Testing Cationic Surface Active Germicides for Use In Industrial Water Systems CAROL HOUCK BOLLENBACK and GEORGE A. CRUICKSHANK National Aluminate Corp., Chicago,
111,
T,
HE inoreased use of and interest in surface aotive germioides
point aut the need for a reliable test method for determining the killing levels of such agents. Frequently the tube methods of testing the billing power of this type of compound give poorly reproducible results. Lack of reproducibility may be attributed t o sampling a t the end of the contact period and lack of suitable inhibitors t o inactivate the germicide a t the end of the contact period. The appearance of “skips,” whereby growth occurs at fairly high concentrations with no growth at intermediate levels, s e e m t o be due t o the methods of transferring the test solution t o a culture medium a t the end of the contact period. Various workers have attributed erratic results t o adherence of organisms to solid surfaces, to improper sampling, and to “carry-over’’ of the germioide. Many methods, modifications, and criticisms of established techniques for evaluating the germicidal action of quaternary ammonium compounds are to be found in the literature. For some time laboratory worlters have observed that the agar plate inhibition test is not suitable far the evaluation of surface aotive materials: agar has “neutralizing” action of its own. This has been oonfirmed by work in this laboratory. At low concentrations surface active germicides, in contact for 1hour with Aerobacter aerogenes, can be completely neutrahaed by the addition of standard tryptone glucose extract agar. To neutralize the surface active germioides a t higher levels, it is necessary to add an inhibitor. The use of inhibitors (or neutralizing agents) in the culture medium is a well established procedure in testing quaternary-type germicides. Weber and Black ( 4 )have investigated several inhibitors. The purpose of the inhibitor is to prevent the carry-over of bacteriostatic quantities of the germicide into the suboulture medium and t o neutralize m y unreacted quaternary on the bacterial cell, thereby definitely stopping any further germicidal action after the specified period of contact. The proposed method attempts t o overcome these difficulties by culturing the entire reaction mixture of chemical and organisms in the test container at the end of the contact period and inactivating the germicide by means of an efficient inhibitor contained in the culture medium. The method has bcen used primarily for the direct comparison
of surface active germicides intended for use in industrial water system where the volume of water to be treated may run into thousands and even millions of gallons. Thus to be economically practical, the dosage levels must be extremely low. Complete sterilization of such systems would be exceedingly difficult and from a practical viewpoint this is unnecessary. Hence, an arhitrary figure of 99.99% kill has been adopted t o represent a satisfactory killing action. Thore is no urgent necessity for rapid killing action in these systems; hence a contact period of 1 hour has been adopted. This constitutes a convenient working interval, eliminates the emor caused by small variations in timing sometimes found when 5- or 10-minute test periods me used, and brings out the maximum effectiveness of the chemical under the conditions of testing It is hoped that adaptation of the method to suitable conditions of time, culture, and other factors will make it useful and convenient in a variety of applications. PROCEDURE
In principle, the test comprises the preparation of serial dilutions of the chemical in a. series of Petri plates, addition of the test organisms, and, after the desired period of contact a t room temperature, flooding the plate with agar containing an excess of neutralieing agent. The plates are incubated for a suitable period and the colonies of surviving bacteria .we counted. The percentage kill is determined by eqlculating against the number of bacteria in the original inoculum as determined by the standard plate count method.
Figure 1. Preparation of Serial Dilutions i n Petri Plates
1355
ANALYTICAL CHEMISTRY
1356 The plunger of the pipet is held in the compressed position while the pipet is transferred to the preceding plate where the contents are mixed in a similar manner. Proceeding in this manner, the contents of all plates are mixed in order. Chemical concentrations in the plates in the amount of 2 ml. per plate cover the range of 250, 125, 62.5 p.p.m., etc. A control plate, containing 1 ml. of water and 1 ml. of culture is included in each series. The plates are now placed flat on the laboratory table and allowed to stand for 1 hour from the time the inoculum was placed in the first plate. Neutralization of Chemical. At the end of this period of contact between the bacteria and the germicide, the activity of the chemical is destroyed by adding a t least 10 ml. of a special neutralizing agar of the following composition: 21 grams of Difco tryptone glucose extract agar; 5 grams of Daxad 11 (polymerized sodium salts of short-chain alkyl naphthalenesulfonic acid, Dewey and Almy Chemical Co., Cambridge, Mass. ); and 1000 ml. of distilled water. The mixture of the germicide and bacteria in the plate is evenly distributed throughout the melted agar by rotating the contents of the plate so that any barteria that may have survived the contact with the chemical will be distributed uniformly throughout the agar. When the agar has solidified, the plates are inverted and incubated for 48 hours a t 37” C., after which time the plates are counted. A count of 100 colonies per plate has been arbitrarily selected as the dividing line between effective and ineffective killing action. This number of survivors represents a 99.99% kill, as there were approximately 1,000,000 organisms in the test plates. Determination of Efficiency of Neutralizing Agar. After an extensive survey of inhibitors for several types of cationic surface active germicides, it was found that 0.5% Daxad 11 in standard tryptone glucose extract agar was entirely satisfactory for neutralization of the germicide a t a concentration of 500 p.p.m. The medium is clear, homogeneous, and noninhibitory to Aerobacter aerogenes. The other inhibitors tested were less desirable for one or more of the following reasons: less effective neutralization, discoloration of the medium, and voluminous precipitates which interfered with colony counts. This is not the first use of sulfonic acid derivatives as neutralizing agents for surface active germicides. Lawrence (a),in studying a series of these compounds, recommended the use of Suramin Sodium, trisodium salt of 3,3’-ureidobis [&(3-ben~amido-p-toluido)-1,3~5-naphthalenetrisulfonic acid], for this purpose. One milliliter of the proper concentration of PROCEDURE. germicide-Le., 10 times the final concentration desired in the agar-is mixed thoroughly with 8 ml. of melted neutralizing agar in a Petri plate. After sufficient mixing, 1 ml. of standard inoculum of Aerobacter aerogenes (approximately 1,000,000 organisms) is added, and mixed again, and the agar allowed to solidify. Plates are incubated for 48 hours a t 37” C. and their appearance is compared with that of a control plate (water or saline) prepared in a similar manner. I n other tests the inoculum was varied to contain approvimately 1,000,000, 100,000,
T a b l e I.
Efficiency of Xeutralizing Agara on ,500 P.P.M. of Surface Active Germicides
Colonies of A . aeravenes DeT-elopine on Neutralizing .4gar Dilution of Inoculum Germicide 1: 1000 1:lOO 1 : 1 6 rndilrited 1220) QAC 1 QAC 2 1252 I 1146 Q.4C 3 11161 QAC 4 Gross appearance of plates identical with 1240 i $.IC 5 corresponding dilutions in control plates 1136 QAC 6 Amine salt 1 1104 926 Amine salt 2 Control (no germicide) 1276 Standard plate count of inoculum 1,240,000/ml. a Neutralizing agar. 0.5% Daxad 11 in standard tryptone glucose extract agar. ~~
1
T a b l e 11. Variations in Killing Levels of Q u a t e r n a r y Determined by Two M e t h o d s Tube Dilution Method 100% killing range, p.p.m. 1 > 2 1 2-4 2 4-8 1 8-16 2 16-31 1 31-62 2 >loo0
KO.of tests
Plate Method 99.99% killing range, p.p.m.
No. of tests 1 6
2
4-8 8-16 16-31
10,000, and 1000 organisms, respectively. I n the plates inoculated with 1000 organisms, direct comparisons were made with the controls by actual counts of the colonies. RESULTS AND DISCUSSION
The efficiency of the neutralizing agar (Daxad-tryptone glucose extract agar) against several quaternaries and two other surface active germicides is shown in Table I. The agar does not interfere with the groath of the test organism and appears to neutralize completely the action of 500 p.p.m. of the quaternary ammonium germicides, but is possibly lees effective at that concentration of the amine salts. Table I1 shows an extreme example of the variation in results encountered when the killing level was determined by a tube method (modified FD.4 phenol coefficient type) using a 1-hour contact period. A comparison of these results with those obtained using the proposed plate method killing test shows a distinct improvement in the degree of reproducibility.
Table 111. Replicate Killing Ranges (99.99%) of Cationic Surface Active Germicides QSC 1 No. of tests P.p.m. 1 1-2 3 2-4 2 4-8
QAC 2 No. of tests P.p.m. 4 1-2 2-4
1 1
4-8 8-16
2
QAC 5
Q.\C 6
QAC 3 No. of tests P.p.m. 1 4-8 5 8-16 1 16-31
QAC 4 No. of tests P.p.m. 3 1-2 1 2-4 1 4-8
Amine Salt 2 No. of tests P.p.m. 3 16-31 3 4-8 1 2-4 3 31-62 1 8-16 5 4-8 rlgar plate method killing test conditions. Serial dilution of chemical: organism, A . aerogenes; contact time, 1 hour; killing range upper level denotes >99.9970 kill, loner level
