T3 BACTERIOPHAGE PENETRATION OF 3HUMAN EPITHELIAL CELLS IN THE PRESENCE OF DIMETHYL SULFOXIDE Thesis for the Degree of M. S. MICHIGAN STATE UNIVERSITY WILLIAM CHARLES WALLEN . 1968 H‘— ‘ LIBRARY Michigan State _-, University k 3—— __ THESIS amnmc BY.” _ TIME 3. sons' : enuxemnmmcs : LIBRARY BIN DE RS I I Illnlnn. unnu--..' (- ABSTRACT T3 BACTERIOPHAGE PENETRATION OF HUMAN EPITHELIAL CELLS IN THE PRESENCE OF DIMETHYL SULFOXIDE By William Charles Wallen Dimethyl sulfoxide. a chemical solvent which penetrates cell membranes. rapidly mediated the uptake of T3 bacteriophage into a foreign host. Cytotoxic studies on the effect DMSO has on the AU human epithelial cell line showed that 10% DMSO was the most effective concen— tration to use. DMSO. in concentrations of 10%.or less. was shown to have no effect on the infectious titer of the T3 bacteriophage. Electron microscope autoradiographs of the beta tracks demonstrated the location of the tritium labeled phage within the cytoplasm and. in a few cases. the nucleus of the cellso T BACTERIOPHAGE PENETRATION OF HUMAN EPITHELIAL CELLS IN THE PRESENCE OF DIMETHYL SULFOXIDE BY William Charles Wallen A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Microbiology and Public Health 1968 Dedication To my wife. in appreciation of her kindness. understanding. and support which contributed immensely to the com- pletion of this work. ACKNOWLEDGEMENTS The author expresses his appreciation to Dr. Walter N. Mack for his guidance and encouragement throughout the completion of this work. I also wish to thank Dr. L. F. Wolterink for his guidance; Dr. William Deal and Dr. Peter Hirsch for their critical review of this manuscript: Mrs. June P. Mack of the Electron Microscope Laboratory. Biology Research Cen— ter. for performing the electron microsc0py; Dr. Gordon C. Spink for guidance and assistance with the autoradiography; and Mrs. Radene B. Winterton for her valuable technical assistance. iii TABLE OF CONTENTS ACKNOWLEDGEMENTS. LIST OF TABLES. . . . . . . . . . . . . . . LIST OF FIGURES LIST OF GRAPHS. . . . . . . . . . . . . . . . . . INTRODUCTION. MATERIAL AND METHODS. . . . . . . . . . RESULTS . . . . . . . . . . . . Cytoxic response of epithelial cells to dimethyl sulfoxide Reduction in number of phage particles from suspension medium when placed in the presence of AU cells and dimethyl sulfoxide Electron microscope autoradiographs of tritium labeled T3 bacteriophage in human epithelial cells DISCUSSION. . . . . . . . . . . . . . SUMMARY . . . . . . . . . . . . . . . . . . . . . REFERENCES CITED. iv Page iii vi viii 13 57 65 66 Table LIST OF TABLES Page Cytotoxic effects of DMSO. in various con- centrations. on human epithelial cells. . . 14 The effect of DMSO on the reduction in pfu of phage particles from the suspending medium in the presence of AU cells. . . . . l6 Scintillation counts of: dialysis samples (20X). virus suspension before concen— tration. and supernatant suspension after concentration . . . . . . . . . . . . 23 The effect of DMSO on the incorporation of labeled T -bacteriophage into AU cells. as shown y counts of beta tracks on the electron microscope autoradiographs . . . . 28 Summary of beta tracks per cell found on autoradiographs . . . . . . . . . . . . . . 29 Figure 10. LIST OF FIGURES A human epithelial cell from experimental group I. exposed to 10%.DMSO and un- labeled T3 bacteriOphage . . . . . . . . A human epithelial cell from experimental group II. exposed to H3-1abeled T bac- teriophage but without any DMSO. I . . A beta track (arrow) located over the cyto— plasm indicating the location of a labeled phage. . . . . . . . . . . . . . . . . . . A higher magnification of the previous (Fig. 3) micrograph. . . . . . . . This beta track (arrow) is located within the cytoplasm. . . . . . . . . . . . . This cell has two beta tracks (arrow) asso— ciated with the cell membrane. . . . . . . A beta track (arrow) is located associated with the cell cytoplasm. . . . . . . . A beta track (arrow) located over the n cleus of a cell exposed to 10%.DMSO and H labeled T3 phage. . . . . . . . . . A higher magnification of the previous (Fig. 8) micrograph. . . . . . . . . . A beta track (arrow) is located over the nucleolus within the cell's nucleus. vi Page 34 36 38 42 44 46 48 50 52 LIST OF FIGURES (Continued) Figure Page 11. This micrograph demonstrates beta tracks made in the nuclear emulsion . . . . . . . 54 12. Beta tracks formed in nuclear emulsion after exposure to a flash of light from a 100 watt overhead lightbulb. . . . . . . . . . 56 vii LIST OF GRAPHS Graph Page 1. Summary of plaque reduction studies . . . . 21 2. Dialysis removal of exogenous tritium label from labeled phage suspension. . . . . . 24 viii T3 BACTERIOPHAGE PENETRATION OF HUMAN EPITHELIAL CELLS IN THE PRESENCE OF DIMETHYL SULFOXIDE INTRODUCTION Dimethyl Sulfoxide (DMSO). a by—product of paper manufacturing. was first produced by Alexander Saylzeff in 1866. The DMSO molecule is composed of sulfur. oxygen. and two methyl groups located at the "corners." The sulfur- oxygen bond being highly dipolar and aprotic. results in DMSO being an excellent solvent (22). DMSO has been shown to penetrate most cells. apparently without destroying the integrity of the membrane (18). Its penetrant and solvent properties make it useful in the topical application of drugs (20). DMSO has also been used to enhance the absorp— tion of drugs in vivo (5). and to preserve cells in the frozen state (9. 4). The penetrating capacity of DMSO has been used to mediate the uptake of molecules varying in size from D o (28). sugars (10). and proteins such as in- 2 sulin (16) to large polypeptides of molecular weight in excess of 300.000 (17). and infectious viral nucleic acid 1 into susceptible host cells (2. 30). Amstey (2) used in- fectious polivirus RNA and African Green Monkey Kidney cell monolayers to demonstrate that a significant amount of the nucleic acid was absorbed into the cell within the first few minutes after exposure to DMSO. The mechanism of ac- tion of DMSO is unclear as yet. but its effect is apparently to increase the rate of movement of certain molecules across membranes into the cell. This effect increases as the size of the molecule being transported increases (3. The present study utilized DMSO as a mediator of bacteriophage penetration into a foreign host. cultured human epithelial cells. A continuous human epithelial cell line isolated by Wheeler (31) was used as the host. The T3 bacteriophage was the virus used because of its foreignness for the host. its small size. and its ease and accuracy in titration. Brief studies were initially performed to determine the toxicity level for the cells when exposed to varying concentrations of DMSO. Plaque reduction studies were then performed to determine if there was any significant reduc— tion of phage in the medium in the presence of DMSO and the foreign host cells. Electron microsc0pe autoradiographs were prepared to demonstrate that the tritium labeled phage had penetrated the foreign host and to localize the posi- tion of the viral particles within the epithelial cells. Electron microscope autoradiography permitted di- rect visualization of the radiation emitted by a radioiso- tope. The emitted beta rays effect the silver salts in photographic emulsion in a manner similar to visible light rays. Upon development. the salts that were sensitized by the beta rays are reduced to metallic silver grains and appear black. The isolated sensitized grains in the emul- sion or. if the beta ray was sufficiently energetic. the tracks of grains made by the beta rays are readily photo- graphed. Thin layer nuclear emulsions of Sn or less pro- vide the best resolution when used with a beta emitter or low energy. The use of H3 as the beta emitter improves resolution because its low energy limits its effects to within In of the labeled particle (14). The T bacteriophage was labeled with H 3 thymidine. 3 The labeled nucleotide is incorporated into the nucleic acid of the bacterium and then during the phage replication cycle. the radioisotOpe is incorporated into the DNA of the bacteriophage. The labeling efficiency of a radioisotope incorporated into the nucleic acid of a bacterium depends on the specific activity to the isotope. the dilution of the labeled compound with nonlabeled intracellular pools. and the bacteria's capacity to metabolize the labeled com- pound (15). MATERIALS AND METHODS The T3 bacteriophage is a small. DNA virus which specifically attacks the bacterium Eacherichia coli. strain E. Its dimensions are: 47x47 mu head. 10x15 mu tall. and a DNA content of 0.9x10-16 grams/particle (27). The exper— imental host cell was the human epithelial cell (AU) iso- lated in cell culture by Wheeler (1956) (31). The AU cells were maintained on a medium of lactalbumin hydrolysate. (LAH) (24). with 2% inactivated calf serum. Monolayers of AU cells were grown in French square bottles to a concen- tration of approximately 1x105 cells/ml for plaque reduc— tion tests. The monolayers were grown in milk dilution bottles to a concentration of 1.2x106 cells/ml for the autoradiographic studies. Cellular toxicity studies were performed by expos— ing three day old cell culture monolayers to varying con- centrations of DMSO. The concentration of DMSO (reagent grade) was varied from 3% to 100%. The DMSO was mixed with the maintenance medium (LAB-2% inactivated calf serum) and added to the cell monolayers. The cells were then placed in the incubator (37C) for 60 minutes. The DMSO media was then replaced with maintenance media lacking DMSO and the monolayers were allowed to grow for 24 hours. The cells were then compared under light microscope examination for apparent alterations that occurred 24 hours after exposure to DMSO as compared to controls not receiving DMSO. The selection of the Optimal concentration of DMSO was based on light microsc0pe examination of the cells to determine which concentration caused the least apparent alteration in metabolism or cell appearance due to exposure for per- iods up to 60 minutes. After determining that 10%.DMSO did not demonstrate toxicity to the AU cells plaque reduction studies were per- formed by exposing cell monolayers to 10% DMSO and T3 bac— teriophage simultaneously for varying lengths of time. Controls involved (a)'T3 phage exposed to DMSO only and (b) T3 phage exposed to AU cells only. After appropriate time periods of exposure. the phage was assayed on E. 291; B by the soft agar technique (19). The assay by Gratia (1936) (12). described by Adams (1950) (l). and modified by Law- rence (l957)(l9). was performed in the following manner: 0.5ml of a 24 hour suspension of growing E. coli B cells was placed into a test tube containing 2.5ml of melted soft agar (7.5 g agar in 1 liter of water) at 45 C. Then 0.1ml of an apprOpriate dilution of the T phage sample was 3 placed in the bacteria-agar mixture. The soft agar—phage- bacteria mixture was poured over a plate of nutrient agar. spread uniformly over the plate surface. and incubated at 37 C. Plaques. clear areas in the bacterial growth due to lysis of the bacteria appeared in about 6 hours. They were counted after 14 to 16 hours of incubation. The number of viable phage particles per ml. was determined by counting the number of plaques. multiplying by 10 to compensate for the 0.1ml inoculum. and then multiplying by the dilution factor. Luria. et al. (1951) determined that this method was extremely accurate for the titration of phage and that one plaque corresponded to one infectious phage particle (21) . All phage dilutions were ten fold dilutions performed in 2% nutrient broth (Difco). Tritiated (H3) thymidine labeled T bacteriophage 3 and AU cells were prepared for-autoradiography in the fol— lowing manner: A thymine deficient E. coli B mutant was selected by the aminopterin solid medium technique (8). The bacteria was grown overnight in a defined medium lack- ing thymine. Approximately 108 bacterial cells were spread over a plate containing supplemented A medium consisting of (in grams per liter): KZHPO4. 10.5; KH2P04: Sodium citrate 5H 0. 0.47; MgSO4. 0.05; (NH4) so 2 . 1.0; agar. 15; casamino‘ 4 acids. 5: adenosine. guanine. cytocine. thymidine. and tryp- tophane. 0.1 each; aminoterin. 0.5; glucose. 2; and thia- mine. 0.001. The cultures were incubated for 24 hours at 37 C. Several of the resulting colonies were selected and tested for thymine dependence. Those mutant colonies which could not grow without exogenous thymidine were maintained on refrigerated aminopterin plates. Tritiated thymidine (29) (spec. act. > 99%{ lZc/ml. diluted to lZOmc/ml) was substituted in fresh aminopterin plates for the non-labeled thymidine. The E. 521; B was incubated for 16 hours at 37 C on the solid medium to label its nucleic acid pool. The phage was mixed thoroughly with the labeled g, 9911 and incubated for an additional 16 hours at 37 C. The labeled phage lysate was harvested from the plates with 2-3 ml of sterile H20 and collected in a sterile 20ml. syringe. The labeled phage was then clarified by two cycles of centrifugation (5.000 RPM; 15 minutes Interna- tional PR-l). It was then filtered through a sterile HA millipore filter (Millipore Corporation. Bedford. Mass.). The lysate was dialyzed against 200ml of 2% nutrient broth. The dialysate was changed at one hour intervals and samples (20A each) were collected to determine the rate of removal of dialyzable H3—thymidine label from the phage suspension. The samples were individually counted on a Mark IV Scintil— lator in a toluene based scintillation fluid. The 50ml of phage suspension was concentrated to 5ml of material in an ultracentrifuge (Spinco; Type 50 ro- tor. 50.800xg. 70 min.). The pellet was resuspended with 5ml of sterile distilled water. low speed centrifuged (5.000 RPM. 15 min. International PR-l). and the resultant 10ml supernatant fluid was concentrated to 2ml of suspen- sion by ultracentrifugation (Type 50 rotor. 57.900xg. 60 min.). Biological (pfu/ml) and Radiological (cpm/ZOA) ac- tivities were determined on the approximately one hundred fold concentrated by volume phage suspension. A 0.1ml phage inoculum was used on each monolayer of AU cells. Three groups of cells were used in the experiment. Group A was used as the control to determine the level of phagocytosis of the phage by the cells. It was inoculated with labeled phage for 60 minutes without DMSO. The 60 minute time period was selected because that was the great- est length of time during the experiment that the cells 10 to DMSO and phage. Two other groups of cells were used. varying in length of exposure to. and the concentration of. DMSO. Group B was inoculated with labeled phage and ex- posed to 10% and 20% DMSO for 1-1/2 minutes. This group was used to determine whether the action of DMSO was in the presence of cells was immediate or delayed. Group C was inoculated with labeled phage and also exposed to 10% and 20%.DMSO for 60 minutes. The controls for the auto- radiograph emulsion consisted of the following: (a) Ex- posure of a test grid to 10 sec of sunlight and a 100 watt lightbulb. and (b) unlabeled T phage. AU cells. and 20% 3 DMSO. All cell monolayers were washed three times with Hanks' basal salt solution and the cells were removed from the glass surface with a sterile rubber tipped glass rod. They were immediately fixed in 6.25% glutaraldehyde in Sor— enson's buffer solution (25). Dehydration was done by a sequential gradient of ethyl alcohol. followed by two changes of propylene oxide. The cells were then placed in a 1:1 propylene oxide: epon 812 mixture followed by em- bedding in epon 812 at 60 C for 48 hours. Ultrathin sections of the hardened cell blocks were cut on a Sorvall MT—Z ultramicrotome at 500 to 700 A. Each 11 section was placed on a colloidion based COpper mesh (100 and 200) grid. The emulsion used was the Ilford L-4 (Il- ford Co.. England) nuclear emulsion. It has a grain diam- eter of 1200 A and a sensitivity to H3 of 132 grains/100 decays (26). Its principle qualities are good autoradio— graphic resolution (about 0.1u)(6). low background. high sensitivity. insensitivity to yellow-green light. good re- producibility on monogranular layering. and easy storage. The loop technique was used to place a thin layer of emulsion on the grids (7). Ten milligrams of Ilford L-4 was melted at 45 C for 15 minutes in 20ml of distilled water in a 250ml beaker. The mixture was constantly stirred. then placed in an ice bath for 3 minutes. and left at room temperature for 20 to 30 minutes. This pro— cedure resulted in a highly viscous emulsion. The grids were placed on circular glass cover slips on aluminum plan- chets. The planchets were in turn placed on corks suffi— ciently large to stabilize them. A 4cm. diameter wire loop was dipped into the emulsion and slowly withdrawn. The film formed in the 100p gelled rapidly. It was quickly placed over the planchets causing a fine even emulsion to fall over the grids. The grids were placed in a light-free box with dehydrant and allowed to develop 4 weeks in the dark. 12 The emulsion was develOped in Kodak D-l9 for l min— ute. rinsed in water for 30 seconds. It was then fixed in Kodafix solution for 1 minute. and rinsed in slowly circu- lating water for 5 minutes. After the emulsion was develOped. the sections were doubled stained in uranyl acetate (11) for 30 minutes followed by 15 minutes in lead citrate-Reynolds buffer so— lution (23). The sections were examined for beta tracks with a Philips 100 electron microsc0pe. RESULTS The toxicity studies were performed to determine the concentration of DMSO which could be used without causing noticable changes in the cells' growth rate of appearance. The toxicity studies were conducted for a maximum at 60 minutes since. as Amstey has shown (2). the action of DMSO was relatively rapid. The results of the cellular toxicity studies are shown in Table 1. DMSO in 10% and 15%.concentration did not alter the rate of acid production of the cells for periods up to 60 minutes. Cellular appearance was slightly granular while the rate of monolayer formation remained the same as the controls. Cells exposed to 20% DMSO showed no effects for periods of 10 minutes or less. A slight reduc— tion in growth rate was noted if 20% DMSO was left on the cells for 30 and 60 minutes. The cells showed a noticable granularity and their relative rate of monolayer formation was retarded. Exposure to 50% DMSO for 1 minute and 10 minutes markedly disrupted cellular rate of acid production although many of the cells survived and were recovered. l3 14 TABLE l.--Cytotoxic effect of DMSO. in various concentra- tions. on human epithelial cells. Time (min) 1 ' 10 '30 60 DMSO Concentration 100% R F F F 75% R F F F 50% R R R F 20% N N R R 15% N N N N 10% N N N N 5% N N N N F=cells became fixed and were unrecoverable. R=cells were recoverable but metabolic rate was reduced. N=cells' metabolism was not altered. The cells were very granular for 3 days thereafter. Ex- posure for thirty minutes of 50% DMSO caused most of the cells to round up. become highly granular. and only a small ratio of the cells were recovered. Sixty minutes exposure to 50% DMSO resulted in the fixation of the cells to the glass wall. These cells were unrecoverable. Cells exposed to 75%.or 100% DMSO for 1 minute were severely al- tered in their rate of acid production. Cells exposed for 10 minutes or longer to either 75% or 100%.DMSO resulted in their irreversible fixation to the glass walls. DMSO in 10% concentration was used for the plaque reduction studies because there was no detectable 15 alteration in the cellular rate of acid production over the 60 minute test period. The plaque reduction studies were performed to de- termine the following: a) The level of infectious unit reduction when the phage was placed in the presence of AU cells and 10% DMSO (Table 2. t). b) The effect 10%»DMSO alone would have on the infec- tious phage titer (Table 2. b). c) The level of infectious unit reduction when the phage was placed in the presence of AU cells alone (Table 2. c) . The reduction in infectious units of phage was determined by counting the number of plaques on a plate covered with .§.qggli B. Each plaque represents one infectious unit or plaque forming unit (pfu). The "input" or stock titer (Table 2.a) was used as a standard and represented 0% reduction of phage (pfu). Control (b) used to determine the effect DMSO has on the phage. shows reduction ranges of 0%.to 4.4% over the tests. 16 Table 2.--The effect of DMSO on the reduction in pfu of phage particles from the suspending medium in the presence of AU cells. W Test Time Ave. No. Phage Per Cent Plaques Titer Reduction a 3 1. Input 95 9.5x103 0 Control 92 9.2x103 3 1 minute 77 7.7xlO3 18.5 2 minutes 66 6.6x103 30.5 5 minutes 65 6.5x103 32.0 60 minutes 57 5.7x10 40.0 2. Input 219 21.9x10§ 0 Control 215 21.5x102 l 1 minute 181 18.1x102 17.4 3 minutes 141 14.1x102 35.6 5 minutes 116 ll.6xlO2 47.0 30 minutes 101 10.1x102 53.9 60 minutes 98 9.8x102 55.3 Controlc 205 20.5x10 6.4 3. Input 178 17.8x10: 0 Control 215 17.4x102 0.3 1 minute 151 15.1x102 15.2 2 minutes 136 13.6x102 23.6 5 minutes 102 10.2x102 42.8 30 minutes 95 9.5xlO2 46.1 60 minutes 87 8.7x102 51.2 Control 172 17.2x10 3.4 4. Input 68 6.8x103 0 Control 72 7.2x103 —6 1 minute 51 5.1x103 25 3 minutes 55 5.5x103 19.2 5 minutes 60 6.0x103 12 30 minutes 49 4.9x103 28 60 minutes 43 4.3x103 37 Control 70 7.0x10 —2 Table 2 (Continued) 17 W Test Time Ave. No. Phage Per Cent Plaques Titer Reduction 5. Input 294 29.4x10: 0 Control 288 28.8x102 2.1 1 minute 253 25.3x102 13.9 3 minutes 239 23.9xlO2 18.7 5 minutes 226 22.6x102 23.2 30 minutes 218 21.8x102 25.8 60 minutes 211 21.1x102 28.3 Control 289 28.9x10 1.7 6. Input 71 7.1x103 0 Control 69 6.9x103 2.9 1 minute 57 5.7x103 19.7 2 minutes 53 5.3x103 25.4 5 minutes 50 5.0x103 29.6 30 minutes 42 4.2x103 40.9 60 minutes 39 3.9x10 45.1 7. Input 70 7.0x10: 0 Control 68 6.8x103 2.9 1 minute 55 5.5x103 21.4 2 minutes 53 5.3x103 24.3 5 minutes 50 5.0x103 28.6 30 minutes 44 4.4x103 37.2 60 minutes 40 4.0x103 42.9 Control 70 7.0x10 0 8. Input 74 7.4x103 0 Control 71 7.1x103 4 1 minute 54 5.4x103 27 2 minutes 51 5.1x103 31.1 5 minutes 49 4.8x103 35.2 30 minutes 41 4.1x103 44.6 60 minutes 37 3.7x10 50.0 Table 2 (Continued) 18 r Vr fl f NO. Phage f Test Time Ave. . Per Cent ' Plaques Titer Reduction 9. Input 68 6.8x103 0 Control 65 6.5x103 4.4 1 minute 60 6.0x103 12.0 2 minutes 54 5.4x103 20.6 5 minutes 50 5.0x103 26.4 30 minutes 47 4.7x103 30.9 60 minutes 43 4.3x10 36.7 a 3 10. Input 65 6.5x103 0 Control 66 6.6x103 -l 1 minute 61 6.1x103 6.2 2 minutes 56 5.6x103 13.9 5 minutes 52 5.2x103 20.0 30 minutes 48 4.8x103 26.0 60 minutes 45 4.5x10 30.8 a 3 11. Input 68 6.8x103 0 Control 67 6.7x103 1.5 1 minute 55 5.5x103 19.2 2 minutes 49 4.9x103 27.9 5 minutes 45 4.5x103 33.8 30 minutes 40 4.0x103 41.2 60 minutes 37 3.7x103 43.6 Controlc 67 6.7x10 1.6 Inputa=T stock phage titer. 3 ControlbéT and 10%.DMSO for 60 minutes. Testt=T 3 3 and AU cells and 10% DMSO. Controlc=T 3 and AU cells for 60 minutes. 19 Control (c) used to determine the effect the AU cells alone would have on the phage in the supernatant fluid. shows a reduction range of 0% to 6.4%. The experimental groups (Table 2. t) show a rapid drop in phage titer from the su- pernatant fluid over the first five minutes of exposure to DMSO. The test shows a reduction of approximately 25% of the phage titer from the suspending medium. After five minutes of exposure the rate of removal of phage from the supernatant fluid appears to reduce itself and hereafter. the action of DMSO was completed as far as reduction of phage was concerned. The results of the studies are shown in Table 2. Summarizing the tests show a consistent reduction in the phage titer (pfu) as the length of time of exposure to DMSO increases. The "Input" represents the titer of phage in a stock suspension that was used for the test. Control (b) represents the titer of phage (pfu) remaining after 60 min- utes exposure to 10%.DMSO in LAH. Control (c) represents the titer of phage (pfu) remaining in the suspension medium after 60 minutes of exposure to AU cells without any DMSO. The test periods for exposure of AU cells to 10%.DMSO with T phage were 1 minute. 2 or 3 minutes. 5 minutes. 30 min- 3 utes. and 60 minutes. 20 Graph I is a composite of all the results of Table 2. Line A shows the per cent reduction of the initial in- oculum in the suspending medium after a given time in the presence of AU cells and 10%.DMSO. The range of plaques for the tests is shown in the vertical line from one through 30 minutes. Lines B and C represent the two controls for the test (see b and c above) respectively. Both lines are represented by the broken line on the graph because they are concurrent with one another. There is no significant reduction if pfu under either of these control conditions. The reduction in pfu when the phage was placed in contact with DMSO and AU cells indicated that the phage may be either (1) entering the cell or (2) attached or associated with the cell membrane. To differentiate which of the two possibilities occurred electron microscope auto- radiography was utilized to localize the phage. A H3- thymidine labeled phage was produced from labeled E 221$ B and extraneous label was removed by dialysis for 18 hours. Table 3 shows the counts per minute obtained for the 9 (20k) samples taken during the 18 hours of dialysis. The counts per minute (cpm) began after the first hour at 68 cpm and were gradually reduced to a relatively constant level of Graph I: Effect of 10% DMSO on Reduction of T3 Phage from Supernatant Suspension in Presence of AU Cells. A = T phage exposed to 10% DMSO in the presence 'o AU cells. B = T3 phage exposed to 10%.DMSO alone. C = T3 phage exposed to AU cells without any DMSO. 22 . _ _ IT _ m _ c. A + m C .m fl 5 4 2 a: to 8:253. a 23 TABLE 3.—-Scintillation counts of: dialysis samples (201). virus suspension before concentration. and supernatant sus-' pension after concentration. m 1 1 , ========= Sample . N Jer Time Cpm Sample 1 1 hour 68.71 dialysate 2 3 hours 58.60 " 3 4 hours 37.53 " 4 5 hours 33.65 " 5 14 hours 29.28 " 6 15 hours 24.39 " 7 16 hours 26.69 " 8 17 hours 27.00 " 9 18 hours 27.48 " 10 - 5714.00 Virus suspension 11 — 36.88 lst ultracentrifuge super- natant fluid 12 - 43.88 2nd ultracentrifuge super- natant fluid 27.00cpm after 18 hours of dialysis. The dialysis proced- ure was halted at this time because it was felt that the rate of removal of label was not great enough to justify further dialysis. Since the dialyzable label was not iden— tified as to source. it was. for statistical purposes. assumed to be extraneous label not associated with intact phage particles.' The rate of removal of the dialyzable label can be seen in Graph II. Line A is the total . Graph II: Dialysis of crude phage lysate to remove dialyzable radiosotope labeled molecules and fragments. A = total counts per minute (cpm) remaining in suspension. H3-thymidine remaining in suspension (cpm). I'D II C = labeled particles. dialyzing at a slow consistent rate. remaining in suspension. 25 '0 Time (hrs) 100 50 IoI 7:an 22:8 3a macaou 26 reduction of label (counts per minute. cpm) over the eighteen hours of dialysis. Line B shows the removal of this molecule was rapid and efficient because of its small consistent size. Graph II indicates that the H3 thymidine molecule was removed after 12 hours. Line C shows the removal of a larger molecule which after approx- imately 18 hours was essentially the only source extraneous label remaining in the suspension. The slow rate at which the label was dialyzed indicates that it was attached to a large molecule which was removed very slowly through the dialyzor. It was necessary to account for the ratio of this extraneous label to labeled phage in suspension when determining the source of beta tracks found on the auto— radiographs. Sample ten (Table 3) shows the activity with— in the dialyzor to be 5.714 cpm. The 50ml suspension. con— taining both the labeled phage and the extraneous large labeled molecule was concentrated in the ultracentrifuge twice. resulting in a one hundred fold concentration by volume of the suspension. After the first ultracentrifuga- tion the upper 45ml supernatant fluid was counted and dis- carded (sample 11. Table 3). The supernatant suspension recorded 36.88 cpm for the 201 sample. The 5 m1 concentrated 27 suspension was resuspended with 5 m1 of sterile water and concentrated by ultracentrifugation again. The upper 8ml of supernatant suspension was counted (sample 12. Table 3) and discarded. The discarded supernatant suspension re— corded 43.88 cpm. The concentration of the labeled phage resulted in further removal of extraneous sources of label. such as disrupted phage or bacterial DNA. in the discarded supernatant suspensions. The results of the autoradiograph study are shown in Table 4. Because there was such a rapid reduction in the number of phage particles in the presence of DMSO in the first five minutes of exposure only the test with 90 sec- onds exposure and the controls were subject to electron microscope examination (Table 2. Graph I). Control group I. consisting of AU cells plus unlabeled T phage plus 20% 3 DMSO for 60 min. showed no beta tracks either inside or outside the cells in a total of 490 cell sections examined. No beta tracks were discernible on any of the sections examined. No beta tracks were discernible on any of the sections examined for this control group. Control Group II. consisting of AU cells plus H3 labeled T phage for 3 60 minutes but without DMSO. was found to lack any beta 28 TABLE 4.-—The effect of DMSO on the incorporation of labeled T3-bacteriophage into AU cells. as shown by counts of beta tracks on the electron microscope autoradiographs. a: J . ' k L.— “L Test Total .__V T 1. Control I-AU cells & unlabeled T &20%DMSO-60min. 3 Sect. No. l 2 3 4 5 5 No. cells 123 47 73 116 129 490 No. tracks 0 0 0 0 0 0 2. Control II—AU cells & HB-T-GO min. Sect. No. l 2 3 4 5 6 6 No. cells 103 74 126 49 113 73 530 No. tracks 0 0 0 0 O 0 0 3. Experiment III-AU cells&H3-T3&lO%DMSO-90sec. Sect. No. 1 2 3 4 4 No. cells 20 92 73 43 228 No. tracks 1 7 5 3 l6 Experiment III-AU cells&H3-T3&10%DMSO-905ec. Sect. No. 5 6 7 8 4 No. cells 38 47 18 20 123 No. tracks 2 5 2 3 ll Experiment III-AU cells&H3-T3&10%DMSO-9OSec. Sect. No. 9 10 ll 12 13 5 No. cells 24 38 36 19 22 140 No. tracks 1 3 2 0 2 8 Totals Sect. No. 13 No. cells. 493 No. tracks 35 4. Experiment IV-AU cells & H3-T3&20%.DMSO-903ec. Sect. No. 1 2 3 4 5 6 No. cells 12 38 29 73 64 28 No. tracks 1 l 3 3 5 6 29 tracks inside or outside of the cells in 530 cell sections examined. Experimental group III. consisting of AU cells plus H3 labeled T phage and 10% DMSO for 80 sec. showed 3 35 beta tracks associated with 493 cells. Experimental group IV. consisting of AU cells plus H3 labeled T phage 3 plus 20% DMSO showed that there were 15 beta tracks asso- ciated with the 244 cell sections examined. Table 5 is a summary of the data presented in Table 4 and shows the results obtained for each group and the resulting number of beta tracks found associated with each cell that was sectioned. Control group I. lacking TABLE 5.~-Summary of beta tracks per cell found on auto- radiographs. — w Test No. of No. of No. of Beta Tracks Sections Tracks Cells per Cell Control I 5 0 490 0 Control II 6 0 530 0 Experiment III 13 35 493 .071 Experiment IV 6 15 244 .061 T labeled phage. shows no beta tracks found on all five of the sections examined. There were no tracks found either inside or outside of the cells. Control group II. lacking DMSO. also showed no beta tracks either inside or outside 30 of the cells in the six sections examined. Experimental group III with labeled T phage and 10%.DMSO and AU cells 3 showed 0.071 beta tracks per cell that was sectioned asso~ ciated with 493 cells examined. Experimental group IV con— sisting of the above constituents except 20%.DMSO was used instead of the lower concentrates showed 0.061 beta tracks per cell section associated with 244 cells examined. A statistical evaluation was used to determine if the beta tracks found on the autoradiographs could be at- tributed to radioisotope that was associated with intact phage particles. The statistical test used was the binomial approximation of numbers (15). The null hypothesis. that the number of beta tracks found on the autoradiographs could all be attributed to extraneous label and not the HB-thymidine labeled T phage. was established to test the 3 values obtained on the autoradiograph. Rejection of the null hypothesis gives strong evidence for some of the beta tracks coming from the labeled phage. Acceptance or rejec- tion of the null hypothesis was based on the following sta— tistical test: Let 8 represent the number of cells exam- ined with beta tracks associated with them and C represent the critical number of cells with tracks associated with 31 them required to reject the null hypothesis.' Then. let the 493 cells in experimental group III (Table 1) be the total number of cells examined for this evaluation. The 0.02 level of confidence was selected for the test. P(B lZC/mM) Tovell. D. R. and J. S. Cotter. 1967. Observations on the assay of infectious viral nucleic acid: Effects of DMSO and DEAE-dextran. Virology ‘32: 84-92. Wheeler. C. E.. C. M. Canby. E. P. Cawley. 1957. Long term tissue culture of epithelial—like cells from human skin. I. Invest. Derm. .22: 383—392.