PRODUCTEQN AND wamcmew OF T 3 BACTEREQPHAGE CONTAINiNG P 32 Thests gov the Degree of M. 5. MECHZGM STATE UNIVERSE” Robert 331. Lawrence 1957 THESIS LIBRARY Micl'xignn State University PRODUCTION AND PURIFICATION OF T3 BACTERIOPHAGE CONTAINING P32 By Robert E. Lawrence A THESIS Submitted to the College of Science and Arts, Michigan State University of Agriculture and Applied Science in partial fulfillment of the requirements for the degree of MASTER OF SCIHVCE Department of Microbiology and Public Health 1957 7/11/57 3 g 57‘ ACKNOWLEDGEENTS The author wishes to express his thanks to Dr. W. N. Mack under whose direction the work for this thesis was done. Thanks are also due to the Biological warfares Laboratory, U. S. Army, Fort Detrick, Maryland whose financial assistance enabled the author to hold the position of special graduate research assistant. TABLE OF CONTENTS INTRODUCTIONOOOOOOOOOOOOOOOOOOOOIOOOO0. 00000000000000000000000000000 1 mulmu‘ MODSCOOOOOOOOOOOOOOOOOO 000000000000 0.... 0000000000 .0 5 Table II.......... ......... .................................... 13 Table III....................... ..... .... ...... .... ............ 16 Chart I................. ................... .. ....... . ......... . 17 Table IV...................... ....... . ..... . ............... .... 20 Charts 11 to V1. .............. .... ...... ... .................... 22 Table V ...... ... ..... . ...... .... ...... . ........... . ........... . 27 DISCUSSION.................... .......... ....... ............ . ....... . 28 SUMMARY................. ........ ....... ............... . .......... ... 37 BIBLIOGRAmYOOOOOOOO00.000.000.00......OOOOOOOOOOOOOO00.00.000.00... 38 INTRODUCTION There are seven types of bacteriophage that are active against W 391.1 strain E. They are designated I and numbered 1 through 7. The 1'3 bacteriOphage is round or possibly of hexagonal shape, and measures approximately 1.5 In in diameter. The "tail" of this virus is small, and in air dried specimens is not visible in the electron micro- scope. (Hillians and Fraser 1953). Frozen specimens do show a small tail. 1‘? bacteriophage is similar morphologically to 1'3. 1'2, ‘11,, and T6 are larger, having a diameter of 60 to 80 mu, and possessing a definite tail about 100 m long. All I bacteriOphages are distinguished by their infectivity of the cannon host, Escherigg up); strain B. (Imria 1953) The availability of artificially produced radioactive isotopes has made possible a large umber of moriments which would have been ex- tremely difficult by other means. The incorporation of a radioactive isotOpe into the system under study allows the investigator to follow changes or to observe movements which would not be detectable by other chemical or physical means. Radioactive phosphorus and sulphur have been most commonly used in studying W 3211 and its associated T bacteriophages. Kosloff and Putnam (1948) Grew E. 3911 and 1'6 bacteriOphage in a medium contain- ing r32 in order to m the origin of virus constituents and the extent to which they were derived from the best. they found that 70% of the virus phosphorus was derived from the mediuu and the remainder from the host cell. The concentration of P32 which they used no 1 on (micro curis) pear I1. Labaw, Mosley, and wyckoff (1950) performed a series of experiments to follow the synthesis of nucleic acids in a culture of 2,. 32;; growing 2 3 to the extent of 105 counts per minute per in a medium labelled with P ml. They showed that P32 uptake and corresponding cell multiplication continued Just as in a non-radioactive culture. Further work by Labaw (1951) showed that the T3 bacteriOphsge derived 78% of its phosphorus from the host cell and only 17% from the medium. In this experiment, which was similar to that perfomd by Kosloff and Putnam, he determined that T1, T3, and T7 derived most of their phosphorus from the host cell. This is in contrast to T2 and T6 which derive most of their phosphorus from the medium. Hershey gt .1. (1951) investigated the rate of inactivation of 12 bacteriophage containing P32. He grew the virus in a medium containing P32 varying in concentration from 0.14 to 2.2 He (millicurie) per ml. Stout and Fuerst (1955) also investigated the rats of inactivation of 1‘2 which contained P32. Both these workers used P32 of a high specific activity. no specific activity being Isasured in no of P32 per total amount of phosphorus present. Stout and Fusrst used P32 with a specific activity ranging fran 250 to 300 Mo per mg of phosphorus. Both papers reported a gradual inactivation of bacteriophage which depended on the specific activity of the media in which they were grown. Iabaw did not use high concentrations of P32. Labaw obtained purified virus preparations by means of the ultra- centrifuge. Hershey and Stout did not find it necessary to obtain a purified virus preparation. While most of the work with E, ggl1_and the associated T bacterio- phage has been concerned with the origin of virus couponents, the fate of these components, or with genetics of bacteriophage (Stout 1955, Lesley and Grahan.1956, Volkin and Astrachan 1956, Stahl 1956, Hershey 1955) there have been at least two reports concerning the use of radioactive bacteria in aerosols. (Goldberg and Leif 1950, Buokland, Harper, and Norton 1950). These two articles were concerned with the use of bacteria containing P32 as a neane of determining the amount and extent of reten- tion of the crganisn.by the tissues of the body. Rather than grinding the tissues and plating or examining the ground up mass these workers achieved the same results by counting the radioactivity of ashed portions of tissue. The experiments described in this thesis were done in an attempt to accouplish essentially the same purpose as those of Goldberg and Bushland. That is, to assay bacteriophage labelled with 232 by counting its radioactivity rather than by determining biologically the nuaber of viable virus particles. work in this laboratory involved the use of the T3 bacteriophage in aerosols. It was found that under conditions of low halidity the re- covery of viable virus was much lower than at high hunddities; often by a factor of 1000. If, as was assumed, a large fraction of the virus population was inactivated under dry conditions, would it be possible to detect both viable and non viable virus particles by some physical or chemicalrlethodY The most obvious leans of accomplishing this purpose was to incorporate a radioactive isotope into the virus and use a Geiger- Mullsr tube to measure the amount of virus in the sample. The work done for this thesis involved: 1) 2) 3) The preparation of T3 bacteriOphage suspensions containing a high concentration of 1’3 2. The treatment of these suspensions to remove free P32. The estimtion of the effectiveness of the removal of free P32. EXPERIMENTAL METHODS P32 is a radioactive isotope of phosphorus and decays to sulfur 32 upon emission of one electron or beta particle. The average energy of this beta particle is 0.68 Mev and the nxim energy is 1.7 hev. P32 has a half life of 14.3 days. The P32 used was in the form of a Sodium phosphate solution obtained from the Abbott Laboratories, Oak Ridge, Tennessee. The specific activity of the P32 varied from 31 to 49 Mc/ag of total phosphorus and averaged about 40 tic/mg. The 1’32 solution was U.S.P. grade and was obtained a1- reedy sterilised. There are amorous methods and techniques used to count samples con- taining radioactive isotopes. If the isotope is of the type which emits particles of low energy these techniques can become quite complicated. Fortunately P32 emits a particle with a aarim energy of 1.7 uev, and is capable of penetrating a considerable quantity of letter. The method of counting used was chosen because of its simplicity. A quantity of the nterial to be counted, usually from 0.01 to 0.1 .1 was put into a round metal dish or planchet one inch in diameter. Micro pipettes were used to meastn-e the radioactive material and the pipette was washed several tines with distilled water which was also put on the planchet. The sample was then dried under an infra-red heat lamp. The samples were counted in a lead chamber with a Geiger Muller tube having a window thickness of 2.4 mg/cn2 and operating at 900 volts. The counts were registered on a Tracerlab 6L scalar. Samples of low radioactivity were counted long enough so that the standard error was less than 5%. The tine each sample was to be counted was estimated by reference to a chart prepared by U. C. Davidon (1953). All the results are expressed as counts per minute per ml of sample. No attempt was made to convert counts per minute to the amount of radioactivity in terms of millicuries. The soft agar technique for the assay of bacteriOphage was original- ly develOped by Gratis (1936) and described in detail.by Adams (1950). The procedure involves placing 0.5 ml of a heavy suspension of growing 5,‘9211_oe11s into a test tube containing 2.5 mls of melted soft agar (7.5g agar in one liter of water) at 45°C. Into this bacteria agar mixr ture is pipetted 0.1 ml of an appropriate dilution of the virus suspension. This bacteriadvirus-egar mixture is poured on a plate of nutrient agar, spread uniformly over the surface of the plate and allowed to harden. After incubation for six hours at 37°C, circular areas or placques appear as clear spots in the otherwise opaque growing bacteria. by counting the number of placques, multiplying by 10 to compensate for the 0.1 ml of virus suspension used, and then multiplying by the dilution factor one can obtain the number of viable virus particles per ml of the original suspension. This was the method routinely used for assay of viable bac- teriophage. Luria 35,5], (1951) have shown that this method is extremely accurate and that one placque corresponds to one virus particle. There are various chrosmtographio methods available to the experi- menter, all of which are described in detail in Block, Durrum, and Zweig (1955). A descending method of chroamtography was used with Hhatnan #1 chromatographic paper out into strips three centimeters wide. 0.01 to 0.05 mls of the virus suspension to be tested was placed about six on from one end of the paper strip. The strip was then suspended from the solvent trough in an air tight jar. A small amount of solvent was put in the bottom of the jar and the entire assembly left overnight while the atmosphere in the jar became saturated with solvent. The solvent was then added to the trough and allowed to move down the paper strip until it had reached the bottom. The strip was removed, dried, and dipped in a solution of 0.3% ninhydrin in 95% alcohol. After develop- ment in the dark for 18 hours a purple color appeared on the paper strip wherever the virus or other nitrogen containing constituents of the mediun had been deposited. The migration of virus is reported in terms of the Rf value. The Rf value is calculated by dividing the distance froa the center of maximum color development by the distance the solvent front travelled. RESULTS Production of T3 BacteriOphage Containing P32 The primary aim of this work was to produce virus suspensions of high concentration; at least 1010 virus particles per ml. The second consideration involved putting as much radioactive phosphorus into each virus particle as possible without decreasing the virus concentration. A limiting factor in this respect was that, due to the physical facili— ties in the laboratory and to reasons of safety, it was decided to have no nore than 10 Me of P32 available at any one time. Non radioactive virus suspensions have been produced which containp ed as many as 1011 virus particles per ml. The method of producing such suspensions is very simple and involves spreading a small quantity of a suspension of E, 9911 over the surface of an agar plate with a sterile glass rod. After incubation for 18 hours at 37°C, 0.5 mls of a virus suspension is thoroughly mdxed with the bacteria with a sterile glass rod. The bacteria-virus mixture is incubated at 37%. when the bacteria have lysed, five mls of distil1ed water is added to the plate, and stir- red vigorously in order to get all the virus off the agar surface. This water suspension of virus is then centrifuged to remove all bacterial debris and filtered through a.Millipore type B A (Hydrosol Assay) mem- brane filter. (sillipore Filter eon-pang, watertown, 72, )hssachusetts). The resulting filtrate is free of all bacteria and contains only virus in a water suspension. hepending on the volume of virus suspension needed, as many plates as necessary may be used. Because the agar will adsorb some water, only four mls of crude lysate will be recovered for every five mls added. Virus concentrations as high as 321011 virus particles per ml have been obtained by this method, with an average con- centration of 5x1010 as the sorrel. In the first experiment the above technique of producing a virus suspension was used. The medium on which the bacteria and virus were grown was a modified form of the synthetic, or "F', medium mentioned by Adams (1950), and contained 0.75; KHZPOA, 1.75g HaQHPO‘, lg NH Cl, 0.1g MgCl, 7.2mls lactic acid, and 153 of agar in one liter ofl‘distilled water. The pH was adjusted to 7 with NaOH. Five No of P32 were added to each of two petri dishes, in which 10 mls of agar medium had been al- lowed to harden. 10 more mls of medium were poured over the P32. Each plate was inoculated with 0.2 ml of an E. 99.11 suspension according to the procedure previously described, incubated for 18 hours, inoculated with 0.5 ml of a virus suspension, and washed with five mls of distilled water. After oentrifugation and filtering, the virus suspension was assayed for the number of viable virus particles by the agar layer method, and found to contain 7x109 virus particles per milliliter. A count of the radio activity gave 3.11107 counts per minute per milliliter. Obviously a great deal of P32 had been scraped from the agar surface and was contaminating the virus suspension. In order to get rid of this contaminating P32, the virus suspension was sedimented in an ultracentrifuge at 110,660 times gravity for one hour, the supernatant fluid drawn off and the sediment resuspended in an equal volume of nedium. This was done twice. The second resuspended virus suspension now contained only 1.11109 viable virus particles per milliliter and had a radioactive count of 2.9x10” counts per minute per milliliter. Although this method of producing a radioactive virus suspension might, in time, have proved satisfactory, the technical difficulties in- volved in handling P32 were such that it was felt advisable to try a method using a liquid medium. The use of a solid agar medium was waste- ful of P32 in that the bacteria would utilise only the fraction of P32 near the agar surface. A liquid medit- would mks all the P32 theoret- ically available. A third argument in favor of wring a liquid medium was that the T3 bacteriophage derives about 70% of its total phosphorus from the bacterial cell and only some 30% from the medium (Labaw, 1951). By washing the bacterial cells it would be possible to eliminate almost all of the P32 in the medium, leaving only that P32 which had been in- corporated into the bacteria. The next experiment was performed using "P" medium without the agar into which varying amounts of P32 were added. The medium was placed in- to s Porton Impinger (Ace Glass Inc., Vineland, New Jersey) and P32 added to obtain the desired concentration. The medium was inoculated with 0.2 to 0.5 ml of a 24.hour broth culture of E. pg;1_and aerated at 37°C in a water bath for 18 hours. The air was introduced through a glass wool filter. The Porton Impinger was used because it was avail- able and seemed ideal for the purpose. It consists of a flat bottom flask four on in diameter and 15 cm high. Into the top of the flask is fitted a long glass tube that extends to the bottom of the flask. The air was introduced through the tsp of the tube and let out through an outlet near the top of the flask. The 18 hour E, 9011 suspension was centrifuged at a relative cen- trifugal force of 1200 times gravity for 15 minutes. The supernatant 10 fluid was discarded and the cells resuspended in the same volume of fresh non-radioactive medium. This procedure was repeated twice. The final resuapended cells were put into another impinger and 0.5 ml of a virus suspension added. The ratio of the number of virus particles to the number of bacteria was about 1:1. Aeration at 37°C was continued for 10 hours, at which time lysis had taken place. This crude lysate was centrifuged to remove gross particles and the supernatant fluid filtered through a Millipore type H A filter. The results of three experiments are summarised in Table I. The phosphate buffer in the modified “I“ medium undoubtedly pre- vented utilisation of much of the r32 by the s. sell. so the remaining experiments were performed using a glycerol-lactate medium which lacked any inorganic phosphates. The new medium contained 1.5g KCl, 5g NaCl, lg NHAQl, 0.25g.MgSoé, 6.33 lactic acid, 2g glycerol, 0.5g Bacto Peptone, 0.5g Bacto Casamino Acids in one liter of distilled water. (Stent and Fuerst 1955). The pH was adjusted to 7 with NaOH. The total amount of phosphorus in this medium was 2% from the peptone and 0.22% from the cassmino acids, or 0.011 mg per ml. The utilisation of P32 by E, 3911 grown in this medium could be expected to be much greater than in the ”F“ medium. The virus was produced in the manner previously described. The only difference was that the P32 concentration was 0.2 MC/ml for all the experiments. The results of these experiments are summarised in Table II. Purification of the Filtered Virus Suspensions Growing bacteria in a highly radioactive medium and harvesting the virus from the radioactive cells was a simple task compared to the 11 12 as}: «R. 89% 0.3.3 8%.: 03.5 some; sonata .H: e H: 8}: 3 an}: em. 8%? 0865 octane Soda...” 203..“ meantm .2 as an}: S as}: «3. 0.3.3. 0832. oodeom Soda .a 203$ moose . 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It was assumed that not all the radioactivity present in the filtered virus suspension was confined to virus particles. Three.methods of accomplishing this were tried. The first has been mentioned already. It involved sedimenting the virus particles in the ultracentrifuge and resuspending them in non-radioactive medium. This is the most common method used to obtain a purified virus suspension. In this case the ultracentrifuge did not prove entirely satisfactory for two reasons. First; every time the sediment was resuspended there was a decrease in the number of viable virus particles. Second; it is diffi- cult to fill the ultracentrifuge tubes without spilling sizable quanti- ties of radioactive liquid. ‘ The second method of removing contaminating P32 was by filtration through a Nillipore virus type membrane filter. These filters have 01h tremely small pores and were reputed to be capable of retaining particles as small as the T3 bacteriophage. The technique involved filtering the virus suspension through one filter, washing that filter with sterile saline solution to remove the virus, filtering the washings through a second filter, and than washing that filter in sterile saline solution. This method, although it sounded promising, proved of little value for one reason. The filters themselves were not of a uniform quality. lost of them would retain the virus, but some would not. Moreover, it was difficult to get more than five or tent-illiliters of liquid through any one filter. One 25 ml volume of virus suspension took six hours to pass through the filter, even though considerable vacuum was applied be- low the filter. The variability in the quality of the filters made them useless for the purposes of this work. The third method was to dialyse the filtered virus suspension against distilled water. The virus suspension was placed into a cello- phane dialysing bag. The cellophane was obtained from the Visking Cor- poration. The dialysing bag was put into a beaker containing 500 mls of distilled water. The supporter for the dialysing bag also held a small glass paddle which was inserted into the virus suspension, and kept it continually agitating. The distilled water was changed at intervals of one hour, and samples taken to determine the radioactivity of the water and the amount of virus, if any, passing through the dialysing membrane. It was hoped that the virus would remain in the dialysing bag, while any P32 in the form of phosphates would pass through the membrane. The counts per minute per milliliter of the dialysing water and the amount of virus passing through the membrane into the dialysing water are given in Table III. Chart I illustrates the relationship between the cumulative radioactivity removed from the virus suspension, and the amount of time which the virus is dialysed. Chromatography of Filtered and Dialysed Radioactive Virus Suspensions There is a considerable decrease in the radioactivity of the dialysed virus in contrast to that of the filtered virus. The problem was whether dialysis had removed any of the free P32 in the medium and, if so, how much had been removed. Uhs the dialysed virus purer with re— spect to contaminating P32 than the filtered virus? 15 TABLE III AMOUNT OF RADIOACTIVIT! PASSING THROUGH DIALYSING MEMBRANE INTO DIALYSING HATER Lot 11* Lot X* Lot XI* Time in Counts per Time in Counts per Time in Counts per hours min. /m1 . hours min . /m1. hours min . /ml . 2 183,800 2-1/2 221,000 1/2 146,400 1. 112,600 3—1/2 40,500 2 127,000 6 36,600 4-1/2 30,000 3 41,600 '7 14,900 5-1/4 15,300 4 9,200 11 19,050 5-3/4 3,100 5 3,300 12 850 9-1/2 37,700 6 7,350 10 3,050 9-1/2 12.550 11-1/2 4,130 10-1/4 4,600 12 200 11 2,530 12 4,970 * No viable virus particles found in Lot IX, X, or XI. 16 accunltuabeu CUILUOB per mnuoe WI m11111ter 400,000 300,000 200,000 100,000 CHART I Amount of Radioactive Phosphorus —- Removed From Three Virus Suspensions ,,,'«v-”""’"" .""9/ "’ --./4~ Ah me.m~ As m.d~ Hos: moo . e no. H.ma ¢.n~ accuses mom . n no. H.na m.o~ HahudohanduOm s N mm. as a. 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Ffi " 0m mm mm .eauaauae .MH you i .\ \ HOME mafia—aha own/IQ \h. x «no .dxm , \ \ ”woosaom unopoawpnoo ow oesopnfia «m cm .eouooaae .~H eon >H sm hmdmo OOOH OOON 000m emnutm sod saunoo nouns 8836 has ”£530. mfl venue undo nuance 33m afloneohau coohaenn NHGQOEdfiGOO a.“ OOflflflflfiQ nouns: oasnvuo own «vooeaom wmm cocoa snag finance convene Lagoon» pooh—Heaps: H> hadmo 4”?” T“? )1 einutm med saunoa TAM V Rf VALUES 0F MAXIMUM COLOR DEVELOPMENT AND MAXIMUM RADIOACTIVITY Exper— Rf value of Rf value of iment Solvent max. color max. radio— Number development activity 1 citrate buffer pH5 a) .7 b) .82 a) .68 h) .81. 2 50% n-propanol . 63 .42 3 50% ethanol .63 .38 1. 10% 111101 a) .8 h) .9 .83 5 34/5 sodium acetate .68 .725 6 citrate buffer p35 a) .7 h) .8 .68 7 citrate buffer p36 .75 .79 8 50% ethanol .58 9 105 NaCl .93 .94 10 W5 sodium acetate .7 .89 ll citrate buffer p115 .74 .79 12 citrate buffer p116 ..74 .8 13 citrate buffer p36 .74 .75 ll. 50$ ethanol .59 l5 citrate buffer 935 .83 16 citrate buffer p36 .73 17 " " " .83 .85 18 " " " .8 DISCUSSION The production of T3 bacteriOphage on solid "Fn medium did not prove to be very satisfactory. Although very high concentrations of the virus were obtained when no P32 was present, the addition of P32 seemed to prevent the recovery of high virus concentrations. The reasons for this can be ascribed to the technical difficulties of working with P32 at such close quarters, and not to an inhibitory effects of the P32. Moreover, the problem of ridding the virus suspension of free P32 would have been extremely difficult, for a great deal of 1’32 must have been washed from the agar surface. Another difficulty was that the bacteria could utilise only the P3:2 at or near the surface of the agar, thus, in effect, wasting the P32 that was too far beneath the surface. It was for these reasons that production of the virus on solid medium was not con- tinued. The three experiments performed using liquid ”F" medium did, in all but one of the experiments (experiment III), produce satisfactory con- centrations of virus. One interesting point to notice is that as the concentration of P32 increased, the number of viable bacterial cells de- creased. (Table I). Since the bacterial concentration was directly responsible for the virus concentration, the rest of the experiments were performed using a concentration of P32 which would not inhibit too greatly the growth of the bacteria. Table II shows the results of using a P32 concentration of 0.2 Mc/ml. All the bacterial concentrations are greater than 108 viable cells per ml. Although the virus concentrations in experiments I and II were satisfactory, the radioactivity expressed in counts per minute per ml was not as high as had been hoped. After 28 (’2. LJ" dialysis, the radioactive count dropped much lower. The count was so low that these virus lots were not considered suitable for use. The reason for this low radioactive count was undoubtedly the fact that the phosphate buffer used in the "F” medium prevented utilisation of the 1’32 by the bacteria. Therefore, the remaining experiments were performed using a liquid glycerol-lactate medium.which lacked any inorganic phos- phorus. Production of T3 bacteriophage in liquid glycerol-lactate medium containing-P32 proved to be very satisfactory. The bacterial concentra- tions in experiments VI through XII were all above 108 cells/ml, and the number of viable virus particles was, with one exception, greater than 1010 particles/n1. The effect of dialysis on the virus is less con- sistent. The number of viable virus particles after dialysis was less in five experiments and greater in two. Neither the increase nor decrease can be accounted for. In only one experiment was any virus recovered from the water in which the suspensions were being dialysed, and the number of virus particles in that instance was not sufficient to account for the decrease in titer. Experiments with non-radioactive virus lots performed previously had indicated that a slight decrease in titer would occur upon dialysis, but that the amount of virus passing through the dialysing membrane was negligible and not sufficient to account for the decrease. The decrease in titer was therefore ascribed either to physical inactiva- tion of the virus while being dialysed, to adsorption of the virus on the membrane and glass stirring paddle, or possibly to both. At any rate, dialysis appeared to be a relatively simple means of removing dialysable components of the medium which might contain P32 29 without losing too nany virus particles. Dialysis was carried out for 12 hours. After that length of time, the amount of radioactivity being removed became too small to profitably continue dialysis. Samples were taken from each batch of dialysing water before it was changed and assayed for the amount of radioactivity present. The results of this assay are given for three virus suspensions, IX, X, and X1 in Table III and in Chart 1. The graphs clearly indicate that after eight or nine hours very little radioactivity was being removed compared to the amount re- moved in the first six hours. Similar graphs for the other virus sus- pensions show the same results. Of course, the question of purity of the virus suspension as regards the presence of 1"32 outside the virus particles has still to be answered. This will be taken up in the next section. An indication of the amount of nitrogen containing compounds re- moved during dialysis was given by determining the amount of nitrogen present in a virus suspension before and after dialysis. Lot III (a) was analysed by the method of Folin and Wu using a Bausch and Lomb monochro- matic colorimeter with a 505 millimicron wavelength filter to measure the degree of color development with Hessler's reagent. The filtered virus suspension contained 0.316 mg and the dialysed suspension contained 0.03 mg of nitrogen per ml. The total nitrogen of filtered and dialysed glycerol-lactate medium was also determined by the same method. Filtered medium contained 0.366 mg and dialysed medium 0.031. mg of nitrogen per ml. The values for the dialysed virus suspension are as low as values found for virus suspensions purified in the ultracentrifuge by other workers in this laboratory. These results, by themselves, do not prove 30 that any contaminating P32 was removed during dialysis, but they do show that dialysis effectively removes nitrogen containing compounds which may have P32 associated with them. Although paper chromatography has many important applications, its applicability to the separation of proteins has been questioned. (Hall and Newalka 1951). However, Franklin and Quastel (1949, 1951) have used the technique to separate blood proteins. They claim that paper chroma- tography can successfully be applied to protein separation when the pro— teins are sufficiently dissimilar. Gray (1952) used paper chromatography to detect tobacco mosaic virus in infected tobacco plants. Using 40% and 50$ ethanol as the solvent he found that approximately 1/3 of the virus remained at the origin while 2/3 of the virus migrated. The virus did not move as a single compact spot but tended to spread out in a streak. This was the situation in the chromatograms described here. The colored portions, after deve10p- ‘ment with ninhydrin, appeared as streaks from.two to four centimeters long.. The color was most intense at the center of the streak and less so at the edges. This was true of the filtered, dialysed, and purified 'virus suspensions. The filtered virus suspensions showed streaks which were a darker purple than those of the dialysed or purified suspensions. This:was to be expected, of course, for the filtered virus was suspended in a medium with a total nitrogen content of about 0.4 mg of N/ml, much of which was contained in protein compounds. A great deal of the color deveIOpment of the filtered virus suspension was due, therefore, to nitrogen containing compounds in the suspending medium, and not to the virus itself. This is illustrated in Table IV (experiments 17 and 18). 31 The medium which had not been dialysed gave a definite color reaction, while the dialysed medium did not. In none of the experiments was any color seen where the spot had originally been placed. It was assumed that the color obtained from dialysed virus suspen- sions after development with ninhydrin was not due to any nitrogen con- taining compounds in the medium.but was due to the virus itself. This assumption was felt to be valid because two virus lots purified in the ultracentrifuge and resuspended in water gave a color reaction which must have been due to the virus. Moreover, the values obtained for the amount of nitrogen remaining in the virus suspensions after dialysis were such that a considerable fraction of the nitrogen must have been virus nitrogen. In a few of the experiments, no color was visible after develOpment with ninhydrin. This was probably caused by the application of too small a volume of the virus suspension. This occurred in at least one chromatogram made with an ultracentrifuge purified virus sus- pension, (not reported) and in experiments 8 and 14 (Table IV). It has been suggested by Franklin (1949) that the movement, or lack of movement, of proteins on filter paper is due principally to adsorption of the protein to the paper and not to partition of the protein between the solvent phase in the paper and the moving solvent front. Gray (1952) alsotnoted that under conditions which caused denaturation of protein, such.as the use of ethanol as the solvent, certain protein constituents from tobacco leaves would not migrate, while the tobacco mosaic virus, which is less easily denatured by ethanol, did not move with the solvent :flront. 32 Any analysis of the chromatography experiments reported'in this thesis must be a cautious one. The experiments were originally under- taken because it was felt that chromatography might prove a rapid and simple method of determining the effectiveness of dialysis in removing free or contaminating P32 from the medium. Ideally, it was heped that the virus would migrate to one definite spot which would be easily do— tectable both visually and by reason of the associated radioactivity, and any contaminating P32 would migrate to another definite spot easily dis- tinguishable from the former. Examination of the experiments performed using citrate buffer at pH 6 (experiments 7, 12, 13, l6, l7, and 18) shows several interesting points. The virus alone has an Rf value of 0.73 (experiment 16), while the medium alone has an Rf value of 0.83 (experiment 17). This would seem to indicate that the color observed in experiments 7, 12, and 13 was due to the presence of virus, since the Rf values of these experi- ments is 0.71. and 0.75. However, the maximum amount of radioactivity does not always coincide with the visible color spot. P32 in associa- ticn with medium.alone (experiments 17 and 18) has an.Rf value of .85 and .8 respectively. The radioactivity associated with virus suspensions has Rf values of 0.79, 0.8, and 0.75 (experiments 7, 12, and 13). These ‘values are not as consistent as those computed from visible color spots, and only in experiment 13 do the Rf values coincide. The Rf values for the radioactivity are so inconsistent that it is not possible to say 'whether the radioactivity is associated with the virus. Experiment 13 seems to indicate that it might be, but experiment 12 seems to indicate the opposite. 33 The experiments performed using citrate buffer at pH 5 show the same inconsistencies, especially as regards the Rf values of the radio- activity of the virus suspensions. (experiments 1, 6, 11, and 15) Only in experiments 4 and 9 is there any indication that dialysis effectively removes contaminating P32. In experiment 4, using a filtered virus sus- pension, the radioactivity is not associated with either of the color spots. However, in experiment 9, using a dialysed virus suspension, the radioactivity migrated to the same position as the visible color spot. These two experiments together with experiment 13 are the only ones that indicate that dialysis removes any contaminating P32, and that the radio- activity is associated with the virus after dialysis. One other observation can be made concerning these experiments. The amount of radioactivity remaining at the origin may be of some sig- nificance. The only solvent that moved nearly all the radioactivity from the origin was the citrate buffer at pH 5. (Chart III). All the other solvents left a considerable amount of radioactivity at the origin. How- ever, citrate buffer at pH 6 did move nearly .11 the P32 that had been added to glycerol lactate medium. If then, the movement of P32 along the filter paper consists mainly of contaminating P32 which might be pre- sent in its original form, then the radioactivity remaining at the origin can be ascribed to P32 which is bound to some nondmovable substance, pre- sumably the virus. However, this neglects the evidence of those experi- ments performed using a purified virus suspension. In these experiments a definite color spot was visible. This color could only have come from the virus. If both the virus and the contaminating P32 migrate down the paper'strip, what significance does the radioactivity that remains at the origin.have: There is no information at hand to answer this question. 34 The most likely answer is that the solvents used, with the exception of pH 5 citrate buffer and possibly M/S sodiumsacetate, did not move all the virus, and the amount of virus remaining at the origin was insufficient to produce a color reaction when develOped with ninhydrin. If this is so, then the radioactivity remaining at the origin would be due to virus, and the radioactivity at the single peak would be due to virus plus any contaminating P32. One reason for the ability of pH 5 citrate buffer to move more of the radioactivity than any of the other solvents might be the fact that it is closer to the iso-electrio point of the virus and would tend to neutralise the electrical charge on the virus. Since ad- sorption to the paper is in part due to the nature of the electrical charge of both the paper and the virus, this might explain the inability of the other solvents which were at or near a pH of 7 to move a large part of the radioactivity of the virus suspension. Unfortunately, no experiments were performed which might have given an indication of the validity of this supposition. Although it is possible to demonstrate that a considerable quantity of radioactivity is removed from the virus suspension during dialysis without a great loss in the infectivity titer of the virus, the chrome- ‘tographic means for testing the purity of the dialysed.virus did not prove to be entirely successful. It is felt that the reason for this Jlies primarily in the fact that the virus is adsorbed to the filter paper and does not migrate as a unit. There are other means analogous to paper chromatography which might prove successful in proving the jpuxity of the dialysed virus suspension. For example, paper electro- phoresis, using either a single paper strip or a vertically suspended sheet of paper, would overcome to some extent the problem of adsorption 35 of the virus to the paper. The added "push" from the electric current would result in a better separation. Another piece of apparatus which might prove useful would be the use of a solid block of starch as the stationary phase. Starch is noted for its low adsorptive power, and would minimise the problem.of adsorption. An electric current applied through the starch block would provide the motive power for the charged virus particles. 36 SUMMARY Three methods of producing_T3 bacteriOphage in medium containing P32 are described. The first, using solid "F" medium, proved to be un- satisfactory because of the technical difficulties involved in working with the P32 and because of the difficulty of ridding the virus suspen- sion of free P32. The second, using liquid "F" medium, was also unsatis- factory because the amount of P32 taken up by the bacteria and virus was too small. The third method, using liquid glycerol-lactate medium pro- duced both high concentrations of virus and high radioactive counts per milliliter of virus suspension. (Tables I and II). The virus suspensions were dialysed against distilled water to rid the suspensions of free P32. Tables I and II show the amount of P32 re- moved from the virus suspensions, and Chart I shows the amount of P32 removed during the dialysis process. Paper chromatography techniques were employed to estimate the effi- ciency of the dialysis procedure in removing free P32 from the virus suspensions. 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Bact. 66; 458-464. 39 map-1.5:! ) Mai glut. fem” Date Due Demco-293 A lllfllllsllultllell‘iuiVETl'lwlUielfiml'es 3 1293 03115 J961 MICHIG