FRANKLAND ON THE DEVELOPMENT OF FUNGI 66 bat that this nitric acid is not necessarily due to tlie oxidation of excretal matter I think the foregoing experiments sufficiently prove and I ven- ture to think in consequence that the “ previous sewage contamination theory” ought to be considerably modified. VIII.-OIP tlLe Development of FumgSi in Potable Water. By E. PRANKLAND Ph.D. D.C.L. P.R.S. IN June 1870 Mr. ITeiscli communicated to the Chemical Society the results of some rery interesting obserrations on the derelopment of cellular and fungoid growths in various waters to which a small quantity of crybtalliiie s u p - liad been added. Having observed this phenomcnon in water to wliich sewage was strongly suspected to have gained access lie procured water from various sewers and after allom- ing the suspended ma,tters to settle six drops of each sample of clarified liquid were mixed with 10,000 grains of West Middlesex and New Rivcr water and to G oz.of each sample thus polluted 10 grains of pure sugar were added n like quantity being mixed with cj oz. of the watcr without tlic sewage. All the samples were placed in stoppered bottles in a window where plenty of liglit could reach tliem. It was founcl tliat the watcr and sugw reinained clear and sweet as did also lhe water and sewage vithout sugar ; but the mixtures of water sewngc and sugar became turbid and on being submitted to microscopic examination were found to contain small spherical cells nith in most cases a very briglit nucleus. After the lapse of some days these cells gradually grouped themselves together in bunches something like grapes ; t,liey next spread out into strings with a wall surrounding and connecting the cells ; the original cell-walls then seemed to break and leave apparently tubular sort of threads branched together.A h . Heisch then describes a number of other experiments which taken together with tlic foregoing led him to tlie conclusion that the cells of these germs whcn thus dex eloped are distinct evidence of sewage contami- nation and that the germs producing tliese cells are not removed by filtration through the finest Swedish paper neither are they destroyed by boiling for half-an- hour. These remarkable results excited in myself and doubtless in other cheinists occupied in the inrestigntion of potable waters the liveliest intercst.h i comeetion with the most generally accepted hypothesis of the cause of the spread of epidemic disease tlirough the agency of water there was liere discovered a much nearer approach to the supposed iiiorbific matter in such water than had before been attained. Another parallel mas apparently opened from which one of the strongholds of 67 IN POTABLE WATER. disease could be a t least reconnoitred if not actually attacked and vanquished. At the earliest possible moment I repeated and extentlcd these cx- periments and a t the outset had the advantage of seeing in Xr. Heisch’s laboratory the cells and fungold growth which lie clcscribcd in the papei- already alluded to. At a subsequent stage of my inquiry I was also much indebted to Mr.Bell of the Inland Revenue labora- tory for information about and specimens of rarious fungoid growths some of which are alluded to in his Faluable paper on “ Fungi and Fer- mentation,” recently published in the Journal of the Chemical Socicty. My own experiments completely coiihm Mr. Heis ch’s obserrations with two important exceptions T-~z. that firstly the fungoid growths are not peculiar t o water contaminated with sewage ; and secondly the germs from which they originate are present in all water 1~111cll has been even momentarily in contact with thc air. Samplcs of xLtm which when mixed with the proportion of sugar wluch he rcco~nmc~ids remained clear and transparent for n eeks rapidly became tnrbicl wllcn a few drops of sewage were added to them; and the consecutive dc- velopment of clusters of cells and threads occurred cxactly as 11e describes.One drop of fi.esli urine in 10,000 grs. of water soon pro- duced crowds of cells and at a later period mycelial threads. Samplcs of effluent water from meadows irrigated with sewage wlicn mixed with sugar presented perfectly similar phenomena as clid a150 samples of well mater yielding the following results on analysis p Besults f Annlysis eqiraa.wd in p r t s p e r 100,000. Gultan’s Row Well > E 2 n - Tellow C l C U . Sliglitly tu I-bid . I Orcliard water; 8 mouth thorue Ea4 ft. Retfoi-d . tow 5 West \Vcll ft. deep . depth Ynr- Cra- to i Jacob’s field .Well Shef- 1 Offcnsir s nivnrnriiig wirli vilirios i l l l d bnctcria. Very turbid. Pump in Sheep Street Ciruncesler - FRANKLAND ON THE DEVELOPMENT OF FUNGI 68 Samples of water taken from the Thames above Streetley and below Reading and from the Kennet below Reading also gave under the same conditions fine mycelial growths. On the other hand the water delivered by the eight London Water Companies gave in but one or two cases slight evidence of cellular or mycelial development whilst the following samples exhibited even after several weeks' contact with sugar no signs of the characteristic cells and fungi nor indeed of any other organisms :- 4 e - 0 '001 ,004 '118 IZe.sults qf Analysis expressed i i L p a r t s p e r 100,000..- B d 1-1- 11'94 0 100.88 '059 deep furnisliihg Harrow Head > Shallow Harrow Well Hill near . top of 1 63'92 '078 Well in chalk. 310 feel) Deep chalk well water at Canterbury softened by Clark's process _. Cerney Springs Tlianies I 33'86 .052 water supply . _. . . . . 1 Hardness. ' 0 0 G 0 '001 Water from bore Iiolc in nasliey Meadows near ' 33'88 ,027 Watford 130feet&vp . Clialk ivell 378 feet deep 29.20 '05? 23.30 '081 - Thus far my results were quite in harmony with those of air. -Heisch and in accordance with what might be anticipated from a knowledge of the sources of the waters and of their previous history as revealcc1 by chemical analysis ; but I nom encountered some reactions which at first puzzled me exceedingly.The first of these was the following :- During a visit to hIr. Hope's new irrigation farm a t Romford on the 25th of November last I collected a smnple of effluent mater from one of tlic drain outfalls. This mater consisted of the sewage of Rom- forcl nhicli lind percolated to the tile drains through some four or fire feet of loose graTelly soil. It was clear but that it still contained a considerable proportion of unosidized sewage mas evident from the Eollowing results which it yielded on analysis- - I i B __ 0 ." z z 8 G -__ 2'10 1'30 22'76 6'30 18.98 1'35 23.71 1'18 21.91 h % .-. - 1 P ." - e 6 a Y - B '438 '299 .087 '127 '325 '083 ,123 ~ - '69 4'17 4'86 Clear.3'88 26'64 8'90 10.37 11.78 32'15 Clear. !8.42 4440 Clear. 3.77 2'1.04 Clear. 3'51 25'42 Clear. 1'00 16'41 3'73 19'14 Clear. IN POTABLE WATER. GD 100,000 parts contained :- Total solid inipurity in solution SS'G0 Organic nitrogen Nitrogen as nitrates and nitrites 1.143 Total combined nitrogen. . 1.419 '933 Organic carbon *844 Ammonia $040 Temporary hardness. . 27.95 Chlorine 8.30 Permanent , 20'GO , 48.S The proportion of organic caiboii mas thrcc times and that of organic nitrogen ten times as great as that found in nnpolluted -ater whilst the comparatively large propor'ion of ainmonin showed that ihc oxidation of the organic matters was still incomplete.Nercrtheless this water when mixed >j-ith the proper proportion of sugar and maintained at a temperature of about 70" Fahr. remainccl perfcctly transparent for weeks The result of the next expeibnent was still morc remmlxable. Two samples of the Grand Junction Company's wntcr wcre mixed with sugar in the usual way ; one of them was drawn from a € 0 ~ 1 nncovcred cistern over and within a water-cloFet ; the othcr from a clcan slatc cistern in passing out of which the water filtcred through some 30 or 40 lbs. of animal charcoal. The x d e r from the foul cistern remaincd transparent for weeks whilst that cli am-n from the clcan cistern through animal charcoal soon became turbid ancl in three dnxs hac1 prodnccd abundant fungoid growths.Reme mbcring Ah. H c i s cli' s statement that filtration through well nimtl animal cliarcoal is effcctnal in pre- venting these growths eren in foul water I nom p a y d n rapid cur- rent of air through the filter for :%bout 1.5 minutes ancl tlitn left it exposed to air for six hours. The result honrwer waq the fiame as before Grand Junction water drawn through it immcdiately after akation behaved exactly as before when niisecl mith sugar. Thcsc experiments seemed to indicate that the presence of a phosphate was in some way connected with the production of thc fungod growths end other living organisms ; for it is lmomn that mntcr dissolrcs traces of calcic phosphate from animal charcoal ancl this supposition mas strengthened Then it was found that the effluent n-atcr from the sewage farm a t Romford contained no detectable trace of pliosphoric acid tlie plants ancl poor soil of this newly cultivated farm 1iaTing cloulitlcss rc- moved all phosphates from the percolating sewage.Thc hypotli the dependence of the fungoid ancl otlier growths upon the presence of phosphates was further supported by the rcsults of thc following - - - .-._- FRANIiLAh‘D ON THE DEVELOPMENT O F FUNGI 70 1. A sample of the Grand Junction Company’s water mixed with mgar remained perfectly clear for twelre days ; minute quailtities of animonic nitrate and phosphate were now added. Three clap later it swarmed with very active vibisios and cells with bright nuclei sobse- qiiciilly very luxuriant brauchcd fungoid threads developed themselves and the mixture emitted a strong ferment odour which a few days later became Iiorribly offensive.2. A sample of the Southwark Company’s water mixed with sugar remained f o r 19 days perfectly clcar and transparent ; small quantities of amlnoiiic nitrate and p1ior:phate were then added. In a few days it was crowded with vibrios and inoiinds ; and later it was found to con- tain tlie charactcristic mycelid fibres. 3. The usual proportion of supr was added t o a snmplc of mater which I collected at the Cerney Springs near Cirencester ; it remailled clear for eigllt days and was then &xed with traces of arnmonic nit1 ate and sodic pliospliate ; it continued turbid erer afternards and soon bccaiiic fi lled wich snmms of yibrios and fine branching tubular fungoid threads ; tlie water subscquently became brownish and emitted D Yery offciisive odour.Il’hese experiments shorn that potable watcrs which stand the sugar test pcrfectly becuine entirely changed in their behaviour with this test ~ ~ l i c i i they are niixed witli ti-aces of ammonic nitrate and Bodic phosphate and the following experiments prove that it is the phosphoric salt which altcrs their beliavionr in this respect. 4. A sample of the Lumbeth Cornpanj’s water after admixture with sngnr rcnmincd perfectly c1e;u. for 19 clays ; aminonic nitrate i m s then d d c d ; after tlic lapse of several weeks the clearness of the sample hnd not been distubecl. 5. Canterbury deep wellxater softeiiedby C l a r k ’ s process remained perfectly clcar during 23 days after admixture with sugar ; traces of nitrate of aiiimonia were then added but after the lapse of two months it was still perfectly trauspareut and unchanged.6. A sample of tlie New Rivcr Company’s water after bcing mixed \vith sugar remained perfectly clcar for 19 daFs ; amnionic nitrate was then added but during a further perioll of 12 days no change took place. 7. The water of the shallow well at Harrow above alluded t o (p. 68) mixed with sugar and remained perfectly clear during 29 days ; one drop of a fresh solution of albumin was then added to about 1) ounce of it. Iu four days it became very turbid aud under the micro- scope showed bpleiidid branchtd tubular fungold threads filled with roulldcd cells pnd closely resembling those developed in mater with mhich sewage had been intentitrnally mixed.Cells with bright nuclei grouped together were seen in abundance together with leptothrix IN POTABLE M'ATER. 71 filaments. The solution of albumin used in this and other cxpcri- ments was made by breaking open a fresh egg and rapidly trniisfei,~~ing the white to a bottle containing about 6 oz. of pnre distillcd water which had been previously boiled for an hour a i d a half. Analysis proved this solution to contain distinct traces of phosphoric acid. 8. A sample of the Grand Junction Company's watcr softencd by Clark's process was mixed with sugar and remained clear during 19 days ; ammonic nitrate was then adclcd b u t no alteration occurrccl during the next six days.When however a few rcry minute frngmcnts of fresh animal charcoal were put into the water i t soon became turbid and exhibited under the microscope groups of cclls \rith bright nuclci and magnificent mycelial threads. It is thus evident that the addition of minute traces of n pliospl~ntc either as sodic phosphate? whit,e 0 egg or animal cllnrcod at. oncc determines these fungoid growt'hs in saccliarinc i n t e r wliicli b2for.c exhibited no tendency t o develop them. An important qucstion now presented itself Are the gcrnis of tlicse organisms nccessarily contaiiiecl only in tlie maters wliicli dovclop fungoid growths or are they present in tlie atmosplicre ? The aiiswer. t o this question was given by the following expcriment :- 9.Small quantities of potassic chloride ammonic nitratc sodic yhosphat,e and sugar were clissolvcd in distillcd wntcr prcviously boiled for many hours with caustic socla and potassic pcrmangnn:rtc and afterwards again distilled. Just. before solution tlie solid ingrc- dients were strongly lieated in a platinum spoon over tho flarnc of a spirit lamp-the potassic cliloride a d sodic pliosphate t o rcdncss tlic ammonic nitrate until a considerable proportion had decomposed into nitrous oxide and water and the sugar until aftcr melting it bcg:m to turn brown. This solution was placed in a stoppered bottle ns in :ill the previous experiments. After a few days' exposure to n. tcmpcra- ture varying between 60" and 70' I?.a magnificent mycelium of t,he characteristic description began t o OW and was soon followed by several others. Under the microscope the tlireacls of tliis fungiis wcrc uniformly tubular branched Tvit~h si~arsely-distributetl cclls inside and a few bulbs. There were also interspersed amongst the m p l i n m abundance of cells wit.h bright nuclei groupcd togcther like bunches of grapes. The liquid W ~ S also crowded with rery minute moring organisms probably monads. A specimcn of real sewcr fungus \vas examined side by side with this and found to bc rcry similar in ap- pearance but more transparent aud somcwliat smallcr. It is thus evident that the purest water wliicli can be obtnincd in contact with the air yields splendid crops of this sewngc mycelimn if it be supplied with the necessary soil ; and further that tlie sugar ancl salts just named contain all the elements necessary f o r its dcvclopmcnt.FRANKLAND ON THE DEVELOPAIEST O F FUKGI 72 Of these elements phosphorus is essential for in a solution made at the samc time exposed t o the same conditions ancl containing the same substances mii~iis f h c sotlr'c 217io~p7inte no trace of mycelium or of ally otlier orgaiiisni made its appearance during the nine weeks it was under observation. If it be true theii that the gel-ms of these fungoid and other organ- isnis be usually present in the air and that they develop in a sacelini-inc solution only when tlie latter contains a phosphate it obviously follows 1 that maters whicli are unaffected by this sngar test ought to be in- capable of propagating semer fungus when they are infected.This conclusion was complctely verified on submitting it to tlie test of ex- periment. 10. Harrow decp well mater (see page G8) which had remained per- fectly clear f o r 24 days after aclmixturc with sugar T V ~ S infected with washed sewcr fungus mliicli I collcctccl a t ah. Hope's irrigation farm at Romforcl. After the lapse of fire days during which time it \vas maintained a t a temperature of betmeen GO" and 70" I?. no growth or tulbiclity had occurred. A single drop of solutioii of albumin wag now added ; four days later the water hail become very turbid and nuincroiis fungoicl growths had commeiiced at the bottoiii of tlie bottle.Thesc examined by tlie inicroscope showed mycelium threads not very well developed also leptotliris and abundance of grouped cells with bright nuclei. No vibrios mere observed. Afterwaids the fungoid growth became more abundant but never SO luxuriant as that ob- tained in Experiment No. 9 which surpassed all others. 11. Water from tlie bore-hole in Bushey Meadon-s (see page 68) wliich after the addition of sugar llacl maintained its transparency (luring 24 days was infected as in the last experiment with x-ashed se\ver fungus. No growtli took place ; neither was tlie tmnsparency of the water distuibecl in the subsequent eight weeks during which it mas repeatedly and closely observed. It is thus evident that the presence of germs in a sample of water is insuficieiit in itself to produce Bfr.H e i s c h ' s reaction when sugar is aclded aiitl further that a short (probably iL momentary) contact with air is sufficient to inipregnste any snniple of watcr Kith the necessary germs which develop on thc acldition of sugnr only in the presence of a phosphate. The reaction is in fact an exceedingly deli- cate test for phosphoric acid. It vioiild probably defy the pomers of tlie most espert chemist to detect in two ounces of water the phos- phoric acid introduced by the adclition of a single drop of a dilute solution of albumin xet these atmospheric germs find i t out appro- priate it and by their growth reveal its presence. As a ready means of discorering traccs of phosphoric acid this method will cloubtless often comnieiid itself to chemists ; but I have considerable hopes that IN POTABLE WATER.- 0 73 in a modified and extended form it may be applied to the exploration of both air and water with far more raluable results. If the germ theory of clisease be true niicl it has of late received considerable experimental support it is not improbable that the germ constituciits of the air may undergo such a marked alteration during the prcvalencc of certain forms of epidemic disease as to be recognisablc in the growths which mould ensue in such a fluid as that used in Experiment No. 9 or in a similar one i o xhich some of tlie constituents of our ordinary food were adcled such as albumin milk and juice of flesh. In order t o expose tlicsc fluids with their contained gcrms t o couclitioiis as closely analogous ab possible t o those wli~cli obtain in the alinien- tary canal tlicy Ought to be kept in the dark ancl maintained at bloocl- heeat during incubation.I have made a few experiments in this direction but it is obyious that Iaborious nncl long-continued obser\ ations will be necessary bcfore the ordinary clcvelupmentd phenomena of a healthy atmosphere can be distinguisheJ from those of infected air ancl IT\ i l l tlicreforc only describe one or t v o of the experiments by way of illustrating tlic mode of operating. Tlic process is also obviously equally applicable to the testing of waters intentionally infected by the cvacuntlons of patients suffering from typhoid fever cholera scarlalina ancl other discascs.1%. A sample of water supplied t o London presented three wccks after admixture with sugar and exposure to a tcmpcrature ranging between GO" ancl 70" I?. faint traccs of fungoid growth. It was now placed in a nearly dark chamber a t a temperature of from 78" t o 85" P. After ten hours the dei-clopmcnt of mycelium had gwatly increased but on raising the temperature to 8Y0-99" 3'. for fourtccn hours tlie fungoid threads nearly disappeared. Lcptothris fibres abounded but no vibrios backria or moring orgnnisrns 17-ere seen. 13. Effluent water from B~rking sewage farm was mixed v i t h s n p r and exposed as in Experiment No. 12 to a temperature of from /8 t o 85" P. for ten hours a t the end of whicli time it had bccome slightly turbid and a fungoicl gromth n as observed.The tomperaturo was then increasecl to 98" F. Fme hours later it 1i:d become very turbid and the fungoid gron th had augmented. After tn-enty-two hours' additional exposure to the same temperature the turbidity had con- siderably diminished and the fungoid growth had not progressccl much. The microscope rerenlcd long slender fragile and jointed mycelium threads very different from those which had developed in similar liquids a t more moJerate temperatures. There were also crox-ds of bacteria -vibrios and other moving organisms. Even a t tlie expira- tion of a further period of three d a p during which time a nearly uniform temperature of 98" F. was maintained tlie fungoid growth had not perceptibly increased.FRhNTrLASD ON THE DEVELOPMENT OF FUXGI DISCUSSION. 74 14. Six drops of sewage and tlie usual proportion of sugar were addcd to about 1+ oz. of Grand Junction Compzny's water and the niixture which smelt vcry offensivcly was exposed as before to a temperature varying from 78" to 85" F. At the end of ten hours no pcrccptiblc change hod taken place. The water was now raised t o blood hcat (9s' F.) and maintained a t that temperature until the close of the cxpeyiment. At thc end of five hours it had become turbid from ihe dcvclopment of crowds of bacteria. Even after the lapse of thirty hours morc no vibrios or mycelium were perceptible ; but after tlie expiration of t h e e more days abundant fine long non-branching tubular thrcads with square cells ciiclosed had developed.Some vibrios and cclls with bright nuclei were also seen. k'rom these a i d a number of other similar experiments the conclu- sion mas impressed upon nie that the temperature of the body is favour- able for the production of bacteria vibrios and similar organisms but ui~favourablc for fungoid growths. As already mentioned however these latter esperhnciits mill require to be greatly extended before any trustworthy coiiclusions con be drawn from them. It mould be especi- ally interesting t o make them with water which had been agitated with the air of the fever &c. wards of hospitals. The following are the conclusions to which these experiments have led me :- 1. Potable waters niixed with sewage urine albumin and certain other matters or brought into contact with animal charcoal subse- quently develop fungoid growths and other organisms when small quantities of sugar are dissolved in them and they are exposed to a sunimcr temperature.2. The gcrms of these organisms are present in the atmosphere and evcry water contains them after momentary contact with the air. 3. The dcvelopment of these germs cannot take place without the presence of phosphoric acid or a phosphate or phosphorus in some form of combination. Watcr howerer much contaminated if free from phospliorus does not produce them. A German philosopher has said '' uliiie Y l i o s y l ~ o ~ ~ heiti GetlniiXe." The above experiments warrant the alteration of this dictum to ohrLe Phosplior gar k e i i ~ Leben.P r e s i d e n t It certainly is striking that the absence of phosphorus should cause the observed cleficiency in the fungoid development but was phosphorus wholly absent ? does not the sugar which is purified by means of animal charcoal contain some ? IN POTABLE WATER. 7 5 Prof. F r a n k l a n d The crystallisation of tho sugar probably excludes any appreciable tracc of phosphorus. Mr. H e i s c h In one or two points Dr. F r a n k l a n d ' s obscrvntions are diametrically opposed to my own results. The one is that water cannot be deprived of its germs by filtration through animal charcoal. Now my observations in this respcct ari? not isolated ones but I halve now for about three years been investigating -wcclc after week watcr which passed through charcoal filtcrs arid have never nict with fungoycl developincnt in such water."lie other point of diffcrcncc betwecn Dr. F r a n k l a n d ' s experimcnts and my o ~ n is that whcreas 110 noticed the same kind of cells whether t h y werc originated tlirougli white of egg or through sewage matte;. I obtained in the two C:LSCS very distinct results. The sewago fungus is rcry small pcrfcctly spherical and excessively transparent ; it grows and clccays rather rapidly ; in about six hours after adclition of tho se-rvagc fluid t o tlic sugar solution tlic splierical cclls most,ly in gnpe-likc b;uichcs appcar which after six more hours liavc deve!opcd tlieinselrcs into mycelia and a few hours later are broken up entirely. During the growth of these orgnnisnis the odour of butyric acid was clcarly pcrccptiblc.Now when white of egg was put into a sugar-solution no sniell of butyric acid was emitted during the formation of the cells which were somewhat larger and lcss t.mnspareiit than those in the former case. Dr. Russell The results of my experiaents mostly coincide with those arrived a t by Mi,. Eeisch. I happoned also to submit to the sugar-test a sample of drainage water froin Romforcl Farm aiid ob- tained like Dr. F r a n k l a n d a negntive result,. Mr. B e l l Perhaps the effluent water saniple examined by Dr. Rus- sell was kept for some time. In t'hat case it mould purify itself and give no fungoid growth on addition of sugar. But if the saniplcs of effluent waters are investigated very soon after t h y Ii:~d bccn collected they mill yield fungi Regarding Dr.Franklancl's shtcmcnts I can say that my experiments fully bear out what lie said with rcspcct to the indispensable presence of pliosphates. I can also fully confirm his remarks as to the animal charcoal-it does not take away the germs from the water ; indeed I am inclined to believc h a t these germs lire in the coal. I found burning to be the best mc:ms for purifying the charcoal. ' Dr. Voelcker Nr. B e l l ' s reinark as to thc rapid changc w1iic.h sewage undergoes is quite correct. A jar of sewage left loosely coverecl for some months had lost almost all its ammonia whilst its nitric acid had increased. Since so much doubt is throvm on the efficiency of animal charcoal for purposes of water purification I may mention t h t iron-sponge mould be a,n excellent substitute for chmcoal Gltcrs.This HUNTER ON THE EWECTS O F PRESSURE 7 G spongy iron is obtained by calcining with coal the residues from copper pyrites. Dr. Thudichum thought that the whole fungus theory was nothing better than a vague and wild surmise. l f r . Wariiigton That water which had passed through iron-sponge does not yield fungi on addition of sugar may be due to tlie removal of the phosphoric acid which had been retained by the hydroxide of iron wherewith the sponge is coverecl. As to the experiments with charcoal I wisli t o observe that water filtering through fresh charcoal takes away from it some phosphates but after the filter has been used for a time this will no more be the case.This circumstance may perhaps explain the difference in the observations of Messrs. F r a n k l a n d and Heisch. Dr. D~iprB asked wliether Dr. F r a n k l a n d and Mr. Heisch had boiled tlieir sugar-solutions bcfore mixing them with the waters to be examined? Whenever he (Dr. DuprB) did so he obtained no fungi. Dr. F r a n k l a n d replying [to Dr. DuprB] My sugar-solutions were not boiled but in Experiment No. 9 the water had been previously boiled for a long time and the sugar and all the other substances heated to a much higher degree than that of boiling mater and I obtained more splendid fungi here than in any other case. [To Mr. HBiscli.1 The difference in our obsermtions regarding animal charcoal appears t o be satisfiictorily explained b~ blr. V a r i n g t o n ’ s observations. As to the two fungi from sewage and from white of egg I merely pronounced them to be similar to but not identical with one another. I paid no particular attention to the emission of butyric smcll. [To Mr. Bell.] 1 investigated the effluent water about 24 hours after its collection. (Here Dr. Russell remarked that he had done the same.) [To Dr. Voelcker.] I have also found that sewage is apt to have its ammonia converted into nitric acid. IX.-Ou the Bfecfa of I”1.essw.e on tlie Absorption of Gases by Charcoal. By J O E N HUNTER N.A. F.C.S. F.R.S.E. Professor of Mathematics and Natural Philosophy University of King’s College Windsor Nova Scotia. IN the following memoir I purpose giving the results of a few obser- vations on the effects of pressure on the quantity of a p s absorbed by cocoa-nut charcoal.