Combustible Fraction of Municipal Solid Waste”, Bureau of Mines Report RI 8244,1977. (7) Marr, H. E., Law, S.L., Neylan, D. L., in Proceedings of International Conference on Environmental Sensing and Assessment, Las Vegas, 1975,IEEE, New York, 1976, Vol. 1, Paper No. 4-3. (8) Law, S.L., Resour. Recovery Conserv., 3,19 (1978). (9) Law, S.L., Ph.D. Thesis, University of Maryland, College Park, 1976. (10) Law, S.L., J . Water Pollut. Contr. Fed., 49, 2453 (1977). (11) Sullivan, P. M., Makar, H. V., Proceedings of the 5th Mineral Waste Utilization, 1976,p 223. (12) Carotti, A,, Smith, R. A,, “Gaseous Emissions from Municipal Incinerators”, EPA Report No. SW-l8C, 1974. (13) Dodson, H., Manager, Alexandria, Va., Incinerator, private communication, 1974. (14) Blum, S.L., Science, 191,669 (1976). (15) Kenahan, C. B., Sullivan, P. M., Ruppert, J. A., Spano, E. F., “Composition and Characteristics of Municipal Incinerator Residues”, Bureau of Mines Report No. RI 7204,1968.
(16) Hegdahl, T. S.,“Report on a Study of the Alexandria, Virginia Incinerator”, USHEW Public Health Service, Bureau of Solid Waste Management, Report No. SW-12ts, 1970. (17) Staff Report, Environment, 13 (4), 24 (1971). (18) Testin, R. F., paper presented at the American Chemical Society Symposium on Energy and Materials, June 1975. (19) Ostrowski, E. J., in Proceedings of 1972 National Incinerator Conference, American Society of Mechanical Engineers, New York, 1972, p 87. (20) Zoller, W. H., Gladney, E. S.,Duce, R. A., Science, 183, 198 (1974). (21) Billings, C. E., Matson, W. R., Science, 176,1232 (1972). Received for review April 24, 1978. Accepted October 27, 1978. The University of Maryland portion of this study was in part supported by the National Science Foundation RANN Program under Grant No. ENV 75-02667. Portions of this research were performed in partial completion of the requirementsfor a Ph.D. in Chemistry from the University of Maryland, College Park, Md.
Inactivation by Chlorine of Single Poliovirus Particles in Water Roger Floyd and D. Gordon Sharp Department of Bacteriology and Immunology, School of Medicine, University of North Carolina, Chapel Hill, N.C. 275 14
J. Donald Johnson* Department of Environmental Sciences and Engineering, School of Public Health, University of North Carolina, Chapel Hill, N.C. 27514
Some kinetic aspects of the inactivation of poliovirus by chlorine in water have been observed in experiments with both HOCl and OC1- using virus preparations in which no less than 99% of the virions were free single particles. In this manner any influence of virion aggregation on the reaction rates observed was minimized. Under these conditions HOCl was clearly superior to OC1- as a disinfecting agent for this virus. Inactivation rates for both agents increased with increasing concentration, but in neither case did this increase continue in a linear fashion. Both forms of free chlorine became less efficient as the concentration was increased. While the decline in log plaque titer was not strictly linear with time for either HOCl or OC1-, HOCl was nearly linear below 0.6 log survival ratio. However, the OC1- inactivation rate slowed significantly survival level. These observations suggest that below a the mechanisms of viral inactivation by these two agents were not the same. Physical evidence of change has been detected by electron microscopy in negatively stained preparations of HOCI-treated poliovirions, even though inactivation occurred first. Some of the virions appeared to retain physical integrity after plaque titer indicated that they must have been inactive.
observed varies greatly with different viruses ( 9 ) ,as well as with the pH and concentration of salts in the virus suspension. We have found it difficult to produce suspensions of viruses without aggregation, although the data in this paper indicate that poliovirus can be prepared with only a very small amount of aggregation (approximately of the particles in small aggregates), but as yet it has not been possible to produce routinely a virus suspension with a given degree of aggregation. However, in those reports of studies of inactivation of viruses with chlorine ( I , I O , I I ) , whether as HOCl or OC1-, no real attempt to control or even to characterize the aggregation has been made. Therefore, in the work to be reported in this paper, we have utilized a suspension of poliovirus as free from aggregation as possible in order to obtain kinetics of inactivation of the virus uncomplicated by aggregation effects. Evidence is presented that the methods of virus preparation used here, as well as in the earlier work with bromine ( 1 2 , 1 3 ) , do achieve the required freedom from aggregation. This evidence, as well as a direct view of the changes produced by the chlorine, was supplied by electron microscopy. The evidence indicates an unexpected complexity in the inactivation of single virus particles. Materials a n d M e t h o d s
There is some evidence from laboratory viral inactivation studies ( I ) as well as field studies of viral contamination of water (2) that concentrations of chlorine that will inactivate bacteria to acceptable levels for drinking water will not always reduce virus concentrations to the same level. There is also evidence that aggregates or clumps of virions survive exposure to both radiation and chemical disinfecting agents which will inactivate singly dispersed particles ( 3 4 ) .Direct observation by electron microscopy ( 7 , 8 ) and differential centrifugation (7, 9) has demonstrated that suspensions of viruses, as they are usually prepared in the laboratory, are very rarely free of aggregation, particularly when the virus particle counts are >lo9 particledml. Furthermore, the degree of aggregation 438
Environmental Science & Technology
Virus. The Mahoney strain of poliovirus type 1 was grown and plaqued in human epidermoid carcinoma cells (HEp-2). Concentration, purification, and storage of virus stocks for these experiments were the same as those for the earlier poliovirus work with bromine (12, 23). Physical assay of virus suspensions was made for virion count and aggregation analysis by quantitative methods of electron microscopy as previously described (8, 9). Inasmuch as some of the experiments required exposure of the virus to chlorine solutions for time intervals as short as 1 s, the fast-flow apparatus was used ( 1 4 ) in all experiments. When exposures of 30 s or more were required, the viruschlorine mixture emerging from the fast-flow apparatus was 0013-936X/79/0913-0438$01 .OO/O
@ 1979 American Chemical Society
-
0
43
8
0 12
-4
16 1
TREATMENT TIME (seccndsl
Figure 1. Inactivation of poliovirus single particles at 2 O C by HOCI, pH 6.0: (0)1.4 pM: ( 0 )10 pM: (A)22 pM; and (0)40 pM
-
0
2
4
6
8
TREATMENT TIME (seconds)
Inactivation of poliovirus single particles at 20 OC by HOCI, pH 6.0: (0)2.2 pM; ( 0 )11 pM; (A)22 pM; and (0)35 pM Flgure 3.
TREATMENT TIME (seconds)
Figure 2. Inactivation of poliovirus single particles at 10 O C by HOCI, pH 6.0: (0)1.5 pM; (0)11 pM; (A)20 pM; and (0)41 pM
caught and held in a beaker maintained a t the temperature of the experiment (13). Samples were taken from the beaker a t proper times for virus and chlorine assay. Reagents. Hypochlorous acid (HOC1) at pH 6.0 and hypochlorite ion (OC1-) a t pH 10.0 were prepared from a 1 : l O stock solution of Fisher 5%reagent grade sodium hypochlorite solution. The stock was added to 21 L of either pH 6.0 buffer or pH 10.0 hydroxide solution to obtain the desired free chlorine concentration. This concentration was determined immediately prior to, and after a run, by amperometric titration with pheriylarsine oxide (PAO) (15). The pH 6.0 buffer (0.01 M phosphate containing 0.1 M sodium chloride) was prepared from Fisher primary standard monobasic potassium phosphate, Mallinckrodt analytical reagent grade sodium chloride, and deionized glass-distilled water. The buffer was made chlorine demand free by adding 2.5 m g b of chlorine. If after 2 to 4 days the buffer still contained 2.0 to 2.5 mg/L of free chlorine, it was dechlorinated with ultraviolet light and considered chlorine demand free. M sodium hyThe pH 10.0 solution (approximately droxide) was prepared from Fisher 50% reagent grade sodium hydroxide. Deionized glass-distilled water was made chlorine demand free as described above. The required amount of 50% sodium hydroxide was added to 21 L of this water to bring the pH to 10.0 just prior to the preparation of the OC1- solution. Chlorine Treatment of Poliovirus for Electron Microscopy. A standard support grid was covered with collodion and then coated by vacuum evaporation with aluminum. A drop of virus was placed on this grid in the kinetic attachment apparatus (8) where virions became attached by Brownian bombardment. Unattached virus was washed away and HOCl solution was applied. When treatment was finished, 5 mM sodium thiosulfate (Na2S203) solution was applied to terminate the reaction. The virions, still attached to the aluminized film, were negatively stained with neutralized phosphotungstic acid (PTA) and examined in the electron microscope.
Results Inactivation by HOCl at pH 6. The inactivation of poliovirus single particles by HOCl at pH 6 was observed at four
T R E A T M E N T TIME ( S E C O N D S )
Inactivation of poliovirus single particles at 30 OC by HOCI, pH 6.0: (0)1.4 pM; (0) 10 pM; (0)20 pM; and (A)42 pM
Figure 4.
different temperatures, 2, 10, 20, and 30 OC, and the results are shown in Figures 1,2,3, and 4, fespectively. The concentrations used in these experiments were in the range 1.4 to 40 pM (0.1 to 2.8 mg/L). Because the virus suspension used in these experiments could not be definitely shown to be free of aggregation at a level below of the particle count (see below), all experiments were termirlated at a survival level no greater than loe4. With single particles at the chlorine concentrations used (above), the survival level was reached in times less than 1 min. It was therefore essential to utilize the fast-flow apparatus (14) to be able to take samples at intervals as short as 1s. The kinetic time curves as shown in Figures 1-4 show a generally consistent pattern of inactivation regardless of temperature. At the lower concentrations of 1-2 pM (0.07-0.14 mg/L as Cl2) the reaction appeared to be linear, although in the times taken, the survival level reached was less than 10-l. At higher concentrations of 20-40 pM (1.4-2.8 mg/L as Clz), and especially a t the highest temperature of 30 OC, there was an initial lag on the inactivation curve which persisted a few seconds to about -0.6 log at most, but after this point the major portion of each curve was linear in character. These results are similar to previous work with single particles in poliovirus and inactivation by HOBr a t 10 and 20 "C (12). In order to compare reaction rates at the four temperatures, we have taken the slope of the straight-line portion of the kinetic curves, calculated by the least-squares method, as a measure of the rate of reaction and this has been plotted against the chlorine concentration in a manner similar to that for HOBr and other bromine compounds (12, 13). These curves are shown in Figure 5. At all four temperatures, the points, when plotted in this manner, fell on a biphasic curve, similar to HOBr a t 2 OC, and to NBr3 a t 4 OC (12,13). It can be seen from these curves that (i) an increase in temperature resulted in an increase in reaction rate, and (ii) a sharp decrease in slope was observed in the first section of the curveb. However, within the second portion of the biphasic curve there was still a slow increase in inactivation rates with increasing HOCl concentration. This was most pronounced at 20 and 30 "C, while at 2 "C, an increase in HOCl from 20 to 40 pM (1.4 Volume 13, Number 4, April 1979 439
"1
IO
H i
I
CHLORINE [&MI ( H O C l , p H 6 0 )
Figure 5. Relation between the rate of inactivation of poliovirus single particles at four temperatures to the concentration of chlorine as HOCI, pH 6: (0)2 O C ; ( A ) 10 OC;(0)20 O C ; and (A)30 O C . A portion of the 30 O C data is shown in the main figure: the entire 30 O C data are shown in the inset. The abscissa and ordinate for the inset are the same as for the main figure Figure 7. Electron micrograph of poliovirus particles deposited by Brownian bombardment from a suspension of purified particles. Some of the aggregates which appear in this micrograph are formed by coincidence, and their number can be calculated by probability as in the text; X 18 000
-4d
30 6b 90 I b O I50 T R E A T M E N T TIME ISECONDSl
Id
Figure 6. Inactivation of poliovirus single particles at 20 O C by OCI-, pH 10.0: ( 0 )3.5 pM: ( A ) 7.5 pM: (0)15 pM; and (0)30 pM
to 2.8 mg/L as Cls) yielded only an increase in rate from 0.165 log/s to 0.2 log/s. These results show that low concentrations of HOCl are the most efficient for inactivation of the virus. Inactivation by OC1- at pH 10. The results of four experiments with hypochlorite ion (OC1-) at pH 10 are shown in Figure 6. The reaction rates under these conditions were considerably slower than HOCl at pH 6, so much so that it was necessary to take samples for virus titration a t intervals of 0.5-1.0 min in order to obtain noticeable inactivation. All tests were made at 20 "C. The overall shape of the kinetic inactivation curve appeared to be OC1- concentration dependent. The lowest concentration of 3.5 WM(0.25 mg/L) yielded a curve having an initial lag of about 1.0 min, then became linear out to -1.8 log inactivation in 6 min with an inactivation rate of -0.34 log/min, or -0.0057 log/s. At 7.5 p M OC1- (0.53 mg/L) the initial activation was linear with an inactivation rate of -0.9 log/min, or -0.015 log/s. After 2.0 min, the reaction rate appeared to be reduced. A t the highest concentrations tested, 15 and 30 pM (1.07 and 2.14 mg/L), the same trend was observed. After a 2-min linear reaction, the rate dropped with increasing time resulting in a retardant dieaway. The initial rates for these two concentrations were -1.78 and -1.95 log/min for 15 and 30 p M OC1- (1.07 and 2.14 mg/L), respectively. Thus, there appeared to be no significant increase in the initial rate of inactivation with increasing OC1concentration from 15 to 30 FM OC1-. These results are similar to those reported for OBr- at pH 10 (13),and they suggest that the mechanism of inactivation by OC1- is different from that by HOC1. In addition, the reaction rate of OC1- was roughly one-tenth that of HOC1. For example, at 22 pM HOCl (1.6 mg/L) at pH 6 the survival ratio was reduced to in 440
Environmental Science & Technology
7.5 s; with OC1- at 15-30 p M at pH 10 it took 70 s to produce the same result, even though the HOCl concentration at pH 10 was only 0.06-0.12 pM (0.004-0.008 mg/L). State of Dispersion of t h e Virus. In view of the curvature of most of the disinfection-kinetic time curves (Figures 1-4, and 6), and particularly because of the decreasing reaction rates with OC1-, the monodispersity of the virus preparation must be clearly established. Figure 7 shows a part of a typical electron micrograph taken for this purpose. The few small groups of particles can be readily distinguished from the single particles even at the low magnification used here. The low magnification is essential in order that a statistically significant number of virions be present and their randomness of distribution be apparent. The number of aggregates actually in suspension must be less than the number observed for the following reason. In the kinetic attachment apparatus, particles are allowed to deposit by random Brownian bombardment on a thin film of aluminum on an electron microscope grid. Thus, during this process, two, or even more, particles may come to rest together forming a pair, triplet, etc. This is most especially true from a concentrated virus preparation and has the effect of causing an artificial increase in the number of aggregates seen on the micrograph. Specifically, when the whole set of 4 negatives of which Figure 7 is a part were projected, 6152 virions of 5 mm diameter were counted on a total projected area of 4 m2. Of this total, 4956 were free (not touching any other). This is 5%more free particles than predicted (16)for coincidence aggregation from a suspension of an equal number of single particles. It appears that if there is any true aggregation in this virus preparation it is too small to be seen by electron microscopy and also too little to show as a decrease in reaction rate at the level of survival in Figure 2. Physical Degradation of t h e Virus Particles. In a previous paper (6),we presented data showing the degradation of reovirus particles after treatment with HOBr a t pH 7.0. Such treatment caused immediate loss of viral RNA, and some destruction of the coat protein, but the number of degraded particles was the same as the number of untreated particles in the controls. This type of physical change appeared to be unique, and hence it was of considerable importance to ex-
amine the effects of HOC1 on poliovirus. Such a process could not he undertaken until the test outlined in Materials and Methods was devised. Because of the small size of the poliovirus particles, attempts to examine particles after HOCl treatment in fluid suspension were unfruitful. The degraded particles would not attach yell to the grid. Therefore, intact particles were allowed to attach to an aluminized grid and the HOC1 treatment was performed in situ. The results are shown in Figure 8. Untreated poliovirus particles are shown in Figure 8A. Most particles are intact and exclude the PTA stain, although a few particles in various stages of disruption are also present. Chlorine-treated particles are shown in Figures 8B and 8C. After 15 s of treatment with 5 pM HOC1 (0.36 m g b ) , very few of the virions were damaged sufficiently to permit penetration by the PTA. This treatment should have reduced the survival ratio helow 1%.After 15 pM HOC1 (1.07 m g b ) , Figure 8C, there were as many as one-third of the particles that permitted PTA penetration, as well as many particles which appeared in further stages of degradation. Some particles seemed to he intact in spite of an estimated survivalratio of Exposure of the virus to 30 pM (2.14 m g L ) for 15 s (not shown) produced debris difficult to identify as virus, although some intact particles remained (estimated survival ratio 10-6.0). Discussion Recent work in this laboratory (12,131 on the inactivation of monodispersed preparations of poliovirus with several different active forms of bromine has revealed inactivation rates much greater than those reported in the literature for chlorine. Now that similar experiments with monodispersed preparations of virus have heen made with chlorine, it appears that the difference between HOBr and HOC1 is not as great as previously believed. For example, hy comparing Figure 3 of a previous paper (12),it can he seen that 22 pM HOBr a t 2 "C reduced the plaque survival level to in 16 s. It took 40 pM HOC1 to produce essentially the same results (Figure 1, this paper). Both of these rates are greater than those reported by Scarpinoet al. (1,171,Weidenkopf (111, or Symons and Hoff ( I O ) . It is possible to draw roughly a tangent line to the semi-log inactivation curve of Symons and Hoff (10) a t zero time, which reveals an initial rate for their virus preparations approximately the same as for our poliovirus preparations given in Figure 1. The fact that their reaction rate decreases with time is probably evidence that surviving plaque-forming units (PFU) are virus aggregates of many sizes. If their virus preparation contained a high proportion of single particles, the initial rate would he expected to equal that of an all-singles preparation, and this is apparently the case. The effects of chlorine and bromine are most conspicuously different when OC1- qnd OBr- are compared with each other, and with HOC1 and HOBr. Hypobromite ion i s more effective than HOBr, whereas HOC1 is far more efficient than OCI-. The relative rates appear to he in the order: OBr- > HOBr > HOCl > OC1-. These findings are a t variance with those of Scarpino et al. ( I , 171, hut are similar in one respect to the data of Weidenkopf ( 1 1 ) . The data from two papers by Scarpino et al. ( I , 171, using the Mahoney strain of poliovirvs type 1, show OC1- inactivating more rapidly than HOCI, the reverse of the results reported here. For instance, with respect to HOCI, Scarpino et al. reported that a t 14 pM (1 m g L ) their time to inactivate to -2.0 log (99%)a t pH 6,5 "C, in phosphate buffer would have heen 117 s. Under quite similar circumstances, although a t 2 "C, Figure 5 of this paper shows that a t 14 pM our rate would have been 0.11 log/s, or 18.2 s to reach -2.0 log. This is in the ratio of 18.2117; our rate is therefore about 6.4X faster than that of Scarpino et al. Conversely, a t 7 pM (0.5 mgL) of OCI-,
Figure 8. Electron micrograph of poiiovlrus particles stained by PTA and examined: (A) in the untreated state, (B) after 15 s of treatment with 5 pM HOCI, pH 6 , and (C) after 15 s of treatment with 15 p M HOCI, pH 6 x43 200
Scarpino's rate to reach -2 log was 50 s. Our time to reach -2 log a t 7.5 pM OC1- (0.53 mg/L) was 153 s. Thus, the rate of Scarpino et al. is approximately 3.1X faster than ours for OCI-. This difference may he related in some as yet obscure way to the fact that Scarpino e t al. ( I , 17) have treated their virus a t pH 10 in borate buffer containing 0.05 M KC1. We were not able to obtain an aggregation-free suspension of virus under these conditions. Even in the presence of the KCI, horatebuffered virus sedimented in the ultracentrifuge by the Single Particle Analysis test (9) faster than single poliovirions; we have avoided for the present any unexpected complications hy adjusting the chlorine solution to pH 10 with NaOH. The pH remained constant during the experiment and there was no evidence of aggregation. In contrast to the data of Scarpino et al., the results ohtained by Kott et al. (18)indicated that OCI- was less effective as a disinfecting agent than HOC1, consistent with our data reported here. With the data from the paper by Weidenkopf ( I I ) , there is not as marked a difference between his data and ours presented here, although his high pH experiments show one similarity to ours a t pH 10. Weidenkopf shows (taken from his Figure 4) 141 s to reach -2 log at 5.5 pM HOC1 (0.39 m g L ) a t pH 6 in phosphate buffer using Mahoney poliovirus. Our data show a rate of 0.042 log/s a t 5.5 pM, or 47 s to reach -2 log. The ratio here is 47:141; our data are therefore about threefold faster. Weidenkopf did not perform any tests a t pH 10 as in our data here, bot the results of his experiments perVolume 13,Number 4. April 1979 441
formed a t pH 6,7, and 8.5 showed a definite reduction in reaction rate as the pH was increased. This suggests that the rate of any test performed a t pH 10 would have been quite slow, consistent with our data. Examination of Figures 1-4 reveals no evidence of decrease in reaction rate with time, a feature frequently attributed to the presence of aggregates. Now that the influence of aggregates has been removed, there is evidence of a short delay before inactivation reaches maximum velocity as though a definite time were required for reaction and penetration of the large number of HOCl molecules with and through the capsid of the virus, as in a diffusion-limited process. In addition to the rate differences between the inactivation of poliovirus by HOCl and OC1-, there are qualitative differences which must be considered. The inactivation by HOCl at all temperatures tested was characterized by a short lag period, followed by a linear semilogarithmic inactivation rate. This was also seen with HOBr a t 10 and 20 OC (12). On the other hand, the inactivation of poliovirus by OC1- a t 15 and 30 yM was characterized by a decreasing rate semilogarithmic curve, as was OBr- (13).It appears that the reaction mechanisms may be substantially different between the two forms of each halogen, but similar when the two acid forms (HOC1 and HOBr) are compared, or when the two ionic forms (OC1- and OBr-) are compared. While the data presented here will allow no conclusion on this possibility, the considerable rate and kinetic curve differences suggest it very strongly. At OC1- concentrations of 15 and 30 yM (1.07 and 2.14 mg/L), the reaction rate decreased with time below a survival level of This would be expected for a virus suspension containing aggregates of many sizes. However, evidence has been brought to define the level of aggregation that remained in the virus suspensions used in this work. Typical pictures, such as Figure 7, have no more virions in groups that can be accounted for by accidental falling of one upon another during their deposition upon the film (16). Thus, aggregation is certainly not enough to influence the survival ratio above the level of 10-2 (19). Doubtless the best evidence that aggregates are not responsible for the decreasing reaction rate with OC1- in the region of survival ratio is that no such decrease was seen in this region with HOCl and the same virus. Electron microscopy of negatively stained poliovirus particles (Figure 8) exposed to chlorine has shown considerable physical damage when applied at a concentration sufficient to cause loss of infectivity to a calculated level of At this level many particles appeared normal, that is, excluded PTA, although about one-half of the particles appeared damaged sufficiently to permit entry of the PTA. At the survival level, some normal appearing particles were found. Since all tests performed for electron microscopy were allowed to incubate for 15 s, these results indicate that the loss of titer precedes the actual damage to the particles. The observed damage, therefore, is not the event which leads to inability to form plaques, but some event which is indistinguishable by electron microscopy is responsible for loss of titer. The effect of HOCl on poliovirus is in contrast to the results of HOBr on
442
Environmental Science 8 Technology
reovirus (6), as reovirus RNA was ejected from the particles, while the total number of particles remained constant. Reovirus capsids suffered some structural damage but remained recognized in the electron microscope. It must be emphasized that the disinfection rates obtained here with carefully prepared suspensions of single virions are for purposes of comparison of one virus with another, one reagent with another, etc. Aggregates must be avoided in this work but aggregates will doubtless be present in practical sterilization of water in the field and so virus inactivation there must be slower. Acknowledgment
We acknowledge the excellent assistance of Dr. Guy Inman for performing the chlorine assays and for preparing buffer solutions, as well as that of William Rumpp and Natalie Moore for their work with the electron microscopes. Literature Cited (1) Scarpino, P. V., Berg, G., Chang, S. L., Dahling, D., Lucas, M.,
Water Res., 6,959-65 (1972). (2) Viswanathan, R., Indian J . Med. Res., 45 (Suppl.), 1-29 (1957). (3) Sharp, D. G., Kim, K. S., Virology, 29,359-66 (1966). (4) Kim, K. S., Sharp, D. G., Radiat. Res., 33,30-6 (1968). (5) Salk, J. E., Gori, J. B., Ann. N.Y. Acad. Sci., 83,609-37 (1960). (6) Sharp, D. G., Floyd, R., Johnson, J. D., Appl. Microbiol., 29, 94-101 (1975). (7) Young, D. C., Sharp, D. G., Appl. Enuiron. Microbiol., 33,168-77 (1977). ( 8 ) Sharp, D. G., in “Proceedings of the 32nd Annual Meeting of the Electron Microscopy Society of America”, C. J. Arceneaux, Ed., p 264, Claitors Publishing Co., Baton Rouge, La., 1974. (9) Floyd, R., Sharp, D. G., Appl. Enuiron. Microbiol., 33, 159-67 (19771.’ (10) Symons, J . M., Hoff, J. C., Proceedings of the AWWA Water Quality Technology Conference, No. 2A-4a, AWWA Research Foundation, Denver, Colo., 1975. (11) Weidenkopf, S. J., Virology, 5,56-67 (1958). (12) Floyd, R., Johnson, J. D., Sharp, D. G., Appl. Environ. Microb i d , 31,298-302 (1976). (13) Floyd, R., Sharp, D. G., Johnson, J. D., Enoiron. Sci. Technol. 9,1031-5 (1978). (14) Sharp, D. G., Floyd, R., Johnson, J . D., Appl. Enoiron. Microbial., 31,173-81 (1976). (15) “Standard Methods for the Examination of Water and Wastewater’’, 14th ed, pp 322-6, American Public Health Association, Washington, D.C., 1975. (16) Sharp, D. G., Buckingham, M. J., Riochim. Riophys. Acta, 19, 1s-21 (1956). (17) Scarpino, P. V., Lucas, M., Dahling, D. R., Berg, G., Chang, S. L., in “Chemistry of Water Supply, Treatment and Distribution”, A. J . Rubin, Ed., pp 359-68, Ann Arbor Science Puhlishers, Ann Arbor, Mich., 1954. (18) Kott, Y.,Nupen, E. M., Ross, W. R., Water Res., 9, 869-52 (1975). (19) Wei, J. H., Chang, S. L., in “Disinfection-Water and Wastewater”, J . D. Johnson, Ed., p p 11-47, Ann Arbor Science Publishers, Ann Arbor, Mich., 1975. Receiued for review April 17,1978. Accepted October 30,1978. Work supported by Grant No. R804635010 from the U.S. Environmental Protection Agency.
