THE USE-DILUTION METHOD OF TESTING DISINFECTANTS Thesis for the Degree of M. S. MECHICAN STATE COLLEGE Marjorie EHen Hanes 1945 This is to certify that the thesis entitled The Use—Dilution methoa of Testing Disinfectants. presented by Marjorie Ellen Henes has been accepted towards fulfihnent of the requirements for degree in Bacteriology M o S o Major profe sor Date September 1, 1845 ~_‘n- L‘ze»~ flMAA‘A *;A-—-_4;_a_.’__ “a THE USE-DILUTION METHOD OF TESTING DISINFECTANTS by Marjorie Ellen Hanee 7- A THESIS 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 Department of Bacteriology 1945 llfn\ (ll ll..'.l|" ACKNOWLEDGMENT I wish to express my gratitude to Dr. W. L. Mallmann whose helpful suggestions and assistance made the develoPment of this method possible. -1- Introduction There is a need for a better method of measuring the effectiveness of chemical agents used in the destruction of bacteria. Many methods have been devised but due to the wide diversity in chemical constituents of the various agents no one method has been satisfactory. The need for testing the efficiency of substances which prevent or retard bacterial action was felt even before it was known that disease and decay are caused by micro—organisms. The development of methods started with the early history of bacteriology; Robert Koch (6) in 1881 was the first to test disinfectants by using a pure bacterial culture as a standard. Silk threads which had been 'impregnated with organisms were exposed to disinfectant action and trans- ferred to a suitable medium to determine the presence or absence of growth. This was a primitive method and the results obtained were not accurate due to the carry-over of the disinfectant into the broth. It, however, aroused the interest of scientists who later worked to develop better methods. Kronig and Paul (7) in 1897 were the first to recognise that time is a function of disinfection~and that bacterial hill is a gradual process and not an instantaneous one. Garnets of standard size were dipped into a culture of micro—organisms and then dried completely before exposure to the disinfectant. After exposure to the disinfectant each garnet was rinsed to eliminate the remaining disinfectant and then incubated in tubes of broth. In 1898 Hill (5) devised a method using long glass rods which were sterilized in stoppered glass tubes, dipped into a broth culture or smeared from a slant culture and then exposed to a disinfectant. The -2- period of exposure and the type of organisms used was purely arbitrary. The rods were placed into tubes of a suitable medium and incubated. I ‘Phenol was introduced as a basis of comparison by‘Rideal4Walker(ig) in 1903. Varyinéfdilutions of the disinfectant and phenol were used so that a mathematical expression could be obtained by a comparison of the dilutions of each required to kill bacteria in a given period. Experi- mental error was minimized by the application of standard temperature, organisms, media and time-periods. Chick and.Martin (2) in 1908 demonstrated that temperature, varia- tions in number and age of organisms, and culture medium have a tremendous effect on the accuracy of results obtained when testing disinfectants. They suggested that one specific bacterial culture should be selected and used for testing all disinfectants to eliminate confusion in evaluating the effectiveness of the disinfectant. A set time period of thirty minutes for exposure of the organisms to the action of a disinfectant was used because it was recognized that the speed of disinfection varies. Sulfide was used to neutralize the bacteriostatic effect of the disinfectant in the testing of'mercuric compounds. Chick and.Martin also suggested a procedure for testing the disinfectant in the presence of 5 per cent organic matter to simulate the actual conditions under which the disin- fectant would be utilized. The Beddish technic‘l) described in 1927 represents the best method of testing devised in the series of procedures based on the original Ridealéwalker method. Since the development of the Ridealalalker method all later deve10pments such as the Lancet Method, Hygienic Laboratory Method, American Public Health.Association Method and the Reddish procedures were merely adaptations pertaining to test organisms, media, and other -3- minutia. All of these technics determine the minimum concentration of disinfectant that will kill under standard conditions. None of these present conditions of testing bees any resemblance to the conditions under which the compounds will be used for disinfection purpose. The attempt to determine a numerical expression in which all compounds are compared to phenol‘is fallacious and misleading. ml mercurial, a quaternary ammonium chloride, and a phenol are not comparable. The important issue to be determined is whether or not the compound under test will kill effectively when applied in the process of disinfection. Jensen and Jensen (5) in 1955 devised the cover-slip method in an effort to improve on the phenol coefficient. This procedure offers an interesting development, not in the fact that it offers another means of evaluating disinfectants numerically in comparison to phenol, but the fact that it suggests a technic more comparable to conditions found in actual practice. In this technic a loopful of a saline suspension of Staphylococcus aureus is placed on a coverbglass and dried prior to use. In disinfection the organisms encountered are dried on the surface of the object being disinfected and piled in layers from'continual exposure of the object to contamination. This technic departs from the Rideal- Walker method and returns to the original technics of Kronig and Paul, and Hill. Killing dilutions of the disinfectants tested are much lower than those obtained hy the Reddish phenol coefficient method. It is evident that present methods of testing disinfectants are not without discrepancies and the necessity of improvement upon these methods is of primary importance. The phenol coefficient method is used as a criterig for evaluating the efficiency of a disinfectant. A compound, therefore, which produces bacterial kill only at low dilutions is desig- nated as a poor disinfectant regardless of the fact that it may be very -4. effective at full strength or at a low dilution and could be used at that concentration with exemplgry results. The manufacturers of chemical disinfectants specify the dilution at which the compound should be used under given conditions. It is this dilution, the use—dilution, which should be used in evaluating the disinfectant. If the disinfectant at the dilution specified by the manufacturer should fail to kill bacteria in a.time period which would correspond to the period of exposure in actual use, the disinfectant could be termed ineffective. A lower dilu- tinn which produces the desired results could then be suggested for use. lhen the phenol coefficient of a compound is used to evaluate its effectiveness a great error may be introduced if the dilution coefficient is not taken into consideration, as demonstrated by Hymn (4). when the concentration of a disinfectant is reduced to oneéhalf of its original concentration the disinfecting ability may be reduced anywhere from two to sixty-four times depending upon the disinfectant. If a disinfectant is tested at its useedilution the problem of the dilution-coefficient is eliminated since this is the concentration actually used by the consumer and no further dilution takes place. The use-dilution method of testing disinfectants was devised in an effort to improve upon the phenol coef- ficient method. ' Death of bacteria is held by most bacteriologists to be the failure to grow after the test bacteria have been planted into a favorable medium and incubated for a reasonable length of time. The reliability of such a concept is questionable due to several factors which.must be taken into consideration; namely: bacteriostasis, extended lag phase and small inoculum. Bacteriostasis of a bacterial cell may result from the adsorption of certain chemical disinfectants upon the surface of the cell. Uhder these conditions no growth occurs but the cell is alive .5. and may remain so for a prolonged period of time. The cell may in time, be able to overcome bacteriostasis naturally or it may require the addition of a neutralizing agent to restore normal cell activity. On- favorable conditions brought about by exposure to a disinfectant may extend the lag phase of the culture so that no growth is evident within the expected period of incubation. If only a few bacterial cells are living after disinfection the number of organisms present may be too small to initiate growth in the volume of medium to which they have been transferred. The enzymes and accessory food products of the culture . become diluted to such an extent that they are no longer of sufficient concentration to support growth and reproduction of the bacterial cells. In this thesis it is recognized that no method has been devised to ascertain death of bacteria so lack of bacterial growth after a reason-— able period of incubation will be accepted as sufficient evidence of sterilization. Terminology used in describing the process of killing bacteria and the killing agents is not only confusing to the laity but also the scientist. A disinfectant is generally defined as a chemical agent that kills organisms capable of producing disease. An antiseptic is defined as a chemical agent that prevents the growth of disease-producing bacteria but does not necessarily 9933112111. The research workers in disinfection and the manufacturers of disinfectants define a disinfectant as a chemical agent used to destroy pathogenic bacteria on inanimate objects. Antiseptics are defined as chemical agents used to destroy or prevent the development of pathogenic bacteria on skin and in tissues of the body. In this thesis the latter definitions will apply to the usage of the terms disinfectant and antiseptic. -6- ggperimental Studies The selection of suitable apparatus for the conveyance of the bacterial culture into the disinfectant and then to the culture medium was the first consideration. In the method devised by Koch the disin- fectant clung to the surface of the silk threads causing a carry-over of the disinfectant into the culture medium. Kronig and Paul selected garnets because of their impregnable surface but the garnets themselves were difficult to handle. The cover-slips used by Jensen and Jensen also presented difficultties in handling. Hill used long glass cylinders which were veny suitable for transfer into tubes of broth. Although the rods were of comparable size the entire surface of the rods were not inoculated with the culture and uniformity of the number of organisms present was questionmeble. Dr. W. L. Mallmann suggested the use of a metal or glass cylinder one inch in length and one-fourth inch in diameter which could be made with a loop at one end to facilitate handling. Cultures dried on a glass rod would be more comparable to the surface of inanimate objects than broth cultures because organisms on such a surface would be dry and piled in layers. In a series of tests made with these rods, a tentative procedure of examination was set up based largely on past experiences with present methods. The rods were dipped into 24 hour broth cultures and placed in sterile Petri dishes to dry. After drying the rods were dropped into medication pots containing 10 cc. of disinfectant solution. After vary- ing periods of exposure at 20°C. the rods were lifted from the tubes with a sterile wire and dipped into a tube of sterile water to remove the excess disinfectant and then dropped into sterile nutrient media. The tubes were shaken vigorously to release the organisms from the rods -7- and suitable dilutions were plated to measure quantitatively the extent of kill. The rods were incubated in the broth for 24 hours to determine any resulting growth and act as a check upon negative plates which showed no colony formation. It is possible that negative plates could be obtained when viable organisms existed in the broth due to the error introduced in random sampling. If the number of organisms present in the broth were relatively small a one cc. amount could be removed which contained no living organisms. Tubes showing turbid growth were indi- cated as innumerable and not plated. Tubes not showing growth were plated to determine the presence of viable organisms. The media selected for these studies were those now used in this laboratory for phenol coefficient determination. Staphylococcus aureug__was grown in Difco disinfectant testing medium and Eberthella typhosa was grown in standard broth described in F. D. A. Bulletin 198 (11). These two media have proved very satisfactory for use in this laboratory, parti- cularly for growing the test organisms. Staph. aureus and E. typhosa were selected as the test organisms because they have been success- fully used in the phenol coefficient determination and no objection to their use could be found. The Staph. aureus cultures when.grown in Difco disinfectant testing medium are always neutralized with Noon to eliminate changing the pH of the disinfectant solution in the usual phenol coefficient procedure. However, in these studies, this was unnecessary because the cultures were always dried and no material change in pH would result when the inoculated rods were introduced into the disinfectant dilution. The test organisms selected showed the following resistance to phenol: .3- Staph. aureus, Dilution 5 min. 10 min. 15 min. 1:60 } - - 1:70 t +, - 1:80 f f f !h_izaaass. Dilution 5 min. 10 min. 15 min. 1:80 - - - 1:90 f - - 1:100 % i a Preliminary teststith these rods were so favorable that the following studies were made to ascertain the feasibility of the proce- dure for the evaluation of disinfectants, particularly in use-dilution. Experiment I -- To determine the adherence of a bacterial culture ‘EEEESEQEEEEEEEEEEEETZZEEEEEE ”‘ ‘ De The first experiments were made to determine whether or not the organisms would adher to the rods in sufficient numbers to give a dependable result when carried from the disinfectant to the rinse and then to the final broth. To measure the loss of organisms, sterile water was substituted for a disinfectant in the first tube to eliminate kill. This tuft tube could be plated to determine the number of organisms removed mechanically by dropping into the liquid.' In like manner, the rinse tube and the final broth could be checked. The results are presented in Tables 1 and 11. These experiments were repeated using sodium lauryl sulfate instead of sterile water to deter— mine the effect of detergent action upon the mechanical loss of organisms. Escherichia cgli was chosen as the test organisms because lauryl sulfate -9- has no inhibitory effect on it. The results obtained are shown in Thbles 111 and 1V. An evaluation of the results obtained demonstrates conclusively that there is a mechanical loss of organisms from the rod into the various liquids with which it comes in contact. The number of organisms removed by deter- gent action is much greater than by water. However, the number of organisms remaining on the cylinders when they are placed in the broth appeared to be of sufficient magnitude to demonstrate the amount of bacterial kill ef; fected by the disinfectant as demonstrated in later experiments. Experiment ll-eggdetermine whether there is any difference in the results when usipg metal or glass rods. ' Both metal and glass rods were used in the first experiments. It soon became evident that experimentation could be simplified if one type of rod were selected for use in further studies. To measure the difference in results obtained, metal and glass rods were dipped in broth bacterial cult- ures, dried, and exposed to disinfectant action for periods of one, five, ten and 50 minutes. The cylinders were rinsed to remove the remaining dis- infectant and then put in broth. The broth was plated and the tubes of broth containing the rods and the plates were incubated for 24 hours at 57°C. Any tubes not showing growth in 24 hours were replated to determine the pre- sence of any viable organisms. Tables V and V1 show the results obtained. Upon examination of results it is evident that the degree of var- iation in results between the two types of rods is negligible and either could be used with accurate results. However, after further investigation it was decided that the glass rods are more practical because they are easier to clean and sterilize, the organisms dry faster on the glass sur- face, and the glass rods are more simple and less expensive to make. 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H55. ,. wooed? .m unease £93m among» .m unease £93m f 500m 30.: r 30m emu-440 H’ 0.335 mo sea. .‘ 3930.3 3% no sense: $550138 .5 e 000.H .ee...05eHu&n.m1aws 435380550 9303 a.“ 053.5.30 me upon 3us H50 H33 mo dosage—woo 4 .. 9 Human. -14- -rosion occurring was negligible the use of glass rods would eliminate any possible oligodynamic action. In the following eXperimental studies the glass rods are used exclusively. Experiment three- To determine the most effective way of drying_the broth bacterial culture on theisurface of the glass rods. In the first experiments the glass rods which had been dipped in a broth bacterial culture were allowed to dry in a sterile Petri dish. The excess broth culture on the rod drained to the surface of the plate and made drying in the alloted time period difficult. To overcome this effect, a piece of sterile filter paper out to fit the bottom of the Petri dish was used. The inoculated rods were placed on this adsorbent surface. Rods which had been dried on the glass surface and those which had been dried on filter paper were eXposed to the action of a disinfectant in the usual manner and the results obtained are presented in Table V11. It is obvious since the number of organisms retained on the glass rods after drying on filter paper is greater than those on the rods dried on glass, that the organisms were well dried and resisted the washing effect of the disin- fectant to a greater extent. EIperiment fourvTo determine the length of time reguired to dry the broth bacterialpculture on the surface of the glass rod. In an effort to determine the actual time period necessary to dry the broth culture on the glass surface of the cylinder the inoculated rods were dried for periods of 50 minutes, one hour, two hours, three hours and 15 hours before exposure to the disinfectant. The results obtained from the rods which had been dried for 50 minutes and the rods which had been dried for 15 hours are shown in Table V111. Upon examination of the results it was found that there is very little difference in the number of organisms retained on the rods after the short and long drying periods. 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It is well recognized that the presence of onganic matter interferes with disinfectant action. Organic matter may 1. react with the disinfectant to form inactive compounds, 2, adsorb some of the disinfectant from solu- tion, 5. neutralize a portion of the disinfectant, or, 4. form a mechanical barrier around the organisms which prevents penetration of the disinfectant. (8). To determine the effect of organic matter upon disinfection, ten per cent horse serum was added to the disinfectant solution. The bacterial count obtained after exposure of the inoculated glass rods to the disinfect— ant containing organic matter was slightly increased as shown in Tables 1X and X. However, it appeared that no appreciable change occurred when organic matter was present at that concentration. In previous attempts to determine the effect of organic matter upon disinfection serum was added to the bacterial culture and the glass rods inoculated with this mixture. When these rods came in contact with the disinfectant the organic matter ”isluffed off carrying the bacterial cells with it into the disinfectant solution. The possibility that the organic matter of the broth in which the test organisms were being grown might act as a protective agent against the disinfectant was questionable. However, to insure accurate results in later studies, broth cultures of the two test organisms were centrifuged and washed several times with sterile physiological saline solution to remove the organic matter. When the washed organisms were dried upon the glass nods and exposed to disinfectant action the results obtained varied little from the results obtained using a broth culture. This is shown in Tables 11 and x11. -13- It may be that the amount of organic matter adhering to the glass rods with the bacterial cells is very small and would therefore be unable to exert an observable degree of protective action. The cells themselves may act as a protective agent. The bacterial cells when dried on the rods are not in a single layer upon the glass surface but form a film several layers thick. The outer layer of cells would form a shield which could protect the inner layers if the disinfectant were not powerful enough to penetrate the barrier and attack all the bacteria. Experiment size To determinethe necessity of using neutralizing ggents Lg counteract bacteriostasfs. ‘—*_~ Many classes of disinfectants have marked bacteriostaic power. This is so marked in the mercurials that even a loopful from the medication! pets to the broth medium in the F. D. A. procedure is sufficient to inhibit growth. With such compounds it has been customary to use the Shippenmlod‘ ification to dilute the compound beyond its bacteriostatic titre or to add some neutralizing agent to the medium to eliminate bacteriostatic activity. It was necessary to test the use-dilution technic to determine whether or not bacteriostatic activity occurred in the test. It is conceivable that sufficient disinfectant might be carried over to the inoculating medium on the rod even though a water rinse were used between the medication pot and the inoculating medium. Two methods of neutralizing the bacteriostatic effect were tried: the use of agar and broth culture media containing the neutralizing agent in the case of mercurials and the addition of the neutralizing compoind to the rinse material when testing cationic disin- fectants. 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Hospeoo o o o o mason em oopsnnoom 0 0 0 0 aHeeeHeeeea empeHa messeHs 0n 0 o o 0 mason em coponsoeH 0 0 0 0 aHeseHeeEEH eessHa messeHe 0H 0 o enanH ssnnH meson 0w emmeQOnH 0 0 0e 0eH aHepeHeeeaH eeeeHm ssoeH ssqu semen ssnnH espeeHz m mason em UmpmpsosH eH we 00H emu aHeseHeeeeH eeeeHm . epseHa H unease .naspm enamoaxm mo osHa smonahv .m mHHmo venues *.esspHse HeHsepeen sweep e use mopeds oHssmeo scam mean mHHoo Hmwheposn some pqopoomnHch a mo poommo shapeesdsoo may HHN manna -25- media when testing mercuric compounds. The active sulhydryl group of the sodium thioglycollate incorporated in these media removes the bacteriostatic power of the metallic compound, thus permitting the growth of the organisms which had been suSpended by bacteriostasis. \ tgeTo counteract the bacteriostatic effect of cationic compounds 0.1% {g . x .- . . J.» }; LV‘ wsoap solution was added to the rinse material. In evaluating the results shown in Tables Xlll and XiV'it was found that complete bacteriostasis did not occur because some bacterial growth appeared when no neutralizing agent was used. The bacterial counts were lower under those circumstances and occurred only after a short period of exposure to the disinfectant. It is apparent that some bacteriostasis occurred and was sufficient to inhibit the small number of viable organisms present after being eXposed to the disinfectant for five minutes and interferes with bacterial growth enough to present a lower count after one minute exposure. Therefore, the use of a neutralizing agent in either the rinse material or the culture media is desirable in testing compound having bacteriostatic power to eliminate a misinterpretation of the disinfecting ability of the compound. Experiment seven- To determine the amount of variation in the number of organisms present on the inoculated glass rods. The accuracy of results obtained when using glass rods as a means for transporting the bathhial culture depends to a great extent upon the main- tenance of a standard number of organisms on the surface of the rods. To determine the variations between the hacterial counts obtained from the inoculated glass rode? the control counts of the tests made were noted and tabulated in Table IV. Controls are run by dropping the-culture-covered rods directly into tubes of broth and plating in suitable amounts. It.is to be expected that there are variations in the bacterial count due to the random sampling but these errors merely account for the variations in results obtained from seemingly identicil tests and do not influence greatly the accuracy of -24- oHnonossssH mHnmnossan 000.0eH 000.0mH 0 0 0 0 0 0 0 0 0 0 0 0 c 0 0 0 0 0 0 0 0 0 0 0 0 0 erasH 0 0 0 meH we awesome .w eschew .nmopm poems mnHaHHsnpsos oz 0 ...-.wd.a. «I .- .mponn use some nH oopmnoauoonH opsHHoohHmownv ssH00m* oHnsnossndH oHnosossqu 000.0eH 000.00H o o o o o 0 0 0 o 0 o o o o o o 0 0 ssenH sanH o 0 00¢ Hm 0 o anmaH EsnnH o 0 eHs nae emondhu .0 opened onompm poems mnHuHHmupsmz mason om novonaomH aHoaeHeesaH eepeHm Hohvnoo meson ea eesepseeH aHepeHeeesH empeHm empeeHz 0m muses om popopsoaH aHeeeHeeeeH eeeeHs movanz 0H mason «N covenaonH aHepeHeeesH empeHm eepeeH: m sheen em steepnesH aHeseHeeesH eeeeHm opan: H .onsmonnm mo osHa * .00mH. H eeesst Haeeeases eeHHesesee oe ope -HoohHonnu ssHpom mnHms HHHM HsHempodn no psomo meHeHHmepson o no poommo one HHHN oHnma -25- .HoHnoums omoHs op copes oOHvsHom deems oHnmnmsnan oHnwaossnsH oHnonossmsH oHnonoasmnH meson om novonsoaH 000.00H 000.0eH 000.00H 000.00H aHeseHeeeeH esseHm Hespeeo 0 0 0 0 0 0 o 0 mason ow poponmonH 0 0 0 0 0 0 0 0 . aHepeHeeeeH eeeeHa eeeseHz 0m 0 o 0 0 o 0 0 o meson em ooaonsoeH 0 0 0 0 0 0 0 0 aHeeeHeeseH eeeeHm 000.H—fig OH 0 0 0 0 0 0 aseeH aseeH sheen em steepeeeH 0 0 0 0 0 0 mmH new aHepeeeeeeH empeHa eeseeHa m 0 0 0 0 0 0 asesH asssH meson an emsensosH 0 0 0 emH 0 0 00mH 000H aHepeHeeasH empeHm . ease-H: H omonmhe .m unease .naepm omonmhv .m eschew .Aaopm mnemoaxm no osHa enema quaHHoepSon oz poems meHuHHonvsoz $.00mNuH Hoaosonm onHHmnpso: o» :OHpsHom doom RH.0 maammsHHHM HsHsopomn no snows mnHuHHonpson o no vacuum one _hHN oHnsa -25- Table XV number of viable organisms obtained from the surface of inoculated glass rods. Staph. aureus E. typhosa 110,000 180,000 120,000 170,000 120,000 180,000 150,000 170,000 120,000 180,000 140,000 170,000 150,000 180,000 140,000 180,000 150,000 180,000 140,000 180,000 150,000 180,000 150,000 180,000 160,000 180,000 150,000 180,000 160,000 190,000 150,000 180,000 160,000 190,000 150,000 180,000 160,000 190,000 160,000 180,000 160,000 190,000 160,000 100,000 170,000 190,000 160,000 180,000 170,000 190,000 160,000 190,000 170,000 190,000 160,000 190,000 170,000 190,000 160,000 190,000 170,000 190,000 170,000 190,000 170,000 190,000 170,000 190,000 170,000 190,000 170,000 190,000 170,000 190,000 170,000 190,000 170,000 200,000 170,000 200,000 180,000 200,000 170,000 200,000 180,000 200,000 170,000 200,000 Average _-_-_ 177,000 Mean :_ 170,000 Average ; 170,000 Mean 2 170,000 -26— of the data from the majority of the tests. Experiment eight- The effect of phenolgresigtance variation of the teat ogganisms on the results obtained_in testing disinfectants. The test organisms M. _a_._u_1_‘§_1_1_s_ and E. M are checked frequently to determine any variations from the pattenn of resistance set for them in the phenol coefficient. In this manner a standard culture may be maintained for use. To determine the effect a slight variation of the phenol resistance of a test organism might have on the results obtained in testing disinfectants, slightly irregular cultures were used in parallel tests with standard cultures. In Table XVI a standard fitgph.‘gggggg,culture and one which is more resistant (phenol kills in five minutes but not in ten at a difuuxdnészlz60) are used in testing tincture of metaphen. The results obtained are comparable. A less resistant E. typhosa culture (1:100 dilution of phenol kills in five minutes but not in ten) and a standard culture present little variation in results when testing merthiolate 121000 as shown in Table XVll. It is evi- dent that these findings present a great advantage. If a bacterial culture which does not strictly adher to the phenol resistance set for it can be used for testing disinfectants'with results comparable to those obtained when using cultures which comply to the standard phenol coefficient, the necessity of eliminating slightly irregular cultures from use is no longer present. It is recognized, however, that a great degree of variation from the phenol resistance is indicative of dissociation of the organism and such cultures must be excluded from use. -27.. 00~.H stresses he oseeesHa veneeeeeeHeHe sees * oHnseossonH.r oHnsAossnoH 000.00H 000.00H o 0 0 o o o o 0 o o o o o o o o esonH ,esmoH aseoH ssooH as H» 0 . be asooH esnoH esooH sonnH . 0mm 00 mNH nHm unease .mospm nsHsmonnH unease .nospm cementum asses em empeneeeH aHoeeHeerH emeeHm Hesseeo 983 S. 03333 aHeseHeeeeH eepeHm e303.- 0... ether «0 seseeseeH aHepeHeeaeH empeHm eeeeeHa 0H eases em easehreeH aHepeHeeEEH tepeHm heeeeHa m asses em eeeepseeH aHeeeHeeeeH eeeeHm 3§§H onsmooxm no osHa *.pmoHonmooo Homage canvases on» seem moHemb noHns.s0Hosmno veep 0 mons some cooHseno oaHsooa mo somansosoo 4 H>M oana 000H.H oeeHeHessez teeeeeeaeHeHe seen * OHDsemssmoH 000.0eH ssnsH assoH « 00 asooH esooH em as ssan asqu as 00 asooH asouH 00H enH enough» .m eeHsmoemH oHnsuoesooH oooaosH ssooH ssqu 0 b snooH aaooH mm mH sonnH ssooH mm 00 ssnoH ssnnH ¢HH NmH steamer .0 eeeeeeem eases em eepeeaeeH aHeseHeeaaH eeeeHm Hosanoo eases em eepeneoeH aHeaeHeeaeH eeeeHm these“: 0» eases e0 eesenseeH .aHeeeHeeeEH eteeHm oopnnHz 0H teach an eeeenseeH aHepeHeeeaH emseHm 000.503”: m ethos em emphasesH aHepeHeeaeH eeaeHm opan: H oasmooxm mo osHa _ *.ooomponon Hosonn unopnsvm one seem moHesb AOHnw amflosmno some a mons some pooHsano mvHsmoh mo neanomaoo 4 HHbN oHpsa -29- . The preceding studies were made to determine a simple efficient method of testing disinfectants._ From an evaluation of these studies the following procedure is suggested: APPARATUS A water bath for the maintenance of constant temperature is used. A lid should be provided with holes of a suitable diameter to hold the medication pots upright in the bath. The medication pots are glass tubes one inch in diameter and three inches in length. Solid glass cylinders one-fourth inch in diameter and one inch in length are pro- vided with a loop at one endfyxAn ordinary wire transfer needle bent at the end is used for manipulating the glass rods. Sterile physiological saline blanks for rinsing the rods and sterile distilled water for making dilutions should be provided in 90cc and 99 cc. volumes..Petri dishes of ordinary size are used. Those used for drying the glass rods should be prOVided with sterile filter paper on the floor of the dish. All pipettes should be graduated and sterilized. Subculture tubeslmrr be of any convenient size. It is preferable to have test tubes of 20 cc. volumes so that the cylinders will be completely immersed in the broth. 0mm ’ The test organisms are 22 to 26 your cultures of g, typhosa and §3§p§.‘ggggg§, of strains designated in the Food and Drug Administration Circular 198 (12), incubated at 37°C. and grown in nutrient broth. Difco disinfectant test medium is used for culturing Staph. aureusand‘§,typhosa is grown in the medium described in the F. D. A. Circular 198 (12). The broth cultures are transferred daily and fresh transfers are made monthly from the stock agar slant culture. The resistance of the organisms to phenol is determined at weekly intervals to ascertain the maintenance of cultures which comply with the standards set for them in the F. D. A. Circular 198(12). PROCEDURE 24 hour broth cultures of Spgph. 323232 and g. typhosa are shaken for fifteen minutes to eliminate bacterial clumps and insure even dispersal of the organisms throughout the medium. The sterile glass rods are submerged in the culture and then placed on sterile filter paper in a covered Petri dish and allowed to dry for thiry'mine utes. Desired dilutions of the disinfectant are made and ten cc. amounts placed in each of the glass seeding tubes. The tubes are placed in.a 20°C. water bath and allowed to come to the temperature of the bath. Since the tests are run in duplicate, two tubes of the disinfectant are needed for each test organism. Controls are run by placing one of the glass rods which has been inoculated directlyrinto a tube of broth. Four of the bacteria—covered glass cylinders are placed in each of the tubes of disinfectant and removed at intervals of one, five, ten and thirty minutes. The cylinders after rimoval from the disinfectant are axes» immersed in sterile physiological saline solution and then placed in 10 cc. «nebuhsr' Mmtéa‘ciumi’ finder of nutrient broth. thbes are shaken vigorously to rgmove the organisms from the glass rods and the broth is plated in one cc. amounts. The con- troltubes are diluted 1:10 and 1:100 before plating to insure an accurate count. The broth tubes and the agar plates are incubated at 37°C. for 24 hours. Any tubes not showing growth in 24 hours are plated. In this manner it is possible to discern growth at time intervals which show no colony formation on the plates made immediately after exposure. -51- PRESENT TESTS ON CHEMICAL DISINFECTANTS Varying Dilutions The practicality of the method developed in this thesis can only be determined by actual use in testing disinfectants. To determine the most effective concentration of a disinfectant varying dilutions of the disinfectant were tested using the procedure Just described. No attempt is made to determine the phenol coefficient of the disinfectant; the exe _periments are done simply to determine the dilution of the disinfectant which produces the greatest bacterial kill in the shortest period of time. One of the most commonly used coal-tar disinfectants, lysol, has a phenol coefficient of five and is designated for use at a dilution of 1:520. From the results shown in Tables XVlll, XVlV and XI it can be demonstrated that even at a dilution of 1:500 lysol fails to prevent the growth of Staph. Eggggg’after 50 minutes exposure. §,typhpsa, a less resistant organism, is killed after ten minutes exposure at this dilution. At a dilution of 1:50 lysol produces immediate bacterial kill and could be used as a disinfectant effectively at this concentration provided that no harmful results to the skin or surfaces being disinfected occurred. \‘The use of phenol as a disinfectant has been.generally discontinued because of its low killing power and toxicity. It has, however, been maintained as a standard for the comparison of disinfectants. The results obtained from testing varying dilutions of phenol are shown in Tables :11 and 1111. Phenol is most commonly used for disinfection at a dilution of 1:20. From the results obtained employing the use-dilution technic it is evident that a 1:50 dilution of phenol produces bacterial kill in less than one minute and could be used as effectively as a 1:20 dilution. -sz- Phemerol, a representative cationic disinfectant, produces immediate bacterial kill at its use-dilution, 1:1000. To determine whether higher dilutions might prove as effective, varying dilutions were tested by the use—dilution technic. After evaluating the results shown in Tables X1111 and I!!! one could conclude that at a dilutions of 1:2500 phemerol could be used for disinfection if the exposure time were extended to ten minutes. manmhmssnnH manseoassnu oooeowa oooeowa o o o o o o o o o o o o o o o o "w o o sedan ssan . o o mam as» o o esnnH InnnH o o 000H 0mm smonnha .m nachos .amspm .. oo»~.a .Hoemssnm mo esoaasawu cansnossnaH ooo.omH uncanny .m J oooH.H cansuosnnnH ooo.o~a meson em nonessenn aflopoaeoeefl oepofim Hosanoo oases em ooeoneodH aaoeoaeoaea ooeoflm season: on meson em popsnuomH aaopeaoeeee sevens nouns“: OH O o o meson em oesopsonH afloeeaeoeaa seeded mousse: m o o oases em ooeehsodH so afloeeaooeea seeded oases: H enamodxm mo made msohnm .nasam msowam>_ov poflamms cannon» noausafipoomn one HHHMN manna cansuosnnsH ooo.omH o o o o ssnuH asnnH 0 av assuH ssnnH one 00> ssnnH sonsH 000H coma mmonmhu .u eanseossnsH 80.8w ssan assaH cows ooom ssnnH ssnuH cows come sunnH abnnH oooo coho sessH assaH comm . ooo.HH 30.5.8 .933 combua mansnossnaH oooeomH ssnnH ssqu Hum mba ssan sunaH cow owe uncanny em ooomua caneeossnsn ooo.oma sauna sedan o as asssH sauna ome sen asssH ssnsH oom coma asssH ssnsH. ooom oomu unease .Amsaw meson em eoeehsonH . aaoeeeooeee sevens ‘ Honesoo meson em ooeoheosH eflosoaeoeea sevens dosage: o» ethos em ooeoneonH haopoeeoeee sevens houses: on meson em oopopeodu aaouoaeoeea oopoam ooesdsa m mason «N wovmnnonn haopdwcossu wmpmHm . manna: H ohnuomsm mo ends .Houosmnm mo escapades ascend» op coaaans cannon» nowvsflwpuoms may pHNN sandy -55- canspmssnnH oooeomH assmH sunsH o o asssH ssnnH own cum asqu asan ooom OOmH ssssH ssnsH come coon uncanny .m sansnoasan ooo.oma Essen asnsH one owe saan sansH coma one ssan sansH comm come eeedH asqu ooom coma unease enemy m canseossnsH ooo.omH ssnnH ssnnH o o asan .ssnsH o o ssnsH snacH Hm me esssH anssH mam mam decades .m sanenmssnsH 000.0ma ssan sonaH mm «a asan asan paw own sansH snasH coma owm ssnaH aunnw ooom comm mashed .seepm eases e~.ooeeoeedH _haovswuossa consam Hogwaoo 933 ea eopehsofl aaoeeeooeen seesaw . mopeds: em eases «a ooeoseosn aaoeoaoosaa ooaeam ooeoeea_ofl mason em coaspsosH hampeaeoeae seesaw @0023”: m eases em ooeensosH anoeeeooeea oopeam 32:: H enamonxm we mean .HOuhq up uncensawp ascend» op voaamms canned» sowusawulom: one xN manna -40- The Use—dilution It is interesting to know the most effective dilution of a disinfectant but it is the determination of the efficiency of the use-dilution which is most important to the consumer. Commercial disinfectants are used by the laity and professional people for all types of disinfection. It is imperative, therefore, that the dis- infectant at the dilution suggested by the manufacturer for use comply with the claims made for it. The use-dilution technic is a method devised to test the disinfectant power of a disinfectant at its use-dilution. Bards-Parker solution is a chemical disinfectant used at full strength for cold sterilization of surgical instruments. From the results obtained from frequent testing of this solution it was found that bacterial kill is effected in less than one minute, as shown in Table XXV. The phenol coefficient of this compound is relatively low indicating that it is a poor disinfectant yet from the results obtained by the use-dilution technic it could be concluded that Bards-Parker solution at its use dilution is an effective disinfectant. Complex compounds of mercury have been given much attention in past years as excellent disinfectants. Their efficiency has been overrated, for although they may produce bacterial kill at low dil- utions they are not bacteribcidal at the high dilutions indicated in earlier investigations of these compounds. Three mercurials were tested at their use-dilution using the use-dilution technic. Tincture of meta- phen, an organic mercuric compound, is designated for use at a dilution of 1:200. This concentration fails to kill Staph. aureus ‘after an exposure time of five minutes as shown in Table XXV. Merphenyl nitrate, -41- a double mercuric salt, at its use-dilution of 1:1500 inhibits g. typhosa immediately but Staph. aureus is able to grow after an exposure of five minutes to this disinfectant. The results are shown in Table m1. Both _E_. tmhosa and S1392. m are able to V resist the bacteri cidal effect of merthiolate at its use-dilution of 1:1000 after 50 minutes exposure to the disinfectant as shown in Table XXVll. A disinfectant which is not able to produce bacterial kill after such an extended period of exposure would be completely ineffective for all practical disifection purposes. Tincture of iodine, employed at a use-dilution of 5%, is cap~ able of killing bacteria in less than one minute. This corresponds to the results obtained by Nungester when using Tincture of iodine to disinfect the skin of a mouse (9). Table XXVl shows the results obtained when testing tincture of iodine by the use-dilution technic. The use-dilution technic was applied in testing hexylresorcinol (8.815?) at its use-dilution 1:1000. As the results shown in Table XXVll indicate, no germicidal effect was evident even after thirty minutes exposure of the organisms to the disinfectant. Dowicide A (orthophenyl phenol) use-dilution 1:100, and Bow:- icide C, use-dilution 1:1000, were tested to determine germicidal ‘* efficiency. Both compounds at their use-dilution were able to effect kill of E, typhosa in less than one minute and Staph. aureus in ten minutes. It is evident from an evaluation of the results shown in Table XXVlll that these compounds would be satisfactory disinfectants if an exposure time of ten minutes were used. a lower dilution which would produce bacterial kill of Staph. aureus upon immediate contact would, however, be preferable. -42- It is evident from the results obtained by the use-dilution technic that disinfectants may be classified as either satisfactory or unsatisfactory for disinfection purposes. The mercurial compounds and hexylresorcinol (S,T. 57 ) were found to be unsatisfactory dis- infectants. Tincture of iodine, phemerol, Bards-Parker solution and the dowicides as! satisfactory for disinfection at their use-dilution. Phenol and lysol are effective as disinfectants only if used at low dilutions. However, lysol, at its use-dilution, would be classed as an unsatisfactory disinfectant. -45— oanmnosssmH mandamanmsH 000.00H 000.05H 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 esssH asan 0 0 0 m 0 0 ssmnH sssmH 0 0 mmw Nb smonahu .m assess .nampm assesses mo assesses canmnmsanH 000.00H mmonmhv .m samenesssmH 000.com mnmnsm .nmmpm Anumaoapm Hasmv eoxasmuoeasm oases em oopspsoeH aHseeHeoeea eoeeHa Honpsoo meson ow oopensoeH aHseoHeoesH oopsHm oneness on mason em coasnsomH aHoesHeoaee eseeHa moaneHa 0H oases em oceansosH aHoeoHooesa eoeeHm oopssHa m mason «N oopmnsosH aHosoHooeeH ooeoHa 3:st H oesmooxm mo made monomers mo composes was soapsaom nexusmaopesm op pefiaams caboose soaasafieuoms one >NN oHnss \ -46- mansuossssH sansnossssH samenessnsH canssossnsH mason em pmesnsosH coo.OeH ooo.oaH ooo.omH ooo.omH aHossHeoesH ooeeHa Hoavsoo o o o o o o o o eases em oceansosH o o o o o o o o eaHoesHooseH eoeeHa noessHs on o o o o o o o o cases on eceoesosH o o 0 Ha o o o o eaHoeeHooseH oopsHa noessHa oH o o sneeH esssH o o o seasH eases am onesesoeH o o as rHe o o 0 Ha .aHoesHeoeeH oopsHa scenes: m 0 0 esssH asssH 0 0 esssH susmH mason «N moumnsosH 0 me How 000H 0 0 so eaH aHoeeHeoeea noesHm oesaHa H anemone .m assess .nmmpm shamans .m assess .nmspm enamomxo mo mafia 000HnH o oeHoason oOHnH a ooHoHaon .o oeeoHsom one a ooHoHeon ea ooHHaoo oHseooe eoHesHHocoos one HHHeax oHeoa -47... SUMMARY 1. A method was developed as a means -of determining the killing powers of a disinfectant at its use-dilution. 2. This use-dilution technic was also found to be effective in evaluating various dilutions of the disinfectant and determining the most.effective concentration. 5. The use-dilution technic is found to be more simple than the phenol coefficient method and is more practical because it evaluates the disinfectant under conditions comparable to actual use. 4. The use-dilution technic makes it possible to divide disinfectants into two categories: satisfactory and unsatisfactory. BIBLIOGRAPHI 1. BREWER, C. M., and REDDISH, GEORGE F.: A Comparison of the Hygienic Laboratory Test With the Method Used by the Department of Agriculture for Testing Disinfectants., Jour.Bacteriol.,17,44, 1929. 2. CHICK, HARRIETTE, and MARTIN, C. J.: The Principles Involved in the Standardization of Disinfectants and the Influence of Ofi.ganic latter Upon the Germicidal Value, Jour. Hyg., 8, 654, 1908. 5. HILL, HIBBERT WINSLOW: A Method of Preparing Test Objects for Disinfect- ion Experiments, Am. Pub. Health Assn., Papers and Reports, 24, 246, 1898. 4. HYHA, ANDREW M.: To Devise a Method of Expressing the Bactericidal Efficiency of a Disinfectant other than the Standard F.D.A. Phenol Coeff- icient Method, M.S. Thesis, Michigan State College, 1940. 5. JENSEN, VILH., and JENSEN, ELSA: Determination of the Phenol Coefficient of Disinfectants by the Cover-slip Method, Jour. Hyg., 55, 485, 1955. 6. KOCH, ROBERT: Ueber Disinfektion, Mitt.a. d. kais. Gesundheit., Vol. 1, 1881, after H. Chick, Jour. Hyg., 8, 92, 1908. 7. KRONIG, B., and PAUL, T. L,: Die Chemischen Grundlagen der Lehre von dre Giftwirkung und Disinfektion, Ztschr. f. Hyg. und Infect., 25, 1, 1897. 8.'I:CULLOCH, ERNEST C., Disinfection and Sterilization, 2nd. Ed. Phila- delphia, 1945. 9. NUNGESTER, W} J. and KEMP, ALICE.) An "Infection Prevention" Test for Evaluating Skin Disinfectants. Jour. Inf. Dis. 71: 174, 1942. 10. REDDISH, G. F.: Examination of Disinfectants, An. Jour. Pub. Health, 17, 520, 1927. 11. RIDEAL; 9., and WALKER, J.T.A.; The Standardization of Disinfectants, Jour. Roy. Sanitary Inst., 24, 424, 1905. 12. RUBHLE, G. L. A., and BREWER, C. M.: United Stttes Food and Drug Administration Methods of Testing Antiseptics and Disinfectants, United States Dept. Agr. Ciro. No. 198, 1951. 15. SHIPPEN, L. P.: A Fallacy in the Standard Methods of Examining Disin- fectants, Am. Jour. Pub. Health, 18, 1251, 1928. 14. UNITED ITATES PUBLIC HEALTH SERVICE: Disinfectant Testing by the Hygienic Laboratory Method, Pub. Health aep., 56, 1559, 1921. .: ‘,-_J in _--.—.- w‘_o._ A—AM‘LA MICH I AN STATE UNIVERSITY LIB llIlllll III! Ill” 0 3 1293 RARIES 3014 8406