Ultraviolet Bactericidal Irradiation of Ice

Transcription

1 APPLIED MICROBIOLOGY, Mar. 1968, p Copyright 1968 American Society for Microbiology Vol. 16, No. 3 Printed in U.S.A. Ultraviolet Bactericidal Irradiation of Ice P. A. LADANYI AND S. M. MORRISON Department of Microbiology, Colorado State University, Fort Collins, Colorado Received for publication 1 August 1967 We investigated the germicidal activity of 2,537 A ultraviolet (UV) radiation on bacteria in ice cubes of varying thickness and in aqueous suspensions beneath an ice layer. The test bacteria used were Escherichia coli, Serratia marcescens, Bacillus subtilis, and Sarcina lutea; aqueous suspensions of the selected organisms were frozen into ice cubes, 2mm to 30 mm thick, at -20 C. The cubes were irradiated for 1 min, whereas the suspensions of bacteria were placed beneath an ice block (19 cm thick) and were irradiated for 0.5 to 15 min. In both groups of experiments, the standard plate count method was used to compare the number of bacteria surviving the UY treatment with the number of bacteria in the untreated controls. The results showed that 1 min of UV treatment killed as many as 97% of the gram-negative and at least 60% of the gram-positive test bacteria (freezing survivors) frozen in ice cubes 30-mm thick. Within 15 min, UV light transmitted through a 19-cm thick ice block inactivated 98% of the bacteria suspended in the buffer solution. We concluded that the UV rays were able to penetrate at least 19 cm of ice and still retain enough energy to kill bacteria. However, the UV penetration depended greatly on the optical quality of the ice. Although it was not the purpose of these experiments to find a practical method for sanitizing ice, the results of this study and of our other unpublished experiments indicate that UV light has adequate penetrating power to be considered practical in certain selected applications. Although freezing is lethal to a variable portion of a bacterial population, a number of bacteria can survive in ice. Foltz (5) showed that commercially available ice made from good quality water can harbor large numbers of viable bacteria. Many of the methods commonly used for bacterial control are impractical for use with ice; however, ultraviolet (UV) irradiation frequencies may provide an effectivemeans of inactivating bacteria. Early observations on the inactivating effect of UV radiation on bacteria were reviewed by Zelle and Hollaender (15). It is believed that a large portion of the bactericidal activity of UV radiation results from the fact that this radiation activates nucleotide dimer formation, especially the formation of thymine dimers which are enhanced by freezing (S. V. Wang, Federation Proc. 24:part III Cryobiol. S-71-S-79, 1965). The altered nucleotides cannot perform their normal metabolic functions, and the bacterial cell is rendered nonviable. The penetration potential and activity of UV light in air, water, and certain food products has been systematically investigated in the past (6, 13). The comparative effects of UV rays on bacteria in water and in ice, the solid crystal state of water, have been less thoroughly examined. Lyons and Stoiber (8) reported that the transparency of clear ice to rays of most of the light spectrum is very great and is like that of distilled water. The light transmissivity of ice appears best in the visible range but not as good for the UV wavelengths. In the infrared range, a high degree of absorption takes place, but accurate measurements are unavailable because of the difficulty in obtaining optically flawless ice (4, 8). Reports by Levine and Cox (7), Levine (Federation Proc. 24:part III Cryobiol. S-85-S- 88, 1965), and Ashwood-Smith and co-workers (2) suggested that inactivation of bacteria and bacteriophages was enhanced with UV light when these organisms were present in a medium 463 passing from the liquid to the frozen state. For irradiation, these investigators used microorganisms contained in frozen saline (3 mm thick), thereby reducing the possibility of any significant energy absorption by the ice itself. The present study was designed (i) to investigate the effect of UV treatment on bacteria contained in ice several centimeters thick (household size ice cubes); (ii) to determine the degree of penetration of UV rays through 19-cm ice blocks; and (iii) to separate experimentally the effects of freezing and thawing from the effects of actual UV inactivation.

2 464 LADANYI AND MORRISON APPL. MICROBIOL. MATERIALS AND METHODS Organisms. Four species of bacteria were used for this investigation. Escherichia coli ATCC and a laboratory strain of Serratia marcescens represented the gram-negative bacteria, and Sarcina lutea ATCC 381 and Bacillus subtilis ATCC 6051 (contained in a mixed spore and vegetative cell culture) represented the more UV-resistant gram-positive organisms (6). Stock cultures of the organisms were maintained on Plate Count Agar medium (Difco) and were incubated for 48 hr at the following temperatures: E. coli and B. subtilis at 37 C, S. marcescens and S. lutea at 25 to 28 C. Liquid test cultures were prepared by inoculating half-strength m-plate Count Broth (Difco) with the growth of a single colony of each stock culture; the cultures were incubated at room temperature (25 to 28 C) on a gyratory shaker (about 130 rev/min) for 36 to 48 hr. Ice cubes. Sterilized buffered water (.0003 M phosphate buffer, final ph 6.8) containing the selected test broth culture (at an approximate concentration of 108 cells per ml) was poured into plastic ice cube trays and was frozen overnight at -20 C. The ice cubes were irradiated from a distance of 12 inches with a 15-w General Electric germicidal tube emitting UV light with a wavelength of 2,537 A. After irradiation, the ice cubes were melted at room temperature and plated in plate count agar by use of the standard pour plate technique (1); after a period of incubation, the viable counts of the test cultures were compared with those of the nonirradiated controls. In some experiments, the size of the ice cubes (30 X 45 X 30 mm) and the length of the UV exposure (I min) were kept constant, whereas, in other experiments, the UV exposure was gradually increased from 0.5 to 15 min or the thickness of the ice cube was increased from 2 to 30 mm. Large ice blocks. To determine the effect of ice thickness on penetration by UV germicidal energy, ice blocks (19 cm thick) were prepared from either sterilized distilled or tap water, and uncovered petri dishes containing bacterial suspensions in buffered water (7 mm deep) were placed directly beneath these blocks. The air temperature was maintained near 0 C to minimize ice melt and contamination of the suspension plates. The blocks and cultures were shielded with black paper so that radiation could reach the suspension only through the ice block. At intervals during the UV treatment, equal volumes of samples were withdrawn from the bacterial suspension and plated to determine the percentage of surviving organisms. RESULTS AND DiscussIoN UV effect on bacteria in small ice cubes. A preliminary exposure-time study provided the results presented in Table 1. UV rays sharply reduced the number of viable E. coli, S. marcescens, B. subtilis, and S. lutea bacteria in ice cubes made from frozen buffered water. UV exposure for periods as short as 0.5 min caused a noticeable reduction in the viable bacterial content of the TABLE 1. Survival of selected bacteria in ice cubes after UV treatment0 Mean percentage of survival UV treatment (min) Escherichia Serratia Bacillus Sarcina coli marcescens sublilis lufea a In this and the following tables, the data are given as the average percentages of survival of organisms in replicate trials. To exclude inactivation due to freezing damage, the percentage of survival of each organism was calculated from the viable bacterial counts after freezing and after UV treatment of the ice. ice, and the percentage of survival decreased with additional exposure to UV energy. A 15-min UV treatment reduced the viable bacterial population by at least 99%, with the exception of the mixed spore and vegetative cell culture of B. subtilis, where inactivation was about 97%. In this experiment, the cubes were removed from the ice-cube trays before UV treatment, and a small amount of moisture collected on the surface of the ice, resulting in a somewhat higher bacterial survival than was found in the continuing experiments. In a series of six experiments, standard-size ice cubes containing test bacteria were exposed to 1 min of UV radiation; the results of these experiments are presented in Table 2. On the average, 2.6% survival was observed for E. coli, 2.7% for S. marcescens, 38.8% for B. subtilis, and 33.3% for S. lutea. Although the experimental standard deviations of survival for the gram-negative organisms were very small, greater inconsistencies were observed in the replicate experiments with S. lutea and B. subtilis. These experimental inconsistencies probably resulted from several factors that were not adequately controlled. The normal reproductive pattern of S. Iutea and the clumping of packets in this organism caused inconsistency in the irradiation effect (11), freezing and thawing damage (9), and break-up during the plating procedure (Morrison and McCarron, unpublished data). The varying quantities of spores in the mixed spore and vegetative cell cultures of B. subtilis produced variations in

3 VOL. 16, 1968 ULfRAVIOLET BACTERICIDAL IRRADIATION OF ICE 465 UV and thawing damage. In addition, it is also possible that the inconsistencies in the replicate experiments with S. lutea and B. subtilis were caused by the varying transparencies of the ice cubes. Repeated experiments with UV penetration in ice made it obvious that the optical condition of the ice directly determined the amount of UV energy transmitted. Air bubbles, cracks, haze moisture, impurities, or any other condition in the ice that could cause the reflection, refraction, or absorption of UV rays would reduce the germicidal effectiveness of UV transmittance considerably. When Lyons and Stoiber frosted a clear ice surface with a breath of air, the transmissivity of visible light through the ice dropped from 90 to 3 % (8). Even when the utmost care was taken to control environmental conditions, production of optically flawless or even optically identical ice cubes was unsuccessful. This inability to produce optically flawless ice introduced an additional variable into our experiments, and this variable had to be considered in evaluating our results. The effect of increases in ice-cube thickness on UV inactivation of the test organisms is shown in Table 3. The survival values of the organisms after UV treatment were decidedly higher when the thickness of the ice layer was increased. The percentage of survival of the viable UV-treated organisms increased proportionally to the thickness of the ice up to about 19 mm; at this point, the per cent survival values seemed to peak and did not increase with additional increases in icecube thickness up to 30 mm. Organism survival was directly related to the penetration of UV energy through ice cubes 2 to 14 mm thick because solid particles, air bubbles, and bacteria, all absorbents of UV energy, were evenly distributed in the thin cubes. However, when cubes were frozen to a depth of more than 14 mm, air TABLE 2. Survival of selected bacteria in ice cubes after I min of UV treatment Expt no. Mean percentage of survival (4 cubes) Escherichia Serratia Bacillus Sarcina coli marcescens subtilis lutea Average TABLE 3. Effect of varying ice-cube thickness on survival of selected bacteria after UV treatment Percentage of survival after 1 min of UV treatment Ice cube thickness (mm) Escherichia Serratia Bacillus Sarcina coli marcescens sublilis lutea bubbles, bacteria, salts, organic molecules, and particulate matter were excluded from the ice crystals and were concentrated in the slowest freezing region of the ice cube, producing an optically dense ice core (3, 10, 12; S. Y. Wang, Federation Proc. 24:part III Cryobiol. S-71-S- 79, 1965). In cubes where ice cores formed (probably containing the test bacteria), the extent of UV inactivation of the test organisms depended on the UV penetration of the ice core and was relatively independent of small changes in the thickness of the ice. The optical properties of the ice core varied with the quantity of air bubbles and with the concentration of bacteria and UVabsorbing debris. These variations in optical properties of the ice core altered the UV survival of test bacteria in individual ice cubes. In these experiments, the viable bacterial counts in the ice cubes before UV treatment were 10O organisms per ml of initial seeding less the organisms destroyed by freezing. The damaging effect of freezing on the bacteria was reduced by using 36- to 48-hr cultures (14; Morrison and McCarron, unpublished data). Freezing and thawing damage was about 40% for E. coli, 34% for S. marcescens, 23% for B. subtilis, and about 12% for S. lutea. Even though the damaged bac. teria were not viable, they absorbed UV energy, thereby shielding the viable bacteria from UV inactivation. UV energy would be more effective if these inert cells were not present. When these experiments were repeated with ice cubes frozen from distilled or tap water, the results were similar to those obtained with the M buffer. A

4 466 LADANYI AND MORRISON APPL. MICROBIOL. TABLE 4. Survival of selected bacteria in buffered-water suspensions UV treated through 19-cm ice blocks prepared from distilled and tap water Mean percentage of survival U treat- Escherichia coli Serratia marcescens Bacillus subtilis Sarcina Iiitea Distilled Tap Distilled Tap Distilled Tap Distilled Tap slightly greater UV inactivation of the bacteria was observed in distilled-water ice cubes. UV penetration of large ice blocks. The results of the penetration of UV rays through 19 cm of ice (made from distilled water) are shown in Table 4. In these experiments, the test bacteria were not embedded in the ice blocks but were placed underneath them in an unfrozen bufferedwater suspension. Inactivation occurred in all four species of bacteria, indicating that the UV energy penetrated the ice block. The gram-negative bacteria responded to the UV treatment more rapidly than did gram-positive S. lutea or the mixed spore and vegetative cells of B. subtilis. These results agree with the bacterial sensitivity differences reported by several investigators (6). When these experiments were repeated with ice prepared from tap water, the UV penetration was reduced, but significant inactivation still occurred in the suspension (Table 4). The reduced UV effect probably resulted from the presence of dissolved and suapended solids in the tap water. In contrast to the reduction in UV action caused by the presence of gas particles and dispersed particulate matter in small blocks (ice cubes), less UV interference was observed in large ice blocks. A bacterial inoculum was not included in the ice, and the migration of gas and other absorbing particles to a concentrated core provided an adequately clear peripheral channel for UV penetration to the cultures beneath the blocks. Irradiation of test bacteria embedded in the 19-cm thick ice block was attempted, and bacterial inactivation occurred. However, we were unable to distinguish between the amount of bacterial inactivation caused by the UV energy and the amount of inactivation caused by freezing and thawing; thus, no quantitative conclusions could be drawn. Considerably more quantitative data are needed before general conclusions can be drawn concerning the nature of UY inactivation of bacteria in ice. This study has shown that UV radiation will penetrate ice to considerable thicknesses and will inactivate bacteria. However, we have not established whether UV rays penetrate ice better or as well as they penetrate water; this is left to future comparative studies. The development of a method of producing optically flawless or at least optically consistent ice would be very helpful in accurately determining the ability of germicidal UV rays to penetrate ice. ACKNOWLEDGMENTS The authors thank James E. McCarron and Kirke L. Martin for their technical assistance and background data. This investigation was supported by Public Health Service research grant EF LITERATURE CITED 1. AMERICAN PUBLIC HEALTH AssocIATION Standard methods for the examination of water and waste water, 12th ed. American Public Health Association, Inc., New York. 2. ASHWOOD-SMITH, M. J., B. A. BRIDGES, AND R. J. MUNSON Ultraviolet damage to bacteria and bacteriophage at low temperatures. Science 149: BORGSTROM, G Microbiological problems of frozen food products, p In E. M. Mrak and G. F. Stewart [ed.], Advances in food research, vol. 6. Academic Press, Inc., New York. 4. CASSEL, E. J Ultraviolet absorption of ice. Proc. Roy Soc. (London) Ser. A 153: FOLTZ, V. D Sanitary quality of crushed and cubed ice as dispensed to the consumer. Public Health Rept. (U.S.) 68: KOLLER, L. R Ultraviolet radiation, 2nd ed. John Wiley and Sons, Inc., New York.

5 VOL. 16, 1968 ULTRAVIOLET BACTERICIDAL IRRADIATION OF ICE LEVINE, M., AND E. Cox The ultraviolet sensitivity of frozen phage. Radiation Res. 18: LyoNs, J. B., AND R. E. STOIBER The absorptivity of ice: a critical review. Sci. Rept. No. 3, AF19(604)-2159, ATCRC-TN Geophys. Res. Directorate, Air Force Cambridge Res. Center, Bedford, Mass. 9. MAJOR, C. P., J. D. MCDOUGAL, AND A. P. HARRISON, JR The effect. of the initial ceu concentration upon survival of bacteria at -22 C. J. Bacteriol. 69: MOORE, E. W Water and its relation to disease, p In K. F Maxcy [ed.], Rosenau preventive medicine and public health, 8th ed. Appleton-Century-Crofts, New York. 11. SHARP, D. G The lethal action of short ultraviolet rays on several common pathogenic bacteria. J Bacteriol. 37: SNYDER, A. E Desalting water by freezing. Sci. Am. 207: SUMMER, W Ultraviolet and infra-red engineering. Sir Isaac Pitman and Sons, Ltd., London. 14. TOYOKAWA, K., AND D. H. HOLLANDER Variation in sensitivity of Escherichia coli to freezing damage during the growth cycle. Proc. Soc. Exptl. Biol. Med. 92: ZELLE, M. R., AND A. HOLLAENDER Effects of radiation on bacteria, p In A. Hollaender [ed.], Radiation biology, vol. 2. McGraw-Hill Book Co., Inc., New York. Downloaded from on November 21, 2018 by guest