Issued May 31, 1907. U. S. DEPARTMENT OF AGRICULTURE, BUREAU OF ANIMAL INDUSTRY. BULLETIN No. 100. ' A. D. MELVIN, CHIKF OF BUREAU. THE GERMICIDAL VALUE OF LIQUOR CRESOLIS COMPOSITUS (U. S. P.). BY C. N. McBRYDE, M. D., Bacteriologist, Biochemic Division. WASHINGTON: GOVERNMENT PRINTING OFFICE. 1907. Property of the United 'Sts Issued May 31, 1907. U. S. DEPARTMENT OF AGRICULTURE, BUREAU OF ANIMAL INDUSTRY. BULLETIN No. 100. A. D. MELV1N, CHIEF of BUREAU. ' THE GERMICIDAL VALUE OF LIQUOR CRESOLIS COMPOSITUS (U. S. P.). BY C. N. McBRYDE, M. D., Bacteriologist, Biochemic Division, WASHINGTON : GOVERNMENT PRINTING OFFICE. 1907. BUREAU OF ANIMAL INDUSTRY. Chief: A. D. M K.I.MS. Assistant Chiif: \. M. FARRIN<;T>S. Chief Clrrk: K. I*. .1. Biochemic Division: M. DOKHKT. chief; JAMES A. EMERY, assistant chief. 7>airy Division: Ki>. II. WKHHTKR. chief; <'. 15. LANK, :i.-.i-t:uit chief. InxiKi-tion Dii"ision: RICE P. STKIIM>M. chief; MOKIUK WOODKS. ;is.*i.tant chief. Pathological l>ii ixinn: JOHN R. MOIII.KU. chief; HENRY J. WAHIIBTRN, assistant chief. Quarantine Division: RICHARD W. HICKMAN, chief. Division of Zoology-' W. H. RANSOM, chief. Erprriment Station: E. C. SCHROEDER, Hiiperinteiulent; W. E. COTTON, assistant. Animal Husbandman: GEORCE M. ROMMEL. Editor: JAMES M. PICKENS. Librarian: BEATRICE OBERLY ROGERS. RIOCHEMIC DIVISION. Chiff: M. Dorset. Assistant chief: James A . Emery. Meat inspection laboratories: T. M. Price, chemist in charge of central lalxirat>ry: A. E. Graham, Ralph Hoagland, C. H. Swanger, \Villiam B. Smith. E. A. H<>\r A. H. Roop. Robert M. Chapin, Clarence T. N. Marsh, Philip Castleman, E. II. IMI;T soil, W. P. Colvin. assistant chemists in branch laboratories. /A-/ cholera investigations: In charge of Chief of Division; B. M. Bolton, bacteri- ologist; C. N. McBryde, bacteriologist; W. B. Niles. inspe<-tor in charge of licld experiments. Bacteriological investigations of meat food products: C. N. McBryde, bacteriologist in charge. Poultry digestion experiments: E. W. Brown, assistant chemist in charge. Investigations of dips and disinfectants: James A. Emery, chemist; C. N. McBryde, bacteriologist; A. M. West, assistant bacteriologist. Preparation of tuberculin and mullein: In charge of Chief of Division; A. M. West and H. J. Shore, assistant bacteriologists; \V. S. Stamper and II. S. McCauley. fatanta. 2 LETTER OF TRANSMITTAL. U. S. DEPARTMENT OF AGRICULTURE, BUREAU OF ANIMAL INDUSTRY, Washington, D. C., April 12, 1907. SIR: I have the honor to transmit herewith, and to recommend for publication as Bulletin No. 100 of this Bureau, a paper entitled "The Germicidal Value of Liquor Cresolis Compositus (U. S. P.)," by Dr. C. N. McBryde, bacteriologist in the Biochemic Division. The substance termed "liquor cresolis compositus" has recently been introduced into the United States Pharmacopoeia, and the results of Doctor McBryde' s experiments show that it possesses a germicidal value which exceeds that of carbolic acid for the bacteria upon which it has been tested. It therefore seems that this solu- tion may be of considerable importance as a disinfectant. Respectfully, A. D. MELVIN, Chief of Bureau. Hon. JAMES WILSON, Secretary of Agriculture. 3 CONTENTS. Pne-p. Introductory 7 Standard methods of testing disinfectants 7 The drop method 8 The rod method 8 Technique of present experiments I 9 Comparison of carbolic acid with lime and carbolic-acid mixture 9 Comparison of carbolic acid with liquor cresolis compositus made from commer- cial cresols 11 Comparative value of solutions of liquor cresolis compositus made from different cresols 14 Separation of the three cresols from commercial cresol 14 Germicidal value of liquor cresolis compositus made from the three cresols. 15 Comparison of carbolic acid with liquor cresolis compositus made with low- boiling cresol 18 Experiments with Bacillus tuberculosis 20 Experiments with Bacillus anthraris 22 Summary of results 24 5 THE GERMICIDAL VALUE OF LIQUOR CRESOLIS COMPOSITUS (U. S. P.). INTRODUCTORY. The following tests were undertaken in connection with the ques- tion of disinfecting cattle cars. Heretofore the United States Depart- ment of Agriculture has recommended the use of a carbolic acid and lime mixture for the disinfection of cattle cars, pens, chutes, etc. As there were certain objections to the use of this mixture it seemed advisable to determine whether a cheaper and more efficient disin- fectant could not be found for this purpose, and "liquor cresolis com- positus," which has been recently added to the United States Phar- macopoeia, seemed a promising substitute. Liquor cresolis compositus is a liquid soap containing 50 per cent of cfesols, which have been shown to possess high disinfectant prop- erties. It is a thick, dark brown, oily looking liquid and is miscible with water, giving clear, saponaceous, frothing solutions. A review of the literature on disinfectants fails to reveal any pre- vious tests with liquor cresolis compositus (U. S. P.), and the object of the tests described in the following paper was to determine its efficiency as a disinfectant in comparison with carbolic acid, which is usually adopted as a standard of comparison in testing disinfectants. In testing the efficiency of a disinfectant the ideal method would be to apply the tests under the same conditions that prevail in practice, according to the purpose for which the disinfectant is to be employed. In the present instance, however, where the object in view was the disinfection of cattle cars, chutes, pens, etc., this would have been quite impracticable on account of the wide variation in conditions which would be met with in practice, and it was therefore deemed best to adopt some standard method. STANDARD METHODS OF TESTING DISINFECTANTS. Of the various methods devised for testing disinfectants, the best known perhaps are the "thread," the "drop," the "garnet," and the "rod" methods. In a recent publication the disinfectant standardization committee of the Royal Sanitary Institute of London reports in favor of the drop method. This report is based on an extensive series of experiments conducted by Firth arid Macfadyen to determine the comparative "Journal of the Royal Sanitary Institute, Vol. XXVII, No. 1, 1906. 7 O1BMICIDAL VALI'K OF LIQUO1 CTOBOLXI COMP08ITU8. vjiluo of the thread, drop, and garnet method*. The committee considered the drop method Miperior to both the thread mid 'jarnet methods, not only becau.se of its simplicity and facility, hut because it yielded sharper and more definite iv-iilt. Tin- committee con- sidered the thread method too elaborate lor general u>e and 'emi- nently unfitted for working with mioTO-OIgailinili at all sensitive to desiccation." As to the garnet method, the committee found that the result- obtained by this method are "too irregular to justify its adoption as a means of obtaining any comparative figure of disinfecting efficiency." In the following paper (lie tesi^ were made according to the drop and rod methods, which may be briefly described as follows: I1IK DROP METHOD. This is one of the simplest and most widely used methods of testing disinfectants, and has been variously modified. The drop method employed in these experiments is that described by Ridcal and Walker, and is the one which was adopted by the British standardi- zation committee, referred to above, as the most satisfactory method of testing disinfectants. The procedure in this method is to add o drops of a 24-hour culture of the organism upon which the disinfectant is to be tested, grown in beef broth, to 5 c. c. of the disinfectant. The mixture is then well shaken, and subcultures are made from it at intervals of 2\ minutes up to 15 minutes. These subcultures arc then incubated at 38 C. for at least 2 days. THE ROD METHOD. This method was devised by Hill fc , and may be regarded as a convenient modification of the thread method. Glass rods th; sixteenths of an inch in diameter and # inches long are etched at one end with diamond ink. Cotton is then wrapped about the rods near one end, and the rods are thrust into test tube* ^ a- t engage the cotton in the mouth of the tube. The tubes containing the rods are then sterilized by dry heat. In making the tests the rods are removed from the test tubes and the inclosed or etched portion dipped into a suspension of the organism, and then held in the air for a few moments until dry. The rods are then transferred to test tubes containing 5 c. c. each of the disinfectant to be tested ;.nd ex|M>sed to the action of the disinfectant for varying lengths of time. After exposure to the disinfectant the rods are dipped in sterile water in order to remove all traces of the disinfectant and are transferred to tubes of sterile beef broth, which are incubated for at least t \voda\- Journal of the Sanitary Inotituto, Vol XXIV, 1908. 6 Report of American Public Health Amociatinn, Vol. XXIV, 1898. PRELIMINARY COMPARISONS. 9 at 38 C. The suspension of the organism into which the rods are dipped is made from a 24-hour culture, grown on ordinary neutral agar, by rubbing up in several cubic centimeters of sterile water enough of the surface growth to give a suspension of approximately the same density as a 24-hour typhoid culture. TECHNIQUE OF PRESENT EXPERIMENTS. In carrying out the experiments the following conditions wore observed: (1) All tests with disinfectants were made at room tem- perature, which was duly recorded for each experiment. (2) The beef broth, prepared from freshly chopped meat with the addition of peptone and salt, had a standard reaction of 1 per cent acid, as recommended by the American Public Health Association. All tubes of beef broth contained 5 c. c. of the fluid. (3) All subcultures, in both the rod and drop methods, were made in beef broth, and were incubated at 38 C. for 6 days, except where growth developed earlier. (4) In the drop experiments the same platinum loop was used for inoculating the subcultures. (5) Fresh dilutions of the disinfectants were prepared for each experiment in sterile flasks with sterile dis- tilled water. COMPARISON OF CARBOLIC ACID WITH LIME AND CARBOLIC- ACID MIXTURE. Before proceeding to a comparison of liquor cresolis compositus and carbolic acid it seemed advisable to compare the lime and car- bolic-acid mixture recommended in the regulations of the Secretary of Agriculture 6 for the disinfection of cattle cars, pens, etc., with car- bolic acid alone, in order to determine whether the addition of the lime, which was added to the carbolic acid solution merely for the purpose of showing where the latter had been applied, had any effect on the carbolic acid ; that is, whether it served to increase or dimin- ish its germicidal efficiency. A mixture of lime and carbolic acid was prepared, therefore, according to the formula given in the aforesaid regulations. This formula was as follows: Lime pounds. . 1J Carbolic acid pound . . ^ Water gallon . . 1 In making up this mixture for the tests which follow, chemically pure calcium oxide and crystallized carbolic acid c were used. The Report of Committee on Standard Methods of Water Analysis to the Lalx>ratory Section of the American Public Health Association, Journal of Infectious Diseases, Supplement No. 1, May, 1905. b Regulations of the Secretary of Agriculture governing the inspection, disinfec- tion, certification, treatment, handling, and method and manner of delivery and ship- ment of live stock which is the subject of interstate commerce. 1905. p. 9. c In this and all of the following tests, crystallized carbolic acid (U. S. P.) was used, 31360-No. 100 07 2 10 UKKMKII'M \ M.I I' "I UtKSnl.1- . < -M I'. - 1 I I -. lime was weighed in a sterile Mask and a sufficient <|iiant it y of the water, which had I ..... n previously sterili/ed l>y hoiling, was addrd i<> insure thorough slaking. The remainder of the water was then added, and the mixture thoroughly shaken and allowed to cool. The carholic acidcrvstaU were then added, and the mixture wa> >haken well and allowed to stand for several hours to insure complete solution of the carholic acid. After thorough shaking, portions of thi> mixture \\cre diluted so as to give dilution-, containing carholic acid in the proportion of 1:100 and 1:125, and those dilution-, were then tested on StapkylococciU jryogenfs avrrus and Bacillus cholery suis in com- parison with aqueous carholic-acid solutions of equivalent strength-. The results of these test*, which were made according to the rod method, are shown in Tahles 1 and '_'. TABI.K 1 . ' '<>mparion of carbolic arid irith mrboUr arid and limr. |lt inin- atm, 7JiPln- iii.-.. II Miii n- iil.--.. 12) in i n- .!. ~. 15 min- ute*. CMrlK>lio ;irii| mill lituriiiixtiin- Cri>ollc cid nd water 1:100 1:100 + + + + + 4. -- + + + means growth; - means no growth. iTriiM-iilmNMl for 6 days at 38 C. TABLE 2. Companion of carbolic acid with carbolic arid and lime. [Rod method. Organism tested. Bacillu* cholrrsr nit, 24-hour agar culture at 3H C. Room temper ture, 26 to 27 C.J Propor- Remiltof exposure! o disinfect itnt for Disinfectant. carl>ollc acid in disinfect- ant. 21 inin- lltl'H. 5 min- utes. Tii.un- ii. -. 10 min- utes. r.'i min- utes. 15 min- utes. Cartxilii- ni-i.l and lime mixture. Crlilic iK-jil and water 1:126 1:125 + + - - - - + means growth: - means no growth. The Milxniltures wrre Incubated for ft days at 33 C. As will he seen from Tahle 1. the carholic acid and lime mixture (carholic acid 1:100) failed to kill N. fnjogenes aureu* in 15 minute-. whereas carholic acid alone (1 : 100) killed in 10 minutes. In Tahle _' we see that it required an exposure of 7J minutes to the lime and carholic acid mixture (1 : 125) to kill n. cholfrx m, whereas an exposure of 2) minutes to carholic acid alone (1: 125) was sufficient to accomplish the same result. It will thus l>e seen that the addition of the lime to the carholic- acid solution materially diminished its ^ennicidal efficiency. This CARBOLIC ACID AND COMMERCIAL CRESOLS. 11 is no doubt due. to the fact that part of the carbolic acid combines with the lime to form carbolate of calcium, which is a weaker dis- infectant than carbolic acid. COMPARISON OF CARBOLIC AGED WITH LIQUOR CRESOLIS COM- POSITUS MADE FROM COMMERCIAL CRESOLS. The United States Pharmacopoeia gives the following directions for the preparation of liquor cresolis compositus: Grams. Cresol 500 Linseed oil 350 Potassium hydroxide 80 Water, a sufficient quantity to make 1, 000 Dissolve the potassium hydroxide in 50 grams of water in a tared dish, add the linseed oil, and mix thoroughly. Then add the cresol and stir until a clear solution is produced, and finally sufficient water to make the finished product weigh 1,000 grams. Two lots of liquor cresolis compositus were prepared according to the above formula from two lots of cresol, designated hereafter as Cresol I and Cresol II. These cresols, when subjected to fractional distillation in a regula- tion Engel flask, gave the following results: Cresol I. Fifty grams of cresol were taken; first drop distilled at 185 C.; the temperature rose steadily and quite rapidly to 189 C., then slowly to 190 C., holding for some time between 190 and 191 C. The fractional distillation of this cresol gave the following result : Below 187 C 1. 60 per cent. This fraction was colorless. From 187 to 189 C 2. 10 per cent. Do. From 189 to 195 C 70. 20 per cent. Do. From 195 to 200 C 17. 90 per cent. This fraction was light straw in color. From 200 to 203 C 4. 40 per cent. This fraction was greenish yellow in color. Above 203 C. (by difference) . 3. 80 per cent. This fraction was dark brown in color. 100. 00 per cent. Cresol II. Fifty grams of cresol were taken; first drop distilled at 185 C. ; the temperature then rose quite rapidly to 195 C., then slowly to 198 C., holding for some time between 198 and 199 C. The fractional distillation of Cresol II was as follows: Water 1. 00 per cent. Below 187 C 1. 40 per cent. This fraction was colorless. From 187 to 189 C 0.40 per cent. Do. From 189 to 195 C 1. 80 per cent. Do. From 195 to 200 C 80. 00 per cent. Do. From 200 to 203 C 14. 80 per cent. This fraction was light straw in color. Above 203 C. (by difference). 0. 60 per cent. This fraction was reddish brown in color. 100. 00 per cent. 12 OKKMH IIAI. YAM K CRKSoUS r.>Mi' (1 -l|i In preparing the liquor cresolis compo^itus made uilh <'re>nl II, tin- soapy mixture of oil and hydrate cleared up as the cresol Mtf added. l>iil clouded on the addition of the last |V\\ cubic cenlimelei the cresol: on the addition of the \\alrr. however. tin- solution became dear a^'ain. In the ease of the liquor c resold eompositus made with Cicxil I, the mixture of oil and hydi. me clear on the addition of the crexol. I. in clouded on the addition of the water: after standing for 'J I hours. however. the solution hecanie clear. The liquor cresolis coinpositus made \\ith ( 'resol II was slightly darker in color than that inade with (Vesol I. In making dilutions of the two lots of liquor cre.xolis coinpositus, prepared as above. it was found that the re>ull int: solutions in hoth instance-, \\ej-e turbid. A 5 per cent dilution of liquor cresolis compositus made with ( 'resol I gave an opaque, milky solution, from which there was no apparent separation, while a similar dilution of the liquor cresolis compositus made with (Ye>ol II t, r aYe a brownish, turbid solution, from which there was considerable separatioiy on standing. In view of the fact that there was some separation in the case of the liquor cresolis compositus made with (Vesol II. it seemed probable that saponilicat ion was not com- plete. so portions oi this preparation were heated for different lengths of time oYer a steam bath, and it was found that by heating for one hour complete >aponifieat ion w as effected and perfectly clear solutions could be obtained upon dilution. The two stock solutions of liquor croolis compositus were therefore heated for one hour on the steam bath, and the following tests were made with these solutions: TABI.K 3.- -Comjiaruon of carbolic acid with lii/nor crtsolit ro//i/>o*i'/i/.hytororrui pyoffene* amrrut. 24-hour broth culturp at 38 C. Room t.'mp.Titture2fiC.) Strcngth I;. Mill nl oxposum 1.. .liMnf.x- tnnt for .:if-tant. 5 soliltinii. 2) niin- iii. >. 5 min- ute*. 7) min- ute*. 10 iinii- \ite*. 12 : lii - ute*. 15min- UtM. CarlMilicarid. . 1:80 1 Hi) 4- 4 4 4 _ _ Do 1 l.'.'i 4- 4 4 4. 4 4- l.iiiuor !i|tu* 1:125 4. 4 4 I.iciiii.r rfu.|i-i i-oiiiixi-ilii- 1:125 4. 4 4 nw-atw growth: iwnnx no growth. TN- riilt . nhiitfil for iUy at 38 C. CarK>lK--iu-i : !,< componituii made with Cn-nol 1-1.40. Carbolic-acid ooefllcicnt of liquor r rraolis oompoaltu* nuuln with Croaol II- l.M-. CARBOLTC ACID AND COMMERCIAL CRESOLS. 13 TABLE 4. Comparison of carbolic acid with liquor cresolis compositus made from com- mercial cresols. [Drop method. Bacillus pyocyaneus, 24-hour broth culture at 38 C. Room temperature, 26 C.J Disinfectant. Strength of solu- tion. Result of exposure to disinfectant for 2J min- utes. 5 min- utes. ?i min- utes. 10 min- utes. 12J min- utes. 15 min- utes. Carbolic acid 1:125 1:135 1:200 1:200 1:200 + + + + + + + + + + + + + + Do Do Liquor cresolis compositus (Cresol 1) Liquor cresolis compositus (Cresol II) + means growth; means no growth. The subcultures were incubated for 6 days at 38 C. Carbolic-acid coefficient of liquor cresolis compositus made with Cresol 1=1.48. Carbolic-acid coefficient of liquor cresolis compositus made with Cresol 11=1.54 (average of 1.48 and 1.60). TABLE 5. Comparison of carbolic acid with liquor cresolis compositus made from com- mercial cresols. Drop method. Bacillus cholerx suis, 24-hour beef-broth culture at 38 C. Room temperature, 26 C.] Disinfectant. Strength of solu- tion. Result of exposure to disinfectant for 2J min- utes. 5 min- utes. 7J min- utes. 10 min- utes. 12J min- utes. 15 min- utes. Carbolic acid 1:125 1:135 1:200 1:200 1:200 + + + + + + + + + + + Do Do Liquor cresolis compositus (Cresol I) Liquor cresolis compositus (Cresol II) + means growth; means no growth. The subcultures were incubated for 6 days at 38 C. Carbolic-acid coefficient of liquor cresolis compositus made with Cresol 1=1.54 (average of 1.48 and 1.00). Carbolic-acid coefficient of liquor cresolis compositus made with Cresol 11=1.54 (average of 1.48 and 1.60). The carbolic-acid coefficients in Tables 3 to 5 are obtained as fol- lows: A. dilution of carbolic acid is first obtained which kills the organism within a given time; a dilution of the disinfectant is next obtained which kills the organism in the same time as the carbolic- acid dilution; the dilution of the disinfectant divided by the carbolic- acid dilution gives a ratio which is called the carbolic-acid coefficient. This coefficient represents the efficiency of the disinfectant expressed in multiples of carbolic acid performing the same work. Taking an average of the carbolic-acid coefficients in the above tables we find that for the three organisms tested the two solutions of liquor cresolis compositus made from commercial cresols are approxi- mately one and one-half times more efficient than carbolic acid. We also find that the solution of liquor cresolis compositus made with Cresol II is slightly more efficient than that made with Cresol I, which may be explained by the fact that Cresol II contains a higher percentage of the high boiling cresols, it being generally conceded 14 GKICMIt ii. M. VM.UK OP LKi..K ORB8OLI8 COMPO8ITU8. by must investigators that the higher l>i.iliii-_ r en-sols, i. e., para- cn-sol ami metacn-sol, an- more efficient disinfectants than orthoci. In an investigation of the en-sol soaps of ( icnnan comnierce l-'ehr- " found that en-sol soaps containing the same amount of crude eiesol varied in disinfectant ellieiency. and he attributed this variation to differences in the chemical compositit>n of the crude cn-so!-, from which the soaps \\ere made: tliat is, to differences in the relative amounts of orthoeresol, paraeresol, and metacresol eontnined in these en-sols. In the course of his investigation Fehrs found that paraeresol and metacresol were both more active disinfectants than oi t hoc-re-, ,1. and ho explains the varying disinfectant values of the cn^ol soap-. <>n this Around. - 'inewhat at variance with these results, however, are the more recent experiments of Schneider/' who prepared a -cries ,.!' <[<-. .1 soaps containing eM \l KIM I \l. < IM'.SOL. Cresol II, which contained less than 1 per cent of impurities in the form of tar oils and bases, was used for this work and was purified in the following manner: Five hundred cubic centimeters of the en-sol was shaken in a separatory funnel with sulphuric-acid solution (1:3) and allowed to stand until complete separation into layers had taken place. The lower (aqueous) layer, containing a sulphuric-acid extract of "organic bases," was then removed. Tin- residue was next made alkaline with 25 per cent sodium-hydrate solution. Ether was then added, and the mixture was thoroughly agitated and allowed to stand over ni^ht to separate. The lower layer, containing the tar acids (phenols, cresols, etc.) in solution in soda as phenylates. en-sylates. etc.. was separated from the upper (ethereal layer containing any tar oils present. The solution of tar acids in soda was then made acid by adding an excess of sulphuric acid '1:3) and allowed to cool; it was then agitated with ether several times until all liberated tar acids were extracted. The ether was driven off from this extract by gentle heating on a steam bath, and the residue of tar acids thus obtained was placed in a distillation flask and subjected to careful fractional distillation. The distillation was repeated three times, and three fractions approximating orthoeresol. paracresol, and metacresol were "CVntralM. fur Bakt., Paraait. und Infectioiwkr. , Bd. XXXVII, Abt. I, 1904. *Zeit. far Hyg. und Infectionskr., Bd., 53, 1906. COMPARISON OF SOLUTIONS MADE FROM DIFFERENT CRESOLS. 15 collected, as follows: (1) A fraction which distilled over constantly between 187 and 189 C. (2) A fraction which came over constantly between 195 and 199 C. (3) A fraction which came over con- stantly between 200 and 203 C. The boiling points of the three cresols are usually given as follows : Orthocresol, 188 C.; paracresol, 198 C.; metacresol, 201 C. There is, however, considerable variation in the boiling points of the three cresols as recorded by different observers, and because of this varia- tion the fractions were collected within the limits stated above. GERMICIDAL VALUE or LIQUOR CRESOLIS COMPOSITUS MADE FROM THE THREE CRESOLS. Solutions were prepared from each of the three fractions described in the preceding paragraph, according to the formula of the United States Pharmacopoeia for liquor cresolis compositus, and the tests which follow were made with dilutions from these solutions. The stock solutions in this instance, as in the case of liquor cresolis com- positus prepared from the commercial cresols, were heated for one hour on a steam bath, in order to insure complete saponification. In the following tests a comparison was made of the drop and rod methods in addition to the comparison of the germicidal value of the different cresols : TABLE 6. Comparison of solutions of liquor cresolis compositus made from cresols which boil at temperatures approximating orthocresol, paracresol, and metacresol. [Drop method. Organism tested, Bacillus typhosus, 24-hour beef-broth culture at 38 C. Room temperature, 30 C.] Disinfectant. Strength of solution. Result of exposure to disinfectant for 2J min- utes. 5 min- utes. 7J min- utes. 10 min- utes. 12J min- utes. 15 min- utes'. Liquor cresolis compositus containing Cresol 187 to 189 C 1:350 1:350 1:350 4- + 4- + 4- + 4- 4- 4- + + Cresol 195 to 199 C. . Cresol 200 to 203 C + means growth; means no growth. The subcultures were incubated for 6 days at 38 C. TABLE 7. Comparison of solutions of liquor cresolis compositus made from cresols which boil at temperatures approximating orthocresol, paracresol, and metacresol. [Kod method. Organism tested, Bacillus typhosus, 24-hour asar culture at 38 C. Room temperature, 28 C.] Disinfectant. Strength of solution. Result of exposure to disinfectant for 2J min- utes. 5 min- utes. 7J min- utes. 10 min- utes. 12J min- utes. 15 min- utes. laquor cresolis compositus containing Crcsol 187 to 189 r 1:450 ]:450 1:450 + + 4- 4- + 4- 4- 4- 4- 4- Cresol 195 to 199 C Cresol 200 to 203 C 4- means growth; means no growth. The subcultures were incubated for 6 days at 38 C. Ill i.l KMI' IDAL VAI.TK >[- l.UjfnK riM-> 'I.I- TMUK S ' .. ;;./!/ -wn njunlulintu <>/'/ir.|i mi-tiiixi. organUni tmti*iirixnn of solutions of /u/i/or i-rmnlis .>//! /KMI/I/.V ;//W/ /Vf/m rrc*ol* which boil at ti in i>i rat tins ni>i>ro.iiinal\nii nrthwrrmd. /niriii-ri snl . nml nu turn mil. |K.I inrtlKMl. Organism t.'.sl.-il, >'//)Aj//ororru* )>yogrnr*avrru*. '-M-hoiir HKHT i-iiltun- t :wC. It. HIM. ' Disinf.'. tiii.t. StrcnKth of snliition. .". niii; UU>8. .'. linn ul.-- 7) min- iii<-. 10 min- utes. r.'j m ii- nti - 1.'. Illlll utea. Litpior TPoli coin posit uii containing Cre)l 187 to 189 C . 1:300 4. 4. 4- 4- 4. 4. ( n-ol l{tttolWC Civftol 200 to 303 C I .tm 1:300 4- 4- means growth; means no growth. Thp MiU-ultiin>s wrro iiK-uhaUni for 6 days at 38 C. TABLE 10. Comparuonof solutions of liquor crrsoli* com poxit us made from rretoln uhirh boil at temperatures approximating orthorresol, paracretol. and metarresol. [Drop method. Organism tested, Baritiu* coli communi*. 24-hour N-.-f i.n.ih niltunc at 38 C. Room trmpi>rature, 28 C.] Strength Rcaultof expostiiv to diainfec tant for Dialnfei-tant. oT solution. 2) min- utes. .". nun utes. 7) in n utes. 10 min- utes l.'i min- utes. 15 111:11- .i. - Liquor cresolia composltiis containing "1 187 to 1W C 1:250 4- 4- 4- 4- 4- Cnwol 1K to 198 C 1 j.-Ji 4. 4- 4- 4. Crvsol200 to 203 C.' 1:250 4. 4- _ _ 4- means growth: moans no growth. The iu>M:ultures w.-rv in. ut>nU-iiro.iiituitiny vrthocresol, paracnxol. nml >iutn< | Hl iix-t h(' 1 300 4. 4. ol 200* to 200* C 1:300 4. _ _ _ 1 4- means growth; - mean* no growth. The subcultures were incubated for days at 38* C. COMPARISON OF SOLUTIONS MADE FROM DIFFERENT CRESOLS. 17 TABLE 12. Comparison of solutions of liquor cresolis compositus made from cresols which boil at temperatures approximating orthocresol, paracresol, and metacresol. [Drop method. Organis.n tested, Bacillus pyocyaneus, 24-hour beef-broth culture at 38 C. Roo.:i t<> uperature, 31 C.] Disinfectant. Strength of solution. Result of exposure, to disinfectant for 2* min- utes. 5 min- utes. 7J min- utes. 10 min- utes. 12.J min- utes. 15 min- utes. Liquor cresolis compositus containing Cresol, 187 to 189 C 1:300 1:300 1:300 + + + + + + + + f Cresol, 195 to 199 C Cresol, 200 to 203 C + means growth; means no growth. The sulxjultures were incubated for 6 days at 38 C. TABLE 13. Comparison of solutions of liquor cresolis compositus made from cresols which boil at temperatures approximating orthocresol, paracresol, and metacresol. [Rod method. Organism tested, Bacillus pyocyaneus, 24-hour agar culture at 38 C. Room tempera- ture, 29 C.] Disinfectant. Strength of solution. Result of exposure to disinfectant for 2J min- utes. 5 min- utes. 7J min- utes. 10 min- utes. 12J min- utes. 15 min- utes. Liquor cresol compositus con- taining Cresol, 187 to 189 C 1:300 1:300 1:300 + + + + + + + + + + + Cresol, 195 to 199 C Cresol, 200 to 203 C + means growth; means no growth. The subcultures were incubated for 6 days at 38 C. From Tables 6 to 13 it will be seen that the germicidal efficiency of liquor cresolis compositus increases with the boiling point of the cresol it contains. It will also be noted that the two solutions of liquor cresolis compositus made from the two higher-boiling cresols (i. e., those approximating paracresol and metacresol) are both very much more efficient and stand apart, as it were, from the solution made from the lowest-boiling cresol, which approximates orthocresol. In comparing the results obtained by the drop and rod methods we find that as a rule the organisms tested were more easily killed on the rods. By the rod method Bacillus typhosus, B. coli communis, and Staphylococcus pyogenes aureus were killed by more dilute solu- tions than were required by the drop method, and it would seem probable that the resistance of these organisms was somewhat low- ered by the drying to which they were subjected in the rod method. B. pyocyaneus was killed by the same strength of solutions in prac- tically the same length of tune with both methods, and it would seem that this organism was probably more resistant to desiccation than the ones mentioned above. From these results it would seem that the same objection that Firth and MacFadyen found to the thread method would hold for the rod method i. e., that it is not well fitted for working with organisms that are sensitive to desiccation. 18 OKKMlUlt.M. VALUE OK LIQUOR CRr>"l .1- COMPARISON OF CARBOLIC ACID WITH LIQUOR CRE8OLIS COM- POSITUS MADE WITH LOW-BOILING CRESOL. In the preceding >e<-ti<>n it \\as round that li<|iior cre^.lU com- pos it us made with a creM>l which lioiLs at a temperature approxi- mating orthocre-.>l i. .. 1S7 to 189 C.) was much the weakest disinfectant of three preparations made from a series of cresols approximating orthocresol, paracresol, and metacresol. In the following tahles this solution of li(piitus. which was made from the lowest-hoiling cresol of tlie scries and foil ml to he the weakest of the three preparations in gcrmicidal value, is compared with carbolic acid: TABLE 14. Comjiari*on of carbolic acid uith /i'//i/or craolu compontm (cretol 187 to 189 (' . i. II>rop nirthrxl. organism tested. Rarillu* coli communi*. 24-hour liccf-broth culture at 38* C. Room tenpermture. 24 to 28 C.] DMnfccUnt. Strpngth of solution. Result of exposure to dlninfrvtant for 2J min- utes. 5 min- ute*. 7J min- n. 10 min- utea. 12} rnln- nu. !.' min- Liquor crpaolis compositus (oreaol 1S7 to 189 C.) 1:175 1:175 1:125 1:100 + + + + + + + + + + + + + + 'ltrl>llr lii'l'l Do... Do + ipeans growth; - means no growth. The subcultures were inculmted for 6 days at 38 C. Carbolic-acid coefficient - 1.67 (average of 1.40 ami 1.75). TABLE 15. Companion of carbolic acid rnth liquor creol\ compositnt (cretol 187 to 189 C.). [Dmp method. Organism tested. Bacillus typh&tut. 24-hour beef-broth culture. Room temperature, 2>to 30 C.] Strength Result of expoHtire to dirinfecUnt for li-inf.rtiiTit. of solution. -M mln- : | Smln- .:. - ; n h - utes. lOmin- I?J min- utes, utes. I.", min- utes. Liquor CresolU rnmpositus (crmol 187 to 18 P.) 1:200 + + (rt, lie acid... 1:200 + + -f + + 4. Do 1:135 + + 4. Do 1:125 4. 1 . 4- means growth: - means no growth. The subcultures were Incubated for fi days at 38 C. Carbolic-Mid coefficient - 1.54 (average of 1.48 and 1.00). CARBOLIC ACID AND LOW-BOILING CRESOL. 19 TABLE 16. Comparison of carbolic acid with liquor cresolis compositus (cresol 187 to 189 C.). [Drop method. Organism tested, Bacillus cholerx suis, 24-hour beef-broth culture at 38 C. Room temperature, 26 C.] Disinfectant. Strength of solution. Result of exposure to disinfectant for 2J min- utes. 5 min- utes. 7J min- utes. 10 min- utes. 12J min- utes. 15 min- utes. Liquor cresolis compositus (cresol 187 to 189 C ) 1:175 1:175 1:135 1:125 + + + + + + . + + + + + Carbolic acid Do... Do + means growth; means no growth. The subcultures were incubated for 6 days at 38 C. Carbolic-acid coefficient = 1.35 (average of 1.30 and 1.40). TABLE 17. Comparison of carbolic acid with liquor cresolis compositus (cresol 187 to 189 C.). [Drop method. Organism tested, Bacillus pyocyaneus, 24-hour beef-broth culture at 38 C. Room temperature, 27 C.] Disinfectant. Strength of solution. Result of exposure to disinfectant for 2J min- utes. 5 min- utes. 7J min- utes. 10 min- utes. 12J min- utes. 15 min- utes. Liquor cresolis compositus (cresol 187 to 189 C.) 1:175 1:175 1:150 1:135 + + + + + | + + + + + + + + + + + Carbolic acid. Do Do... + means growth; - means no growth. The subcultures were incubated for 6 days at 38 C. Carbolic-acid coefficient = 1.23 (average of 1.30 and 1.16). TABLE 18. Comparison of carbolic acid with liquor cresolis compositus (cresol 187 to 189 C.. [Drop method. Organism tested, Staphylococcus pyogenes aureus, temperature, 27 to 30 C.] 24-hour beef-broth culture. Room Disinfectant. Strength of solution. Result of exposure to disinfectant for 2J min- utes. 5 min- utes. 1\ min- utes. 10 min- utes. 121 min-, utes. 15 min- utes. Liquor cresolis compositus (cresol 187 to 189 C.) 1: 150 1: 150 1: 100 1: 80 + + + + + + + + + + + Carbolic acid Do Do + means growth; means no growth. The subcultures were incubated for 6 days at 38" 1 C. Carbolic-acid coefficient =1.63 (average of 1.50 and 1.87). 20 OK.UMlt il.\l. VAI.I'K OK UijroK CKKSoUrt OOMPO8ITU8. Talking an average of tin* carbolic-acid coefficients in Tables I I Is, it will be found I lint for the I'm* organisms tested the gcrmicidal elli- ciency >f liquor cresolis composing made \vitli cre^.l which boils between 1S7 and ivi ('. is nearly one and one-half times greater than that of carbolic acid. \Vc thus s<><> that a solution of liquor cresolis compositus made from the lowest-boiling and least effective cresol (i. e., cresol 187 to 189 C.) has a higher disinfectant value than carbolic acid, and we may safely conclude, therefore, that a solution of liquor cresolis compositus made from any cresol \\hieh boils at the temperatures specified by the United States IMiarmacopu-ia ( !'."> to -J05 G.) would surpass carbolic acid in disinfectant eflieiency. EXPERIMENTS WITH BACILLUS TUBERCULOSIS. A culture of Bacilhift tubfrmlozis grown at 38 C. on beef broth con- taining peptone, glycerin, and acid potassium phosphate was selected for this experiment. The culture was 17 days old, and showed a well-developed pellicle covering the entire surface. Several bits of the growth were removed by means of a platinum loop, placed in the bottom of a dry, sterile test tube, and thoroughly macerated by means of a heavy platinum rod 'flattened at the end. Three cubic centi- meters of normal salt solution was then added, and some bits of sterile glass, and the tube was thoroughly agitated. The tube was then allowed to stand for about half an hour, at the end of which time the coarser particles had settled out, leaving a homogeneous sus- pension of about the density of a 48-hour typhoid culture. This sus- pension was carefully drawn off by means of a sterile pipette, and transferred to a dry, sterile test tube. Sterile glass rods with etched ends (Hill's method) were then dipped in this suspension, and allowed to dry for a few moments in the air. The rods were then exposed for varying intervals of time to 1 per cent solutions of carbolic acid and liquor cresolis compositus (cresol 187 to 189 C.). After exposure to the antiseptic each rod was dipped in a large test tube containing 50 c. c. of sterile water in order to get rid of all traces of the disin- fectant and the bacilli were then carefully washed from the rods in 2 c. c. of sterile salt solution in a small sterile beaker. A small glass rod, tipped with rubber tubing and sterilized by boiling, was used in washing the tubercle bacilli from the end of the etched rod, the latter being thoroughly nibbed in the salt solution with the rubber- tippet! rod, so as to remove all of the tubercle bacilli adhering to the etched portion. The salt solution containing the tubercle bacilli which had been exposed to the action of the disinfectant was then injected into n guinea pig beneath the skin of the flank. Guinea pigs were also inoculated from check rods which had received a film of bacilli and had then been dipped in sterile water and washed off in EXPERIMENTS WITH BACILLUS TUBERCULOSIS. 21 salt solution in the same manner as those which were exposed to the disinfectant. The room temperature at the time the rods were exposed to the action of the disinfectant was 28 C. The guinea pigs used for these experiments were of uniform size and weight, the average weight being about 375 grams. TABLE 19. Showing effect of 1 per cent carbolic acid on Bacillus tuberculosis. [Rod method. 17-day beef-broth culture No. 128 at 38 C.] Days No. of guinea Material injected. Date of inoc- ulation alive after Result. Pig- inocu- lation. 7708 Culture not exposed to disinfectant July 16,1906 35 Died. Tuberculous. 7711 do ... do 51 Do. 7709 Culture exposed to disinfectant for J minute ...do... 12 Died. Pneumonia. 7707 Culture exposed to disinfectant for 1 minute do 83 Died. Tuberculous. 7710 Culture exposed to disinfectant for 2 minutes do ... 91 D>'ed. Nontubercu- lous. 7584 Culture exposed to disinfectant for 5 minutes do 91 Do. The autopsy on guinea pig 7709, which died within 12 days after inoculation, showed consolidation of the anterior lobes of both lungs and this animal probably died from an intercurrent pneumonia. Had the animal lived it would in all probability have developed tuberculosis, as the animal next above it in the series guinea pig 7707 inoculated with tubercle bacilli which were given a longer exposure to carbolic acid, developed tuberculosis and died with well- marked lesions. It will be noticed that guinea pig 7707 lived for 83 days after inoc- ulation, whereas the two checks guinea pigs 1708 and 1711 died within 35 and 51 days of the inoculation. Taking an average for the two checks it will be seen that guinea pig 7707 lived nearly twice as long as did the check animals and it would seem from this that the tubercle bacilli with which guinea pig 7707 was inoculated had been considerably attenuated by an exposure of 1 minute to carbolic acid, or else that a large number of the bacilli were killed, thus reducing the dose of live bacilli which the animal received and consequently prolonging the course of the disease. At the time guinea pigs 7710 and 7584 died a number of guinea pigs were dying from unknown causes, but careful autopsies on these animals failed to reveal any traces of tuberculosis and it would see'm that the tubercle bacilli with which these animals were inocu- lated had been completely killed by the carbolic acid. \ M.IK OK LUJI "K CRKSOI.IS COMI'OSITCS. TMII I 'JU , il'x-l i>J I JUT n nt liijtinr rrrsnlm COWI/KMI/I/* (crttol 187 to on linrilluit tiilxrnilosu. |ltiH| inplhiMl. |7^!ny lvf-l>r..tli ciilturr N.. IJMit 38 C.) (\ > No. of . pig. MntiTiiil Inj- ' I l>tof inoculation. Dm live aft. r Inncti- 1. Hi. .n. Remit. I'llUlirr not eXpnmM Id .IiMiifii't.uil Juno fl,180 41 KIlliMl. TuUTviil.Mi- 7517 do 41 7530 Culture vxpoaed l<> 'liiinfii-tunt (<>r ; nun. do 41 Kllll-l NnlltlltHTT-U- 7516 do ...do... 41 llMIH. |... 7523 7515 Ciiltun- i<.\|x>fted lodlalnfivtiitit f>r 1 nun . do do ...do... 42 42 [)., Do. 7534 Ciiltun- oxposrd to disinfectant tr J minute*... ...do... 42 !>.. 7514 do ...do... 42 Do 7513 Culture exposed to diainfivtatit fur 5 minutes.. . .do.. do do 33 40 Died. NontuU-n-u- lou*. Do. In the experiment recorded in Table 20 the two check animals guinea pigs 7525 and 7517 both showed well-marked lesions of tuber- culosis at the end of 6 weeks, whereas none of the animals inoculated with tubercle bacilli which had been exposed to liquor cresolis com- positus (cresol 187 to 189 C.) showed any trace of tuberculosis. Comparing Tables 19 and 20, we find that it required an exposure of two minutes to 1 per cent carbolic acid to kill B. tuberculosis, whereas an exposure of one-half minute to 1 per cent liquor cresolis compositus was sufficient to accomplish the same result. In other words, liquor cresolis compositus (cresol 187 to 189 C.) killed B. tuberculosis in about one-fourth the time required by the same strength of carbolic- acid solution. EXPERIMENTS WITH BACILLUS ANTHRACIS. In all of the preceding tests purely vegetative i. e., nonspore- bearing micro-organisms were used, and it was shown that liquor cresolis compositus Is strongly germicidal for these organisms, viz, Bacillus pyocyaneus, B. cholerse suis, B. coli communis, B. typho&us, B. tuberculosis, and Staphylococcus pyogenes aureus. The spore-bearing micro-organisms, by reason of their spores, possess a 1 1 UK 1 1 lii-jliiT derive >f rr-Ul a lire t> Imt li clirinical ami |ih\ -- ical agents than the purely vegetative micro-organisms, and for this reason it seemed advisable to determine the. germicidal efficiency of liquor cresolis compositus for such an organism. The following tests, therefore, were made with Bacillus anthracis, the spores of which are highly resistant to most chemical disinfectants. The cultures used in these tests were examined microscopically before the tests were made, and showed an abundance of spores. EXPERIMENTS WITH BACILLUS ANTHRACIS. 23 TABLE 21. Showing effect of liquor cresolis compositus on Bacilhts anlhracis. [Rod method. 10-day agar culture at 38 C. Room temperature, 28 to 30 C.] Disinfectant. Strength of solution. Result of exposure to disinfectant for 5 minutes. 30 minutes. 1 hour. 3 hours. 6 hours. 24 hours. Liquor cresolis compositus con- taining Cresol I 5 per cent.. 6 percent. 8 per cent. 5 per cent. 6 per cent. 8 percent. + + + + + + + + + + + + + + + + + + + + + + + + + + Do Do . Cresol II Do Do + means growth; means no growth. The subcultures were incubated for 10 days at 38 C.. TABLE 22. Showing effect of liquor cresolis compositus on Bacillus anthracis. [Rod method. 10-day agar culture at 38 C. Room temperature, 25 C.] Disinfectant. Strength of solution. Result of exposure to disinfectant for 5 minutes. 30 minutes. 1 hour. 3 hours. 6 hours. 24 hours. Liquor cresolis compositus con- taining Cresol 187 to 189 C 6 per cent... do + + + + + + + + + + + + + + + - Cresol 195 to 199 C. Cresol 200 to 203 C do + means growth; means no growth. The subcultures were incubated for 10 days at 38 C. From Tables 21 and 22 it will be seen that for the destruction of anthrax spores much stronger solutions of liquor cresolis compositus would be required than for the purely vegetative micro-organisms. In these tests, owing to the well-known resistance of anthrax spores, the lengths of exposure to the disinfectant were made longer than in the case of the purely vegetative micro-organisms, and the results, therefore, were not so sharp and definite as with the latter. Thus, in Table 21, we fail to see any difference in the germicidal efficiency of solutions of liquor cresolis compositus made from Cresol I and Cresol II, whereas in the case of the vegetative inicro-organ- isms liquor cresolis compositus made from Cresol II was found to be the more efficient. Also, in Table 22 we fail to note any difference in the germicidal efficiency of solutions of liquor cresolis compositus made from cresols of different boiling points, whereas these solutions showed marked differences in germicidal efficiency when tested on the vegetative micro-organisms. With stronger solutions and shorter lengths of exposure, differences in the germicidal efficiency of the various solutions of liquor cresolis compositus tested would no doubt have been noted for the anthrax spores as well as in the case of the vegetative micro-organisms. 24 1. 1 i:\lirm.\L VALUE OF Mi/l>K KI-X'i.is OOMPO8ITU8. A-^ llu- carbolic-acid coefficient ut li<|in.r crc^oli-, OOmpOflltlM for anthrax spores \\ns not determined, \\e can not make a companion in thi> instance between lujnor cresol is compo-itus and carhoiic arid. SUMMARY OF RESULTS. (1) Liquor |I> compoit i;v i> ^i rongly ^'cnnicidal for tlic fol- lowing iiiicro-or^anisins: /lurillns jtiftH-i/nni UN, J>. clmlcrx suit, /'>. coli com m mi i*, I!. t>/]>ho8U8, Ji. tuberculosis, and Staphylococcus pyogtm* (iur> (2) The gennickU] ellicicncy of li(|iior crosolis compositus increases with tin' boiling point of the cresol it contains, and solutions of liquor cresoli^ compositus maU which approximate paracresol and inetacresol arc both much stronger germicides than liquor cresolis compo>itus made from a lo\v- boiling oresol, which approximates orthocresol. (3) Liquor cresolis compo>itus made from commercial cresols may be expected to vary somewhat in germicidal elliciency, owing to varia- tions in the boiling points of the cresols it contains. This variation, however, does not render it unreliable as a disinfectant, as a solution of liquor cresolis compositus made from the lowest-boiling and lea-t- active cresol was found to possess stronger gennicidal proj>erties than carbolic acid. (4) The gennicidal value of liquor cresolis compositus made from cresol which boils at a temperature approximating orthocresol (187 to 189 C.) is nearly one and one-half times greater than carbolic acid.