AN WALUAYIOH G? EHREE AMESEHECE USED IN I‘HE PREaSTURGICAL PREPARATION OF PATIENTS AT THE MICHEGAN STATE UNIVERSETY VETERiNAR‘Y CLENEC Tfiests for H19 Deqvae oi: MfS,1 MICHIGAN STATE UNWERSITY Thomas M. Ford 1959 THESIS University [J LIB R A R Y I" Michigan State ' F ‘v AN EVALUATION or THREE ANTISEPTICS USED IN THE PRE-SUFGICAL PREPARATION OF PATIENTS AT THE MICHIGAN STATE UNIVERSITY VETERINARY CLINIC by THOMAS M. FORD A THESIS Submitted to the College of Veterinary Medicine Michigan State University of Agriculture and Applied Science in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Surgery and Medicine 1959 Approved: @1134!ch 3: ‘9 NL\_~’¥LDLVJVL Date: 4&6” l. 834 ‘75" 3'5'5‘? ?‘ gcfigx J ACKNOWLEDGMENTS I wish to express my sincere appreciation to Dr. A. P. Drury for his advice and encouragement in the preparation of this work. My association with him has been a most memor-' able and pleasant experience. To Dr. W. V. Lumb for his guidance and explanation on the preparation of a thesis, I am very grateful. I am thankful to Dr. G. H. Conner for his valuable sug- gestions on context and content of this work. I wish to thank Mrs. Betty Leiby for her coOperation and willingness to assist with any task in the laboratory. ABSTRACT Laboratory and clinical tests were performed on three antiseptics that were being used in the presurgical prepara- tion of patients at the Michigan State University Veterinary Clinic. Two of the antiseptics (Weladol and Triocil) were on a trial basis, while the third (Liquid Germicidal Deter- gent) has been in use for a number of years. Phenol coefficients of 2.83 for Weladol and uuu.u for Liquid Germicidal Detergent were determined. Triocil did not give results from which a phenol coefficient could be established. The speed of disinfection and use dilution tests were employed to determine the per cent reduction of broth cultures of Salmonellg,txppgsa and Staphylococcus pygggneg var. auzggs upon exposure to the particular anti- septic. These cultures were standardized by at least three transfers at 2% hour intervals. Samples from the cultures were taken within one half hour of the 2% hour time interval so the number of organisms present would be consistent. A swab technique was used to ebtain culture material from the skin of cattle, swine, andvdogs after they had been prepared for suréery. The cotton swab was placed in 10 cc of sterile saline and shaken well. Five cc of the saline were plated on five per cent blood tryptose agar plates for h8 hours, and then the number of colonies was counted. From the results obtained by these testing methods it was determined that Liquid Germicidal Detergent was the most effective of the three antiseptics. II. III. IV. TABLE OF CONTENTS INTRODUCTION . . . . . . . . . . REVIEW OF LITERATURE . . . . . . . A. History . . . . . . . . . . 8. Origin of Laboratory Tests . .. . . C. Selection of an Antiseptic . . . . . . 1 . . 2 . . 2 . . 6 . . 8 D. Literature of Triocil, Weladol, and Liquid Germicidal Detergent . . . . . . MATERIALS AND METHODS . . . . . . . A. Laboratory Tests . . . . . . . l. Phenol Coefficient Test . . . . 2. Use Dilution Test . . . . . . 3. Speed of Disinfection Test . . . B. Clinical Testing . . . . . . . C. Antiseptics Tested . . . . . . . l. Triocil . . . . . . . . . 2. Weladol . . . . . . . . . 3. Liquid Germicidal Detergent . . . RESULTS . . . . . . . . . . . A. Laboratory Testing of Triocil Solution 8. Laboratory Testing of Weladol . . . C. Laboratory Testing of Liquid Germicidal gent e e e e e e e e e e e D. Clinical TQSting e e e e e e e . . 10 . . 1h . . 1h- . . lb . . 16 . . 18 . . 19 . . 21 . . 21 . . 22 . . 22 . . 2h . . 2h . . 25 Deter- . . 26 . . 27 VI. VII. DISCUSSION . A. Triocil . B. Weladol . D. Comparative Evaluation of Antiseptics Tested . CONCLUSIONS BIBLIOGRAPHY o. Liquid Germicidal Detergent 60 6o 61 62 TABLE II. III. IV. VI. VII. VIII. IX. X. XI. XII. XIII. LIST OF TABLES PAGES Phenol Coefficient Tests on Triocil Solu- tion e e e e e e e e e e e e e 28-29 Use Dilution Tests on Triocil Solution . 30-32 Speed of Disinfection Tests on Triocil SOlution e e e e e e e c e e 0 33-35 Phenol Coefficient Tests on Weladol . . 36-37 Use Dilution Tests on Weladol . . . . 38-h0 Speed of Disinfection Tests on Weladol . hl-h6 Phenol Coefficient Tests on Liquid Germici- dal Detergent . . . . . . . . . . h7-h8 Use Dilution Tests on Liquid Germicidal Detergent e e e e e e e e e e e ”9-50 Speed of Disinfection Tests on Liquid Germi- Cidal Detergent e e e e e e e e e 51-5h Clinical Testing of Triocil Solution . . 55 Clinical Testing of Weladol . . '. . . 56 Clinical Testing of Liquid Germicidal Deter- gent e e e e e e e e e e e e e 57-58 Comparison of Cultures of Clinical Samples 59 I. INTRODUCTION Aseptic technique may be symbolized as a pillar of modern successful surgery. The veterinary practitioner is often compelled to work alone and under difficult field conditions, where the opportunities for contamination are enhanced. Therefore, it is a matter of prime importance for him to know which antiseptics are most effective on the skin, and how they may best be used, so that the likelihood of infection frem this source may be reduced to a minimum. In gathering laboratory and clinical data on the effi- ciency of an antiseptic, one must recognize that there is not yet available a single universal method of testing which is applicable under all conditions. Efficiency under condi- tions of use is, in the final analysis, the important cri- terion. In this work, an attempt was made to evaluate the effec— tiveness of three antiseptics that are being used for presur- gical preparation of patients in the large and small animal clinics at Michigan State University. The effectiveness of the antiseptics was based on both in_v1§zg studies with known organisms and on swab cultures obtained from the sites prepared for surgery. II. REVIEW OF LITERATURE A- HIEIQEI. The earliest precursors to present-day antiseptics and disinfectants were the preservative measures of drying, salt- ing, freezing and the use of spices, wines, and vinegars to prevent the Spoilage of food. The ancient Egyptian's art of mummification evidences the effective use of salts, spices, oils, and dehydration in the prevention of decomposition. Many references in the Bible indicate an understanding of the effect of what is now known as bacterial infection. Such dangers were known to Aristotle (1h), who advised Alex- ander the Great to require his armies to "boil the water and bury the dung". At an early date, the Greek physician, Hippocrates (1h), realized the possility of wound infection and demanded that attending physicians wear clean robes, keep their fingernails trimmed, and cleanse the patient's wound with wine. During the Middle Ages, various efforts were made to check the plagues which were sweeping across all of Europe. In 1365, during the pneumonic form of the plague, John of Burgundy (7) instructed that the air in the infected rooms be purified by burning of incense to provide fumes for the patient to inhale. It was not until the eighteenth century, however, that quantitative tests were made to determine the effectiveness of disinfectants. In 1750, Sir John Pringle (7). the founder of modern military medicine and sanitation, presented to the Royal Society some experiments comparing the preservative and disinfectant action of various substances. Although the drugs he tested are no longer of interest, his experiments have remained outstanding as the first scientific comparison of disinfectants. His procedure consisted of cutting beef into pieces of equal size, adding equal amounts of water, and then known amounts of the chemical he wished to test. The water, meat, or air provided the necessary bacteria. Taking as a standard the action of 60 grains of sea salt dissolved in two ounces of water, he was able to determine the relative value of many different substances. One of the great problems confronting surgeons at that time was the mortality resulting from puerperal septicemia. Women confined to hospitals during Childbirth were seven times as apt to die as those cared for in private homes. In 18h3 Oliver Wendell Holmes published his paper on "The Contagiousness of Puerperal Fever" (1“). Holmes aroused violent criticism from his colleagues by suggesting that women in childbirth should not be attended by doctors who had been performing autopsies on women dead of puerperal fever. It was not until l8h7, when the Austrian, Semmelweis, discovered the cause of puerperal fever, that steps were taken to reduce hospital mortality from this cause (b). As obstetrical assistant in the Allgemines Krankenhause in Vienna, Semmelweis noticed that the mortality rate in the First Division of the hospital was much higher. This section was utilized for the training of medical students. The rest of the hospital, which utilized nurses, had a much better record. He observed that after a colleague cut his finger while performing an autopsy, the man exhibited identical symptoms as the women dying of puerperal fever. Ht also observed that many of the medical students performed dissec- tions on cadavers prior to visiting the wards. His conclusion was that the disease was transferred from the cadavers to the obstetrical patients in the hospital via the medical students. Thereafter Semmelweis insisted that his students wash their hands in a solution of chlorinated lime before attending a patient. Mortality rates drapped more then twenty per cent after this procedure was adapted. This doctrine of prophy- laxis preposed by Semmelweis was not readily accepted in Vienna, and was unknown in other places in EurOpe. After his death it was so completely forgotten that Lister did not know of it until long after he developed his own ideas of anti- septic surgery. Even after the introduction of anesthetics in the early nineteenth century, post-operative infection was very common. Operations were limited to surface wounds and amputations, and these were extremely hazardous because of the almost inevitable infection which resulted. The existing conditions of that time can best be illus- trated by the mortality following major amputations. In military cases it ran from 65 to 90 per cent, while civil cases ranged from 26 to 60 per cent. The medical men believed that the suppuration of wounds was due to some constituent of the air. They suggested that this could be controlled by eliminating air from the area of the incision. It was not until 1863 that Joseph Lister, professor of surgery at Edinburgh University, familiarized himself with the researches of Pasteur. He came to the conclusion that the organisms described by Pasteur in his fermentation reac- tions were likewise responsible for the putrefaction and suppuration of wounds. He became convinced that it was not the oxygen in the air that caused putrefaction, but airborne microorganisms settling on the wound were causing the infec- tion. Lister sought measures by which he could kill these organisms before they entered the open wound. His original practice was to apply crude carbolic acid to the incision or wound. He reasoned that this powerful agent would destroy each organism as it was deposited from the air. To complete the arrangement, Lister introduced the practice of spraying the air with dilute carbolic acid during an Operation or when changing a dressing. Although the first attempts at anti- sepsis were crude, the results were apparent from the start, as mortality rates steadily decreased. Lister met with strong opposition and his practices were slow to be accepted. The germ theory on which he based his practices was new. Many debates about the value of antisepsis turned into argu- ments over acceptance of Pasteur's germ theory. with con- tinual improvement in technique, antiseptic surgery gradually became an accepted practice, and at the time of Lister's death in 1912, his precepts were commonly followed. The second period in the study of disinfection may be said to have started around 1881 with the semiquantitative studies of Koch. He tested various chemical substances in the presence of pure cultures of test organisms to determine their disinfectant properties. George Sternberg (7), a military surgeon and pioneer American bacteriologist, began his study of the comparative action of commercial disinfec- tants in 1878. His method was simple: to mix equal parts of his cultures of bacteria in broth with a disinfectant for two hours. He would then subculture, using the absence of growth in the subculture as the criterion for the bacterici- dal properties of the disinfectant. This was thet>eginning of a long and useful series of investigations which have led to the present methods of testing antiseptics and disinfectants. mmmwm A number of methods have been devised for the laboratory testing of antiseptics. The results of these methods permit the comparison of one antiseptic with another in terms of their effects upon bacteria in culture media. £3,2i522 tests have reached a considerable degree of standardization, while clinical tests are more difficult to perform and to evaluate. The first method of evaluating the germicidal value of an antiseptic was develOped by Robert Koch in 1881 (lb). The method was based on the use of bacteria impregnated threads as the test material. In 1889, Geppert (lb) proved that the antiseptic carried by the thread in Koch's method was responsible for the exceptionally high values obtained with mercuric chloride. To eliminate this factor, Kronig and Paul in 1897 employed bacteria coated garnets as test objects and in their thorough study formulated tenets which have served as a foundation for procedures subsequently de- vised. In 1903 Rideal and Walker (1b) devised a test tube me- thod of examining chemicals for their killing action. In 1911, Anderson and McClintic published a procedure designed to eliminate some of the objectionable features of the Rideal- walker method. In 1916 Shippen combined the best features of these two tests. In 1927 this was published by Reddish (1h) as the Rideal-Valker method. This test, with very few changes, is known today as the Food and Drug Administration method. This method is the standard phenol coefficient test by which thousands of antiseptics and disinfectants intended for all sorts of use have been arbitrarily measured. The most recent U. 5. version is known as the "A.O.A.C. Phenol Coefficient Method”. The A.O.A.C. (Association of Official Agricultural Chemists) method (9) is being used routinely by a regulatory agency operating under the provis- ions of the Federal Insecticide, Fungicide, and Rodenticide Act; and to this extent it enjoys a quasi-legal status. Anderson and Mallmann (1) showed that penetration of the bacterial cell by the antiseptic or disinfectant is essential to the ultimate destruction of the cell. They also showed that this power to penetrate rapidly is a prime requisite of all compounds designed to kill bacteria within a short period of time. They prOposed the speed test as a means of measur- ing the penetrative power of antiseptics and disinfectants. Mallmann and Hanes (11) presented a technique of measur- ing antiseptics and disinfectants under conditions of prac- tical application. It was designed to measure and evaluate the dilution of an antiseptic that could be successfully used under field conditions. It is called the use dilution method. C. SELECTION QE_A§_ANTISEPT1C As yet, no single chemical agent has been found which embodies all the desirable characteristics of a good anti- septic, and at the same time is practical under most condi- tions. A generalized list of properties which such an anti- septic should possess can be made, and used as a reference when selection of an antiseptic is made. Such a list would include: 1. §2§§IE1§IIX~ An ideal antiseptic should have a wide killing range. Many of the germicides which are only slight- ly toxic to tissues are very specific in their actions, and are effective against only a few types of organisms. Since the chances of finding a pure culture in the area being scrubbed are slight, an antiseptic should be sufficiently broad in its action to give reasonable assurance of success. 2. EEIIQIENCX I§_1fl§ EBESENQE 91 QEGAEIC MAIEEIAL, An anti- septic should not have marked affinity for the organic matter which may be present, or the antiseptic will be used to satis- fy the organic material and its ability to destroy bacteria will be dissipated. 3. QOEEEICIENT Q£_QILQEIOX. Antiseptics are rarely used with- out further dilution of the solution. A good antiseptic must, therefore, be effective not only in its original concentration, but also in the dilution existing upon application. h. flQflQQEflEIII,AEQ,§IA§1LIII. A good antiseptic should also possess the qualities of homogeneity and stability. Homo- geneity is important in that every drOp should be just as effective as every other drOp. Many compounds would be good antiseptics if it were not for the fact that they form emul- sions in water rather than homogeneous solutions. Stability until the actual time of use is important. Many compounds deteriorate on standing, and the decomposition products which result have little or no effect. 5. II§SQE TQXICITI. The toxic effect an antiseptic has on tissue must be considered when a selection is being made. Unfortunately, the relationship between the protoplasm of bacterial cells and tissues is so close that there is no chemical known which is highly toxic to bacteria and non in- jurious to the body tissues. Some substances exert a speci- fic action against certain types of organisms which they kill or inhibit in dilutions that can be tolerated by the tissues. 6. EEELIBAEILIIX. Penetrability is also a factor to be con- sidered when selecting an antiseptic. In order to obtain a complete kill of the bacteria the antiseptic must be able to penetrate the organic material surrounding it. If an anti- septic can penetrate organic matter, then it generally can penetrate the bacterial cell. Thus it can effect a quicker kill than antiseptics that merely coat the surface of the cell 10 and interfere with its metabolism. Hence the preperty of penetration is an important measure of the practical value of a compound and should be used as one measurement of its efficiency. There are, in addition to the above mentioned, several other factors which may well be considered in characterizing an ideal antiseptic. Such properties as detergent action, solubility, chemical compatibility, odor, and cost are all important. D-WTE EQHW.__DQ_WELA maximum DETERGENT 1. 131921;. Triocil* (hexetidine) has been recommended for several uses. Kral (8) states that it has been found effec- tive in pyogenic otitis and some types of dermatomycoses. He successfully treated superficial pyogenic dermatosis by local application without any systemic treatment. Green (6) report- ed on the use of Triocil in the treatment of 316 cases of derma- titis, including fungus infections, acute and chronic eczemas, and non specific dermatoses. He obtained excellent results in 211 cases, 89 were satisfactory, and in only 15 cases were results unsatisfactory. Ripps (16) reported on the use of Triocil in the treat- ment of a variety of skin conditions, which included derm- atoses of many types, lacerations, cuts, and otitis. A total of 6h cases were treated. Fifty five were relieved or showed marked improvement, five were improved, and four showed no improvement. Sixty two of these cases were dogs. One horse * Warner-Chilcott Laboratories, Morris Plains, New Jersey. 11 and one parakeet were treated. In a personal communication with the Company, it was stated that: "Triocil has been used with complete success as a presurgical scrub." (5) In the Company's summary of research data, clinical abstracts on the treatment of many primary and secondary infections associated with skin condi- tions were given, but no laboratory or clinical work was in- cluded on its use as a presurgical scrub. 2. WELAQQ . Weladol* is an iodine preparation containing one per cent available iodine. Iodine is known as an active and reliable antiseptic and disinfectant. Since the latter part of the nineteenth century, much has been written extolling the virtues of iodine as an antiseptic. Iodine has been used in various ways as an antiseptic for the skin, wounds, and mucous surfaces of the body; for sterilization of the air and of inanimate objects such as catgut and surgical instru- ments; as a prophylactic and therapeutic agent in diseases caused by bacteria, viruses, and fungi. The factors which have often made iodine preparations unsuitable for use as a skin antiseptic are that they often stain badly, leaving a characteristic brown stain when applied to the skin, and re- peated application may cause blister formation. Iodine may be irritating to the tissues and painful to the patient. Weladol rep:esents an attempt to retain the antibacter- ial properties of iodine without its toxic effects. It con- tains a surfactant-iodine complex, polyalkyleneglycol, usually referred to as an iodOphor or "iodine carrier". This combina- * Pitman-Moore Co. Division Allied Laboratories, Indianapolis, Indiana e 12 tion of iodine reduces iodine vapor pressure to a low level, reduces the toxicity of iodine, and changes certain iodine absorption equilibria and chemical rates. As a result, free iodine is liberated slowly when diluted with water and is available for antibacterial action without its characteristic toxic, irritating, corrosive, odorous, and staining proper- t1.3 (19)e Terry and Shelanski (20) reported results of bacterio- logical testing comparing iodine in Lugol's solution versus an iodine-iodophor complex against Staphylococcus pyogeggs var. aunasao Tug; A, 5.0 ml iodine (Lugol's solution3 100 ppm) plus i, pyggggeg var. aggggs (2 x 10 ) after 1 minute, no viable bacteria remained. Then h x 108 fi, nxgggggg var. augeus were ad ed to the same tube, and after 1 minute, 2 x 10 organisms still remained. lg§§,§, 5.0 m1 iodine (iodine-iodophor complex, 100 ppm) i- W var. Emma. (2 x 108% After 1 minute no viable bacteria remained. Then h x 108 §, pyogeggs var. aggggg were added and after 1 minute no viable bacteria were found. These results indicate that Lugol's solution does not maintain its efficacy upon repeated exposures to bacteria as well as an iodine-iodophor complex. This shows that the avail- able iodine in Lugol's solution is utilized immediately, while the iodine-iodOphor complex, which is releasing iodine slowly, will maintain its germicidal properties after repeated exposures to bacteria. Merrill (13) showed by use dilution tests that 50 ppm of aqueous iodine would give complete kill in 60 seconds. 13 Research data by the Pitman-Moore Co. on Veladol showed that on presurgical scrub tests of h2 human subject's hands, 10 showed 100 per cent reduction of bacteria, 18 were between 95 and 100 per cent, and 1h were below 95 per cent reduction (19). The length of time the subject's hands were exposed to the Weladol in the scrub tests was not stated. 3. LLQQIQ,G§RMICIDAL DETERGEE . Liquid Germicidal Detergent* has been in use in the field for a number of years. In l9h8 Byran and associates (3) reported on studies conducted at the Michigan State College Veterinary Clinic. By plating swabs taken from the skin of dOgs and horses they showed that it was a satisfactory skin cleanser and antiseptic. Research by the Parke-Davis Company (15) showed that when Liquid Germicidal Detergent was tested against such common skin contaminants as Staphylococcus pyogeges var. guzgug, Sglmgggllg tyngggg, a hemolytic streptococcus, figghggigpig,ggli, and nggdomgggs agggginggl it was effective in relatively high dilutions. The maximum dilution that was effective var- ied with the organism tested, ranging from 1:200 against Eggugo- mggas 2;;uginggg,to 132000 against a hemolytic streptococcus. * Parke-Davis and 00., Detroit, Michigan 1h III. MATERIALS AND METHODS A. LABORATORY TESTS Since it is impractical to test a particular antisep- tic in the presence of all the organisms found in connection with the bacterial flora of the skin, two of the more repre— sentative species were used. These were Salmonella typhosa (representative of the Gram negative, non sporulating bacilli), and §taphylococcgs pyogeges var. ggregs (representative of the suppurative group, and a Gram positive cocci). The cultures were supplied by the Department of Micro- biology and Public Health, Michigan State University. The cultures were transfered at least three times at twenty four hour intervals. This was done so that a constant growth curve was established, and the number of bacteria present in the broth was relatively consistent., The tubes were shaken vigorously by hand for fifteen minutes prior to use so as to break up the clumps and make the culture as homogeneous as possible. 1. PHENCL CCEFFICIEVT TEST The Federal Drug Administration test for the determina- tion of phenol coefficients was used. This method was devel- Oped by Lloyd P. Shippen, George F. Reddish, C. M. Brewer, and 3. L. A. Ruehl and identified as the "Food and Drug Ad- ministration Phenol Coefficient" in the U. S. Department of Agriculture Circular No. 198 (18). 15 The Federal Drug Administration test for the determina- tion of phenol coefficients is conducted as follows: five milliliter amounts of a five per cent stock solution of phenol are prepared in dilutions of 1:70, 1:80, 1:90, and 1:100. Dilutions of 1:70, 1:80, and 1:90 are used for Staphylococcus pyogenes var. aureus. For Salmonella txphosa, dilutions of 1:80, 1:90, and 1:100 are used. Five milliliter amounts of the antiseptic to be tested are prepared in dilu- tions arranged in a series of decreasing concentrations. Both the phenol and antiseptic dilutions are placed in a water bath at 20 degrees C. The 2h hour broth cultures are shaken by hand for 15 minutes to break up the clumps. Using a sterile 2 ml pipette, add 0.5 ml of the 2h hour broth cul- ture of the test organism to one tube of each dilution of the antiseptic at intervals of 30 seconds. At the end of five minutes from the time of each inoculation a transfer is made with a 100p (h mm inside diameter, No. 23 B and 5 gauge) from the proper seeding pot to a tube of nutrient broth. These Operations are repeated at intervals of 10 and 15 min- utes. As soon as all transfers from the seeding pots are completed, a second set of transfers from the broth tubes just seeded is made. This is called Shippen's modification (17). Four 100pfuls are transfered from each tube of inocu- lated broth to another tube of broth. This additional trans— fer dilutes the antiseptic beyond the point where it will be able to exert bacteriostatic action. Thus it can be deter- mined if negative growth in the first set of tubes indicates actual kill, or if the first set is negative, but the second 16 is positive, it would indicate that the action of the anti- septic is bacteriostatic. All cultures are incubated at 37 degrees C. for U8 hours. The phenol coefficient is computed by dividing the high- est dilution of the disinfectant which will kill in 10 min- utes but not in 5 minutes by the highest dilution of phenol which will do the same. 2. USE DILUTION TEST Mallmann and Hanes (11) proposed this test as a means of measuring the actual dilution of an antiseptic or disinfectant that can be used for field use. In this method a 2h hour culture of Staphylocogggg pyo- gg2g§,var. agrgus is used. Sterile glass rods one inch in length and a quarter inch in diameter, having a loop at one end for handling, are dipped in a broth culture of the test organism and then laid in petri dishes on sterile filter paper to dry. Care is taken to avoid rolling the rods while drying. A drying period of 30 minutes is used. A medication pot (one inch by four inches) containing 10 ml of the disinfectant in the dilution being tested, and four pots each containing 10 ml of sterile saline rinse, are placed in a 20 degree C. water bath. At the end of the drying period, four rods are dipped simultaneously into the medication pot containing the test disinfectant. At intervals of one, five, ten, and thirty minutes, a rod is removed and immersed for one minute in a tube containing 10 ml of sterile saline. A neutralizing sub- stance may be incorporated into the saline to control bacterio- 17 static action. For Liquid Germicidal Detergent the neutrali- zer consisted of a mixture of azolectin and Tween 80. Sodium thiosulfate was used for Weladol. Tween 80 was used to neutra- lize Triocil. At the end of one minute, the rod is transfered to a tube containing 10 ml of nutrient broth. This tube is shaken vigor- ously to remove all remaining viable organisms adhering to the rods. One milliliter amounts of the nutrient broth are then plated in tryptose dextrose agar to measure quantita- tively the extent of the kill. The tubes containing the rods,” and the plates are incubated at 37 degrees C. for #8 hours. Lack of growth in both the tubes and plates at the end of this period is accepted as evidence of complete kill. Controls are prepared by dropping a rod covered with the dried organisms into a pot containing 10 ml of sterile saline for one minute, and then into a tube containing 10 ml of broth. One milliliter of the nutrient broth is used to make dilutions of 1:100, 1:1000, and l:l0,000, which are plated in tryptose dextrose agar. The plates are incubated at 37 degrees C. for US hours. The number of organisms surviving on the rods exposed to the antiseptic is determined by counting the number of colonies on the plate and multiplying by ten, as only one tenth of the nutrient broth is used. In order to determine the control number a plate is chosen having between 30 and 300 colonies. The number of colonies is multiplied by the dilution factor of that plate, and then by ten, as one tenth of the broth is used. 18 3. SPEED OF DISINFECTIOY TEST In order to test the Speed of action of a particular disinfectant, the following procedure is followed (1). Add 0.5 m1 of a 2h hour broth culture of §tgphylococcus pxgggggg var. auregs to a seeding pot containing 10 ml-of a particular antiseptic dilution. Also place 10 m1 of sterile distilled water in a seeding pot. Place seeding pots in a water bath at 20 degrees C. At intervals of 15, 30, h5, 60, 120, and 180 seconds a loop transfer is made from the seeding pot to tubes of melted agar in a #5 degree C. water bath. After thorough mixing the agar is poured into sterile Petri dishes and incubated at 37 degrees C. for US hours. After that time the plates are counted. Controls are run to determine the original number of organisms present before addition of the antiseptic. Controls are made by adding 0.5 ml of the 2“ hour broth culture of the organism to the seeding pot containing 10 ml of sterile dis- tilled water. Transfer one loopful to a tube containing 10 ml of nutrient broth. One milliliter of this nutrient broth is then used to make dilutions of 1:10, 1:100, and 1:1000. These dilutions are also plated and incubated at 37 degrees C. for #8 hours. In order to determine the control number a plate is chosen having between 30 _nd 300 colonies. The number of colonies is multiplied by the dilution factor of that plate, and then by ten, as one tenth of the broth is used. The per cent of organisms remaining is determined by dividing the number of colonies found on the test plate by 19 the control number. The per cent reduction is determined by subtracting the per cent remaining from 100 per cent. B. CLINICAL TESTIflG This phase of the testing employed animals in the Michigan State University Veterinary Clinic. These animals were hOSpi- tal cases that were being prepared for surgery in the manner 'routinely employed. No special measures were taken, as it was desired that the samples be representative of the stand- ard procedure used in the h05pital. A swab technique was used to obtain measurements of the number of organisms present on the skin after the presurgical scrub. It is rec0gnized that this technique does not measure the absolute number of organisms present because the resident bacteria living deep in the skin are not reached. However, this procedure does allow the accumulation of data relative to the comparative number of transient bacteria present follow- ing the use of different antiseptic preparations. Cotton swabs on wooden applicator sticks were used to obtain the samples. Immediately after use, they were placed in screw cap vials containing 10 ml of physiolOgical saline and a neutralizing compound. Both the swabs and the saline had previously been sterilized by the use of the autoclave. To prevent the possibility of bactericidal or bacterio- static action by the antiseptic which might be carried over to the saline on the cotton, appropriate neutralizers were added to the saline. For Liquid Germicidal Detergent the neutralizer consisted of 2.22 grams of azolectin and 15.6 ml of Tween 80 added to one liter of distilled water. Then 1.25 ml of a phos- 20 phate buffer, potassium acid phOSphate-disodium phOSphate, were added and the solution heated until clear. After the preparation was autoclaved, it was stored until used. To neutralize Weladol, sodium thiosulfate was used. Three hundred and twenty milligrams of sodium thiosulfate were added to one liter of distilled water, and 1.25 ml of the phosphate buffer were added. For Triocil, a one per cent solution of Tween 80 in distilled water was employed. All three preparations were sterilized in the autoclave at 2&8 degrees F. for 20 minutes at 15 pounds pressure. Cne milli- liter of the apprOpriate neutralizer was added to the saline after the sample was obtained. Cattle, swine, and dogs were checked at the termination of the surgical preparation. The sample was obtained from the approximate place the incision was to be made. The following procedure was used for the preparation of cattle and swine for surgery. The hair was clipped from a liberal area around the prOposed incision line. Stewart Clipmaster clippers with a No. .ShAU head were used. The area was wet with water from a hose. The antiseptic to be used was then liberally applied from a polyethylene squeeze bottle. The area was scrubbed vigorously with a stiff brush. The lather was washed away with tap water, and the process was repeated. Total scrubbing time ranged from four to six minutes. The scrubbing procedure for the dog usually entailed five applications of the antiseptic to be used. The surgi- cal area was clipped using a model A-z Oster clipper, with a size no head. The antiseptic to be used was then applied 21 from a polyethylene squeeze bottle. The scrubbing was done by hand and the lather was removed between each application with several cotton wipes. The cotton was from a roll of non-surgical bleached cotton (non-sterile). The procedures described above for cattle, swine and dogs remained the same no matter which antiseptic was employed, or what type of Operation was performed. The following procedure was used in obtaining samples: 1. The swab was removed from the glass vial that was used as a container while it was being sterilized. 2. Approximately two square inches of skin were thoroughly wiped at the termi- nation of the presurgical preparation. The swab and applica- tor stick were immediately placed in the tube containing the sterile saline. The tube was shaken vigorously, and the con- tents plated within one-half hour after the swabs were taken. Five per cent blood tryptose agar was used as the culture medium. Plates were prepared by adding a sufficient amount of sterile bovine blood to previously prepared and sterilized tryptose agar to make a five per cent suspension. The blood agar was then poured into disposable plastic Petri dishes and five m1 of the original 10 m1 saline sample added. The plates were incubated for 2h hours at 37 degrees C. After that time the plates wenaexamined for growth and hemolysis. The colony count obtained was doubled because only half the sample was used. ammw 1. TRIOCIL Triocil is Warner-Chilcott's trade name for hexetidine 22 (bis-l, 3-beta—ethy1hexy1-5 methyl-5 amino hexahydropyrimidine). The preparation contains 0.5 per cent of the active ingredient. It is a colorless oil that is soluble in organic solvents such as alcohol, acetone, and ether. It is soluble in water only to the extent of one part in 10,000. The manufacturer's recommendations for its use as a pre- surgical scrub (15) are as follows: a. Triocil Spray (0.5%) can be used as the sole agent cleansing the surgical area. b. In physically dirty dogs, Triocil Solution (0.5% with a detergent base is diluted from 1:2 to 1:5 and is used as a cleansing agent. This is removed with water and Triocil Spray is applied. c. Triocil Spray may be used as the final step if other preparations are used initially. d. Triocil Solution may be used initially if other pro- ducts are used as a final step. 2. WELADOL Weladol is a product of the Pitman-Moore Company. It contains a surfactant iodine complex, polyalkyleneglycol, commonly known as an iodOphor or "iodine carrier". This pre- paration contains one per cent available iodine. As a presurgical scrub, Weladol is used in the same manner as any other antiseptic detergent; that is, by thoreughly scrub- bing the site with Weladol and warm water for an adequate length of time (19). 3. LIQUID GERMICIDAL DETERGENT Liquid Germicidal Detergent is Parke-Davis and Co.'s trade 23 name for a suspension of high molecular weight alkylamide hydrochlorides containing 2.5 per cent of a quaternary ammon- ium chloride compound (phemerol)*. It has wetting and emul- sifying preperties, mixing freely with water of any temperature and hardness. The product is recommended for use at either full strength, or a 1:5 dilution. Optimal scrubbing time with Liquid Germicidal Detergent will vary, depending on the charac- ter of the skin surface and the amount of gross contamination. In general, however, a period of five to ten minutes is recom- mended (10). * Benzethonium chloride, Parke-Davis and Co. 2h IV. RESULTS A. masuus o; W TESTING gm TRIOQIL sngxon 1. PHENOL COEFFICIENT TEST Table I-A shows that when Triocil was employed in the phenol coefficient test, absence of growth occurred only after 15 minutes exposure at full strength with Salmonella typhosa as the test organism. Against Staphylococgus pyogenes var. agreus growth occurred in all tubes (Table I-B). 2. USE DILUTION TEST Tables II-A through II-E give the results when Triocil was employed in the use dilution test against Staphylococcus pyggoggs var. 33:323. At full strength a 100 per cent re- duction was not obtained until five minutes exposure. In Tables II-B, II-D, and II-E complete reduction was never obtained. The greatest reduction was achieved after 30 minutes exposure. This was 99.98h per cent for the 1:2 dilution, 99.06? per cent for the 12h dilution, and 99.133 per cent for the 1:8 dilution. 3. SPEED OF DISINFECTION TEST Tables III-A through III-C give the results when Triocil was used in the speed of disinfection test. When used un- diluted, complete reduction was obtained in the minimum time of exposure (15 seconds). Table III-B shows that when Triocil was diluted 1:“ a reduction of 99.7h0 per cent was obtained after the minimum time of 15 seconds. This does not differ 25 appreciably from the reduction obtained after the maximum exposure time of ten minutes, which was 99.91? per cent. Similarly, in Table III-C where a 1:8 dilution was used, a reduction of 99.1h5 per cent was obtained in the minimum time of 15 seconds, while a 99.508 per cent reduction was obtained in 10 minutes. Sglmonella typgosa was the test organism used. 8. EESQLTS QE,LA§0RATQBY TESTING QX‘EELADQL 1. PHENOL COEFFICIENT TEST Tables IV-A and IV-B indicate the results when Weladol was used as the test antiseptic in the phenol coefficient test. A phenol coefficient of 2.83 was established, using §Ilmonella typhosg as the test organism. The Shippen's modification indicates that there was no bacteriostasis in the tubes showing no growth. 2. USE DILUTION TEST When Woladol was tested by the use dilution method (Tables V-A through V-E) a 1:25 dilution gave 100 per cent reduction in the minimum time of one minute, while a 1:75 dilution gave a 100 per cent reduction in a minimum time of five minutes. Dilutions of 1:225, 1:250, and 1:300 required ten minutes to elicit a complete reduction (Tables V-C, V-D, and V-E). 3. SPEED OF DISINFECTION TEST Woladol was used in the speed of disinfection test at dilutions ranging from 1:2 through 1:300 (Tables VI-A through VI-K). §tgphylococcgs pyogggos var. gureus was the test orga— nism used. At dilutions from 1:2 through 1:32 all plates were 26 negative (Tables VI-A through VI-E). At dilutions of 1:100, 1:150, and 1:200 complete reduction was obtained in a minimum time of 30 to 60 seconds (Tables VI-G, VI-H, and VI-I). At dilutions of 1:250 and 1:300 a 100 per cent reduction was not obtained until the culture was exposed to the antiseptic for two minutes (Tables VI-J and VI-K). C- Biéflklé.Q£.LA§QBAIQBI.IEéIIEQ.Q!.LIQ!12.§§BMI§12AL.DEIEBQEEI 1. PHENOL COEFFICIENT TEST When Liquid Germicidal Detergent was used in the phenol coefficient test, with Salmggglla txpgggg as the test organism, a phenol coefficient of “Uh.h was established. The Shippon's modification indicates that no bacteriostatic action occurred (Tables VII-A and VII-B). 2. USE DILUTION TEST In the use dilution test all the plates were negative when Liquid Germicidal Detergent was used at full strength (Table VIII-A). At a 1:5 dilution a minimum time of five minutes was needed for complete reduction, while at dilutions of 1:7 and 1:9, ten minutes was needed for complete reduction (Tables VIII-B, VIII-C, and VIII-D). 3. SPEED OF DISINFECTION TEST In the speed of disinfection test (Tables IX-A through IX-D) dilutions from full strength through 1:? gave 100 per cent reduction on all plates. At a dilution of 1:9 a mini- mum time of 60 seconds was required for 100 per cent reduction (Table IX-E). When using a sample of unknown concentration taken from a clinic dispenser, 100 per cent reduction was of- fected in a minimum time of 15 seconds (Table IX-F). . 27 D. CLIEICAL iEfiTIflG l. TRIOCIL In the clinical testing of Triocil (T‘ble x) at a dilu- tion of 1:8 a completely negative sample was never obtained. Numbers of colonies ranged from a low of 12 (canine ovarie- hystoroctomy) to a high of 3&0 (porcine umbilical hernia). Three of the plates contained over 300 colonies. Four of the plates contained hemolytic colonies, with the highest being 12 per cent. 2. WELADOL In the clinical testing of Weladol (Table XI), used at full strength, a negative sample was never obtained. With one exception, the number of colonies ranged from 2 (canine ovariehystorectemy) to 3&2 (porcine inguinal hernia). One case (canine perineel hernia) had a count of 7076 colonies. Hemolytic colonies were demonstrated in eight of the samples. One plate had 50 per cent hemolytic bacteria. 3. LIQUID GERMICIDAL DETERGENT Clinical testing of Liquid Germicidal Detergent at a 1:5 dilution (Table XII), showed that seven of the samples taken were negative. The greatest number of colonies cultured from one sample was 66. In positive samples counts ranged from a low of 2 (canine ovariohysterectomy) to a high of 66 (bovine rumonotomy). Only one sample contained bacteria that were hemolytic (bovine rumenotomy). 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