod ) feet hole \ used 9 hole tained ve 30. ere is its of a at one id have water unsuit- by one winter, actively from an ible tor |no men- gtlogues ', jly with 8. I form of lination and rapid enough in its working to enable me to obtain a large number oi' iiidividual Hamples. My first attempt was made with the assistance of Dr. R. F. Ruttan. We prepared a set of wido-moutbed bottles, fitted with perforated corks in which two open glass tubes were fitted. To one of the tubes a long rubber tube was attached, the end being guarded by a stop-cock. The bottles, with their glass tubes attached, were sterilized by dry heat and the rubber tubing was stemmed separately for several hours. After sinking the bottle to the required depth by attaching a weight, upon opening the stopcock the water displaced the air in the bottle. The method seemed to give accurate results, and in each case a- bit of the tubing reserved for the control teat of washing it out with sterilized water yielded no bacteria colonies. The sterilization seemed to be perfect, but the method was abandoned as it was found too troublesome to sterilize a separate length of tubing for each sample that was to be taken. I had obtained by this time some collecting bottles from Messrs. Eimer k Amend of New York. These were made of very heavy glass and held about a pint, the stoppers cjn« sisting of a rubber ring fitted round to a glass rod which lay within tho bottle, and was so arranged that the stopper could be pulled up against the lower part of the neck from within by means of a wire attached to the glass rod. In using this bottle one line was attached to the neck of the bottle and one to the wire fastened to the rod shaped stopper. The bottles were lowered by this second line, thus holding the stopper tightly against the neck of the bottles and so preventing the water from entering, until, at the desired depth the strain was taken off the stopper by pulling in the line attached to the neck of the bottle allowing it to fill, the stopper being heavy enough to fall from its own weight. This was open to the obvious objec- tion that the neck of the bottle above the rod shaped stopper filled with water from the surface, most of which was afterwards naturally washed into the bottle. Besides 22 Canadian Record of Science. this the precautions neoessaiy to guard against contamina* tion of the wire while attaching the string, and the neces- sity of having a separate bottle for each sample collected, rendered them inconvenient for field use. The method of using sealed tubes or flasks with a tapering end bent at right angles to be broken off under the water, has been recommended by Escherisch of Munich. This is much more free from technical sources of error than the apparatus last mentioned, but the trouble of pre- paring such flasks is considerable, as one has to be manu- factured for every sample to be taken. In the last edition of Bohrbeck's catalogue I find an ap- paratus figured for collecting bacteriological samples at different depths. From the impression conveyed by the illustration it seems too complicated to be easily handled, and the entire apparatus evidently requires to be re-sterilized before a second sample can be taken. At this stage my attention was directed to a most ingenious apparatns invented by Prof. Ellis of Toronto University, which differed from all the others in principle. This was a device by which sterile glass stoppered bottles could be placed in a weighted frame and lowered to the required depth. By pulling a string the stopper could then be raised sufficiently to allow the water t.o enter. By releasing this end the stopper was instantly replaced by means of a spring. Any number of samples could be taken, as the bottles could be placed in the fi-ame one after another with very little lo-js of time. The advantages of this as compared with the plans described above are very greai. There is absolute cei*tainty that no water is obtained from any except the required depth. There is n^ limit to the number of samples which can be taken, and all the preparation necessary is limited to sterilizing the bottles. It is also far more economical, as a single sinking frame contains iu itself the attachments for opening and closing the bottles. The instrument I am about to describe is a modification of that devised by Prof. Ellis, and I can claim no origin- ality whatever with regard to the principle of opening ■n ^'s"- i '( • Tirma Water for Bacteriological Analffsis. 28 and olosing glass stoppered bottles under water. My apparatus, though a modification of Prof. Ellis', contains improvements of my own which render it specially adiipted to taking large numbers of samples by making it simpler in construction and more rapid and accurate in action. All who have worked at water analysis know the great importance of making a very large number of separate observations before drawing conclusions. My outfit consists of one collecting fra me, shown (reduced to one-half its linear dimensions) in Plate III., into which the bottles can be successively fitted. It was made under my direction by Mr. 0. Wendell, of 170 Gonrsol Street, Montreal, and cost about eight dollars. It may be briefly described as a sinking frame, to which the bottle is attached by a fixed clamp, while a movable clamp is used to raise and lower the stopper. The frame is made of brass and has for its base a hollow cylindrical box B, 2^ inches deep and 2 inches in diameter. The box contains two pounds of shot and can be filled at a small hole E, which is closed by a screw. Attached to the top of this box are two flat brass bars F F, in the upper part of which a slot is cut allowing the movable cross bar A sufficient vertical play (1 inch) to admit of the bottle being opened beneath the water. The neck of the bottle is grasped at B by a brass clamp, the jaws of which are lined with soft rubber, fastened on by rivets. These jaws work on pivots and are attached to the upright bars F. F. by means of a brass rod bent outward so as to bring the neck of the bottle into the line of traction. The pivots allow some lateral play. The clamp is kept closed upon the neck of the bottle by a brass spring C made of No. 18 wire. The stopper H of tlje bottle is in the form of a tapering glass rod which is grasped by another clamp K and kept closed by the brass spring C. This clamp is secured to the sliding cross bar A by a horizontal pin working in slots which allow of sufficient backward and forward play to per- mit the stopper to adjust itself to the bottle. At the point i^vfe^^'iZiiij;" ^S^fet.*^' r n ^^^^h^k Ij^^>-^i4ife*>' jA j''fcfi)»*^ • '/.I'^J [ 24 Canadian Record of Science. where the pin is fastened the cross bar is bent outwards to bring the jaws of the clamps into the line of traction. The shoulder thus formed bears the entire strain in opening and closing the bottle as both ends of the clamp are balanced beneath it. It will be seen that these attachments are not rigid, thus preventing any straining or jamming of the stopper. A loop of heavy brass wire L connects the two side bars F. F. above and another loop M is attached to the cross bar A. To these loops strings are attached enabling apparatus to be worked under water. A pail' of spiral springs Q. G. made of No. 23 wire are hooked over the ends of the cross bar A above and fastened to the foot of the upright bar below. They close the bottle when it has been opened and keep it closed at other times. To place a bottle in the frame the ends of the clamps Cand C are compressed between the thumb and fore- finger sufficient!^ to open the jaws. The frame, with bottle in position, is then lowered by means of a heavy string N attached to the loop L, when the desired depth is reached the stopper is raised by pulling a lighter string N, attached to the ci'oss bar loop O. On releasing this again the springs close the bottle. The movement of raising the stopper can easily be felt at a depth of 15 or 20 feet. The bottle fills in about 20 to 30 seconds and the bubbles of displaced air can usually be seen. It is better not to fill the bottles quite full, but to leave some space for subse- quent shaking. In very swift currents or when the sample is to be taken at a greater depth than 30 feet an additional weight in the form of a small bag of shot may be tied to the lower part of the frame. To prevent any tendency of this frame to rotate while being lowered in a current, and thereby tangle the strings, I allow one string to glide in each side of my forefinger or else hold one in each hand. Before placing a bottle in the frame it is well to ascertain nffiniiifiilBiiiiiwfli'Twii ■*■> -1. /i '--m •ds to The g and anced re not of the e bars )SB bar paratus ive are 'astened 086 the at other of the ind foro- th bottle jtring N tched the ached to ) springs ipper can |e bubbles not to fill br subse- be taken [ht in the ler part of Itate while \e strings, refinger or asoertun Water for Bacteriological Analysis. 25 that the stopper is not jammed in the neck of the bottle » from unequal expansion in the hot air steriliser. In working at considerable depths I have found it con- venient to use a screw at 0. This increased pressure upon the wings' of the clamps holds the stopper more firmly. At other times the screw is not needed. By substituting a wiiNs for the string attached to the cross bar, the opening and shutting can* be readily controlled at very great jdepth. A bottle is removed by simply compressing the wings of the clamps and lifting it out from the jaws. The ease and rapidity with which the apparatus works will be understood fVom the fact that I am able to collect 10 separate samples of water at a depth of 20 feet in from 10 to 15 minutes. The bottles made use of are those dropping bottles fitted with groand glass pipettes now in common use for holding histological reagents. Both ends of the pipettes are sealed up in a gas flame, thus converting them practically into glass rods. As these bottles are kept in stock in the laboratoiy, one can always be replaced if it happens to be broken. The ones I employ hold 50 0. cm., but I would have preferred 100 c. cm. bottles had they been obtainable. The method of clasping the bottle by the neck admits of various sizes being employed in the same frame as there is space to spare between the cros^ bars. The difi'erences between the model here described and the original form introduced by Dr. Ellis are that the bottle is grasped by the neck ' instead of being forced into a socket from above. The use of spring clamps to hold the bottle, enables bottle and stopper to be brought into position by a single act instead of taking them apart and putting them in separately. The chief advantage of using the dropping bottles described lies in its giving a long tapering stopper, the lower end of which remains in the neck of the bottle when open, and guides it back into position, and It seemed preferable to use a bottle readily obtainable rather than to order a special form, which could not be repUced if broken. The little sinking frame 1 have just dei^cribed was ori- 2 & Su PiiPPlR ^t. >f. 4 V-. P^- S6 CwttKnm MBo&td'^ Settifct. , ginally designed to enable a Mmple to be collected at any required depth with the same safety and procisi4»n as at the saifaoe, bat as it also fhltils all the precautions for collect- ing samples in general and saves one the necessity of re- peatedly plunging one's arm into the water, I employ it whenever a sample is to be collected fram an open body of water. In securing samples by hand from a stream I was previoaaiy under the necessity of either securing the ser- vices of a boat or else taking the sample from off the bank, with the great chance that in the latter case the shallow water near the shore might not be typical of the general body of the 8ti*oam. Bat from this apparatus, which can be lowered into the water from a bridge, or by a rod, much more uni- form results are obtained. As the apparatus left little to be desired, as far as regards the rapidity and safety with which the act of collecting is performed, itonly remained necessary to ensure the necks of the bottles against o^ntarainatlon previous to using them. Instead of using steriUaed rubber caps for each bottle, A constant source of trouble and annoyance, I had a tin box made which holds forty bottles at once, each kept in posi- ^on by cross partitions of tin. The bottles are numbered serially, before sterilizing, by writing in pencil upon the groDud glass of the stopper, and by noting where each bottle is used the use of labels is unnecessary. Instead of a simple lid, the cover of the box is a tray four inches deep, in which a lump of ice is placed in warm weather. A small tube at one of the comers of this tray conducts the water away as fast as the ice melts. I find this keeps the temperature within the box below 8° C, even in the hottest weather. A handle across the tray serves to carry the box, and a small padlock in ttont guards it against an ever too inquisitive; public. Though I have not yet had cause to use it Ibr this' pur- pose, I think that my box, with its lump of ice on top, would form a better means of sending samples of water by express than any I have seen recommended. The temper- ature is kept down to a point where no increase of tiie •''* }'"■ »> WattrfBt BtuMrMogiM dntdpsis. 9t lis ptir- In top, |t6r by »mper- I of the bMtsria can go on, uid the ioe oould be repleniehed by the oAoiaki from time to time, while the padloek or a hoaI woakl pferent its being tampered with. The npaoe occu- pied by this box (18"xirx8") admits of its being placed in a large hot-air sterilizer and heated together with its contents to ISO" C. A small piece of fine string placed in the neck of each bottle permitted the escape of any mpis* tvre, so that it is nnnecessaiy to dry the bottles thoroughly before beating them. As the box is quite dust-tight the ■eeks of the bottles remain sterile until the time comes to use them, doing away entirely with the employtnent of rubber caps. In some cases when it seem^ of interest to examine great stretches of water I took my samples fVom off the bow of a pflsseng^r steamboat in a very simple manner. By using a stout fishing rod and about twelve feet of line a saimple can be seonred well outside the " wash " of the boat, eiven at a speed of 12 to 15 miles per houi*. To ensure the bottle sinking I wrapped a piece of sheet lead round it. By making the erst well ahead the bottle usually sank 6 to 8 teeta v As these examinations were always made in duplicate the accidental encounter of any extraneous source of pollu- tion would infallibly have been shown by an abnomal excess of growth in one of the two samples. A striking proof of the delicacy of the method is that the duplicate Samples always gave practically the same number of colonies. This " fishing " was often found a convenient method of obtaining a sample from the banks of a stream. To ensure the accuracy of the result in estimating the number of bacteria in a water it is of great importance to curtail to a minimum the time *vhioh elapses between the collection of the sample and the plating of the cultures, to guard against a possible increase of bacteria in the interval. It is also advisable to make the cultures in some flat vessel which permits of their being counted from time to time without exposing the gelatine to the danger of receiving ■^^!^''^^':w Vte;^«>;«SM 28 B' t ' I Canadian Record of Scimee'. additional bacteria ft'om the air. Both of these objects are met by the flattened glass flasks designed by Petmsohky. Those flasks contain the nutrient gelatine ready for nse, so that it is only ncceseary to warm them gently and so melt the gelatine, drop in the proper amount of water and after shaking them gently to lay them on their side till the gelatine stiffens. i . As these flasks are expensive and not always easy to oh* tain, it may be of interest to those who work under condi- tions which make it difficult to obtain apparatus to know that I have found ordinary flat sided, common, white glass via)8, obtainable anywhere, answered the purpose admir* ably. Owing to the ^mall size of the bottle necks I And it best to plug them by wrapping the cotton wool about the end of a wooden toothpick, which is then broken off short. By doing this the plug can be readily inserted and removed. The colonies ai-e readily counted with a lens, and to facilitate this I rule with a writing diamond a couple of parallel si ratbhes bn the flat side of the bottle in the axis. Cross lines are not usually nec« ssiary. Any of the colonies can bo flshed out with as much ease as from a Petru»ohky flask. The only ii-spect in which these bottles are not stUisfactory is that, being made of rather thick glass, when using a low power microscope, the object ap- pears somewhat blurred. This also could probably be obviated by using a correcting lens. They possess, how- ever, a distinct advantage over the Petruschky flasks in being much stronger. They also pack closer, owing to their flat sides, and having flattened bottoms they can be stood up. For summer fleld work I was able to pack 160 of the bottles in a small double walled tin chest or portable refrigerator, measuring 20"xl6"xl8", and this included a space of 8" x 8" x 1 8" for the ice chamber. «,.<« > \ - •jj * ■ i »; ap- be in %n to be the ftable |to|ij jji^^*\i^jfe^^ iV i' ; *.-'■ '■* 1 A'-^K'-f'isif -ai'-j^ ^^fe i^ MHHM ig. ^HB % '•^■:^'