A New Method for Determining the Value of Disinfectants - Industrial

Ind. Eng. Chem. , 1914, 6 (3), pp 198–200 ... Publication Date: March 1914 .... Chemical Society, CAS, and ACS Publications in Liverpool from August...
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T H E JOURNAL OF INDUSTRIAL A N D ENGINEERING CHEMISTRY

inert gases or a n y excess air present to the ignition temperature of the mixture. Having given the heat of the reaction a n d t h e ignition temperature, the calculation of t h e lower limits of combustible a n d explosive mixtures of gas is comparatively easy. These theoretical calculations, if based on the temperature a t which a reaction will take place, give results for the amounts of combustible gas required lower t h a n t h e results f o u n d b y experimental means, as the experimental results necessarily include enough additional combustible gas t o overcome t h e effect of radiation a n d conduction losses. T h e values by theoretical calculations while lower t h a n actual values are useful in t h a t they show t h e potentially explosive properties of a mixture a n d a n y potentially explosive gas is t o be regarded as dangerous. Assuming ignition temperatures of combustion of 600 a n d 7 5 0 ’ a n d ignition temperatures of explosion of 7 0 0 a n d 8 j o o for mixtures of hydrogen in air a n d of methane in air a n d determining t h e amount of each required t o satisfy t h e thermal requirements of t h e reaction for these temperatures, a n d assuming combustion as occurring a t constant pressure a n d explosion as occurring a t constant volume, t h e theoretical lower limit of a combustible mixture of hydrogen in air is 6.8 per cent a n d of methane in air is 2.8 per cent a n d the theoretical lower limit of a n explosive mixture of hydrogen in air is j . 9 a n d of methane in air is 2.4 per cent. I n a succeeding paper entitled “Partial a n d Intermittent Combustion of Gas,” the writer gives t h e results of some experiments undertaken in order t o obtain further experimental data upon these limits a n d also t o t r y t o harmonize the different experimental values found in t h e literature, which for air a n d hydrogen range from j t o I O per cent a n d for air a n d methane range from 3.2 t o 6 per cent. T h e writer desires t o express appreciation a n d indebtedness t o Dr. W. E . Henderson of the Department of Physical Chemistry for advice a n d sugiestions. DEPARTMENT OF METALLURGY

OHIOSTATE UNIVERSITY COLUMBWS

A NEW METHOD FOR DETERMINING THE VALUE OF DISINFECTANTS By C. A. DUYSERAND W. K. LEWIS Received Dec. 26, 1913

Since the chief function of a disinfectant is to kill bacteria or other micro-organic growth, its commercial value may be measured in terms of either of two quantities--first, the time required for a disinfectant of definite dilution t o destroy a predetermined bacterial culture; second, t h a t certain dilution necessary t o kill t h e bacteria of this culture in a definite interval of time. B u t bacteria are living organisms having more or less a n individuality. Not only are there many different strains or types of each organism, b u t the same culture of bacteria may differ in many of its characteristics from d a y t o day. Hence i t ‘is impossible to employ as a n analytical standard for determining the killing power of disinfectants a n organism which m a y vary in its vitality, or a culture which

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m a y be heterogeneous as t o the vitality of the individual bacteria present. The other alternative is t o agree upon a certain chemical compound of known composition, a n d which may be obtained with ease, as the standard disinfectant, a n d t o measure all other disinfectants in terms of t h e killing power of this standard. Phenol is t h e substance more generally employed for the purpose, a n d t h e ratio of t h e ability of a disinfectant t o kill t h e bacteria of a certain culture t o the ability of phenol t o kill the same bacteria under absolutely t h e identical conditions is called t h e “Phenol-coefficient.” There are a t present three methods for measuring t h e bactericidal value of disinfectants, all using t h e above principles, b u t differing in details of manipulation; these are t h e Rideal-Walker, the Lancet a n d t h e Hygienic Laboratory Methods. Each of these, however, gives unsatisfactory results, not only when carried on by different experimenters in different laboratories, but by the same operators when carrying on his work in duplicate. Blythe’ has called attention t o this fact most forcibly, a n d has made a strong appeal for a more chemical method for testing disinfecting materials. We believe t h a t t h e methods now in use are not sound for the following reasons: T h e mechanism of t h e reaction b y which a disinfectant kills bacteria is not definitely known, but it is generally conceded t h a t t h e concentration of t h e disinfecting solution falls in proportion as the number of living bacteria present is decreased. It is possible t h a t the number of molecules of disinfectant is so great in proportion t o t h e number of bacteria present t h a t the change in t h e concentration of t h e disinfectant as t h e living bacteria disappear is negligible. B u t i t is generally true in carrying out these methods t h a t so large a number of bacteria is used t h a t t h e strength of t h e disinfectant is materially changed as t h e killing of the bacteria proceeds a n d before t h e last, or more hardy individuals are killed, the disinfectant is appreciably exhausted. Misleading results are therefore obtained. Each of the above methods provides for removing what is supposed t o be a perfectly constant volume of the culture from a tube or flask b y means of t h e so-called standard loop. This is a circular loop made b y winding a platinum wire of determined size around a rod having a definite diameter. There are so many physical conditions which apparently would influence t h e volume of liquid such a loop would carry t h a t i t was thought of interest t o determine this volume. A strong solution of iodine of known strength served as the liquid t o be transferred, a n d a dilute solution of thiosulfate was used t o measure the amount of iodine contained in each loopful. A number of loopfuls were withdrawn with great care a n d placed in cold distilled water. T h e weight of iodine in each was then determined by titration, and from this d a t a t h e volume of the loopfuls calculated. When great precautions were used, t h e volume of one loop varied a s much as 30 per cent from the mean of 2 j loopfuls, a n d when hurriedly done the variation rose as high as 80 per cent. T h e volume transferred by the standard 1

Orig. Communications, Intern. Cong. Applied Chem., 1909.

Mar., 1914

T H E J O U R N A L O F I i V D G S T R I A L A rA\ D E N GIN E E RI X G C H E M I S T R Y

loop is on t h e average 0.003 cc. I t m a y be easily seen t h a t if t h e number of living bacteria has been largely reduced b y t h e action of t h e disinfectant it is very possible t o transfer a loopful from t h e disinfected culture t o a new culture t u b e which m a y contain no living bacteria, even though there are a n appreciable number present. B u t even assuming, notwithstanding t h e law of chances, t h a t each loopful contains a number of bacteria proportioned t o t h e volume carried b y t h e loop, since this volume m a y vary b y a t least 3 0 per cent from t h e mean, a n d m a y easily v a r y as much as 80 per cent, concordant results cannot be expected. The use of a tube of sterile broth into which t o transfer t h e loop of disinfected culture t o determine whether

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D lL U T l O N S Parts Water per One Port Disinfectant

or not all bacteria have been killed, is also unsatisfactory. If t h e t u b e should prove fertile there is no means of determining whether t h e inoculation was due t o one stray survivor which happened t o be in t h e loop a n d whose presence was accidental, or t o a considerable number of bacteria which t h e disinfectant had failed t o destroy. T o recapitulate, t h e present methods are unreliable because: (a) The use of a n excessive number of bacteria depletes t h e disinfecting solution before t h e culture is rendered sterile. ( b ) An unknown volume is withdrawn for testing, i n t h a t t h e volume of t h e standard loop is not constant. ( c ) I t is impossible t o determine from t h e broth tube inoculated, hoT3- com-

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plete t h e killing was a t t h e time t h e sample was withdrawn . A method developed in this laboratory which seems t o eliminate these sources of error, a n d which is not more laborious, is, in principle, as follows: The disinfectant t o be analyzed for i t s relative bactericidal power is diluted with water t o three or four definite concentrations, t h e extent of dilution depending upon its strength. Pure synthetic phenol is diluted in a like manner. I n t o a series of these known concentrations of both phenol a n d the disinfectant under consideration is placed a n equal volume of a standard bacteria culture a n d t h e mixture allowed t o remain a n exact number of minutes. At t h e end of this time a n aliquot p a r t of each mixture is plated o u t in Petri dishes on nutrient agar a n d incubated until t h e colonies representing t h e surviving bacteria can be counted. At t h e same time plates of equal dilutions of t h e culture b u t without t h e disinfectant are incubated. T h e ratio of surviving organisms t o t h e number present on t h e undisinfected plates is then plotted against t h e dilution, a n d two curves, one for t h e standard phenol a n d one for t h e disinfectant are obtained. From these t h e relative strength of t h e t w o disinfectants can be read off a t a n y desired point on t h e curves, a n d a coefficient either for total killing or a n y determined percentage of killing m a y be calculated. T h e exact manipulation recommended is as follows: One gram of t h e disinfectant t o be analyzed is weighed accurately a n d diluted with sterile water t o a volume of I O O cc. From this stock solution a series of dilutions is made. Since a n equivalent part of each of these is later t o be mixed with a n equal volume of a dilute water suspension of bacteria, t h e strength of t h e series should be twice t h a t of t h e final dilution desired. When nothing is known of t h e relative strength of t h e disinfectant in hand, dilutions of one part disinfectant in 2 0 0 , 400, a n d 800 parts sterile water have been found advisable F r o m a 5 per cent solution of synthetic phenol a series of dilutions of one part phenol t o 100,1 2 j a n d I jo parts water will give a curve covering a considerable range of killing when B . coli comnzztitior is used. Tubes of sterile broth a n d nutrient agar culture medium are prepared according t o t h e standard methods, a n d a number of I O cc. a n d I cc. pipettes and ordinary test tubes are provided. Ten cc. of each of t h e dilutions of disinfectant a n d phenol are added t o properly marked test tubes a n d placed in a water b a t h a t z o o C. Ten cc. of a broth culture of B . c o l i , so diluted with sterile water t h a t I cc. contains from 20,ooo t o 60,000 bacteria, are placed b y means of a straight graduated pipette i n test tubes, one more t h a n t h e number of disinfectant a n d phenol tubes just described, a n d these are also placed in t h e water b a t h . This is a twenty-four hour broth culture of reaction + I , which has been transformed daily for not less t h a n three days. When t h e whole has come t o a temperature of 20' C. the contents of one of t h e tubes of disinfectant is poured into one of t h e tubes of bacteria a n d well shaken. After one-half minute this procedure is repeated with t h e second t u b e a n d so on through t h e series. .%t t h e end of five

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minutes, I cc. is withdrawn from t h e first t u b e of disinfected bacteria a n d added to a bottle containing 99 cc. of sterile water; one-half minute later t h e second minute intervals tube is so treated a n d t h e others a t in the same way. T o t h e tube of bacteria in which no disinfectant was placed, I O cc. sterile water are added a n d I cc. is transferred t o a bottle containing 99 cc. sterile water. These bottles are well shaken, a n d duplicate Petri dishes are poured from each one, I cc. solution being first added t o the dish followed b y enough nutrient agar t o make a satisfactory culture plate. When t h e dishes set they are inverted a n d allowed t o incubate a t 3 7 . j o C. for 48 hours. At t h e end of this time t h e plates are counted a n d the ratio of t h e number of surviving bacteria t o t h e number of colonies on t h e non-disinfected plate is determined. When plotted against t h e dilution t w o curves are obtained, types of which are shown on the accompanying plate. If a number of disinfectants are examined a t t h e same time, it is of course not necessary t o repeat t h e phenol series a n d t h e bacteria blank with each. T h e coefficient of total killing for Disinfectant A is seqn t o be 2.88, while for B it is 4.76. 4 study of a large number of these plates shows t h a t t h e curves for t h e so-called emulsion disinfectants are almost straight lines; a t most there is b u t a slight curvature. T h u s a small number of points will locate a line with a fair degree of accuracy a n d t h e necessity of using small increments of dilution as obtains in t h e older method is not here present. As is clearly shown in this plot, though desirable, i t is not necessary, as in t h e other methods, t h a t t h e most concentrated solution of t h e disinfectgnt used should produce a sterile tube or plate, t h a t is, show total killing. If a t least three points have been found t o lie fairly well upon a smooth curve, t h e line may safely be interpolated until i t cuts t h e axis representing complete killing a n d t h e coefficient calculated from this intersection. T h e results obtained may be duplicated with sufficient precision t o warrant confidence in them. This is true not only of a single operation carrying on duplicate determinations, b u t b y two analysts working separately. 1 ........................ 2 ........................ 3........................ 4 ........................

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4.82 5.17 6.95 9.64 4.28 2.04

4.58 5.05 6.64 9.30 4.08 2.24

T h e above duplicate results obtained by t h e same analyst will give a n idea of the degree of accuracy which may be readily attained. RESEARCH LABORATORY OF APPLIEDCHEMISTRY MASSACHUSETTS INSTITUTE OF TECHNOLOGY

BOSTON

users of this pigment. Both among rubber manufacturers a n d producers of paints, i t is being found essential t h a t t h e contents of lead oxide a n d lead sulfate be known, so t h a t advantage m a y be fully t a k e n of its characteristic properties. This control necessitates a n analysis of t h e compound in t h e laboratory. I n analyzing sublimed white lead b y t h e usual method, it is found t h a t t h e percentage composition can be determined only b y a n analysis entailing lengthy manipulation, in which t h e content of lead oxide is directly dependent upon t h e accuracy of t h e other determinations, owing t o t h e necessity of estimating its percentage b y a calculation based upon t h e percentage of t h e other constituents present. T h e steps in t h e procedure must therefore be closely watched for slight inaccuracies a t all times. As is well known, t h e average composition of sublimed white lead is given as follows: Lead sulfate.. . . . . . . . . 78.5 Lead o x i d e . . . . . . . . . . . 1 6 . 0 Zinc oxide., , , , , , , , , , , 5 . 5

T h a t its composition varies only slightly from t h e above analysis during a long period of time, is shown by its comparison with a n average of the entire o u t p u t of t h e Picher Lead Company extending over, five months time, a n average embracing 2 7 0 total analyses. This average shows t h e composition t o be: Lead sulfate.. . . . . . . . . 7 6 . 6 8 Lead oxide.. . . . . . . . . . 17.23 Zinc oxide.. . . . . . . . . . . 5 . 7 9 99.70

A slightly higher lead oxide a n d zinc oxide content a n d a correspondingly lower lead sulfate content is found, t h a n in t h e usually stated formula. It shows, however, only slight variation. T h e average total percentage, consisting of lead sulfate, lead oxide, and zinc oxide, was found t o be 99.70 per cent. T h e remaining 0.3 of a per cent is only rarely determined, a n d when actually sought is found t o consist of moisture, occluded gas a n d ash. A definite ratio exists between t h e total lead content a n d the lead sulfate a n d lead oxide contents, a n d advantage m a y be t a k e n of this relation for a rapid a n d accurate determination of t h e lead constituents in sublimed white lead. I n order t o arrive a t t h e short method for t h e analysis which is based upon a direct calculation of the lead a n d zinc contents, i t is necessary t h a t the usual method of analysis be considered. USCAL

THE LEAD CONTENTS IN SUBLIMED WHITE LEAD-A CALCULATION B y JOHN A. SCHAEFFER Received December 1 1 , 1913

T h e composition of sublimed white lead, the basic sulfate of lead, has become a most important factor to

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METHOD ADOPTED

FOR

THE A N A L Y S I S O F SUB-

LIMED WHITE LEAD DETERMINATION O F TOTAL

of t h e sample with beaker. Treat the boil gently for ten hours. Dilute t h e

SULFATE-Mix

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3 grams of sodium carbonate in a mixture with 30 cc. of water a n d

minutes. Allow t o stand for four contents of t h e beaker with hot