Jar
916
T H E JOURNAL OF I N D U S T R I A L A N D ENGINEERING CHEMISTRY
I n conclusion, the writer desires t o express his indebtedness t o Dr. F. E. Tuttle, head of t h e School of Chemistry, State University of Kentucky, not only for his general advice and suggestions during this investigation, b u t for his assistance in surmounting experimental difficulties t h a t at t h e time seemed insurmountable. CHEMICAL LABORATORIES, STATE UNIVERSITYOF KENTUCKY LEXINGTON. KENTUCKY
THE PHENOL COEFFICIENT OF GERMICIDES’ By
FREDB. KILMER. A. WAYNB CLARK
AND
POWELL HAMPTON
The desire for a method of comparing t h e disinfecting or germ-killing power of disinfectants with some standard and of expressing t h e results i n definite figures is one of long standing. The “Rideal-Walker” and t h e “Lancet” methods have been a prolific source of discussion in England a n d in our country as well. The problem herein discussed is a n attempt t o show some of t h e experiences involved in a n attempt t o follow t h e so-called hygienic laboratory method a n d arrive a t results concurrent with t h a t of another laboratory. The problem is a t once trite a n d practical for t h e reason t h a t disinfecting preparations are a p t t o be examined in several laboratories under somewhat varying conditions with t h e consequent liability of discordant figures. T H E HYGIENIC LABORATORY METHOD
45
so t h a t notes and results could be compared in order t o arrive at a thoroughly satisfactory and safe conclusion. This work was done by t h e Lederle Laboratories of New York City, and will be referred t o later. CULTURE MEDIUM
A critical examination of t h e document containing the official method, with t h e object of proceeding with t h e work, immediately brought out t h e fact t h a t there was therein no positive statement as t o the formula of the culture medium used. This statement is made on t h a t subject: “Standard extract broth is used. . . .The broth is made from Liebig’s extract of beef and is in exact accordance with the standard methods adopted by the American Public Health Association for water analysis. It i s important that the reaction of the media is just +1.5.” Careful reading of the above mentioned A. P. H. A. publication for 1912 shows it t o contain these statements: “Infusion of fresh lean meat, and not meat extract, shall be used as the basis of various media” and “the standard reaction of culture media shall be f 1 . 0 per cent.”
It is evident, therefore, t h a t there is no standard method for water analysis in which there is used broth made of beef extract or having a reaction of f 1 . 5 per cent. The reaction is, of course, easily adjusted b u t we call attention to t h e exceedingly loose statements made in this Bulletin, No. 82, and t h e consequent impossibility of complying accurately with its requirements on the information furnished. The formula for nutrient broth for water analysis given in the A. P. H. A. publication for 1912 is: Water . . . . . . . . . . . . . . . . . 1000 Meat . . . . . . . . . . . . . . . . . . . 500
Under date of April, 1912,there was published by t h e U. S. Public Health a n d Marine Hospital Service a pamphlet known as Hygienic Laboratory Bulletin No. 82. This contained a “Method of Standardizing Disinfectants,” b y John F. Anderson a n d Thomas Peptone.. . . . . . . . . . . . . . . . 10 B. McClintic. T h e method has since become known As ordinary broth is seldom used in water analysis as t h e “Hygienic Laboratory Method” according t o we examined t h e formulas for gelatin and agar media, the suggestion of t h e writers. I t has, we believe, been generally recognized t h a t this method has become these being the commonly used media. We find t h a t t h e official U. S. procedure for determining t h e value of gelatin t o be: Water . . . . . . . . . . . . . . . . . 1000 Meat . . . . . . . . . . . . . . . . . . . 500 of disinfectants. A casual examination of this publicaPeptone . . . . . . . . . . . . . . . . . 10 Gelatin . . . . . . . . . . . . . . . . 100 tion is sufficient t o indicate t h a t t h e subject has been thoroughly gone over a n d t h a t t h e older methods have and for agar: Water . . . . . . . . . . . . . 1000 M e a t . , . . . . . . . . . . . . . . . . . 500 received attention and criticism. The authors have 20 Agar . . . . . . . . . . . . . . 10 to 15 Peptone. . . . . . . . . . . . . . . . . attempted t o retain the good points of each of these older methods a n d a t the same time t o improve a n d It is plainly evident, therefore, t h a t i t is impossible revise in those points which seemed to them to be open t o choose t h e formula intended, b u t as t h e agar fort o objection. On t h e whole, t h e points brought mula is t h e one used where t h e culture is incubated forward seem to be well taken and the improvements a t 3 7 ” C., it was decided t h a t t h a t one should be used t o be along the line of making t h e method more practi- for this work on disinfectants because t h e incubation cal, more easily carried out by t h e average operator, temperature is stated t o be a t t h a t figure. Also it iess liable t o variations due t o t h e personal equation, was thought t h a t t h e exactness of t h e formula of the a n d capable of being carried out without t h e use of broth was probably n o t ‘ a very important factor. How far wrong was this belief will be seen later. Note unusual or special apparatus. The testing laboratory of Johnson a n d Johnson has t h a t S A L T is not mentioned in any of these formulas, tried out t h e method thoroughly and applied i t t o and also t h a t in t h e 1905 edition of the A. P. H. A. those substances manufactured by Johnson a n d Johnson “Methods of Water Analysis” it says: “Sodium chloride shall not be added to any culture medium which lay claim t o germicidal properties. This being a new a n d not yet well-tried method, herein specified.” i t was determined beforehand t o have t h e results The amount of beef extract t o be used was presumed carefully checked by a n outside independent laboratory t o be 3 grams and our formula was: 1 Presented a t the 50th Meeting of the American Chemical Society, 1000 grams 3 grams Water., . . . . . . . . Liebig’s beef extract.. . New Orleans, March 31 to April 3, 1915. Peptone (Witte) ....... 20 grams Reaction.. . . . . . . + 1 . 5 per cent
T H E JOLrRiVAL O F I N D U S T R I A L A N D E N G I N E E R I N G C H E M I S T R Y MANIPULATION
T o a novice in the manipulation of this method it seemed t o be very difficult t o keep a correct record of t h e position and contents of each of the many tubes, for t h e work must be done rapidly and practically automatically. As referred t o later, we decided t o make tests only a t t h e 2’/2 and I j-minute intervals a n d we constructed a time table for working by t h e clock from a n y even hour so t h a t the attention need not be taken from t h e actual manipulation. This time table is printed herewith. TIMETABLE FOR WORKIIG BY THE CLOCK PHEKOL-COEFFlCIENT BY HYGIENICLABORATORY METHOD 2’/2 and 15-minute tests O’CLOCK PHENOL DISINFECTANT 0.00 SBEDS SEED-4 0.01 SEEDT SEEDB 0.02 SEEDU SEEDC Inoculate S 2’/2 0.021/2 Inoculate A 21/2 0.03 SEED V SEEDD Inoculate T 21/2 0.031/2 Inoculate B 2’/2 0.04 SEEDW SEEDE 0.041/2 Inoculate U 21/2 Inoculate C 21/2 0.05 SEEDX SEED F Inoculate V 2112 0.05’/2 Inoculate D 2 1 / 2 0.06 SEEDY SEEDG Inoculate W 21/2 O.O6’/z Inoculate E 21/z 0.07 SEEDZ SEEDH O.Oil/z Inoculate X 21/2 Inoculate F 21/2 0.08 SEED I 0.081/2 Inoculate G 21/2 0.09 SEED J Inoculate H 2112 0,09‘/2 0.10 In&iate I 21/2 0.10’,/2 .... 0.11 I n k i i a t e J 21!2 0.1 1’/2 Inocuiate S 15 Inoculate A 13 0.15 Inoculate T 15 Inoculate B 15 0.16 Inoculate U 15 0.17 Inoculate C 15 Inoculate V 0.18 Inoculate D 15 Inoculate W Inoculate E 15 0.19 Inoculate X Inoculate F 15 0.20 Inoculate Y Inoculate G 15 0.21 Inoculate Z H 15 Inoculate 0.22 Inoculate I 15 0.23 .... J 15 Inoculate .... 0.24
T h e meaning of the words “seed” and “inoculate” need explanation. “Seed” is used as in the original paper. The tubes containing t h e various dilutions t o be tried are called seeding tubes, so we have used the word “seed” as meaning “ t o add t h e culture t o t h e diluted disinfectant.” By “inoculate” we mean t o transfer a “loop” from t h e seeding t u b e t o t h e subculture t u b e a t the expiration of t h e given time; thus, for example, at 2 : o j o’clock t h e culture is added t o t h e dilution in seeding tubes marked X a n d at z . 0 7 ’ / ~ o’clock a loopful of t h a t mixture is removed and dipped into sub-culture t u b e marked X 2 l / * . I t seems t o us t h a t a time table of this kind is a great help t o an operator who is only occasionally doing this work. Also, an ordinary large test-tube rack can be used instead of specially made blocks. DILrTIom-The making u p of t h e dilutions of t h e disinfectants brought out t h e necessity for a n interpretation of the meaning of t h e figures’given in t h e very useful and complete table printed in this pamphlet. The figures are expressed thus: r : 2 0 , I : 2 j, etc. Not until we gave considerable study t o i t was it made clear t h a t r : z o meant I t o 19 or I in 2 0 . It seems t o us t h a t a statement t o this effect might well have been made. especially as the peculiar situation develops t h a t I:I means full strength (no dilution) and 1 : 2 means j o per cent. TILIE-Further critical study of the details of t h e method fails t o explain why tests are made for 2 ’ / 2 , 5, 7 1 / * , I O , r 2 1 / 2 and 1 5 minutes, whereas only t h e
1701.
8, NO. I
2 l / 2 - and 15-minute figures are used in calculation of results. T h e only conceivable advantage would be t h a t a single mistake or outside infection could be shown t o be irregular and out of place in t h e table. As probably no one would rest on a single set of tests, this does not seem worth while. T H E PHENOL-There are two other substances entering into a series of tests of this character, which might be possible sources of differences in results: one is the phenol and t h e other the typhoid organism. The original publication states t h a t Merck’s “silver label” phenol was used. T e had none of this on hand and used (as in Table 1) a supply of Powers-WeightmanRosengarten Co. phenol t h a t we had on hand and which appeared t o be in good condition. This was made up j per cent b y assay as directed. Later we secured a supply of t h e Merck “silver label” phenol as in Table 11. THE ORGANISM-In Bulletin No. 82 no specific reference is made t o the source of t h e typhoid organism used in tests. We had on h a n d a culture of B typhosis originally from H. K. Mulford Co. We used this in all t h e work, each series of tests being made with a culture t h a t had been transferred t o fresh tubes of broth on three consecutive days a n d incubated a t 37’ C. for t h e z4-hour periods between t h e transfers. After this work was about finished we were informed b y Lederle Laboratories t h a t t h e so-called Hopkins strain of B. t y p h o s i s used b y t h e m was t h e one also used b y Anderson and McClintic. There is no statement t o t h a t effect in t h e original paper and our experience is not sufficient t o allow us t o state t h a t it is necessary t o use a certain strain. Perhaps i t is sufficient, as required in Bulletin KO.82, t h a t t h e culture
TABLEI-FIRST TESTSOF “HYGIENICLABORATORY METHOD”BY JOI~NSON
and JOHNSON LABORATORY ORGANISM, . , , , .Typhosis 24-hr. culture filtered ORGANICMATTBR-NO~~ 1.5 MEDIUM. . . , , . , , , .Ext:act 3, peptone 20, water 1000. Reaction CULTURE... . . . , . .0.1 cc. t o 5.0 cc. disinfectant (P.-W.-R. Co. phenol) SUBCULTURES.. , . . . . . . . . . . . , . , , . . .One 4 mm. loop to 10 cc. TEMPERATURES., . . . . . . . . . . .Contact 20’ C.. incnbatlon 3 7 ’ C. RESULTS... , , , , . . , .x = growth (not killed). 0 = no growth (killed) CAMPHENOLTime of coniact Time of contact PHENOL Dilutions 2 1 / 2 min. 15 min. Dilutions 2 1 / 2 min, 15 min. 1-100 0 0 1-80 0 0 1-200 0 0 1-110 0 0 1-225 X 0 1-120 0 0 1-250 X x 1-izn Y n 1-275 X X . _”_ 1-300 X X 1-140 X 0 X X 1-325 X X 1-150 1-350 X X 1-160 X Y 1--77F 1-180 X x 1-400 Y X
..
. . . . . .. . . . . ... . . ...
+
. ...
..
~
Phenol-coefficientof Camphenol =
(’00 120 + ”‘5) 140
i 2 =
1.60
shall be grown for 2 4 hours in each of three successive tubes of broth of definite composition. The typhoid organism was specifically selected because of its supposed uniformity, and t h e Hygienic Laboratory Method would lose some of its value in our opinion, if t o t h e already serere requirements was added t h e requirement t h a t a specified strain of organism should be used. S O U R C E O F SAMPLEs-The samples of Camphenol a n d Synol Soap used b y 1,ederle Laboratories were purchased in t h e open market from sources unknown t o us. The samples used in t h e Johnson and Johnson tests were taken from the shelves of the regular stockroom a t t h e works.
T E E J O U R N A L OF I N D U S T R I A L A N D ENGINEERING C H E M I S T R Y
Jan., 1916
47
TABLE 11-PHENOLCOEFFICIENTS OF CAMPHENOL AND SYNOLBY HYGIENIC LABORATORY METHOD RESULTS -~~ .~ . . . . . . . . . . . . . . . . x = arowth JOHNSON and JOHNSON LABORATORY ORGANISM, . . . . . . . . . . . . . . . . . . . . . . . . . . . Typhosis, 24-hr. culture, filtered ORGANIC MATTER . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . one MEDIUM., . . . . .Extract 3, peptone 10, salt 5, water 1000. Reaction +1.5 . . PHEKOL...................................... Merck’s “Silver Label” . . . . . . . . . . . . . ,o. . 1 CC. t o 5.0 CC. PROPORTION CULTURE _ ~ _ . TO DISINFECTANT, SUBCULTURES ................................ One 4 m m . 1 0 0 ~t o 10 cc. Incubation 37‘ C. TEMPERATURE., , . , , , , . . , , , , , , , , , , , ,Contact 20° C. CAMPHENOL Time of contact PHENOL Time of contact Dilutions 21/2 min. 15 min. Dilutions 2 1 / 2 min. 15 min. 1-100 0 0 1-180 0 0 1-110 X 0 1-200 0 0 1-225 0 0 1-120 X 0 1-250 0 0 1-130 X X 1-275 0 0 1-140 X X 1-300 0 0 1-325 0 0 X X 1-150 1-350 X 0 X X 1-375 X 0 1-160 1-180 X X 1-400 X X 325 375 Phenol-coefficient of camphenol = (--) i 2 = 3.20 100 120 SYNOL Time of contact PHENOL Time of contact Dilutions 2112 min. 15 min. 2 1 / ~min. 15 min. Dilutions 1-30 0 0 0 0 1-90 1-100 X 0 1-35 0 0 1-40 0 0 1-110 X 0 1-45 X 0 X 1-120 0 1-50 X 0 1-130 X X 1-55 X 0 ... 1-60 X 0 1-140 X X X X 1-65 X X X X 1-150 1-70 1-160 X X 1-80 X X ~
~
~~
~
~~~~
~
~~
+
Phenol-coefficient of synol =
40 (-90
+0 ) 120
i 2 =
0 47
RESULTS
I n reference t o the results given here, all were checked by duplicate series, both in t h e Johnson a n d Johnson a n d t h e Lederle laboratories. T h e results were considered sufficiently concordant a n d t h e tables are t h e ones considered most accurate. Table I gives t h e results of our original work on camphenol. Knowing t h e composition of this preparation, it seemed incredible t h a t the results should be SO low (1.60). T h e receipt of information from Lederle Laboratories t h a t their results showed i t t o have a coefficient of 3.46 (see Table 11) indicated at once t h a t there was need of comparison of methods pursued in t h e manipulation. A conference on t h e subject developed t h e fact t h a t Lederle Laboratories were using a broth containing sodium chloride a n d only I O grams peptone, instead of 2 0 , t o each 1000cc. Following up t h e statement of Lederle Laboratories t h a t theirs was t h e correct formula for this work, we addressed a letter t o Dr. Anderson in Washington asking for t h e formula used in his original work and received a reply t h a t t h e broth contained Liebig’s extract 3, Witte’s peptone IO, salt 5, water 1000. I n other words, we h a d reasoned wrongly so we made up new broth and in t h e meantime, having secured some Merck’s “silver label” phenol, used t h a t also in all future work. Table I1 gives t h e results on camphenol; we consider t h e 3.20 t o be sufficiently concordant with t h e Lederle result of 3.46. We are unable t o offer a n y rational explanation in t h e results on camphenol of t h e great difference between t h e tests when salt is used in a n d omitted from the broth. It seems hardly possible t h a t t h e minute amount in 0.1cc. of t h e culture used in contact with t h e 5 . 0 cc. of diluted camphenol should produce such difference on account of chemical reaction with t h e camphenol. Also, such reaction would seem likely to consist in a certain amount of camphenol being
(not killed). 0 = no growth (killed) LEDERLELABORATORIES ORGANISM., . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B . typhosis (Hopkins) ORGANIC MATTER . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . None AGE OF CUI.TURE., . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .24 hrs. a t 37’ C. MEDIUM.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .Standard beef extract broth DOSE.,. . . . . . . . . . .,O.l cc. of filtered culture t o 5 cc. diluted disinfectant SUBCULTURE.. ..................... .One 4 mm. loop to 10 cc. of broth TEMPERATURES. . . . . . . . ..Medication 20° C. Incubation 37’ C. (48 hrs.) MINUTESEXPOSURE TO DISINFECTANT DILUTIONS21/2 5 71/1 10 121/2 15 PHENOL . . . . . . . . . . . . . . . 1-80 0 0 0 0 0 0 1-90 x 0 0 0 0 0 1-100 x x x 0 0 0 1-110 x x x x x x 0 0 0 0 0 0 CAMPHENOL. . . . . . . . . . . 1-250 0 0 0 0 0 0 1-275 0 0 0 0 1-300 x 0 1-350 x x x 0 0 0 x x x x x x 1-400 x x x x x x 1-450 275 350 Phenol-coefficient of camphenol = (-) i 2 = 3.46 80 100 MINUTESEXPOSURE TO DISINFECTANT 5 71/2 10 121/2 15 DILUTIONS21/2 PHENOL . . . . . . . . . . . . . . . 1-80 0 0 0 0 0 0 1-90 x 0 0 0 0 0 1-100 x x x 0 0 0 1-110 x x x x x x SYNOLSOAP.. 1-25 0 0 0 0 0 0 1-30 0 0 0 0 0 0 1-35 x 0 0 0 0 0 1-40 x x 0 0 0 0 1-45 x x x x 0 0 x x x x x x 1-50 1-60 x x x x x x 30 45 Phenol-coefficient of synol = (loo) + 2 = 0.41$ 80
+
.........
+
removed from active participation in germ-destruction a n d hence would reduce, not increase, t h e figures. Sodium chloride, in common with many other salts, we of course know, throws out of broth, etc., certain organic constituents of animal fluids, but this suggestion, in view of t h e small amount of broth used, does not supply a satisfactory explanation. However, such action, removing as i t would a certain quantity of organic matter from t h e broth, would seem t o tend toward t h e increase in the figures. The use of less peptone might also influence the results in t h a t direction. As t o phenol, we think i t hardly possible t h a t t h e clean crystallized chemical could be held accountable for such widely divergent results. We regret t h a t we have not found time t o thoroughly check u p these factors. T h a t t h e camphenol result in Table I1 is exactly double t h a t in Table I is merely a coincidence-not a mathematical error. DISCUSSION
I n considering these results we must always remember t h a t the high accuracy of a chemical analysis cannot be expected because we are dealing with a living organism under conditions t h a t are as well controlled as t h e state of t h e science permits b u t yet may be relatively quite variable. Also t h e two laboratories were using different samples of the disinfectants and different strains of t h e typhoid organism both of which may or may not have a slight effect upon the result. On the whole, these results are probably as nearly concorda n t as a n y two operators could secure in tests of this kind. AS t o t h e Hygienic Laboratory Method we feel fairly well satisfied with the results and with t h e method except for t h e unfortunate omissions referred t o above. We see no reason why this method should not continue t o be satisfactory a n d of sufficient accuracy t o remain as a standard American method for determining the phenol-coefficient of disinfectants. JOHNSON AND JOHNSON LABORATORIES N E W BRUNSWICK, N. J.
.
