r h I" H lllll le ‘E‘ES‘TS OZ? CCKWOUNBS WHICH MIGHT 8?. USES TC? P2189524? NAVE'L AND SURGICAL ENFECTEONS IN YOUNG LAME-5 Thai: for that Degree cf M. 5.. MICHMAN SKATE. COLLEGE i 5. “W "W. . filCK, be misfit? igéfit ..§" {“5}; 2.", ygL'..’\}-fi~r.\} -. .. _ ‘ 1v gm“: T'- I :4 ". -:.ld” . ’t 2“ .‘l . d T‘. '. lr .' .w-‘iv-nfim 3 x' fi‘m’hLMI-M v ‘“ 6-, .l'."'- 'w.‘ . . ' . .' I u -.' l‘,'- U." m“ .w' «’4.» ~ 2 . . '1; a 'é'.;15§3.’1a‘1-t "’3 . : w. 4... r ' H» ‘_"."‘.® 4&7“ ,m' I 4‘ . "f ‘ I . _ I ”357",]; 'y ‘ . ‘ 7“."“é” , (K: a '. 3.1' ’ “I p L «a H .A L.” n “5 |,\.V 1 , (0:? “'.5s’§' yv " 1 ~ " ‘ ‘1':f~ '1: “)1 "I. N If"; i . . _ ‘ ‘ . ‘..:"_.‘~‘.}' .3 7-149th -' -‘ w ‘ ‘j‘ . _ .' I ‘ A l V I .‘ ' , . . . , I “ l . A ‘A' "‘ ‘I 5.. ‘. O I I L0 \' if». "Javsz. 'J'k' I.“ . ~ 9 gm ~ 5451' afl'g 74" .3 5 . , '. '1 ’ ' ‘r {A . . n“: " l‘ 1" Jr 1 I' - 1’ ‘ln; . h\ ‘M '1. x. A ‘..' A . . ‘ . _ . ”r. s ' ~ . , . ~;r=a"“.v - - “' ,. '. . -'..~ A. —. 50‘ ' "'33 .<. \ ' .. , - .-..;.;:-:sfa'ut - - v -_ -C' b . . eta. EB. . ‘; . ' “'0‘ l . .gl ,. 'k ., -r- ‘b k . - km ”"9” .“ gin; L- V . a". ' 'I . ..r> ' {“‘.'Y".~"“ h ‘3‘ _ .. .~5‘:"-'} . q \Ly'fltylZ-‘lg‘klfl ' I v if? “7‘9 :1 . ‘Q‘c \kivflr'i'fi. I . .~._ '1‘.“:‘:.‘ .. I“\ - . . , A . "<\-', _ _ .‘fi!- 5" ' o > ~ I . _ _,.I ‘_ _ .. < . . < H ‘ ~ ‘ '. _. ‘ . ' -Lt ‘ . .- This is to certify that the thesis entitled Tests of Compounds which Might be Used to Prevent Navel and Durgicsl Infections in Young Lambs. presented by Jack D. Tiner has been accepted towards fulfillment of the requirements for .__L§.zdegree infiniflriljg tholo gy 'fiVLFrEgfij Major professor Date 7" 30 "((6 TESTS OF COMPOUNDS WHICH MIGHT BE USED TO PREVENT NAVEL AND SURGICAL INFECTIONS IN YOUNG LAMBS TESTS OF COMPOUNDS WHICH MIGHT BE USED TO PREVENT NAVEL AND SURGICAL INFECTIONS IN YOUNG LAMBS by mm D. TINER w ATHESIS Submitted to the Graduate School of Michigan State college of Agriculture and Applied Science in partial fulfilment of the requirements for the degree of MASTER OF SCIENCE Dopartment of Animal Pathology 1946 was 7/1b/flg AMOWMENT The writer is indebted to Dr. Frank Thorp, Jr. and especially to Dr. W. L. [alhann for nuch rundenentel prelininery knowledge and many helpful suggestions. The tireless and sympathetic aid of Dr. Tharp on more than one occasion doubtless extended the performing of the exp perinents presented here further than one person alone could have car- ried than. Sincere thanks are offered to the my others who contributed ideas or value in the conduct of this inwestigation and the preparation of this report. Dr. I. S. Feenstra, Dr. C. 1'. Clark, and Ir. Donald A. Schnidt deserve more credit than nerely nentioning their mes gives. Thanks are expressed to Dr. R. 1'. Langhen for taking all photographs. pa. SE (‘3 t“ +9.10 F... TABLE OF CONTENTS Page mmmoN O O O O I O O O O O O O 0 O O O O O O O O O O O O 1 HISTORICALREVIEWOFEXPERIMENTALMNDISINFECTION..... 2 EE‘I‘HDDSANDBESULTS...................... 13 DISCUSSION...............-........... 35 WANDGONCLUSIONS................... u. BIEIOGRAPM O O O O O O O O O O O O O O O O O O O O O O O O O 43 INTRODUCTION Statistical studies on the causes of death in lambs are al- nost nonsexisteht. Gal-on knowledge and.data fro-.the breeding and experinental flocks of Iichigan State College for the year 1943 indi- cate that a significant number of lambs die each year frcn.navel and docking infections. Since no work had been done to indicate the rela- tive efficiency of various skin disinfectants as an aid in preventing these losses, the present study was undertaken. HISTORICAL REVIEW 92 WW SKIN DISINFECTION The earliest account of the recovery of organians from skin after the application of a disinfectant to which this writer had access was one by Welsh (1892). "Scrapings from the epidermis and beneath the nails were made with a sterilized knife constructed for the purpose, and were transferred to tubes of liquified agar which were then rolled or poured into Petri's dishes." The danger of bacteriostasis was recognized and hands treated with bichloride of mercury were subjected to mania sulphide to precipitate the mercury. By thus neutralizing the disin- fectant Welsh showed that a previous investigator, Purbringer, was not getting the degree of sterilization which had been reported. A search for organims in deeper layers of the skin was also nade by Welch. An area on a human subject was thoroughly disinfected with potassium pemanganate and oxalic acid so that surface scrapings showed no growth. Sterile silk threads were then drawn through the skin from one to five times and dropped into melted agar. A white staphylo- coccus grew in several colonies along the side of the thread. Also, the stitches from laparotony wounds were moved by aseptic methods and dropped into melted agar. Fifty to sixty colonies of the white stapl'qlo- coccus grew around each. Grossich (1908) has been frequently credited with introducing tincture of iodine as a skin disinfectant, but his priority was disputed by Gershenfeld and liner (1932) in their review of the literature. Sev- eral persons who used iodine prior to this date are mentioned. Gershen- feld and Miller state that "As a disinfectant for the skin, particularly for the treatment of injuries and for the field of operation, iodine came prominently to the attention of everyone during the year 1905.” Tinker and Prince (1911) showed that tincture of iodine was not entirely reliable. They rubbed W m into the skin of the finger and applied tinc- ture of iodine after allowing the culture to dry for six hours. Sterile threads were drawn vigorously across the skin and dropped into bouillon. Thirty minutes later iodine was again applied and the threads drawn across the area and cultured. Additional scrapings were made with a sterile scalpel and dropped into bouillon culture medium. A 'pronpt and positive” growth was obtained in every case. Robb (1913) stated that Bovee deployed somewhat similar nethods and was able to report sterilization of the skin. This was attributed by Robb to the fact that “the cultures were all taken from the skin still coated with iodine.” Runoving the iodine with 10 per cent potassium iodide enabled the recovery of W W, coli, and W $112113. from the skin of dogs. Silk threads drawn through the area revealed B... m in one of the two trials. Turner and Oatto (1911) tested the extent of surgical disinfec- tion by relieving a thin strip of skin inediately after the first inci- sion. This was taken to the laboratory in a sterile test tube and culti- vated in nutrient broth for '72 hours. The culture was examined every 24 hours, and if turbidity was observed, a hanging drop slide was made. All specinens not showing growth at the end of 72 hours were routinely ex~ anined necroscOpically and microscOpically. When microorganism were found they were transferred to agar, studied, and identified. Bonney and.Browning (1918) made cultures of surgical areas on patients prepared for Operation with a combination of crystal violet and brilliant green. The skin was deeply scratched with a sharp needle and the surface of an agar plate inoculated. The needle was then resteril- ized and the process repeated until feur streaks were obtained. The re- sults enabled that to report favorably on the mixture. A fallacy in their method, however, was dunonstrated by Colebrook (1941): ”Experhment I. A.circu1ar area 6 cm. in diameter on the thigh.of a normal man was sampled in two ways: 39 EW'making four scratches two inches in length which almost drew blood. The scalpel point used was then streaked across a blood agar plate after each scratch. so colonies could be detected by naked eye or by low power microscOpe after 48 hours incubation. 33. The same area was vigorously rubbed with a gauze swab moistened in a sterile broth, the swab being immediate- ly shaken in 20 cc sterile broth and the bacteria in this enumerated by subcultures of an aliquot part. This count showed that 5,000 organisms were present on the area swabbed. A conclusion based on method 3 would clearly have been misleading." In advocating the use of picric acid, Farr (1931) suggested that antiseptics be tested by scraping and culturing the edge of the would immediately after the first incision. A complete cross section of the skin would thus be obtained. By doing this he had observed growth in 11 of 27 trials. Scott and Hill (1925) rubbed a sterile moist swab on the skin and than on an agar plate. The disinfectant, aqueous mercurochrcme, was applied and the culturing process repeated. Since 100 per cent re- sults were obtained, bacteriostasis was obviously present. Tinker and Sutton (1926) tested on human skin the then commonly used antiseptics. The compounds were bichloride of’nercury, tincture of iodine, trinitrOphenol, mercurochrome, chloramine T, chlorinated lime, and acriflavine. The method.employed is not very clearly stated. Appar- ently a sporulated culture of W W was applied to the skin, allowed to dry, treated with antiseptic, moistened by sterile distilled water, and scraped deeply with a sterile scalpel. IOOps of the scrap- ings "deeply stained with antiseptic” were used to make cultures. Pene- tration was tested by excising a 3 mm. strip of skin from.a patient pre- pared for Operation. Ilany different regions of the body were represented, but how they were cultured is not stated. NO growth was obtained only with acriflavine, and this was later questioned.by Leonard (1930). Reddish and Drake (1928) used rabbits to compare mercurochrome- 220 solublewith tincture of iodine. The abdominal skin was shaved, washed with.warm.water, and allowed to dry. “The Operations were carried out with ordinary aseptic technique, but no special precautions were taken against contamination.” Staphylpggggn§.§n;§g§*was applied with a sterile cotton swab over the shaven surface and allowed to dry. The rabbit was removed from the table and placed in a cage. Two hours later (5 tests) and 24 hours later (4? tests) squares were marked off and antiseptics applied to the center of each. .An effOrt was made to apply in the same manner each thme. One area treated with distilled water was used as a control. Five minutes after the antiseptics had been applied the excess was removed with a sterile moist swab. The control area was also swabbed. One square centimeter in the center of each area was scraped with a ster- ile scalpel until capillary bleeding occurred. All scrapings were re- moved in 1 cc. of sterile water in a petri dish. Fifteen cc. of cooled melted agar was poured to make a plate. If no growth was observed in 24 hours, W m was streaked on the plate, and if it pro- duced colonies, bacteriostasis was considered to be not present. No un- seeded and untreated area was cultured to establish the necessity for seeding the skin with W m instead of using merely the skin flora. A completely new method was introduced by Simone (1928). The skin of the abdomens of a series of white mice was shaven and contami- nated with a 10 day old sporulated culture of W W and painted liberally with antiseptic. Ten minutes later (2 tests) and 2 hours later ('7 tests) the skin from the center of the area was excised, inverted, and the wound sutured together with horsehair sutures. If the animal died, cultures were made from the heart blood. Surviving animals were observed for eight weeks. Simone used the skin of rabbits in a manner similar to the methods of other investigators which have been described. An area was dry shaven. and cultures 01' Was sums. Wars masses. W assassins. Maharishi: .2911. and W 191.0211 applied. After being allowed to dry for an hour, 5 a. squares were marked Off and each painted liberally with an antiseptic. After 5, 10, and 20 minute intervals (30 and 60 minutes for £1. W cotton swabs moistened in sterile broth were rubbed over the painted areas and cultures made on a plate of nutrient or blood agar and a flask contain- ing 100 cc. Of broth. A second set of cultures was then made from a seller portion of the same area. Deep skin cultures were made by scrap- ing a still smaller portion until capillary bleeding occurred and plat- ing the scrapings in 15 cc. of melted agar. Superficial and deep cul- tures were made for each of the tune intervals used. Simmons secured best results with U. S. P. tincture of iodine, stating that it destroyed all bacteria in every case. Rodriguez (1928) compared iodine with.norcurochrome on the nat- ural flora of the ora1.mncosa and obtained very good results with 3.5 per cent tincture of iodine and 1.75 per cent iodine in glycerin. Roberts (1929) took cultures by holding a sterile swab of stand- ard size moistened in sterile media against the treated and control areas.. There was no rubbing and no more pressure than necessary was used to hold the swab in place. At the end of exactly thirty seconds the swabs were rinsed.in a tube of nutrient media and discarded. The tubes were then in- cubated at 37° c. for five days. Antiseptics were tested on the skin of rabbits and humans by this technic. This would obviously not give any idea of the depth of the disinfection. This procedure can merely show that a film.of disinfectant was placed on tap of the skin, and perhaps on top of the bacteria as well. Raziss, Severac, end loetsch.(l930) used a technic based on that of Roberts. The abdominal skin of rabbits was depilated with.barium sulphide and the area washed for three minutes with soap containing Dieta- phen. Afterward Metaphan l-l,OOO was applied to the skin and removed with alcohol and ether. The area in question was then rubbed 30 seconds with a sterile cotton swab moistened in sterile broth. The tip of the swab was then clipped and allowed to fall into a test tube of sterile bouillon. They concluded that "this procedure effected complete sterilization of the abdomen as evidenced by cultures.” The publication of 100 per cent results drew some criticism. Leonard (1930) in a letter to the editor of the American ledical Associa- tion Journal questioned the validity of Tinker and Sutton's results with acriflavine and those of Raziss, Severac, and.lostsch.with.metaphen. He stated that: ”One hundred per cent results involving the use of biologic material such as living tissue or bacteria always invites close inspection on the part of those who have been unable to duplicate such results by their own experimental methods.” .Hs further pointed out that acriflavine will inhibit growth of spores and bacteria in high dilution, and that metaphan according to Raziss et al. was more than bacteriostatic enough to prevent growth in the dilutions used. White and Hill (1930) preceded to demonstrate that Raziss and co-workers were actually dealing with‘bacteriostasis by repeating their experiments, and innoculating the tubes in which no growth had been ob- tained with.§§gphylgggggggmgnzgn§. The staphylococci failed to grow, thus demonstrating that enough metaphan had been carried over to produce bacteriostasis. When swabs were drapped.into 900 cc. of'media dilution was such that microorganisms grew. Kelser and Mohri (1932) made comparative tests of ”Mercurochrome- 220 Soluble" and tincture of iodin on the normal skin flora of the horse. An area 6 by 9 inches on the side of an.animel was dry shaven and rectan- gles 1% by 2 inches were marked off. The disinfectants were rubbed on. At the and of ten minutes moist sterile swabs were rubbed briskly over the area, drapped into sterile test tubes, carried to the laboratory, and streaked on blood agar plates. Good percentages of growth were obtained in spite of the obvious risk of bacteriostasis. In the same paper an apparatus for washing the organisms to free thm from bacteriostatic quantities of the disinfectant was des- cribed. It consisted of Berkefeld filters with an attaclment for forc- ing a backflow of distilled water. Unfortunately, they used it only for test tube cultures of W m. They pointed out that some of the organians stick in the pores of the filter. Whether the method would be practical for washing skin scrapings would have to be determined by actual trial. Birkhaug (1933) compared phenyl-nerouric-nitrate, mercurochrome, and metaphen using rabbits. The animals were strapped on Operating tables and the abdomen depilated with a sanieliquid mixture of 1 part barium sulphide and 2 parts flour. After rinsing with water 25 squares with about 20 mm. sides were marked off with a red wax pencil. Cultures of Wm WM (sporulated). W W, and W 2911 were neared on and dried. The disin- fectant was then applied. Exactly three minutes later an area approximate- 1y 3 by 3 by 2 mm. was pulled up with forceps, out off, and deposited in 100 cc. of Douglass broth. Ninety-two per cent sterility was obtained with metaphen and 96 per cent with penyl-mercuric-nitrate. Norton (1920) showed that in handwashing neither antiseptic nor plain soaps were of any value other than for mechanical cleansing. How- ever, it was not until 1938 that Price published a method for measuring the quantitative effect that this mechanical cleansing had on the bacter- ial flora of the hands. By washing in a series of sterile basins and plating the water, Price (derived a mathsnatical formula for the rate of removal which was found to decrease logarithmically. He was able to esti- mate with accuracy the numbers of bacteria on the forearms and hands of -10- various individuals. The terms ”transient" and ”resident” bacteria were introduced. It was demonstrated that persistent exposure to pathogenic organisms will incorporate th- into the individual's flora as Welch had shown (1892). Pohle and Stuart (1940) and Sears et a1. (1941) used Price's procedure and found it quite reliable. Base (1939) prepared rabbits in the same manner as Birkhaug. However, he did not add organisms to the skin, stating that "in no case was it ever sterilized by depilation.” Hercurials, tincture of iodine, and picric acid were tested. Seventy per cent etl'wl alcohol served as somewhat of a control. The antiseptics were placed in sterile test tubes, each! containing a sterile swab. The soaked "swabs were rubbed on the area for 25 seconds, and at the end of three minutes a piece of skin was snipped off and drOpped into a test tube containing 100 cc. of infusion broth. This was incubated at 39° 0, and at the end of 48 hours, if no growth was observed, 50-l,000 organise from untreated skin were dropped in to prove that there was no bacteriostasis. Barnes (1942) tested a synthetic detergent, cetylo'trimethyl- amonium bromide by marking out an area on the forearm 4 by 6 inches. It was first swabbed a minute with a cotton swab. This was drapped into 100 cc. of broth. One cc. of this was plated in 10 cc. of nutrient agar and resulting colonies counted. The detergent was then applied by rubbing three minutes with moistened swabs. These were rubbed briskly so as to obtain a good lather. A one minute rinsing with water under the tap was used to remove the disinfectant. Plates were then made as before and col- oniss counted. Barnes cited Price's results as sufficient evidence that -11.. swabbing did not remove an appreciable amount of the total flora and used as a control previously untouched areas. The effect of an equal amount 1 of rubbing using ordinary soap followed by rinsing was not determined. In an effort to estimate with reliability the true value of a disinfectant against pathogenic organisms, Numgester and Kamfp (1942) em- ployed an infection prevention test in mice. Cultures of organisms of "high and constant virulence for mice" were rubbed on the tail, the tail was dipped into an antiseptic, and a half inch piece was removed and in- serted into the peritoneal cavity. The same pathogenic organisms were recovered with a high degree of frequency from the heart blood of those animals which died of the ensuing peritonitis. Various disinfectants showed different abilities to protect animals. Alcoholic iodine two per cent was the only antiseptic which gave 100 per cent survival, and [er- thiolate the only one which failed cOInpletely. Cultures were also made directly from a second portion of the tail. The percentage of growth obtained from these was slightly lower than the number of’mioe infected. A similar procedure was develoPed by Sarber (1942). The ab- dominal skin was contaminated with W W, disinfected with various substances, and then inserted into the peritoneal cavity. Eamolytic streptococci were recovered from all mice that died. Gershenfeld and Witlin, (1941) tested disinfectants on the skin flora of rabbits. They were able to show that "in not one instance was there complete sterility”, but unfortunately theirvmethod allowed no studies as to the relative merits of the various compounds. The question of the location of bacteria on or in the skin has always confronted workers in skin disinfection. Welch (1892) was probably -12- the first to investigate this problma. Price (1938) found that the transient organisms were "relatively scarce on clean, unexposed skin . . . . These bacteria lie free on the skin, or are loosely attached by grease and other fats along with dirt. [any collect under the nails. . . The resident flora is entirely on the skin, attached by adhesion, adsorption, or possibly in some special way.” Apparently he was unable to demonstrate organisms 'in sebaceous and sweat glands. Lovell (1945) incubated human skin samples in a moist atmosphere and at the end of six hours fixed and sectioned tha. With specially stained slides, masses of staphylococci were dancnstrable in the sebaceous glands. In the literature to date there seems to be no report of anyone's being able to dauonstrate a bacterial flora in sweat glands. -15- METHODS AND RESULTS The disinfectants used in these experiments were as follows: Wfilodinsi’fl’zll- mmmtflnlormhm. ‘ m W, Merck and Co. Inc., Rahway, N. J. A 4 per cent solution will be referred to in this paper as W m. In some instances it was used in combination with a wetting agent, Naccanol, National Aniline Chemical Co., New York, N. Y., in order to reduce sur- face tension and enhance penetration among densely matted wool fibers. um mm W PEP; 11... Jensen-Salaam Laboratories. Kansas City, No. A 2 per cent solution was given a limited trial. m, Winthrop Chunical Co. Inc., New York 13, N.‘Y. A quarternary ammonium compound. 1922, Rolls and Bees 00., 222 Washington Square, Phila- delphia, Pa. A quarternary ammonium compound, obtained through the kindness of Dr. W. L. lallmann. The purity of the sample used in making up the solutions was unknown, and the concentrations consequently could not be determined. damn m am am asshol- Wgfi W, Parke Davis and 00., Detroit, Michigan. A quarternary amonium salt. The tinted form of the tincture was em- ployed. W m, Winthrop Chemical Co. A quarternary «- monium compound with the same structural formula as Roccal, but marketed as a stainless tincture. At the outset the immediate problan was to find a method of show- ing whether it would be advisable to replace the generally recommended Tincture of Iodine USP with some other compound. Since the worth of a laboratory test is largely related to the degree to which natural condi- tions are simulated, the initial trials became an empirical search for a method that would approximate field usage and yield quantitative data. -14- Sheep wool is unique in that it contains lanolin, a fat like secretion from the sebaceous glands. The question of what effect it might have on the efficiency of the disinfectants had to be answered. An attempt to study this directly was made by seeding tryptose agar* plates with a rapidly growing Staphylococcus isolated from.a lamb dying of a naval infection. .A cost of sterile lanolin was then poured over the surface and a drOp or two of the compound in question was placed on tsp of the lanolin after it had hardened. The plates were incubated for 48 hours and the extent of the zone of inhibition was measured. Trials indicated that colloidal iodine was quite efficient and that an aqueous solution of Eyamine was of little if any value for penetrating a heavy coat of lanolin. Tincture of iodine was considered not comparable since it persistently etched through the wool fat and diffused throughout the agar‘below, thus allowing no growth over large areas of the plate. The methods of Price (1938) seemed to have some promise of being useful in evaluating the disinfection of sheep skin and wool. According- ly, a sample of wool was clipped from a sheep in the barn with sterile scissors and placed in a sterile jar. One gram.samples were weighed out and exposed to tincture of iodine and colloidal iodine for the intervals of 30 or 60 seconds. They were then rinsed in 500 cc. of distilled water to which 10 grams of sodium thiosulfate had been added before sterilizing in the autoclave. Samples were then transferred to two-quart Jars contain- ing one gram of soap which was dissolved and sterilized in one liter of distilled water; Two agar platings of 0.5 cc. each.were made from the wash 1 \ *Bacto Tryptose dehydrated, Difco, Detroit, Mich. -15- water and incubated 48 hours before counting. On some of the plates an excessive, growth of spreaders made counting difficult. In order to surpress as many variables as possible until more was known of the method and its possibilities, it was decided to substi- tute wool which had been sterilized and seeded with the Staphylococcus mentioned above by immersing in inoculated broth, incubating for 24 hours at 37° C., draining, and drying. One change was made in the procedure: The shaking was done with a Killer Paint Mixer“ for three minutes in this and all succeeding trials. The data in Tables I and II indicate the re- sults obtained. When the application of iodine was not followed by rins- ing in sodium thiosulfate, 100 per cent results were obtained. A ten second immersion in iodine with a 40 minute delay in neutralization did not achieve a complete kill. Aqueous hyamine neutralized by soapy water failed to significantly reduce the nmnbers of bacteria. The Staphylococci on the seeded wool died out in approximately ten weeks and it was decided to try measuring the effect of the disin- fectants on skin samples taken from young lambs coming to autOpsy. These lambs represented several breeds and had died of various conditions as in- dicated in Table In. and constituted a more or less random sampling of the lamb crap. A rough estimate was made of the total number of bacteria per lamb. This was done by detemining the average number that were moved from a square centimeter (Table III) and multiplying by the area of the whole skin as calculated by direct measurement when spread out. The re- sults of such computations on ten lambs are recorded in Table III. The variation in the total number of organisms per lamb might be tamed ex- I"Miller Mfg. Co. , 3238 Bryn Nawr Ave. , Chicago. - 15 - treme -- from 432 million to in excess of 43 billion. Distilled water was found to be as effective as soapy water for removing bacteria from skin and wool and was used in these and in all subsequent experiments. It was found that the skin samples could be stored in the freezing com- partment of the electric refrigerator at a temperature of -3 to -5° C. with no appreciable quantitative change in the bacterial flora over periods of several months. . The data recorded in Tables VI and VII were obtained by using the procedure outlined in.Tigure 1. Small skin samples of measured area were immersed in 30-50 cc. of disinfectant for ten seconds, allowed to drain for fifty seconds, and rinsed in 500 cc. of a sterile solution of a neutralizing agent for thirty seconds. Sodium thiosulfate was used in the case of iodine and 0.2 per cent soap in the case of the quarternary ammonium compounds. This was followed by washing for three minutes. Counts for the "control” listed in Table VI were obtained by drapping skin samples directly into the washing Jar and shaking. ‘That such counts were too high is shown by Table IV in which percentages of organisms lost in disinfecting and rinsing varied from.56.4 to 93.7. Approxnmate per- centages of the flora removed by the first three minutes washing and rinsing was determined and recorded in Table V. They appeared high enough that no further washing was considered necessary. ‘Use of soapy water ap- parently gave no significant increase in efficiency. The altered.method outlined in Figure 2 was used to obtain the results in Tables VIII and IX.upon which the conclusions of this paper are largely based. Except in the case of Lamb 7989 (Table VII) , the neutral- izing agent was replaced by 500 cc. of sterile distilled water. The con- -17- trol runs were subjected to an equal amount of agitation in volumes of sterile distilled water equaling the amounts of disinfectant and water that the skin samples were exposed to. More uniform skin sample‘sizes were obtained by measuring the area of a large piece of skin, for example 50 sq. om., dividing it into perhaps 70 small pieces which were made as uniform as possible, and then. taking three of these aturandom to make samples of about 50/70 x 3 = 2.15 sq. cm. for each trial. In order to show the relationship of the compounds extensively tested to some of those that are very commonly used by the veterinary profession, some data were obtained on Liquor Cresclis Saponatus USP 11 and 70 per cent ethyl alcohol. An additional experiment was done to determine the extent of any neutralizing action which oxalated blood might exert on tincture of iodine and colloidal iodine. The procedure is depicted in Figure-3, and the results recorded in Table II. The 5 minute stop in oxalated sheep blood between the rinsing and washing Jars is the only change from the procedure outlined in Figure 2. In order to observe the location of bacteria on lamb skin, samples were taken from near the navel of recently dead newborn lambs, fixed in Zenker's solution, «bedded, sectioned, and stained with Gram Weigert stain. PhotomicrOgraphs (Figures 4 and 5) were made of some bacterial forms among the wool fibers. The red blood cell present in Figure 4 is interesting to note. It hints as to the nature of the medium in which bacteria establish themselves on the skin of a newborn lam -- a mixture of fetal fluids and blood. - 13 - During the performance of the above tests the observation was made that areas of the skin stained as long as a week previously with iodine had extranely low numbers of bacteria. It appeared that the dura- tion of disinfection might offer some basis for the comparison of disin- fectants. Accordingly a group of five sheep was placed in the barn, areas marked off on their sides, control samples taken, and 24 hours later disinfectants applied. The first control samples represented 1/5 gram per area per sheep. A control patch was interspersed among the disinfected areas. The day following disinfection the samples listed for September 15 in Table I were taken. The experiment was run a second time 28 days later, using different areas on the same sheep. Samples were taken Just before and shortly after the disinfectants were applied. Results of both experiments are recorded in Table 1 in which the logs- rithm of the number of organisms is plotted as a function of the time in days. The iodine compounds achieved the most imediate and lasting sur- pression of the bacterial flora. When the samples were still wet with the solutions it was necessary to dry tha in a vacuum. The areas treated with the tinctures were apparently dry within 24 hours, but the aqueous compounds produced wet areas which dried out more slowly. No effort was made to reach a constant weight. The samples were considered dry when the petri dishes containing them no longer became cold after being re- moved fron the vacuum chamber, wanned, and returned to the vacuum. Neither was it determined whether ordinary wool, e. 3. control samples would suffer a change in weight if placed in a vacuum. These factors would probably not affect the results significantly, but should be checked were the method to be much more extensively used. -19- When lambskin was placed in Roccal, a visible precipitate often immediately fomed. It was easily seen by all observers who happened to be present. Its presence was confirmed by use of the photelometer“. A liter of distilled water and a liter of Roccal l-l,000 were checked for percentage of light tranmission. lashings from the control sample which had just been used a few minutes previously to “obtain the bacterial count for October 13, 1945 (plotted in Table I) were then added to each in 10 cc. amounts and the percentage transmission again checked. Results are recorded in Table 31. The increased light absorption on the part of the Roccal solution indicates the presence of the precipitate. The mean and standard deviations of a few series of samples were detemined. Table XIII contains a series of eight consecutive control areas from Lamb 8030 and Table XIV nine consecutive areas need in de- temining the total bacterial count for Lamb 7989. '“Sheard-Sanford type. new blSleECTANT' 'Naso orSOAP' 'SIERILE Hzo' 50'50cc. 500 . 1000 , .' M“ e. ——“‘n~ . \\ ». _4'\ .1...“ Figure 1. Apparatus for disinfectant tests with rinse in neutralizing agent. ‘2“ 'DISINFECTANT' .STERILE H30. 'SIERILE H-0' 50‘50cc. 5006c. 1000... ‘ Figure 2. Apparatus used in disinfectant tests with rinse in distilled water. - 21 - .-.—.€‘~ - i\ 1 .. ct — V > w a o .— O ‘- A 'DBINFECTANT'SIERILE mobxmmfo mom’srtmu H,O' ~ \ 50‘50cc. 500cc. 60cc. lOOOcc. .— ‘ .7 ' ‘ Figure 3. Apparatus used for disinfectant tests in- cluding sterile oxalated blood for study of its neutralizing effect. - 22 - Figure 4. Photomicrograph of section of lamb skin show- ing wool fibers, bacterial forms, and a red blood cell. x 838 Figure 5. Photomicrograph of section of lamb skin show- ing wool fibers and bacterial forms. 1 838. Table 1. ONE GRAN SAMPLES 0F STERILE WOOL SEEDED WITH STAPEYIDCOCCI AND EXPOSED TO TING- T0123 01‘ IODINE AND SODIUM THIOSULFATB Minutes hposed to Iodine 0 t t 1 N Minutes in O 2 Colonies per Plating .5 cc. 24,945 1, 745 3,095 2,908 521 see 751 -25- Tablo II. ONE GRAM SAMPLES OF STERILE WOOL SEEDED WITH STAPHYIDCOCCI AND EJQDOSED TO: Iinutes llinutes in Colonies per cc. Plating Exposed Ha23203 from Soapy 820 a. TINC‘IURE OF IODINE FOLLOWED BY SODIUM THIOSULFATE 0 39,375 0 o (3 in 320) 0 14,957 4,855 2,437 1,365 145 Bgsawwwo b. COLLOIDAL IODINE FOLLOWED BY SODIUM THIOSULFATE o 0 39,375 1 o o 1 3,415 a 5,123 10 7,600 so 1,337 c. AQUEOUS HYAMINE, l-l,000 3011mm) BY SOAPY urn: Minutes in amyfio 0 0 39,375 1 Immuwh 3 38,000 10 43,000 20 . 36,000 40 (Plated from distilled H20) - Innumerable moo; oom.sn men 89.: m3 89.3 3m.» as 8a.? conga . .8 was main: Siam n2; sac.» pm».m ”on; film macro. $1» o3.m eons scrim in ma It) e '0 ma DH new." when n.” canon—Beam shoe 3” oases—fish odmho scanner. who“. am done Janos—neg as 9 e3.” sexesm eaobo non—Show shed on some 633505 choc e ewmnnhoaon Hensoqu when. 9 3355mm when a sauce—seam whee m $3.398: Hogans”— hsc .n ewmnnnosn awakens” ngOOHQw choc an msownunmsem .43. .93 «a aeaafieeeeg coco 93m congeveosb nmoo UHOHHO 0&8 chafing—now Sow anemoanm onom enEmmoanm. «mow Sadness 82. £333 82. 23326 82. 33386 some 32.38 $2. on «sunfism $2. Saigon-um as? 823928 82. 2338.5 nv\m\n 9\H\m 9}}. 3).}. nexexe ne\QA\n nexmaxn nexaa\n ne\ofi\n ne\m\n his? Exam} ne\mm\m woo neg ragga 8 coonm dawned go even awn mead 20 203.30% mbgzqaomg g 9,500 dgog ASHE .HHH 0."an - 27 - fl Table IV. WMECHANICALF LOSS OF‘BACTERIA.IN DISIN- FECTING AND RINSING Lamb Total No. Organisms No. per 0.2 Indi- 7989 7990 8024 8069 8030 per 0mg - Without Previous Washing 4.26 x 106 907,000 177,000 1.23 x 10" 7e 45 x 106 cated by Control Plates 1.86 x 105 104,500 28,010 765,000 1.23 x 10‘ fiflLost 56.0 88.5 84.2 93.7 33.5 Table V. EFFECTIVBVESS OF WASHING PROCEDURE IN REMOV- ING BACTERIA FROM SKIN OF YOUNG LAMB Lamb 8050: 21.121311121112322. llinutes 3 6 9 12 15 No. 0.2 1.09 x 107 299,000 34,860 11,860 11,300 87.l$ 1.13 x 107 = No. per mg rmoved by first 15 min. washing Total No. per 012 1.13 x 107 of which 97.1 removed in first 3 min. Subtract approx. 83.5% lost in disin- fectant and rinsing to leave approx. 13.6% of flora used in actual test. Lamb 8030: a Wat Minutes 3 6 9 12 15 No. One 3.421105 28,000 4,000 14,550 3,110 98.7% 3.47 x 106 = No. per mg runoved by first 15 min. washing Total No. per 0112 3.47 x 106 of which 98.7% removed in first 3 min. subtract approx. 83.5% lost in disin- fectant and rinsing to leave approx. 15.2% of flora for actual test of disinfectant. Table VI. mow 0N museum FLORA OF ONE MINUTE EXPOSURE TO mucus stmmc'rmws 1011011313 BY NEUTRALIZATION , 700111301." occurs T00 HIGH -- SURVIVAL pmcmmcs T00 1071 001. I Lamb ”Control" Tr. I 001. I a; Nae. Roccal 11633203 Nazszos M23203 Soap 7932 #Sanmles 4 7 2 2 6 #062 1.2x10 200,000 2.4.210 %Survival 100.0 2.3 19. 7 7944 #Samples 4 1 l #an 214,000 53,000 49,000 9101mm 100.0 2.47 23.0 7982 #Samples 5 l 1 #an 2.01107 11,830 1.11106 $Survival 100.0 0.01 5.6 7988 #Samples 7 3 3 3 2 #0112 2.2::106 356,000 . 576,000 780,000 965,700 %Surviva1 100.0 16.0 26.0 35.1 43.4. Table VII. HIT-CT 0N LAMBSKIN FLORA OF ONE MINUTE MOSURE TO VARIOUS DISINFECTANTS FOLLOWED BY NEUTRALIZING AGENT 001. I Cont. Tr. I 001. I & Nac. Roccal Lamb E20 11625203 N828203 N828203 Soap 7989 #Sanmles 5 7 4 8 5 2 6 6 , 6 6 #(h 2.0x10 585,000 1.12x10 . 86x10 1.08x10 $Survival 100.0 29.1 55.5 77.5 53.8 Table VIII. EFFECT ON LAMBSKIN FLORA. OF ONE MINUTE EXPOSURE TO VARIOUS DISINFECTANTS 001. I Lugol 70$ Liquor Lamb Cont. Tr. I 001. I a Nac. Iod. Roccal Ethyl Cresclis Alcohol Sapona- tus 2% 7990 #Samples 3 5 5 3 3 #an 104,200 1,500 3,010 972 9,100 anl 100.0 1.44 2.89 0.93 8.72 8024 #Samples 4 4 4 4 #ca2 23,400 1,685 1,500 375 1686va 100.0 7.2 6.4 1.6 8069 #Samples 15 12 12 12 12 10 4 4 #012 735,000 9,650 7,300 7,100 10,650 21,800 78,200 71,300 6030 #Samples 8 8 a a #m2 1.99:106 11,950 21,200 15,350 $Survival 100.0 0.60 1.06 0.77 -31- Table II. EFFECT ON W FLORA OF‘ ONE MINUTE EXPOSURE TO VARIOUS DI SINFECTANTS -:_. -— Lamb Cont. Tr. I 001. I 001. I Lugol Roccal & N8°e IOde 8069 #Samples 15 12 12 12 12 10 #0n2 735,000 9,650 7,800 7,100 10,650 21,800 $Survival 100.0 1.81 0.99 0.97 1.45 2.97 + ADDITIONAL 5 MINUTE STOP IN 0mm) BLOOD Tr. I 001. I #Samples 3 5 #082 28,400 28,800 %Survival 3.18 3.85 Table X. EFFECT OF VARIOUS DISINFECTANTS ON WOOD FLORA OF ADULT SHEEP -33.. Table XI. PRECIPITATION 0F AQUEOUS ROCCAL BY WOOL WASHINGS 1 liter Water 1 liter Roccal Distilled 1-1,000 Photelometer 100 100 Washings obtained by shaking 5 grams sheep wool in 1,000 00. dis- tilled water added at rate of 10 00. between photelometer readings: 98.6 98 97.2 96 96 94.4 Table III. AVERAGE NUMBER OF ORGANISIlS PER (112 880771170 VARIATION IN A SERIES OF LAMBS Lamb Organisms in Number Thousands 7928 . . . . . . . 246 7932.......12,200 7944 . . . . . . . 214 7946 . . . . . . . 7,430 7982 e ‘e e e e e e a),000 7988 O O O 0 0 O 0 2,220 7990 O O O O O O O ”7 ”89 O O O 0 O O 0 4,260 3030 e e e e e e 0 7.460 W31 0 O O O O O O 234 8069.......12,200 8085 O 0 O 0 O O O m 12170.96: Mean: 5,839 Standard deviation: 5,985 Table XIII. A SERIES OF CONTROL SAMPLES SEEKING VARIATION W Lamb 8080 * Organisms per Caz in thousands 855 Mean: 1,865 Standard deviation: 775 Table XIV. VARIATION IN 'IOTAL BACTERIAL COUNT PER SAMPLE Lamb 7989 2 Organisms per Om in thousands 9 . IMean: 3,912 Standard deviation: 2,552 DISCUSSION Since the time of’Lister various sUbstances have been intro- duced as skin disinfectants. More than once the initial claims for a new product have implied that actual sterilization was accomplished. Evidence from experimental results often seemed to substantiate these claims. Closer examination has, in every instance of which the writer is aware, revealed that only varying degrees of disinfection had been accomplished. ZPerhaps it is time to recognize the apparent reality and instead of seeking an agent which will give absolute sterility, effort should be made to develop germicides which are very highly efficient in reducing the Skin flora to a.minimum. In the past, conclusions that any agent is 100 per cent genui- cidal have invariably been due to a faulty method of evaluating its action on the skin. Before attempting any further work in this direction a careful examination of the procedures of other investigators and the potentialities of each method was considered necessary. Two approaches to the problem of evaluating the action of dis— infectants on the skin have been used. By one, a pure culture of the organism, usually a pathogen, is applied, allowed to dry, and treated with the compound in question. Various techniques are then anployed to recover the surviving organisms. The second.method uses the normal skin flora. In both, a most difficult problem.seamed to be to separate re- covered organisms and antiseptic so that bacteriostasis did not give a false impression of germicidal value. Net too adequate fundamental knowl- edge as to when bacteria are really "dead" increases the difficulty of reaching an agreanent on how this separation may be accomplished. Table II shows 100 per cent disinfection with iodine compounds when the seeded wool sample was transferred without rinsing directly from the disinfectant to the washing Jar. When transferring from tincture of iodine a deep iodine color was produced in the latter. This seemed to indicate that the carrying over of any appreciable quantity of iodine in- to the solution from which the plating is to be done is to be careflllly guarded against. The data in Tables 71 and VII indicate that a neutralizing agent which is specific for a disinfectant is able to "revive" supposedly ”killed” organisms. The work of Sears, et al. (1941) contains evidence that they too could have been ”reviving" some organisms when they followed 3.5 per cent alcoholic iodine with sodium thiosulfate solution in experi- mental pre-Operative hand scrubbing. Welch, (1892) stated that a large percentage of the flora on supposedly disinfected skin could be recovered if the bichloride of mercury was followed by ammonium sulfide. Bachem (1913) mentions that the usual method of applying tincture of iodine as a skin disinfectant at that date, in Germany at least, was to allow it to dry and ranove it within a minute with sodium thiosulfate. To the best of the writer's knowledge, Tables VI and VII present the first evidence that this procedure is not experimentally justifiable. In per- forming the experiment it was noticed that frequently the iodine stain in the wool fibers was visible even after the percentage of survivors had been markedly raised by the thiosulfate solution. It is interesting to note that the color produced by tincture of iodine scanned to be more com- pletely ranoved by the thiosulfate solution, whereas colloidal iodine which was consistently much more susceptible to neutralization in or on the bacteria produced the more lasting stain in wool fibers. The data presented can leave very little question that solutions of iodine are among the best skin disinfectants available at the present if they are not fellowed by neutralization. Just which iodine compound should.be used depends largely on the task at hand. Tincture of iodine, because of its alcoholic content and resulting lowered surface tension is able to diffuse very rapidly among wool fibers and foreign material. wab ever, there is as yet no experimental evidence to indicate that there is any Justification for its choice over the:more economically prepared aqueous Lugol's solution. Colloidal iodine, according to Anderson (1939), has unusually high penetrating power and consequently it may be superior to the other two for certain purposes. Nonetheless, it was noticed that when colloidal iodine was applied to the wool of adult sheep the stains remaining a few days later were highly irregular. Apparently the col- loidal particles did not disperse among the wool fibers as readily as the aqueous iodine solution in which these particles were suspended, and con- sequently were left concentrated in the areas of original application. The results of this particular experiment (Table X) indicated that it did not suppress the flora in the disinfected areas quite as lastingly as tincture of iodine. When flaccanol was added to colloidal iodine an apparently stable mixture with lowered surface tension resulted. It gave promising results (Table VIII) as a disinfectant. However, the neutralizing effect of the sodium thiosulfate solution appeared to be markedly raised by the presence of the Neocanol (Tables 71 and.VII). Since the trial with oxalated blood indicated that blood and probably other body fluids might have some neutralizing effect on iodine compounds, it was not possible to reach any conclusions as to the relative worth of the colloidal iodine -- Naccanol mixture. There is no evidence as to what might result if the iodine con- tent of the usually prepared solutions were varied. Work on the question of what might approximate the Optimum strength of the various iodine com- pounds and in fact all disinfectants is indicated. The writer is indebted to Dr. N. A. Fattu of the Dopartment of Psychology and Philosophy, Michi- gan State College, for calling to his attention the dosage-mortality curves which were uployed by C. I. Bliss in determining the strengths of insec- ticides to be used. More references on the subJect are found in Bliss (1985) and Broadbent and Bliss (1986). quarternary ammonium compounds have been given their first con— trolled trial on the skin of sheep. Although the percentages of survival recorded from lamb skin flora seen favorably low in the'trials using Roccal, they are not dependable. In the performance of the experiment it was noticed that when lambskin samples were immersed in aqueous Roccal a cloudy white precipitate was often immediately apparent. Several trials in which there was semingly 100 per cent survival were not averaged in because of the possibility of mistaken use of distilled water. In the light of the work on adult sheep it seems probable that actually no such error was committed. The sustained rise in the count in Roccal treated areas can probably be attributed to the fact that the moisture left after the quarternary amonium salt was inactivated facilitated bacterial repro- duction. Confirmation that some substance on sheep does precipitate -39- quarternary ammonium compounds was obtained with the photelometer read- ings recorded in Table II. Convincing evidence has thus been secured that aqueous Roccal is not useful for disinfecting ovine skin. The work of Nungester and Kemfp indicates that Tincture of Phemerol 1-500 is decidedly more effective than 70 per cent alcohol or an acetone and alcohol mixture. The experiments reported here, however, offer no means of determining what percentage of bacterial mortality was due to the action of the alcohol and/or acetone in the tincture. Some disinfecting action was observed with the 2 per cent solu- tion of Liquor Cresclis Saponatus USP 11. The very limited trial on lamb skin indicated that 90 per cent of the organisms were killed by a one minute exposure. The experiment on adult sheep suggests, however, that it is a rather slowly acting compound. Table I shows that the number of organisms declined for two days or more. The low point seems to be around 2,000,000 organimns per gram. As compared with the original count this may seem to be a significant reduction. Nevertheless, when one considers that the wool was still wet with the solution 24 hours after application and had a characteristic cresol odor, and had to be dried in a vacuum be- fore samples could be weighed, and that its organisms represented a sam- pling of the environmental flora, cresol may not be as satisfactory for barn and stable disinfection as the U. S. Dept. of Agriculture Famer's Bulletins 926 and 954 (Dorset 1918 and Pepe 1918) some to imply. The writer and those with whom he has associated during the per- formance of these tests feel that the results obtained are quite a reli- able basis for drawing conclusions as to the relative values for use on lamb skins of the types of disinfectants in question. Experience showed that some lambs had a flora which was more evenly distributed than others, and also, some seaned relatively free .from the surface spreaders which tended to make counting difficult. It was with these skins, selected by trial and error, that the data upon which the conclusions are based were obtained. The results of a series of consecutive samplings, recorded in tems of organials per square centimeter are listed in Table XIII for Lamb 7989 (total number bacteria per (h.‘?') and in Table XIV for Lamb 8030 (the figures listed were among those used for controls for experiment). These may be considered, in the light of the greater variation found in most of the others as almost ideal for the purpose of testing disinfect- ants. Usually the standard deviation exceeded the mean. The fraction of the skin flora actually used to test the disin- fectants is surprisingly small. Table IV indicates that from 56.4 to 93.7 per cent of the total flora was washed off before} the Jar from which the plating was to be done was reached. Since the washing procedure failed to remove perhaps three per cent of the organises, an additional though small fraction was lost by not being removed from the skin. Table 7 indicates that in the case of Lamb 8030 approximately 13.6 to 15.2 per cent of the total flora was used in the test. Extensive comparison with other commonly used substances was not made because the supply of skin salnples stored from the lambing season became exhausted. The performance of such tests would probably have lit- tle bearing on the problem at hand. The compounds which sealed most val- uable on the basis of Dr. Hallmann's years of experience were scinpared in a fairly thorough manner with the tincture of iodine now in use. SUMMARY AND CONCLUSIONS Experimental work comparing disinfectants under conditions sim- ulating actual use in veterinary medicine has been quite limited- The present study was undertaken to compare quarternary ammonium and iodine preparations for possible use in disinfecting ovine skin. The compounds given extensive testing were as follows: Tincture of Iodine, U.S..P. XII Colloidal Iodine Colloidal Iodine with 1 per cent Naccanol added to reduce surface tension Lugol's Iodine, 0.5.13. XII Roccal A new method for testing skin disinfectants was devised. The technic involved the use of skin taken from lube Just prior to autopsy. Samples of known area possessing the natural skin flora were treated with the compounds for one minute intervals, rinsed, and then washed in sterile distilled water. Platings with tryptose agar were made in order to calcu- late the nunber of organians per square centimeter. The percentage of survival was ‘detemined by comparison with control samples subjected to equal volumes of sterile distilled water. The number of surviving micro- organias could be raised markedly if the rinsing was done in a neutraliz- ing agent specific for the disinfectant. Best disinfection was obtained with the iodine preparations. Ox- elated blood counteracted to a slight degree the effect of iodine compounds. Naccanol and colloidal iodine do not seem chemically incompatible. As yet we do not wish to advocate the replacement of tincture of iodine as a skin disinfectant. -42- A.test was also made of the relative efficiencies of some of the compounds in depressing the numbers of microorganisms composing the flora of the wool of adult sheep. Tincture of iodine was most effective for this purpose. Evidence has been obtained that aqueous Roccal is precipitated by some substance on adult sheep and had no value as a disinfectant for unwashed wool. BIBLIOGRAPHY Anderson, L. P. 1939. Penetratise Powers of Disinfectants. Thesis, School of Graduate Studies, Michigan State College. Also . . . and w. L. hallmann. 1948. Tech. Bul. 188. Section of Bacteriology, Agricultural Experiment Station, Michigan State College. Bachem, C. 1913. Ein- Haltbarer Ersatz der Jodtinktur in Foster Fom. Em- m- m- 9952625- Barnes, J. M. 1942. CTAB: A New Disinfectant and Cleansing Agent. 11232.91 243531-532. Bass, A. D. 1939. An Experimental Comparison of Certain "Stin- Sterilizing" Agents. 1. W. 56:279-288. Birkhaug, K. E. 1933. Phenyl-mercuric-nitrate. 1. mm. m. &: 250'2614 Bliss, C. I. 1935. Estimating the Dosage-mortality Curve. 1. m. m. 285646.647. Bonney, Victor and 0. s. Browning. 1918. Sterilization of the Skin and grin Surfaces by a Mixture of Crystal Violet and Brilliant arcane me “e lo 1:562’563e Broadbent, B. M. and C. I. Bliss. 1936. Comparison of Criteria of Susceptibility in the Response of Drosophila to Hydro- cyanic Acid Gas. 1. m. M0 22:143-155. Colebrock, Leonard 1941. The Disinfection of Skin. Ell].- Ea; m. 2:73-79. Dorset, M. 1918. Some Common Disinfectants. Farmer's Bul. 926, Re- 71806. O. Se Depte Agriculture. Farr, C. E. 1921. Picric Acid in Operative Surgery. gym. fluid. Gershenfeld, Louis and R. E. Miller. 1982. The Bactericidal Effici- ency of Iodine Solutions. 1. m. Egan. £1993. 21:894-903. Gershenfeld, Louis and B. Witlin. 1941. Surface Tension Reducents in Bactericidal Solutions: Their 13 mm and 11 11.19. Ef- ficiencies. £3. 1. man. 113:215-236. -44- Grossich, Antonio 1908. Eine Neue Sterilisienlngsmethode der Haut bei Operationen. W. L 99.12. 41:1289. Kelser, R. A. and R. Pl. Mohri. 1932. Comparative Germicidal Tests of Mercurochrome and Tincture of Iodin. {,A,1,E,A, 83:87-95. Leonard, V. 1930. Pitfalls in the Interpretation of Skin Disinfection. g, A, g, A, 24:1524. Norton, J. F. 1920. Soaps in Relation to Their Use for Hand Washing. I, A, u, A, 15:302-305. Nungester, W. I. and A. H. Kempf. 1942. An ”Infection-prevention" Test for the Evaluation of Skin Disinfectants. 1. 132.953.; 211,. 11:174-178. Pohle, W. D. and L. 3. Stuart. 1940. Germicidal Action of Cleaning Agents--A Study of a Modification of Price's Procedure. lo want... us... 91:275-281. Pope, G. W. 1918. The Disinfection of Stables. Farmer's Bul. 954. Revised. U. S. Dapt. Agriculture. Price, P. B. 1938. The Bacteriology of Normal Sun: A New Quantita- tive Test Applied to Study of Bacterial Flora and Disin- fectant Actionin Mechanical Cleansing. 1. mm. 213g 63: 301-318. ' Raiziss, G. W., Severac, M. and J. C. Moetsch. 1930. Metaphen as a Germicide and Skin Disinfectant. g, A, M, ,5, 24:1199-1201. Reddish, G. F. and W. E. Drake. 1928. Mercurochrome-220 Soluble and U. S. P. Tincture of Iodine. A Comparison of Germicidal Efficiency in Skin Disinfection. I, A, g, A, 2],: 712-716. Robb, Hunter, 1913. Iodine in Sterilization of the Skin. @113. 91.1.1.0 92,. 1:824-827. Roberts, K. 1929. Value of Methylene Blue-Gentian Violet Five Per Cent in PreOperative Skin Preparation. Ann, @4289. 83:183—190. Rodriguez, F. F. 1928. Mercurochrome and Iodine as Disinfectants of Mucous Manbrane of Mouth. J. A. M. A» 2],: 708-712. Sarber, R. W. 1942. An In-vivo Method Used for the Evaluation of Sub- stances Used in Skin» Disinfection. _J_. W and m. 11131183. 31:277-281. -45- Scott, W. W. and J. R. Hill. 1925. Presentation of 8 Preoperative Skin Disinfectant, an Alcohol-Acetone-Aqueous Solution of Mercurochrome. _J_. 11:91. 15:132-152. Sears, H. .T., W. E. Smick, G. C. Schauffler, and R. C. Shoemaker. 1941. A Quantitative Test to Evaluate Methods of Hand Steriliza- tione wegie lo Me 9_b_e andgme i2:458-464e Simmons, J. S. 1928. Bactericidal Action of Mercurochrome-220 Soluble and Iodine Solutions in Skin Disinfection. ,1, A, M, A, 21: 704’708e Tinker, M. B. and H. L. Prince. 1911. Common Fallacies Regarding Skin Disinfection with Special Reference to the Iodine Methods. 3333,. Qfl. 91;. 13:530. Tinker, M. B. and H. 3. Sutton. 1926. Inefficiency of Most of the Commonly Used Skin Antiseptics. J. A- M. 9» 813347-1350. Turner, Phillip and H. C. Catto. 1911. The Iodine Method of Steriliz- ing the Skin. 1422211 1:733. Welch, W. H. 1892. Some Considerations Concerning Antiseptic Surgery. 12. 9.282. 211- Madman. 51-28. A189 . - . . Isn- 13nd no lie a: 45-47e White, E. C. and J. H. Hill. 1930. Inefficiency of Metaphen as a Skin Disinfectant. J. A. M. A- 25:27-34. 5 0M ’9 "' ' .'. R“ c ‘ “S; 1'..." I . . ‘1 3 ‘1 'n'.‘ 2 . 4., r .i h I " I. I P ‘ I |.‘, r. ;‘ .' '- ' "1‘ .. . 5"" \qu' 7 4') .- ’4‘ M." I ff. '. r W '. I ‘i . .‘V‘ v ‘. _ H: .J . . ‘5 .I‘ .‘.--. 3“ ~24- . 'PN' ’. fix) _ at; ‘9 .. t , .. ‘3 rr‘ K )- .‘IU - v‘t‘ . -. L ..~w -- . j.) ' ‘ \s P ‘ . a. . . .Q‘ . a: P Q.’ T. l' ‘ ': ‘ ‘- ”A v - '1 4 ‘ , -' "p f; ' "\ f 7 x ' ' . " I u 'l (q '- t: ., ’ ' H? . ’4-YHFLS)’: M‘ ' ',"' tyra‘. \' “L“ Av " l t xiv? 7.1; '* Lag-153?} . ,7 | I. I ‘ F l‘ . . 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