REMOTE STORAGE L P NOTES ON THE BACTERIOLOGICAL ANALYSIS OF WATER. BY L. H. PAMMKL. The recent epidemic of typhoid fever at the college is of interest to us and especially the methods now in vogue 1 with reference to the examination of water for various organisms. During the recent epidemic and previously the well waters in the vicinity of Ames as well as the college water supply were examined at various times. An I examination has also been made of water coming from wells of the parties who have furnished milk to the college. It should be stated here that this report is not completed owing to the fact that some of the species have not been sufficiently determined. From the nature of the case it requires a great deal of patient and careful work to run out the different species, so that the biological examination was not completed. Thanks are due to Mr. F. W. Faurot, Mr. A. D. McKinley, Mr. H. H. Thomas, Miss Nellie Nicholas, ; Miss Estella Paddock, and Mr. L. R. Walker for assistance I in carrying out this work. In the paper on the Iowa State College Sewage Disposal j Plant will be found a brief note on the water of the deep well previous to this spring. Examinations have been made from time to time, and as a result of our woi’k, we I found that the water during the winter months varied from r no bacteria to 50 per cubic centimeter, thus showing an i unusually good supply of water. A Marslon, J. B. Weems and L. H. Pammel. The Iowa State Collejje Sewage Disposal Plant and Investigations. Proc. la. Engineering Soc. IVOO. Contr. la. State Coll. Argrl. & [ Mech. Arts. 1:19. — 2 — BRILEY SHALLOW WELL. Depth, 45 feet; 18-inch glazed tile, cemented at the joints, covered with boards on top. The well has not been used since October 20th. DATE-. GAS TEST. Total number bacteria per CC. GELATINE. Agar. LITMUS AGAR. Lique-fy- ing. Non-lique- fying October 17th October 18th Present 18.000 12.000 6. 000 1,440 2.000 2,400 125 125 Used. Used. Used. Used. Used. Used. Some acid- pro- ducing germs. October 25th .. October 25th .... October 25th October 25th October 29th . . . First pumping One-half barrel pumped One barrel pumped 1 % barrel pumped Present BRILEY DEEP WELL. Depth, 185 feet; 2-inch pipe and casing. October 17th 60 Used. No acid-produc- October 17th .... None 30 ing germs. October i8th ..... None 30 October 27th 30 Used October 29th 30 PRITCHARD WELL AND TANK — WELL. Depth, 170 feet; 3-inch casing well and inside a 2-inch pipe. October i8th 20 October 22d 30 3c Used. October 29th 20 20 Non - acid - pro- October 18th 60 20 40 ducing. TANK. Open tank used for watering stock, above well. October 29th October 29th 33^ CC October 31st 225 225 October 29th 3^ CC Acid reaction. — 3 — PETERSON WELL AND TANK. — WELL. Depth, 185 feet; 120 feet down to cylinder. Cased. Two inch iron, with about four inch of casing. Located two miles north of Ontario. TANK. Open tank for watering stock. October i8th 3 cc 2, 600 4,200 25 Acid producing October 29th Acid 360 2, 240 October 31st 10 molds 4, 200 320 100 acid. October 18th 220 non acid. SKELTON WELL. Thirty-five feet deep, ten inch casing. October i8th 0 0 30 Non-acid. October i8th October 27th 3 cc Acid. October 31st 200 200 October 27th 2 molds 90 6.33 October 27th NO. 2 633 RIVER WATER. DATE. SKUNK RIVER WATER. DATE SQUAW CREEK WATER. Total num- ber germs per cc. Medium used agar. Total num- ber germs per cc. Medium used agar. April 30th 1,800 1,800 916 1,800 27, 000 May 19th 300 11.200 16.200 8,520 2,400 May 7th July 2nd May 9th August 8th May 19th Augu.st 8th July 6th October 4th _4 — Investigations carried on with the water supply of var- ious wells in the vicinity of Ames by Messrs. McKinley and Thomas and Mr. Faurot gave the following results: fatjrot’s well. DATE. Number germs per cc. REMARKS. April 23d 1,600 4,500 9,360 9, 480 220 Collected after a rain April ^^d May 22d May 29th July 6th Average 5,032 OTIS HOUSE WELL. May 7th — May 2ist — May 28th July 2d August 8th.. August 8th.. October 4th. October 4th. October 23d. October 23d. 80 3 200 54,000 120 None 120 360 3,000 2, 400 Collected without ice. Collected without ice. Collected without ice. Indication of something in pipes. Indication of something in pipes. After pumping 15 minutes, collected with ice. With ice— first pumping. With ice— after pumping. First pumping— no gas. Second pumping— no gas. Average 6,028 LABORATORY TAP. May 7th None. None. 360 520 700 80 Poured immediately. Poured immediately. Poured immediately. Poured immediately. Poured immediately. Poured immediately. May 2ist October 4th October 17th October 17th November 6th Average 276 PARSON S WELL. May 7th 3,600 Failure. Well full. May 2Tst Well full. May ii8th 1,300 90 150 170 Well full. With ice. First pumping. Very little water in well. With ice. First pumping. Very little water in well. With ice. Second pumping. Very little water in well. J 111 y ^Tid August 8th August 8th October 23d 50 Without ice. Second pumping. No gas. October 23d 380 Without ice. First pumping. Average . 643 — 5 — illsley’s well. DATE. Number germs per cc. REMARKS. May 7th 8,000 Failure. 600. 1, 200 590 220 80 800 Without ice. First pumping. With ice. Second pumping. No gas. Second pumping. First pumping. May aist May 28th Tulv 2d August 8th August 8th October 23d October 23d Average 1, 642 WELL AT HOaSE, NEAE BRICK YARD. May 2ist 300 330 TO, 800 7,800 1.400 5.400 First pumping. With ice. Second pumping. With ice. First pumping. With ice. Second pumping. August 8th — August 8th . . October 4th October 4th. Average 4.338 CREEK WATER. May 19th July 2d 300 11,200 16, 200 8,520 2 , 4 C 0 Without ice. With ice. With ice. August 8th August 8th October 4th Average ... 7,724 OLSEN’s WELL. May 28th ... August 8th.... . August 8th October 4th October 4th . ... October 23th -October 23th Average 10 60 120 620 240 • ' With ice. Wind mill in operation one-half day. Wind mill in operation one-half day. With ice. First pumping. With ice. Second pumping. Without ice. First pumping. Without ice. Second pumping. No gas. 286 FOUNTAIN WATER IN PARK, STORY CITY, IOWA. October 7th 4,500 20 Without ice. Poured in laboratory. No gas. Poured immediately. October i3ih HIGH SCHOOL, STORY CITY, IOWA. October 7th 0 0 Collected without ice. No gas. » Poured at well. October 13th — 6 — HENRYSOn’s well, story city, IOWA. DATE. Number germs per cc. REMARKS. October 7th 280 230 Collected without ice Produced gas. Poured at well. October 13th HYDRANT, STORY CITY, IOWA. October 7th October 13th 0 0 Without ice. No gas. Poured at hydrant. C. & N. W . WELL AT WEBSTER CITY, IOWA. October 6th 310 Without ice. Gas. A. J. HAVILAND’s well, fort dodge, IOWA. October 5th 150 Without ice. 30 moulds. WILL HAVILANd’s WELL, FORT DODGE, IOWA. October 5th 5,400 Without ice. The records kept by Miss Nicholas were as follows : munn’s well. May 5th 570 Agar used. September 24th 300 Agar used. October nth 80 Agar used. pammel’s well. September 9th 1,300 Agar used. August nth 400 Agar used. September 27th 510 Agar used. budd’s well. May 5th 50 Agar used. i September 27th.. .. 40 Agar used. | October 8th 30 Agar used. October 27th 20 Litmus agar used. Non-acid producing. — 7 — eeed’s well. DATE. Number germs per cc. REMARKS. May 17th 2, 500 Agar used. May 31st 1,200 Agar used. September 19th.. , . 7C0 Agar used. October 27th Litmus agar used. Acid and non-acid. miller’s well. May 17th May 31st 0 0 0 Agar used. Agar used. Paxton’s well. May 17th September 19th September 27th,. . . 1,900 1.300 2,4CO Agar used. Agar used. Agar used. Hardin’s well. May 31st 30 Agar used. Lincoln’s well. May 5th May 31st September 27th 300 400 100 Agar used. ) Agar used. >No gas at any time. Agar used. ) hunt’s cistern. May 17th 150 Agar used. • hoover’s spring. May 17th October 27th 2,400 40 Agar used. Litmus agar used. Non acid producing. — 8 — The following are the results of Miss Nicholas of exam- ination of samples, the second after discarding a few pails- full. The medium used was ordinary agar. DATE. Well Fjrst pumping. Second pumping. September 9th Lincoln. .. Munn 460 240 30 180 1,700 2, 800 330 230 20 170 1,600 6,000 September 9th October 8th Budd October 8th Lincoln. . . Reed October 8th Kinkade .. The Kinkade well is very shallow and the second sample was collected after several barrels of water had been pumped out, therefore the much greater number of bacteria in the second sample may be due to sediment. All of the shallow wells examined contained gas-produc- ing germs. The Paxton well produced 30 cc. of gas in the fer- mentation 4:ube, 10 cc. of which was 00^ and 20 cc. CH^ . The Reed well produced 100 cc. of gas (40 cc. CO^ and 60 cc. CH 4 ). The water from the Kinkade well produced a very great amount of gas. The Briley Shallow Well . — In conjunction with Dr. Weems and Mr. McKinley on another occasion the writer collected samples of the water at the Briley well, and later Mr. Faurot also collected this water twice. The second time when Mr. Faurot collected these samples we got an unusually large number of germs per cubic centimeter. That collected by the writer on October 17 had 18,000 and that by Mr. Faurot had 6,000. It is worthy of note in this connection that the samples collected by myself on October 17 contained 18,000 germs per cc., that in one of the samples collected by Mr. Faurot on October 25, the number of germs had diminished very materially, the largest number found was 6,000. On October 29 the highest number obtained was 125 per cc. y — In regard to the last plates poured it is a singular fact that but a very small development occurred, and this is strange since we had such an unusual development before running from 6,000 to 18,000 per cubic centimeter. In regard to the condition of the well it looks as though the water could easily have drained off from the surface,, but nevertheless upon removing some of the boards from the top of the well I found that the water might easily have entered between the cracks of some of the boards. In fact I found moisture on the inside on the upper tile,, showing the water had run down. One can readily see how B. coli-communis or other foreign organisms could get into the water. Gas was produced in one tube poured by Mr. Faurot and a slight amount in another. In this case we made the usual test. We also obtained gas from the first plates that I poured. The samples collected on October 29 were kept for forty days in the laboratory and then were examined by Mr. McKinley and Mr. Thomas with the following results: WELL. Depth. No. of germs. Briley Shallow Well 45 feet. 185 feel. 200 20 10 None. 30 340 1. 000 30 Briley Deep Well Laboratory tap. Same source Kitchen Tap Skelton’s Well Peterson Deep Well 35 feet. 185 feet. Peterson’s Trough Pritchard Well 170 feet. Various species were found. Some of these have been excluded as having no connection with Bacillus typhosus or B. coli-communis. On the other hand there are a number of species that belong to the typhosus group culturally so far as has been carried out. Our work was interrupted although cultures of all of the species were made and placed away for further study. Fire destroyed the entire labora- tory so no further study can be made. One peculiar pearly white Bacillus developed in consid- erable quantity, in fact at least three-fourths of the colon- — 10 — ies belonged to this species. This Bacillus though actively motile had none of the cultural peculiarities of B. typhosus. Two species are quite commonly found in surface waters, namely the B. cloacm first detected by Jordan in sewage. I am inclined to think that both B. coli-communis and B. cloacce occurred in the Briley shallow well, but the definite separation was not carried far enough to determine this point to my satisfaction, though Dr. Eli Grimes states B. coli-communis was found. THE COLLEGE WATER SUPPLY. It is certainly worthy of mention in this connection that all of the species found in the college w’ater supply in the tank are non-liquefying, and the fact that gas was found on one occasion does not argue that the college water sup- ply was contaminated. The simple fact that the species here found did not produce gas in the proportion given for B. coli-communis, namely, of two parts of H. to one part of CO2, but represented by formula one to two. It is also a significant fact that morphologically none of the species found indicated either B. coli-communis or B. typhosus in the college w^ater supply. Of the oft-repeated statement that sewage contamina- tion might have occurred, I wish to state that the writer, together with Professor Marston, climbed to the top of the towmr and investigated conditions, and everything was found in its usual good condition. There was certainly no indication of growth of algae on the water, nor were there any indications of other filthy conditions. In fact, the water, and everything connected with it, seemed to be in an ideal state. The statement has also been made that owdng to the fact that the college at different intervals used the supply from the spring, and in this way became contaminated. An investigation made of the college spring water, as well as the different hydrants and cisterns, those of Professor Stanton, Professor Curtiss, and the old Sexton well, indi- Experimental Investigations St. Brd Health, Massachusetts, 1889 - 1890 ; 836, and later found by Moore to be widely distributed in the soil. Russell and Bassett. Trans. Amer. Pub. Health Asso., — 11 — cate unusually good water, with the exception that in the Curtiss well and the Sexton well gas was produced, but this undoubtedly came from the surface soil. The spring water showed no gas whatever, nor was any obtained from the hydrant which was next to the spring. The samples and plates were carefully plated. BACTERIA FOUND IN OTHER WATER SUPPLIES. We have found quite commonly in all of our waters the B. liquefaciens-fiuorescens. The Tyroth’ix of Duclaux is certainly also common. Most attention has been given to the chromogenes. The common genera of Bacillus and Micrococcus were represented, and of the these the Micro- cocci were found more frequently than the Bacilli of these Micrococcus roseus-flavus, Hefferan, M. agilis, A. Cohn, and others were found. BACILLUS TYPHOSUS IN WATER. Now, as to the relative vitality of Bacillus typhosus in water; many determinations have been made, and it would not be strange if the Bacillus typhosus should not be found in water. It is usually held by sanitarians that water is the most frequent source of infection. The evidence of B. typhosus in water, in most cases, is circumstantial; but I recall a case where Dr. Ravold found it in Mississippi river water, and bacteriological journals report cases of its occurrence in wells and streams, but the reported findings of the organism under such circumstances are not numerous. It is very evident that the typhoid fever bacillus will not grow in the ordinary media with other pathogenic o-gan- isms, nor are the special media much more satisfactory. It is evident from the results obtained from several investi- gators that not much can be expected from the organism after four weeks. It is certain that the typhoid fever organism will not multiply freely in water. MILK AS A SOURCE OF CONTAMINATION. As to the bacteria found in the milk supply, an investi- ^ gation has been made, but this work was not completed, -- 12 — owing to the destruction by fire of all of our cultures. We found present in the milk a large number of chromogenes^ but none of these, of course, can be referred to, or are in any way related to the typhoid fever bacillus. On the other hand, we did find B. coli-co.mmHnii>,h\xi\tdioeBnot necessarily follow that the B. coli-communis comes from human dejecta, as this organism is very commonly found in connection with cow stables, and the organism being found quit frequently in the intestinal tract of animals as well as man. Therefore this cannot be considered to be the cause, nor as an argument against the use of milk. This work, however, was not completed, and hence a final statement cannot be made. COMPARISON WITH THE SEWAGE BACTERIA. The results of the work carried on on the College Sew- age Plant show the following conditions with reference to the purification, and it is of interest to compare these results with the water obtained from the Briley well. It will be seen that in every case, excepting the last one, that the Briley w^ell contained many times more organisms than the effluent of either filter bed. DATE From Air Water September September September September September September September September September September September September September September September September September September September September September September September September September September September September ist 2d 3 d. 4th 5th 5th 5th 6th 7th 8th 9th loth loth loth nth i2th 13th 14th 15th i6th 17th 17th 17th 18th 19th 20 th 2 ISt 22d. W. E.... W. E E. E W. E E. E Tank' 90 degrees 72 degrees 62 degrees 63 degrees 82 degrees Manhole . W. E 83 degrees W. E 82 degrees W. E 90 degrees E. E..... Tank 87 degrees Manhole . E. E 68 degrees E. E 69 degrees W. E 70 degrees W. E 84 degrees W. E 85 degrees W. E 55 degrees E. E 65 degrees E. E Tank 68 degrees Manhole . W. E 50 degrees W. E 66 degrees E. E 72 degrees W. E. ... 71 degrees E. E 79 degrees 75 degrees 71 degrees 73 degrees 72 degrees 72 degrees 68 degrees 68 degrees 72 degrees 74 degrees 74 degrees 74 degrees 62 degrees 72 degrees 73 degrees 74 degrees 72 degrees 74 degrees 74 degrees 72 degrees 70 degrees (34 degrees 68 degrees 64 degrees 65 deg rees 67 degrees 67 degrees 66 degrees Manhole Tank Effluent 242, 400 1,363,000 424, 200 696, 600 484,600 960 , 4CO , 100 390 230 i,8oO' 460 230- 310 2 ia 440. no 1 , 200 480 100 320 3.600 460 340 420' 340 480 — 13 — From September 23cl to September 28th, inclusive, the sewage effluent pipe was under water, hence no samples. From Air Water Manhole Tank Effluent E E 69 degrees 68 degrees 72 degrees 64 degrees 64 degrees 65 degrees 67 degrees 980 460 360 W E W E Tank 568, 400 Manhole . WE . . 896, 600 80 degrees 75 degrees 81 degrees 80 degrees 72 degrees 63 degrees 40 degrees 67 degrees 67 degrees 67 degrees 67 degrees 68 degrees 68 degrees 68 degrees 62 degrees 61 degrees 70 degrees 1, 200 360 1, 800 450 1,200 2, lOp 1,800 E E W E E E E E W. E E E Tank 260, 000 Manhole . W. E 1,333, 200 63 degrees September 29th September 30th October ist October ist October ist October 2d October 3d October 4th. . . . October 5th October 6th October 7th.... October 8th October 8th October 8th October 9th From 10th to 13th, inclusive, the beds were being <;leaned and the sewage was turned directly into the creek from the tank. October 14th, October 15th 'October 15th October 15th October i6th October 17th October i8th W. E W. E Tank Manhole . W. E W. E E. E 63 degrees 63 degrees 60 degrees 55 degrees 63 degrees 63 degrees 63 degrees 63 degrees 64 degrees 62 degrees 62 degrees 61 degrees 2r2, 000 360 210 120 120 130 * Too thick to count. Estimated at 5,000,000. CONCLUSION. It may be stated that so far as the analysis show the col- lege water supply may be considered excellent. It is true that in a number of instances more organisms were found than at other times, but an examination made from time to time shows that the number is not unusually large, and on the whole that we may consider our water supply practi- cally pure, and I should also state that the water from the spring supply is unusually good. We should bear in mind that the failure to find the typhoid fever bacillus in the water supply or milk of the Briley well is not at all surprising. It is a well known fact that the saprophytic species grow so readily in the nutrient media that the typhoid fever bacillus has not the same chance to grow. The same may also be said with reference to milk, only here we are dealing with such a large — 14 — number of species that it would be a mere accident to dis- cover the organism. As said heretofore it seems to me to be reasonable that the milk formed a favorable medium for the growth of the organism, and be it specially remem- bered that Mr. Briley, from his own testimony, failed to wash the cans with boiling water as should have been done. The milk cans could easily have been contaminated, and the failure on his part to wash the cans, it seems to me, made it not only possible but probable that these germs propagated in the milk. A comparison of the water of the Briley well and the college effluent shows that the Briley well had a greater amount of contamination than the college effluent from the sewage filter beds.