608 Journal o(Food Protection, Vol. 44, No.8, Pages 608-613 (August 1981) Copyright©, International Association of Milk, Food, and Environmental Sanitarians Antimicrobial Activity of Halogens 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 chlorine 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 iodine 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 acid (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 hydrogen ion (H + ) and hypochlorite ion (OCl - ) as shown below. The halogens include the following group of Cl 2 + H 20 HOC! + H + + Cl - nonmetallic elements: chlorine, iodine, fluorine and Ca(OClh + 2Hp---+ Ca(OHh + 2HOCI bromine. 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 halogen 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 molecule 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 cell 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 nutrients, and that chlorine­ treatment programs, sanitizing equipment and surfaces treated cells were unable to take up labeled carbo­ and treatment of foods such as poultry (25) and sea food hydrates and amino acids. (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 calcium and sodium 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 sodium hypochlorite 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.