<<

608

Journal o( Protection, Vol. 44, No.8, Pages 608-613 (August 1981) Copyright©, International Association of , Food, and Environmental Sanitarians

Antimicrobial Activity of

THERON E. ODLAUG

TravenolLaboratories, Inc., Morton Grove, Illinois 60053

(Received for publication June 19, 1980)

ABSTRACT agents to various degrees. The mode of action of

The bactericidal and sporicidal effects of halogens are as an antimicrobial agent has not been fully determined, Downloaded from http://meridian.allenpress.com/jfp/article-pdf/44/8/608/1654444/0362-028x-44_8_608.pdf by guest on 30 September 2021 reviewed. Chlorine and are the halogens most widely although a number of theories have been presented. used for inactivating microorganisms. Compounds containing There seems to be general agreement that hypochlorous chlorine and iodine are, in general, equally effective in (HOCI) is the most active form. destroying vegetative cells, but chlorine compounds are more When liquid chlorine (C1 2) and hypochlorites are effective in inactivating spores. These relationships are added to water, they hydrolyze to form hypochlorous illustrated graphically from the data available in the literature. acid. In water, hypochlorous acid will dissociate to form ion (H + ) and hypochlorite ion (OCl - ) as shown below.

The halogens include the following of Cl 2 + H 20 HOC! + H + + Cl - nonmetallic elements: chlorine, iodine, and Ca(OClh + 2Hp---+ Ca(OHh + 2HOCI . There have been numerous studies of the HOC! ~ H+ + OCI- antimicrobial effects of these agents. The relative proportion of hypochlorous acid to Fluorine, a highly toxic element, is an irritant and is hypochlorite ion is dependent on pH, temperature and highly corrosive. There are few reports of the use of this ionic strength (27). Chlorine compounds are more as an antimicrobial agent. Bromine, also very effective at lower pH values where the presence of toxic, has been used occasionally in water treatment hypochlorous acid is dominant. At higher pH values, the programs. Chlorine and iodine are the most widely used hypochlorite ion (OCl - ). not effective as an antimicro­ halogens (1 3). Therefore, most of the information in this bial agent, predominates. In contrast, chlorine dioxide report is for these two halogens. does not hydrolyze in aqueous solutions, and the intact The antimicrobial effectiveness of each halogen is considered to be the active agent. compound varies with such factors as availability of the A number of theories have been proposed to explain element to react with the populations, pH. how chlorine inactivates cells. Initially, researchers concentration, contact time, temperature, organic matter thought that the formation of toxic N-chloro compounds. and the type and form of microorganism. In addition, inhibition of glucose oxidation, or oxidation of sulfbydryl different methods of evaluation produce different results, groups were responsible for its effect. Freiberg (14.15), making comparisons difficult in some instances. The using radioactive 35Cl, found that all free available objective of this paper is to review some of the available chlorine but not combined chlorine was taken up by the data on the effectiveness of various halogen compounds vegetative cell. He concluded that the first contact in destroying bacterial cells and spores in suspensions. oxidation reaction with the microbe results in activity, and to develop some empirical relationships from these and that formation of chloramines in the cell protoplasm data. does not cause initial destruction. Using 32 P, Freiberg also showed that, in the presence of chlorine. there is a CHLORINE destructive permeability change in the bacterial cell Chlorine has been used for many years in the home as membrane. Recently, Camper and McFeters (7) showed well as in food. drug and other industries. Some typical that chlorine impairs membrane function, especially uses are treatment of drinking water, waste water transport of extracellular , and that chlorine­ treatment programs, sanitizing equipment and surfaces treated cells were unable to take up labeled carbo­ and treatment of such as poultry (25) and sea food hydrates and amino . (16). Chlorine compounds have also been used widely in Wyatt and Waites (40) have suggested that chlorine­ the health industry for controlling microorganisms. releasing compounds stimulate spore germination and There are five major groups of chlorine compounds: then inactivate the germinated spore. Kulikovsky et al. (a) liquid chlorine, (b) hypochlorites. (c) inorganic (21) showed that chlorine alters the spore permeability chloramines, (d) organic chloramines and (e) chlorine through changes in the integument, resulting in release dioxide. All of these compounds are active antimicrobial ofCa + 2• DPA. RNA and DNA.

JOURNAL OF FOOD PROTECTION. VOL. 44. AUGUST 1981 ANTIMICROBIAL ACTIVITY OF HALOGENS 609

Benarde et al. (J), using chlorine dioxide and labeled TABLE 2. Inactivation ofBacillus spores by hypochloritesa. 14C amino acids, showed that, for Escherichia coli, the mechanism of action is disruption of protein synthesis. However, they did not determine the level of activity at Time fora FACC 90%reduc· which protein synthesis is disrupted. Organismb pH (ppm) tion (min) B. cereus (10) 6.5 so 1.5 Hypochlorites B. cereus (38) 7.0 100 0.9 Hypochlorites are the most widely used chlorine B. coagulans (24) 6.8 20 6 compounds, with and hypochlorite the B. macerans (26) 7.0 15 6.4 major compounds, in this group. These agents are very B. metiens (32) 7.0 25 1.8 effective in inactivating bacterial cells in aqueous B. subtilis (5) 7.2 2.5-2.6 20.5 suspensions, requiring short contact times. A summary B. subtilis (8) 7.0 1000 0.12 of some published data for the antimicrobial etTective­ B. stearothermophilus (8) 7.0 1000 1 Downloaded from http://meridian.allenpress.com/jfp/article-pdf/44/8/608/1654444/0362-028x-44_8_608.pdf by guest on 30 September 2021 ness of is shown in Table 1. Because aTests were done with NaOCI or CaOCI in water or buffer at the experimental methods vary from study to study, only 20-25 c. general statements can be made about these data. All of bReferences are in parentheses after species name. the microorganisms indicated are very sensitive to CfAC =free available chlorine. chlorine. There is a 90 o/o reduction in cell population for TABLE 3. Inactivation of Clostridium spores by hypo· most of the organisms in less than 10 sec with relatively chloritesa. low levels of free available chlorine (FAC). Because the inactivation of vegetative cells in chlorine is very rapid, it is difficult to make meaningful comparisons between Time tor a organisms. However, Vibrio parahaemolyticus, Pseudo­ FACe 90% reduc· monas aeruginosa and Bacillus cereus cells appear to be Organismb pH (ppm) tion (min) more resistant than the other organisms. C. botulinum · Type A (18) 6.sd 4.5 2 TABLE 1. Inactivation of bacterial cells by sodium hypo· C. botulinium · chloritea. Type E (18) 6.5 4.5 0.9 C. perfringens (11) 8.3e 5 >30 C. bifermentans (11) 8.3 5 4.5 Time fora C. sporogenes (11) 8.3 5 8.5 FACe 90%reduc· Organismb pH (ppm) tion (sec) aTests were done with N aOCI in buffer or water. bReferences are in parentheses after species name. S. derby (28) 7.2 12.5 1.5 CfAC =free available chlorine. E. coli (1 7) 8.5 3 2.7 dTest temperature 25 C. E. coli (37) 7.1 1 30 eTesttemperature 10 C. E. coli (30) 7.5 0.6 <6 E. coli (28) 7.9 12.5 3.5 S.foecalis (30) 7.5 0.6 11.9 Figure 1 shows the empirical relationship of the time S. lactis (17) 8.4 6 3.5 required for a 90%reduction in Bacillus and Clostridium L. plantarum (1 7) 5.0 6 3.4 spores versus chlorine concentration as developed by P. cerevisiae (17) 8.5 6 7.1 Odlaug and Pflug (29) from the available literature. They V. parahaemolyticus (16) 7.0 13 15 calculated the amount of hypochlorous acid based on the P. aeruginosa (37) 8.0 2 7.5 FAC and pH for each data set to enable comparison of B. cereus (38) 7.0 5 "-100 results from different investigations. aTests were done in distilled water, hard water or a buffer If we disregard data for Bacillus stearothermophilus. solution at 20-25 C. then for both Bacillus and Clostridium spores at the low bReferences are in parentheses after species name. effective HOC! concentration, the logarithm of the time CfAC free available chlorine. for a 90% microbial cell reduction is a linear function of the logarithm of the HOCl concentration. As the HOCl Compared to vegetative cells, bacterial spores are very concentration increases (to where the time for a 90% resistant to the hypochlorites. In Table 2, some data on microbial cell reduction is 1 to 2 min.), the rate of resistance of spores to chlorine are summarized. The microbial cell destruction increases. The lines drawn in time required for a 90% reduction in cell population is Fig. 1 represent the general conditions for destruction of longer (minutes opposed to seconds) and the concentra­ Bacillus and Clostridium spores. tion of free available chlorine needed for inactivation is Figure 2 was prepared to illustrate the large differ­ higher than specified for vegetative cells. Similar data for ences in resistance between vegetative cells and spores. Clostridium spores are presented in Table 3. Clostridium The vegetative cell data in Fig. 3 were taken from spores are not as resistant as Bacillus spores. Table 1 after the hypochlorous acid concentration was

JOURNAL OF FOOD PROTECTION. VOL. 44, AUGUST 1981 610 ODLAUG 0000~

100 ":;.. a; I" .0 e E .!::! z= 10 70 10 :; e C. botulinum· Type A .E Q. c 0 • .!::! Q. B. SUbtii!S u r:: r:: i" :::"'o!hjmophHu' .!::! a: t; :I ;;: "0 0 .. "' a: V. parah&emolyUcus ,g s. lactis e •

.. e P. cerevisiae, L planatarum Downloaded from http://meridian.allenpress.com/jfp/article-pdf/44/8/608/1654444/0362-028x-44_8_608.pdf by guest on 30 September 2021 E 0.1 -..·---r j:: I I I I E. coli e • E. coli 0.1 10 100 1000 10,000 S. derby• Concentration of Hypochlorous Acid (ppm)

Figure 1. Suggested relationship between the concentration 0.01 -+------,,..-----..,.----.,------,------, of hypochlorous acid and the time to reduce the bacterial 0.1 10 100 1000 10,000 population of Bacillus and Clostridium spores by 90o/o. Data summarized by Odlaug and Pflug (29). Concentration of Iodophor (ppm) Figure 3. Summary of data comparing the bactericidal and 1000 sporicidal effects of iodophor expressed as the time to reduce the cell population by 90%as a function of the concentration of iodophor. Data from Tables 6 and 7. en 1oo :;" e I There have been a number of published reports on the .ge antimicrobial effectiveness of Chloramine T (sodium

Concentration of Hypochlorous Acid (ppm) dichloroisocyanurate was more active than NaOCl Figure 2. Summary of data comparing the bactericidal and against E. coli. Staphylococcus aureus, Klebsiella sporicidal effects ofchlorine expressed as the time to reduce the aerogenes. Salmonella typhi and P. aeruginosa. cell population by 90% as a function of the concentration of Chlorine dioxide hypochlorous acid. Data adapted from Odlaug and Pflug (29) Chlorine dioxide is another chlorine compound that amJTable L has received more attention in recent years. Its use has calculated based on the FAC and pH. Bacterial spores increased due to new chemical formulations permitting are about 10 to 1000 times more resistant to chlorine shipment to areas of use rather than requiring on-site than vegetative cells. The data indicate that in situations generation of the compound. In contrast to other where the concentration of hypochlorous acid is low and chlorine compounds, there have been very few published contact times are short. vegetative cells would be reports on the antimicrobial effects of this compound. inactivated rapidly, but there would be little effect on the Chiorine dioxide is reported to have 2.5 times the spores. oxidizing power of chlorine Its eftect on E. as Chloramine:; compared to th~t of chlorine, is shown in Table 5. At pH Inorganic chloramines are compounds forr.:ted as a wag not as: effective as but the reverse is result ofthe reaction of chlorine with . true at pH Benarde e~ zL a!sc Organic ch!oramines are formed the reaction sterile. unchJorina ted is more hypochlorous acid with , ~mines or effective imides, Recently that 5 ppm of is

JOURNAL OF FOOD PROTECTION. 44~AtJGt;Sl ANTIMICROBIAL ACTIVIIT OF HALOGENS 611

TABLE 4. Sodium hypochlorite and Chloramine T as bactericidal agents.

Organism a Chemical form pH ppm Time (min) Reduction ( o/o)

Chloramine 200 240 37 NaOCl 9 0.5 120 so C. bifermentans (11) Chloramine 9b 200 120 22 NaOCl 9 0.5 120 99.8 B. metiens (9,32) Chloramine 6C 1000 900 99 NaOCl 6 25 2.5 99 E. coli (,30) Chloramine 6.4c 2.4 >10 90 NaOCl 7.5 0.6 2 > 99.9999 S.faecalis (.30) Chloramine 6.4C 2.4 > 10 90 NaOCl 7.5 0.6 0.5 > 99.9999 Downloaded from http://meridian.allenpress.com/jfp/article-pdf/44/8/608/1654444/0362-028x-44_8_608.pdf by guest on 30 September 2021 a References are in parentheses after species name. bTest temperature 10 C. CTest temperature 20-25 C.

TABLE 5. Chlorine dioxide and chlorine as bactericidal iodine and are the active agents. The major iodine compounds are -iodine solutions, aqueous iodine solutions and iodophors. The aqueous Chemical and alcoholic solutions are usually used as skin forma,b pH Time (min) Reduction (o/o) . The iodophors are used for cleaning and CI2 6.5 0.5 99 disinfecting equipment and surfaces, in water treatment 8.5 5 99 and as a skin . Cl02 6.5 1.0 99 Iodophors, one of the most popular forms of iodine 8.5 0.25 99 compounds used today, are prepared with iodine and a a Tests were done with E. coli in buffer at 24 C with 0.25 ppm of carrier such as polyvinylpyroliodone (22), or Cl 2 orCl02• such as nonyl- ethoxylates (1,34). These com­ bBenarde et al. (2). pounds are bactericidal and, in comparison to aqueous and alcoholic suspensions of iodine, have greater equivalent to 20 ppm of Ch (generated from ) in solubility in water, are nonodorous and are nonirritating maintaining low bacterial levels in poultry processing to the skin (23). water. From these few studies it appears that at higher Some investigators have indicated that the amount of

pH values Cl02 is not affected to the degree that Ch or free available iodine determines the activity of iodophors hypochlorites are affected by pH or organic matter. More rather than the type of present (39). However, studies on the effects of this compound on microbial cells other investigators have shown that the surfactant can and spores in different situations are obviously needed. affect the bactericidal properties of iodine (16,28). In general, chlorine compounds used in solutions or on Reported activity of iodophors against some bacterial surfaces where available chlorine can react with cells will cells is summarized in Table 6. At concentrations of 6 to be bactericidal and sporicidal. Vegetative cells will be 13 ppm of available iodine, the time to reduce the killed more easily than spores, and Clostridium spores bacterial cell population by 90o/o ranged from about 3 to will be killed more easily than Bacillus spores. The lethal 15 sec. The effectiveness of iodophors against bacterial effect of most chlorine compounds will increase with an spores is summarized in Table 7. Spores are very increase in free available chlorine, a decrease in pH and an increase in temperature. However, there is a point at TABLE 6. Inactivation ofbacterial cells by iodophorsa. which solutions of high chlorine concentration and/or low pH can be corrosive to . Also, the solubility of chlorine in water decreases with increasing temperature. Time fora Cone 90%Reduc­ Organismb pH (ppm) tion (sec) IODINE V. parahaemolyticus (16) 7.0 13 15 Iodine, like chlorine, has been used in water treatment E. coli (17) 6.9 6 5 programs and in sanitization of equipment and surfaces E. coli (28) 6.6 12.5 4 (19). Iodine compounds have also been used in place of S. lactis (I 7) 6.8 6 13 antibiotics for disinfecting skin in the treatment of L. plantarum (17) 6.7 6 11 \vound infections (22), and on some (16). P. cerevisiae (17) 6.9 6 10 In contrast to chlorine, there have been few studies on s. (28) 6.7 12.5 3 the mode of antibacterial action of iodine. It is generaUy aTests were done in water or buffer at 20-25 C. thought that, under most conditions, free elemental bReferences are in parentheses after species name.

JOURNAL OF FOOD PROTECTION, VOL. 44, AUGUST 1981 612 ODLAUG

TABLE 7. Inactivation ofbacterial spores by iodopho~. chloramine compound was more effective at pH 6.5 than the organic bromine compound, but chloramine with bromine was the agent least affected by pH, as indicated Time for a by its activity at pH 8.5. The organic chloramine with Cone 90o/o reduc- Organismb pH (ppm) tion (min) bromochloramine was just as effective as NaOCI against Streptococcus faecalis. Other investigators (12,20,35) B. cereus (10) 6.5 50 10 have shown that addition of bromine to solution with 6.5 25 30 chlorine compounds increases effectiveness, and in some 2.3 25 30 studies, the effectiveness of bromine and chlorine B. subtilis 09) 25 >S solutions was shown to be synergistic. C. botulinum Type A (18) 2.8 100 >6 a All tests were done in distilled water at 15-25 C. CONCLUSION hReferences are in parentheses after species name. Our knowledge of the antibacterial activity of halogens Downloaded from http://meridian.allenpress.com/jfp/article-pdf/44/8/608/1654444/0362-028x-44_8_608.pdf by guest on 30 September 2021 resistant to iodine, as compared to vegetative cells. Sykes has increased considerably in the last 20 years. However, (36) reported that iodophor is more effective against there are some areas that need further investigation. The Bacillus subtilis spores than the alcoholic or aqueous mode of action of these agents has not been fully solutions of iodine. However, there appeared to be little elucidated. Another area of halogen activity that has difference in effectiveness of the three compounds received very little attention is injury of microbial cells. against Bacillus pumilus spores. Because vegetative cells are so sensitive to halogen A comparison of the resistance of spores and compounds, low concentrations of halogens with short vegetative cells to iodophor is shown in Fig. 3. As with exposure times and low temperatures are required to chlorine, spores are about 10 to 1000 times more produce cell injury (33). Concentrations of halogen resistant than vegetative cells. If we compare the data in solutions used for sanitizing should be bactericidal to Fig. 3 with those in Fig. 2. it appears that iodine and most of the population. Injured cells that recover could chlorine are equally effective in inactivating vegetative cause misinterpretation of test results and other cells (16.17.28). However, iodine does not appear to be as problems when the control of microorganisms is critical effective as chlorine in inactivating spores (10.18). (6). For this reason, recovery methods for bacteria exposed to halogens should be validated so that there is BROMINE confidence that both noninjured and injured organisms Bromine has been used alone, or in combination with are enumerated. other chlorine compounds, in some water treatment The halogen compounds have played a vital role as programs. Data comparing chlorine and bromine antimicrobial agents in a variety of applications for many compounds are shown in Table 8. Against B. cereus years. As we learn more about their effects on spores, NaOCl was the most effective agent. The organic microorganisms, we will able to optimize their use.

TABLE 8. Bromine and chlorine as bactericidal agentsa. ACKNOWLEDGMENTS This paper was presented at the 80th Annual Meeting of the American Society for Microbiology. Miami Beach, Florida, May 13. 1980. Time fora o/o 90 reduction REFERENCES Organismb Compound Conditions (sec) 1. Bartlett, P. G., and W. Schmidt. 1957. Surfactant-iodine B. cereus (10) NaOCI 1.5 complexes as germicides. Appl. Microbiol. 5:355-359. 2.5 2. Benard e. M.A .. B. M. Israel, V. P. Olivieri, and M. L. Granstrom. Dichloro- 50 ppm, pH 6.5 4.5 1965. Efficiency of chlorine dioxide as a . Appl. dimethyl and 34 Microbiol. 13:776-780. hydantoin 100 ppm, pH 3. Benarde, M.A., W. B. Snow, V. P. Olivieri, and B. Davidson.1967. Dibromo- 13.5 Kinetics and mechanism of bacterial disinfection by chlorine dimethyl 12.5 dioxide. Appl. Microbiol. 15:257-265. hydantoin 4. Bloomfield. S. F., and G. A. Miles. 1979. The antibacterial S.Jaecalis (30) NaOCl 0.595 ppm, 0.1 properties of sodium dichloroisocyanurate and sodium hypochlorite fom!Uiations. J. Appl. Bacteriol. 46:65-73. pH7.5 5. Brazis, A. R., J. E. Leslie. P. W. Kabler. and R. L. Woodward. Dichloro- 0.594 ppm, 0.16-2 1958. The inactivation of spores of Bacillus ~lobit:ii and Bacillus dimethyl pH 6.2 anthracis by free available chlorine. Appl. Microbiol. 6:338-342. hydantoin 6. Busta, F. F. 1976. Practical implications of injured microorganisms Bromochloro- 0.602 ppm, 0.1 in food. J. Milk Food Techno!. 39:138-145. dimethyl pH 7.3 7. Camper. A. K .. and G. A. McFeters. J 979. Chlorine injury and the hydantoin enumeration of waterborne coliform bacteria. Appl. Environ. Microbiol. 37:633-641. a Tests were done in buffer or water at 20-25 C. 8. Cerf. 0 .. J. L. Berry. M. Riottot. andY. Bouvet. 1973. A simple bReferences are in parentheses after species name. apparatus for the determination of the efficiency of quick acting

JOURNAL OF FOOD PROTECTION. VOL 44. At:GUST 1981 ANTIMICROBIAL ACTIVITY OF HALOGENS 613

disinfectants and sterilizing solutions. Application to the activity of 632-634. sodium hypochlorite against bacterial spores. Pathoi. Bioi. 25. Lillard. H. S. 1979. Levels of chlorine and chlorine dioxide of 21:889-894. equivalent bactericidal effect on poultry processing water. J. Water 9. Chariton, D. B .. and M. Levine. 1937. Germicidal properties of Sci. 44:1594-1597. chlorine compounds. Eng. Exp. Sta., Bull. 132: la. 26. Mercer, W. A., and I. L Somers. 1957. Chlorine in tood 10. Cousins. C. M., and C. D. Allan. 1967. Sporicidal properties of sanitation. pp. 129-160. In Advances in food research, Vol. 7. some halogens. J. Appl. Bacteriol. 30:168-174. Academic Press, Inc. ,New York. 11. Dye, M., and G. C. Mead. 1972. The effect of chlorine on the 27. Morris, J. C. 1966. The acid ionization of HOC! from so to 35°C. viability of clostridial spores. J. Food Techno!. 7:173-181. J. Phys. Chern. 70:3798-3805. 12. Farkas-Himsley, H. 1964. Killing of chlorine-resistant bacteria by 28. Mosley, E. B .. P.R. Elliker, and H. Hays. 1976. Destruction of chlorine-bromine solutions. Appl. Microbiol. 12:1-6. food spoilage indicator and pathogenic organisms by various 13. Forwalter, J. 1980. 1980 selection guide to cleaning and sanitizing germicides in solution and on a stainless steel surface. J. Milk Food compounds. Food Process. 46:40-44. Techno!. 39:830-836. 14. Freiberg, L. 1956. Quantitative studies on the reaction of chlorine 29. Odlaug, T. E., and I. J. Pflug. 1976. Sporicidal properties of with bacteria in water disinfection. Acta Pathol. Microbiol. Scand. chlorine compounds: Applicability to cooling water for canned 38:135-144. foods. J. Milk Food Techno!. 39:493-498. Downloaded from http://meridian.allenpress.com/jfp/article-pdf/44/8/608/1654444/0362-028x-44_8_608.pdf by guest on 30 September 2021 IS. Freiberg. L. 1957. Further quantitative studies on the reaction of 30. Ortenzio, L. F., and L. S. Stuart. 1964. A standard test for efficacy chlorine with bacteria in water disinfection. Acta Pathoi. Microbiol. of germicides and acceptability of residual disinfecting activity in Scand.40:67-80. swimming pool water. J. Assoc. Off. Agric. Chern. 47:540-547. 16.Gray. R. J .. and D. Hsu. 1979. Effectiveness of iodophor in the 31. Rode, L. J., and M. G. Williams. 1966. Utility of sodium destruction of Vibrio parahaemolyticus. J. Food Sci. 44: 1097-1100. hypochlorite for ultrastructure study of bacterial spore integuments. 17. Hays, H., P. R. Elliker, and W. E. Sandine. 1967. Microbial J. Bacteriol. 92:1772-1778. destruction by low concentrations of hypochlorite and iodophor 32. Rudolph, A. S., and M. Levine. 1941. Factors affecting the germi­ germicides in alkaline and acidified water. Appl. Microbiol. cidal efficiency of hypochlorite solutions. Eng. Exp. Sta., Bull. 15:575-581. 150:1a. 18. Ito, K. A., M. L. Seeger. C. W. Bohrer, C. B. Denny, and M. K. 33. Scheusner, D. L .. F. F. Busta, and M. L. Speck. 1971. Injury of Bruch. 1968. The thermal and germicidal resistance of Clostridium bacteria by sanitizers. A ppl. Microbiol. 21:41-45. botulinum Types A, B, and E spores. pp. 410-415. In Proc. of the 34. Schmidt, W., and M. Winicov. 1967. Detergent/iodine systems. 1st U.S.- conference on toxic-microorganisms, University of Soap Chern. Spec.43:61-64. Hawaii. 35. Shere, L., M. J. Kelley, and J. H. Richardson. 1962. Effect of 19. Kinman, R.N., A. P. Black, and W. C. Thomas, Jr. 1970. Status bromide hypochlorite on microorganisms. Appl. of water disinfection with iodine. In Proc. of the national specialty Microbiol. 10:538-541. conference on disinfection, American Society for Civil Engineers, 36. Sykes, G. 1970. The sporicidal properties of chemical disinfectants. New York. J. Appl. Bacteriol. 33:147-156. 20. Kristofferson, T. 1958. Mode of action of hypochlorite sanitizers 37. Trueman, J. R. 1971. The halogens. pp. 137-180. In W. B. Hugo with and without . J. Dairy Sc;i. 41:942-949. (ed.) Inhibition and destruction of the microbial cell. Academic 21. Kulikovsky, A., H. S. Pankratz, and H. L. Sadoff. 1975. Press. London. Ultrastructural and chemical changes in spores of Bacillus cereus 38. Wang, M. Y., E. B. Collins, and J. C. Lobben. 1973. Destruction after action of disinfectants. J. Appl. Bacteriol. 38:39·46. of psychrophilic strains of Bacillus by chlorine. J. Dairy Sci. 56: 22. Lacey. R. W. 1979. Antibacterial activity of providone iodine 1253-1257. towards non-sporing bacteria. I. Appl. Bacteriol. 46:443-449. 39. Wilson, P. W., and P. E. Nelson. 1979. Improving the efficiency of 23. Lav.Tence, C. A., C. M. Carpenter, and A. W. C. Naylor-Foote. chemical sanitizers used in bulk storage tanks. J. Food Sci. 1957. Iodophors as disinfectants. J. Am. Pharm. Assoc. 46: 44:251-253. 500-505. 40. Wyatt, L., and W. Waites. 1973. The effect of hypochlorite on the 24. LeBree, T. R., M. L. Fields. and N. W. Derosier. 1960. Effect of germination of spores of Clostridium bifermentans. J. Gen. Micro· chlorine on spores of Bacillus caa.fiulans. Food Techno). 14: bioi. 78:383.

JOURNAL OF FOOD PROTEC110N. VOL. 44, AUGUST 1981