September, 1941
I N D U S T R I A L A N D E N G I N E E R I N G CHEMISTRY
tread stocks, this precaution decreases the average deviation from the mean value in any series of experiments. I n the case of extrusion tests, the effect of adsorbed moisture is so great that the use of a standard moisture content, or a t least the determination of moisture content a t the same time as the black is milled for extrusion, is necessary if reproducible results are to be expected.
Acknowledgment We gratefully acknowledge the assistance of B. S. Phillips and other members of Continental Carbon Company’s tech-
1185
nical staff for carrying out various tests in connection with the experiments.
Literature Cited (1) Allen, R. P., and Schoenfeld, F. K., IND. ENG.CHEM.,25, 994 (1933). (2) Amon, F.H., and Estelow, R. K., Ibid., 2 4 , 5 7 9 (1932). (3) Dewey, C. S.,and Lefforge, P. K., Ibid.,24, 1045 (1932). (4) Johnson, C.R., Ibid., 20, 904 (1928). (5) Schoenfeld, F. K., and Allen, R. P., Ibid., 25, 1102 (1933).
PRESENTED before the Division of Rubber Chemistry at the lOlst Meeting of the Amerioan Chemiral Society, St. Louis, Ma.
Effect of Cupric Oxychloride Cement on Microorganisms M. A. FARRELL AND R. T. WOLFF The Pennsylvania State College, State College, Penna.
This study reports the action of cupric oxychloride cement, “Hubbellite”, on the growth of eight mold and eighteen bacterial cultures representing microorganisms associated with various skin infections, such as athlete’s foot, ringworm, etc., as well as bacteria of different chemical compositions. Cupric oxychloride cement brings about a marked reduction in the number of bacterial cells within a 15-minute interval, and the growth of all the mold cultures was completely inhibited within this period. Covering the cement with dirt or washing with water for 6 hours did not diminish the antiseptic properties. Similar results were obtained with this cement under actual service conditions over a 6-month test period. Throughout the study cupric oxychloride cement had a marked antiseptic effect on the mold cultures, while the growth of bacteria was influenced to a less marked degree. S A RESULT of investigations to overcome certain of the disadvantages of magnesium oxychloride cements, Hubbell (3) developed a new material, “Hubbellite”, by the incorporation of finely powdered copper. During and for long periods following the initial hardening, the metal powder reacts to form a new phase in the cement composition which has been identified (4) as a cupric oxychloride, closely resembling the natural mineral atacamite. This new cementing material was found to possess an unusual combination of
A
properties of advantage for a variety of applications-for example, in the use of the cement as a floor surfacing. The known bactericidal and fungicidal properties of certain of the copper compounds suggested that the new cupric oxychloride cement might possess inhibitory or destructive powers against microorganisms. Preliminary studies by Emig ( 2 ) gave definite indications that this was the case. The present study mas undertaken to obtain further quantitative data on the influence of this cement on the growth of microorganisms; such an influence may be significant in connection with practical uses for the cement which inhibitory or destructive powers suggest, such as floor surfacing in locker rooms and showers, around swimming pools, in certain food industries, and in similar places where sanitation is of paramount importance.
Experimental Procedure The microorganisms selected for this $project included eight mold and eighteen bacterial cultures. Table I shows the microorganisms employed and the environment with which they are usually associated. The types of bacteria listed contain representative species of microorganisms of different chemical composition as evidenced by their reaction t o a differential stain; certain species grow in the absence of air while others do not, and certain forms possess protoplasm which is more resistant to destruction by chemical agents. These microorganisms were grown by the generally accepted methods on different substrates. I n studies dealing with the influence of the environment on the growth of microorganisms, numerous factors play important roles. As a preliminary t o this investigation, rather extensive exploratory studies were conducted, including (a) the influence of temperature on the rate of destruction of microorganisms in the presence of Hubbellite, (b) the influence of various media adjusted to different p H values on the survival of microorganisms, (c) the influence of different temperatures of incubation in demonstrating the presence of living cells, and (d) the effect of the type of surface on the
1186
I N D U S T R I A L AND E N G I N E E R I N G C H E M I S T R Y
O F CUPRICOXYCHLORIDE CEhlENT FIGURE 1. EFFECT IMMEDIATELY
ADJACENT TO
THE
Vol. 33, No. 9
IK P R E V E N T I S G THE GROWTHOF Epidermophyton THE CENTER O F h P E T R I DISH
interdigitale
CEMENT I N
survival of microorganisms. The information obtained was employed as a guide in conducting t h e t,ests reported.
Survival Time of Microorganisms after Contact with Water-Extracted Cement Hubbellite was tested in direct comparison with magnesium oxychloride and portland cements. T h e survival time of the bacterial cultures in contact with water-extracted cement was determined by t h e following procedure: Four blocks of each of the three cements, approximately 2 inches (5 om.) square, were used in testing each culture. These blocks were washed for 6 hours by a stream of running water flowing over the surface t o be tested. Then 0.1 cc. of a n 18-hour aqueous suspension of the culture under test was placed on the surface of each block and spread in a thin film by means of a sterile platinum loop over the entire surface of the block. A sterile glass slide was treated in the same manner as the blocks and used as a control. Sfter 15-minute intervals of contact over a period of 1 hour, the bacterial suspension was thoroughly washed from a block of each of the cements by using a 10-cc. water blank and n sterile Petri dish, and dilutions made directly from these washings were placed in suitable mcdia t o determine the number of viable microorganisms. T o lessen the probable error associated with making total counts, all dilutions were plated in triplicate, and the arithmetic average of the three plates was used. All the experiments were carried out at approximately 25' C.
The molds were studied by a modification of the methods of Burlingame a n d Reddish ( I ) , as follows: Sterile glucose-yeast extract agar was poured into sterile Petri dishes t o a depth of 0.5 inch (1.27 cm.). The plates were allowed t o harden, and their surfaces were inoculated by streaking the mold cultures with a sterile swab. The plates were incubated at room temperature for 7 days and then cut into 1-em. squares. A s uare of each was placed, culture side down, on individual bloc?& of the respective cements and kept in contact with the cement for periods u p t o one hour. At the end of each 15-minute interval a mold culture was removed with sterile forceps from the face of one of the blocks in each series and spread with the culture side down over the surface of a sterile plate of glucose-yeast agar. These plates were, in turn, incubat,ed at room temperature for one week and observed for growth. Tables I1 and I11 show representative results obtained with a bacterial and a mold culture, respectively. Similar
data were determined o n t h e action of t h e three cements against t h e eight mold and eighteen bacterial cultures listed. Table I1 shows t h a t t h e cupric oxychloride cement has the ability t o cause a marked reduction in t h e number of cells
TABLE I. &fICROORGlNISMS TESTED Name of Organism Environment Molds 1 Epidermophyton floccosum '1 2 Epidermophyton interdigitale 1 (Weidmann strain) 3 Epidermophyton interdigitale Epidermophytosis or athlete's (Holland strain) foot 4 Epidermophyton interdigitale I (Weidmann strain) i) Epidermophyton rubrum 6 Monilia bonordeni Pathological cutaneous conditions Ringworm of fingernails 7 Trichophyton violaceum Ringworm of scalp, beard, etc. 8 Trichophyton tonsurans
No.
i
1
Bacteria 9 Streptococcus lactis 10 Streptococcus citrovorus 11 Lactobacillus bulgaricus 12 Escherichia coli
\
Pseudomonas vendralli Pseudomonas fluorescens Bacillus subtilis Aerobacter aerogenes 17 Clostridium sporogenes
7
13 14 15 16 18 19
20 21 22 23 24 25 26
clostridium botulinum Salmonella. enteritidis Salmonella pullorum Salmonella s u i s Eberthella typhi Streptococcus pyogenes Staphylococcus aureus Diplococcus pneumonia, type 3 Saprophytic acid-fast bacterium
1 )
Souring of milk Intestinal tract of warm-blooded animals Soil forms
I
Soil anaerobe, intestinal tract, gmeous gangrene Soil anaerobe, botulism Food poisoning Bacillary white diarrhea of fowls Hog cholera Typhoid fever pus
Pneumonia Resistant type similar to Mycobacterium tuberculosis
INDUSTRIAL AND ENGINEERING CHEMISTRY
September, 1941
TABLE11. Escherichia coli SURVIVING AND PERCENTAQE REDUCTION AFTER CONTACT WITH THREECEMENTS After 15 Min.
a
After 30 Min.
re1;ction
No. Material cells/oc. Hubbellite 300 Mg oxychloride 22,900,000 Portland oement 22,700,000 Control 76,000,0005
99.9 69.9 70.2
0
No. cells/cc. 300 10,400,000
re2ction
14,200,000 75,200,000
81.4 2
After 45 Min.
No. oells/co.
99.9 86.4
100 2,000,000
99.9 97.4
7,500,000
50.3 21.1
60,000,000
Initial number of cella per cor
c
TABLE 111. INFLUENIX OF CEMENTS UPON THE GROWTH= OF Epidermophyton interdigitale Material Hubbellite M g oxychloride Portland cement Control 0, no growth;
15 Min.
+ +++ +++ + + +
30 Min. 0
+++ +++ +++
45 Min. 0
+++ +++ +++
After 60 Min.
regotion
60 Min. 0
+++ ++c +++
+, +++ indicate increasing amounts of growth.
of Escherichia coli within a 15-minute period of contact under the conditions established for this test. These results are rather typical of the findings with the other bacterial cultures tested; the reduction in the number of microorganisms on
No. oells/cc. 100 400,000
1187
ran to thirty extractions and was sufficient to produce definite evidence of inhibitory powers.
Effect of Soil, Sawdust, and
reduc% tion
Milk
99.9 99.4
I n actual use a cement may have a 500,000 98.3 certain amount Of dirt in contact with 50,000,000 34.4 it a t one time or another. I n a cement embodying an inhibitory property, it is necessary to determine if soil, refuse, sawdust, etc., will react in any way with the cement to destroy this property. Cupric oxychloride, magnesium oxychloride, and portland cement blocks and a sterile glass slide as a control were covered for 6 hours with a thin layer of dry soil, a little fresh milk, or some sawdust. At the end of this time the soil, milk, or sawdust were thoroughly washed off with distilled water. The cement blocks and control were then placed in a sterile Petri dish, and the procedure employed in the preceding study was repeated. Tables IV and V are representative of the results obtained with eight cultures. No reduction was apparent in the antiseptic power of Hubbellite after it was covered with soil, sawdust, or milk.
REDUCTION IN CEMENT ON Escherichia coli SURVIVINQ AND PERCENTAGE TABLEIV. EFFECTOF IMPURITIES After 15 Minutes Soil Sawdust % re% re% reNo. cells/cc. duction No. cells/cc. duction No. cells/cc. duction 99.9 20,000 99.9 2,000 99.9 6,000 Milk
Material Hubbellite Mg oxychloride Portland cement Control
5
9,600,000
27,000,000 50,000,000a
80.8 46 0
14,500,000 29,600,000 50,000,000
70.4 40.8 0
5,400,000 16,000,000 50,000,000
83.2 68 0
After 30 Minutes Soil Sawdust % re% reNo. cells/cc. duction No. cells/cc. duction No. cells/cc. c?%in Milk
2,000 8,100,000 25,400,000 49,500,000
99.9 83.3 49.2 1
10,000 9,600,000 21,200,000 49,000,000
99.9 80.8 57.6 2
200 6 000 000 19:000:000 49,000,000
99.9 82 62 2
Initial number of cells per cc.
TABLEV. the cupric oxychloride cement was never below 95 per cent after the 15-minute contact in any case. Results with the magnesium oxychloride cement varied with the different microorganisms. The reduction was as low as 16 per cent and never higher than 97 per cent after 15-minute contact, except with Eberthella typhi where a 99.9 per cent reduction was attained. The portland cement showed considerably less reduction of microorganisms than either the Hubbellite or the magnesium oxychloride cement. The control never showed a total reduction greater than 50 per cent with any of the organisms studied. The cupric oxychloride cement inhibited all the mold colonies within 15 minutes (Table 111). The magnesium oxychloride and portland cements failed to inhibit the growth of any of the fungistudied. An antiseptic is a substance that opposes sepsis, putrefaction, or decay, and prevents or arrests the growth or action of microorganisms, either by inhibiting their activity or by destroying them. The antiseptic potency of the cupric oxychloride cement measured in these experiments was that retained after 6 hours of continuous washing of the surface with water. Emig (a) studied the retention of toxic properties of the cement when subjected to daily extractions with distilled water. He reported some decrease in potency, evidenced by results obtained on the fresh cement as compared with specimens after the first two 24-hour extractions. After the second extraction, however, the potency of the cement remained constant for the length of the experiment which
EFFECTOF SOILON CEMENTS UPON
Material Hubbellite Mg oxychloride Portland cement Control
THE
GROWTH OF
45 Min.
60 Min. 0
Epidermophyton interdigitale 15 Min.
+ t+++ tt
30 Min. 0
t+++ tt
0
$ 22 +++
tT-t +++
~~~~~~
The number of Escherichia coli cells was reduced almost completely on those blocks which had been covered by milk and sawdust and to a slightly less extent on those covered with soil. Table V shows results with blocks covered with soil and then treated with the mold. I n this instance no growth of Epidermophyton interdigitale was demonstrable after a 30minute period of contact.
Degree of Inhibition for Various Organisms I n order to determine the degree of inhibition possessed by the three cements, suitable agar media were inoculated with a suspension of the microorganisms tested and poured as a plate about a block of the cement located in the center of a sterile Petri dish. I n this test inhibition is evidenced by the presence of an area free from growth immediately,surrounding the cement (Figure 1). Triplicate plates were made of each test, and an average measurement of the inhibition was taken in millimeters. A clear zone of no growth adjacent to the cement block plus an area of slight growth a t the periphery of the clear zone gave the total zone of inhibition. The results are shown in Table VI.
INDUSTRIAL A N D ENGINEERING CHEMISTRY
1188 TABLE VI.
DEGREEO F INHIBITION OB’ CEXEKTS MICROORGANISXS
Hubbcllite >g Oxychloride Total Total Clear Clear zone none of area of area of no of no inhibiinhibition, grow-th, tion, growth, Organism mm. mm. mm. mm. 17 10 E p i d . inferdigitale 12 9 T k h . tonsurans 12 8 Trich. violnceum 25 15 Strep. lactis 20 16 B . subtilis 15 10 Salmonella suis 25 10 Stanh. aweus Sa -1 ophytio aoidFait 2.5 12 15 5
FOR >vhRrOUS
Portland Cement Total zone of
inhibition, mm.
Clear area of no growth, mm.
0
0
Cupric oxychloride cement inhibited all the organisms studied within a minimum radius of 12 mm. The zone of inhibition consisted of two parts. One was a clear area of no growth immediately adjacent to the cement block, while the other was a larger area extending farther out from the clear area and having only a few colonies present. A much smaller zone of inhibition was found 1vit.h the magnesium oxychloride cement with the bacteria studied. The two molds tested were not inhibited. The portland cement exhibited no inhibitory powers. Sections of agar in the cleared areas immediately adjacent to the cement were removed aseptically and placed into suitable media. S o growth resulted. Determination of Microorganisms under Service Conditions To determine the influence of a Hubbellite environment upon microorganisms under actual conditions of use, several pieces of rubber-backed cement were placed in the passageway of a gymnasium between the shower room and the locker room for 6 months. Weekly examinations were made over this interval by determining the number of microorganisms present on these cupric oxychloride pieces and on the portland cement which makes up the passageway floor. TABLEVII.
UMBER OF MICROORGANISMS FOUND UPOS CEMEKTSIN A GYMNASIUM
Hubbellite Cement
TWO
Portland Cement
The procedure was t o determine the total number of inicroorganisms on 1-inch (2.s-cm.) square areas of the two cements. One-inch squares were cut from a red rubber inner tube. They were sterilized in a Petri dish, the bottom of which contained enough glycerol to wet the underside of the rubber. The squares were then used to pick up the organisms on one square inch of cement surface by placing them on the surface, covering them with a piece of sterile paper a little larger than the rubber, and rolling over this paper with a sterile photographer’s print roller, in order to contact all the surface and crevices as closely as possible. This upper piece of sterile paper was then removed, the rubber was quickly placed in a dilution bottle containing 100 cc. of sterile water, and the plates were prepared within 30 minutes. Aseptic technique was observed, and forceps used were dipped in alcohol and then burned off in a flame between each operation. Table VI1 shows some of the typical results obtained when square-inch areas on the two cements were tested for microorganisms, several hours after classes had used the showers and while both cements were dry as well as damp.
Vol. 33. N o . 9
The data support the information gained in the laboratory (Table 11) that apparently a greater rate of destruction of microorganisms occurs on the surface of cupric Oxychloride cement than on the surface of portland cement, since the total number of microorganisms found on the latter is many times as high as the number found on Hubbellite. While no differential counts were made with respect t o bacteria and mold colonies, it was observed that mold colonies appeared only on the portland cement. This checks with the laboratory resiilts shown in Table 111. Discussion In this study of cement blocks with regard t o their inhibitory or destructive properties for microorganisms, new procedures were employed which did not lend themselves to completely controlled conditions. One of these problems n-as the recovery of the inoculuin from the surface of the cement blocks. The physical and chemical characteristics of the surfaces studied undoubtedly varied somewhat, but in view of the similar treatments received throughout it is believed that results are comparable. In a durable product such as cement the question regarding the retention of the antiseptic properties of such a product is natural. This study is in agreement with Emig’s inveatigation in that the inhibitory property of Hubbellite was not removed after washing with running mater for 6 hours. It was also shown that n-hen this cement was covered with dry soil, sawdust, etc., for 6 hours, and then placed in a locker room and examined over a 6-month interval, it retained its ability to inhibit or retard the growth of microorganisms. To what extent this toxic property will bc retained beyond this period of time was not determined. The germicidal action of copper salts on algae has been known for many years. It was especially interesting to note the marked inhibitory effect of cupric oxychloride cement on mold cultures. I n the experiments with the eight mold cultures, only one, Xonilia bonordeni, was not markedly inhibited. On account of the specific inhibitory property of Hubbellite for this group of microorganisms, such a cement may find practical use in the control of athlete’s foot and similar skin disturbances. The antiseptic action of this cement on bacterial cultures was less marked in that much longer exposure was often required to attain complete destruction. In view of the fact that these studies were conducted under optimum conditions for the destruction of the cells (lack of organic matter, etc.), it is not possible a t this point to conclude further than the fact that the Hubbellite cement does not provide a favorable environment for bacterial growth. Summary 1. The growth of molds was markedly inhibited after contact with cupric oxychloride cement. 2. The growth of bacteria was inhibited by a longer time exposure and to a less marked degree. 3. The toxic properties of this cement were found to be retained after washing for 6 hours in running water. Acknowledgment The authors wish to thank B. F. Percival for assistance in the completion of study of performance of the cements under service conditions. Literature Cited (1) Burlingame, E. M., and Reddish, Q. F., J . Lab. CZim Med.. 24. 765-72 (1939). (2) Emig, W. H., Univ. Pittsburgh, unpublished studies, 1938. (3) Hubbell, D. S., IND.ENG.CHEM.,29, 123-32 (1937). 14) Huhhell D. S., J . Am. Chem. Soc.. 59, 216 (1937).
