OAK RIDGE NATIONALLABORATORY LIBRARIES

3 H4Sb 0571bD4 4

AppendixORNL-3942

Special

APPENDIX TO

SEMIANNUAL CONTRACT PROGRESS REPORT

TO THE NATIONAL INSTITUTE OF

ALLERGY AND INFECTIOUS DISEASES

SEPTEMBER 1, 1965, TO MARCH 1, 1966

OAK RIDGE NATIONAL LABORATORY

operated by

UNION CARBIDE CORPORATION

for the

U.S. ATOMIC ENERGY COMMISSION

LEGAL NOTICE

This report was prepared os an account of Government sponsored work. Neither the United States,

nor the Commission, nor any person acting on behalf of the Commission:

A. Makes any warranty or representation, expressed or imp Iied, with respect to the accuracy,

completeness, or usefulness of the information contained in this report, or that the use of

any information, apparatus, method, or process disclosed in this report may not infringe

privately owned rights; or

B. Assumes any liabilities with respect to the use of, or for damages resulting from the use of

any information, apparatus, method, or process disclosed in this report.

As used tn the above, "person acting on behalf of the Commission" includes any employee or

contractor of the Commission, or employee of such contractor, to the extent that such employee

or contractor of the Commission, or employee of such contractor prepares, disseminates, or

provides access to, any information pursuant to his employment or contract with the Commission,

or his employment with such contractor.

APPENDIX

OR NL-3942

Special

Interagency Agreement: 40-30-64

APPENDIX*

TO

SEMIANNUAL CONTRACT PROGRESS REPORT

TO THE

NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES

September 1, 1965, to March 1, 1966

Norman G. Anderson, George B. Cline, Richard M. Jamison, and Warren W. Harris

Biophysical Separations Laboratory, Biology Division,Oak Ridge National Laboratory

MARCH 1966

*The material in this Appendix is not to be released without prior approval of theOak Ridge National Laboratory. Research jointly sponsored by the VaccineDevelopment Branch, National Institute of Allergy and Infectious Diseases, NationalInstitutes of Health, Bethesda, Maryland, and the U. S. Atomic Energy Commissionunder contract with the Union Carbide Corporation.

A. Gradient Resolubilization Studies.

I. Objectives

When cells are infected with Adenoviruses, they produce several virus-specific

antigenic materials in addition to the Adenovirus virions. These antigenic materials

have been termed "soluble antigens." The soluble antigens of Adenovirus can be

separated into three classes by chromatography or electrophoresis. As a group, these

antigens constitute the greater portion of virus-specific material produced by the

infected cell; the virions produced account for approximately 20% of the total

antigenic mass. It is obvious that the loss of this "soluble" antigenic mass from

materials destined to be used as antigens for production of Adenovirus vaccines represents

a serious problem. Previous studies have shown that attempts to isolate Adenovirus

antigens by continuous-flow centrifugation in the B-IX rotor generally leave large

amounts of antigens in the supernatant. Dr. Erling Norrby has indicated that the

soluble antigens of Adenovirus Type 3 have sedimentation coefficients in the range of

13 S to 56 S. This factor would seem to account for the lack of efficiency of the B-IX

continuous-flow system. In studies of the B-IX rotor, it has been shown that the

removal of particles with sedimentation coefficients < 100S is inefficient, if not

impossible.

This study was begun as an attempt to provide means of isolation from tissue

culture harvests of the Adenovirus soluble antigens by continuous-flow centrifugation

in the B-IX rotor. It was felt that aggregation and/or precipitation of the soluble

materials in tissue culture fluids prior to isopycnic banding would facilitate the

removal of these antigens from the flow-through stream and allow their isolation in

the rotor. Several different means of accomplishing this goal were investigated.

Each will be briefly described below. The Adenoviruses used as test systems were

(a) Adenovirus Type 5 (made available by Parke-Davis Inc.), and (b) Adenovirus

Type 3 (made available by the National Institute of Allergy and Infectious Diseases

through the National Drug Co.). Both of these virus preparations were in the form

of formalin-inactivated tissue culture fluid.

II. Materials and Methods

Methods used for precipitation of the Adenovirus soluble antigens were:

(1) Precipitation of the soluble components of the tissue culture material with

40% ethanol prior to introduction of the specimen into the B-IX rotor. In this

manner, preformed aggregates were centrifuged out of the flow-through stream and

toward the interior of the rotor.

(2) Precipitation of the soluble components of the tissue culture fluid by passing

the material over the surface of an ethanol-sucrose or ethanol-cesium chloride density

gradient. In this manner, as aggregates were formed, they were centrifuged out of

the flow-through stream and toward the interior of the rotor.

(3) A combination of the above two experimental systems, i.e., precipitation

of the tissue culture fluid prior to flow-through over a gradient which contained

ethanol. It was thought that incorporation of varying concentrations of ethanol into

the density gradient (Fig. 1) might increase the resolution of the system; that is, as

aggregates of precipitated proteins were centrifuged through the density gradient,

they might find points such that the ethanol concentration in the gradient might allow

for selective resolubilization of some component(s) and the aggregate be resolubilized.

The protein components of such a resolubilized aggregate would then separate on an

isopycnic basis in the density gradient.

All centrifuge runs were performed in the B-IX rotor using standard conditions

(i.e., the material was introduced into the rotor at 2 liters/hr; banding was for

2-1/2 hr at 35,000 rpm). At the conclusion of each run, the gradient in the rotor

was pumped through a spectrophotometer, monitored for absorption of ultraviolet light,

collected as 40-ml fractions, dialyzed against phosphate-buffered saline and examined

in the electron microscope. Aliquots of each fraction were then sent to Parke-Davis

for titration of Adenovirus complement-fixing antigens.

III. Results

Resolution, as defined as the maximum number of peaks obtainable, was found

to be highest when method three was employed. In addition, antigenic analysis

showed maximum removal of antigenic materials from the flow-through stream by this

method. Results of a representative experiment in a cesium chloride-ethanol system

are given in Fig. 2. It can be seen that four major peaks of ultraviolet-absorbing

material can be resolved. In addition, each of these peaks was shown to contain

levels of Adenovirus complement-fixing antigens significantly higher than the areas

without the peaks. The fractions containing the peaks each had a complement-fixing

titer of 1:32-64. All other fractions contained complement-fixing activity at a

level of ' 1:4-1:8.

Results of electron microscopy can be seen in Table 1. Peak 1, occurring at a

density of 1.08 gm cm , was found to contain amorphous particulate debris in

amounts which precluded demonstration of more regular particles. Upon dilution of

the specimen to remove the debris, no Adenovirus virions could be demonstrated.

6

TABLE 1. — Results of examination in the electron microscope of fractions

from an ethanol-cesium chloride gradient

Fraction Results

Start Few easily recognizable particles. Much debris.(after alcoholprecipitation)

5 Adenovirus virions per 10 fields at Tap 10.

Fraction 1 Amorphous particulate debris.(Peakl)

Fraction 5 Amorphous particulate debris. Possibly 1

Adenovirus virion per field at Tap 9.

Fraction 8 Three particle types noted: (1) Adenovirus virions

(2) Smaller particle, approximately 40-45 mp in

diameter. Regular hexagonal shape. (3) Small

regular particle. Apparently icosahedral,

approximately 20-25 mp in diameter. About

10 Adeno per Tap 8 field; 2 of Type 2 per 5 Tap 9

fields; 5 of Type 3 per Tap 8 field.

Fraction 13 Same general appearance as Fraction 8. Numbers(Peak 4) - ., - _., . . , ., L .

ot three types ot particles varied. About 4

Adenoviruses per Tap 8 field; 1 Type 2 particle

per 10 Tap 9 fields; 15 of Type 3 per Tap 8 field.

Effluent Much fine granular debris. No Adenovirus

virions seen.

Particles which resemble Adenovirus virions could be found in the peaks which

were found at densities of 1.29, 1.36, and 1.42 gm cm . In addition, the peaks

occurring atdensities of 1.36 and 1.42 contained large numbers of small (approximately

22 mp) regular particles. A few regular, apparently icosahedral particles

approximately 40 mp in diameter were also present.

Upon the basis of results such as these, it was concluded that mixing of the

components of the gradient might be occurring during centrifugation orduring unloading

of the gradient at the conclusion of the run. If so, this mixing might account for the

presence of three types of particles at the same places in the gradient and for the

occurrence of Adenovirus virions at a density of 1.42 gm cm . Accordingly,

experiments were designed to investigate this possibility. Since mixing of the

density gradient during unloading of the rotor has now been demonstrated (see Part B

of this report), further experimentation of the gradient resolubilization has been

halted pending study of the rotor system.

B. Gradient Recovery from the B-IX Continuous Flow Rotor.

Results of experiments using Adenovirus harvest solution suggested that cesium

chloride-ethanol gradients recovered from the B-IX zonal rotor were not truly

representative of the gradients in the rotor during banding of the virus. The finding

of Adenovirus particles at densities higher than previously reported suggested that

some of the displacing cesium chloride was mixing with the gradient during unloading.

The possibility of gradient mixing was of interest since considerable time and effort

has been put forth in utilizing this rotor system for the isolation and purification of

Respiratory Syncytial (RS) virus. Since the RS experiments had been carried out

using sucrose gradients, experiments were carried out to determine the degree of

mixing of sucrose density gradients by the displacing sucrose solution. The extent

and location of mixing were determined by using displacing solution dyed with

phenol red. The amount of the red color in the recovered gradient fractions was

subsequently determined by titration of similar volumes of gradient fractions with

displacing solution and comparison of color to the fractions from the rotor. It was

found that there is one zone of mixing which is centered at about 200 ml from the

rotor core. Additional red color was observed at the boundary between the gradient

and the displacing material. This color is attributed to the diffusion of the color

into the gradient at this point. Since the unloading time is about 30 minutes,

considerable diffusion could occur within this time. Similar studies were carried out

with the B-VIII rotor with similar results. However, the mixing was much more

extensive and the main zone of mixing was centered at about 400-ml volume in the

gradient.

The results of the studies on gradient mixing in the B-IX zonal rotor show that

some mixing of the gradient with the displacing material does occur. These findings

do not suggest that this rotor cannot be used in the present form for continuous-flow

operations for particles such as RS virus. Since the volume of displacing solution

mixing with the gradient was of the order of 5 to 10 ml, little disturbance of a zone

of particles at that point would be noted. Theoretically the increase in zone volume

would be only by the amount of displacing solution being added. If mixing is

considered to be a problem, the density gradient can be designed to stop the particles

at a smaller or larger radius in the gradient and thereby not be subjected to the

possible mixing.

Experiments using density gradients containing cesium chloride indicate that the

degree of mixing is considerably more extensive than with sucrose gradients. In

addition, the high diffusion coefficient of cesium chloride allows the peripheral portion

of the density gradient to be permeated by the displacing solution during the 30-minute

unloading procedure. Since the width of the gradient is only about 1 cm, considerable

diffusion could occur into the outer few millimeters of gradient. This width in

gradient would be equivalent to about 1/4 of the rotor volume or about 200 ml of

solution. Virus zones falling within this "diffusion zone" would thus have an

indicated density greater than densities determined for that particle under more stable

conditions. For this reason, experimental Adenovirus runs in the B-IX rotor using

cesium chloride gradients suggest that the density for this virus is about 0.10 density

units higher than published values. As a result, values of virus zones obtained from

the B-IX rotor in the peripheral portions of cesium chloride gradients cannot be

interpreted as real. If analytical data is desired from such a run, the virus must be

10

banded near the rotor core away from the "diffusion zone." Gradient mixing with

the displacing solution during unloading of the rotor will also affect the density of

every fraction so that this aspect must also be considered.

The mixing problem can be solved hopefully by a design change in the B-IX core

to insure that displacing solution does not come out of the effluent holes. There are

two solutions now being checked. The first is to install check valves in the effluent

holes to permit the harvest solution to flow out of the rotor through the effluent

holes but inhibit the reverse flow of displacing solution through these same holes.

The displacing solution would thus be forced to the rotor wall without mixing.

These alterations are being made on a B-IX core at the present time and initial tests

indicate that the mixing is somewhat reduced. The second solution is to utilize

a distributor baffle system to make sure fluids flow in the required direction. Since

this distributor baffle system works well in the K-I rotor, it will soon be installed for

testing on the B-IX rotor.

11

C. Zonal Analysis of Disrupted Flu Virus in the B-XV Rotor.

I. Objectives

The finding that subunits of flu virus may be useful as a vaccine material

suggests that the antigen(s) of interest are particulate in nature and withstand

disruption of the intact virus. It was of interest to separate this potential vaccine

material into its various components on sucrose density gradients for the purpose of

identification and purification of each of the subunits.

II. Materials and Methods

Tween-Ether-disrupted PR-8 flu virus was prepared by Dr. Fred Davenport,

School of Public Health, Ann Arbor, Michigan. Preliminary Schlieren analysis

of the material was made in the Analytical Ultracentrifuge using dialyzed material.

Size estimates of particulate fractions observed by Schlieren analysis were used in

deciding how long to run the zonal rotor. The PR-8 concentrate (20 ml) was

loaded onto a 10-30% sucrose density gradient into the B-XV rotor with a 55%

sucrose cushion on the rotor wall. An overlay of 200 ml of phosphate-buffered

saline pH 7.5 was used on top of the sample zone. The duration of the run was

about 5 hours at 20,000 rpm giving a total of 75,000 uu2 X10° at the time of

deceleration. The effluent stream was monitored at 280 mp and collected in 20-ml

fractions. Samples of each of the peak fractions were saved for electron microscopy.

Grids of the material were prepared by first dialyzing the sucrose from the fractions

on a spot plate and negatively staining the material on the grid with 2%

phosphotungstic acid.

12

III. Results

Schlieren analysis indicated that particulates which had sedimentation rates

ranging from about 100 S to 1000 S were present in the preparation. Accurate

determinations of the various particulate fractions were not made since the particles

were photographed during acceleration of the rotor. The sedimentation values were

estimated by extrapolation from polysome sedimentation rates. Figure 3 shows the

absorbance profile of the B-XV rotor contents and indicates the presence of .at

least four distinct fractions. In addition, considerable material was left at the

starting boundary, and considerable material penetrated both the gradient and the

cushion and pelleted to the rotor wall. Antigenic analysis and electron microscopy

of the various fractions have not been completed.

13

D. The K-I Zonal Rotor.

I. Objectives

The K-series of zonal rotors represents the second generation of centrifuge rotor

for the isolation and concentration of small particles. Specifically, the K-I rotor is

a prototype continuous-flow rotor of the B-IX design which operates on the conventiona

air-driven Sharpies centrifuge. This series of rotors is designed for the rapid processing

of large volumes of virus suspensions and is of interest to various commercial firms for

use in vaccine production. The K-I rotor is, however, primarily a proof-of-principle

rotor and is not designed for production capacities. It will, however, be useful for

up to 10-liter volumes of solution in cases where the total content of suspended solids

is of the order of a few milligrams. It is the first step in the design of a large-scale

K-II rotor. This section describes the physical characteristics of the K-I rotor, the

operational designs, and the results of preliminary experiments on the isolation and

banding of ragweed pollen and Type 3 bacteriophage. In addition, the final product

of the K-I rotor evaluation, namely the K-II production rotor, is described and

discussed.

II. Materials and Methods

Simplicity of operation and maintenance are of greater importance in the design

of a production-type system than they would be for a research-oriented machine.

The only changes made in the Sharpies centrifuge system were the rotor and its

bottom damper bearing assembly, as shown in Fig. 4. Design speed for the rotor is

50,000 rpm, and it holds 240 ml of liquid. Stress calculations indicate that speeds

on the order of 60,000 rpm are quite safe; however, the characteristics of the

14

drive system at speeds over 50,000 rpm are not known. Yield strength of the

titanium alloy used is 137,000 psi, and at 50, 000 rpm the bowl wall safety factor is

approximately 3.2.

Friction damping in the bottom bearing allows the rotor to spin freely about its

own axis during changes of center of rotation caused by the rotor contents. The

damper takes a minimum of energy out of the spinning rotor and characteristic

vibration amplitudes of a properly designed rotor are quite satisfactory. Effective

lubrication of the bottom bearing is attained by applying a small amount of light

grease to the shaft before each run. Typical runouts of the rotor as a function of

speed, as well as the mode shapes of the two rotor critical speeds, are shown in

Fig. 5. It is interesting to note that precession from the more dominant first-rotor

rigid-body critical is re-excited at higher operating speeds if the friction damper at

the bottom of the machine seizes.

Loading and unloading of the rotor take place along the same fluid path.

Initially, a buffer solution or water is introduced through a feed jet to the 0.25-inch

tube running through the center of the core. The fluid spins Its way up the central

hole to the top of the core and is centrifuged to the rotor wall. Half of the rotor

volume is filled in this manner, then a dense solution is pumped into the rotor by the

same route to fill the rotor. The sample solution containing the virus follows this

path also, eventually leaving the rotor through the shaft exit holes at the bottom of

the rotor. A hydrostatic pressure of about 30 inches of water is sufficient to obtain

flow rates of up to 4 liters per hour. However, optimum flow rates for desired

cleanout must be determined for each type of particle. The density gradient is

removed from the rotor by pumping more displacing solution to the rotor wall, thus

15

backing the density gradient and its banded particles out through the shaft exit holes.

As the gradient leaves these holes, it is tunneled into fraction tubes through the

collection chamber and drain line. During unloading, the gradient may also be

monitored if provision is made to bleed the air out of the effluent stream. The fluid

remaining in the rotor is unloaded by decelerating the rotor to rest; the rotor then

drains itself by gravity.

III. Results

The results of cleanout and banding of ragweed pollen in a sucrose density

gradient are shown in Fig. 6. The figure shows that a density gradient of sucrose

was recovered from the rotor and that the pollen was concentrated in a zone within

the gradient. The composite results of two cleanout experiments using Type 3

bacteriophage and lysate are shown in Fig. 7. The solid line represents one

experiment of phage cleanout from 2 liters of lysate as a function of flow rate

through the rotor. The dashed line represents a second experiment of the same

type in which 2 liters of lysate flowed through the rotor at the speeds indicated.

a

The titer of starting material in Run No. 1 was 7X10 plaque-forming units per

9ml. The second experiment used lysate containing 1X10 plaque-forming units

per ml. The efficiency of cleanout at each flow rate is based upon the amount of

virus remaining in the effluent stream after the flow rate has been established. It

is apparent from these results that the K-I rotor can be used effectively for the

isolation of T3 bacteriophage from liter volumes of fluid.

The second aspect of particle separation in the continuous-flow —isopycnic

banding rotors involves removal of the virus zone from the rotor. Ragweed pollen

16

was found to be banded into a zone within the sucrose density gradient. However,

T3 phage was not found to be banded into a distinct zone in a cesium chloride

gradient even after an hour or more of banding time after completion of flow through.

Analysis of the gradient in such runs indicated that the displacing material was mixing

with the gradient. Similar studies using sucrose gradients indicated the same results.

A modification of the rotor core has been performed which reduces the amount of

mixing during unloading of the density gradient. This modification is a baffle

arrangement which insures that the dense material can get to the rotor wall directly.

Mixing of the displacing solution with the gradient is thus limited to the swirling of

the displacing solution as it flows down the rotor wall. The presence of the baffle

also limits this swirling action. Although it can be shown that little or no mixing

of displacing solution with gradient solution occurs, no information is yet available

to indicate that virus bands can be isolated without mixing. T3 phage banding

studies in cesium chloride are being carried out at the present time. If these studies

show that a zone of T3 can be recovered after flow through, additional preliminary

studies will be carried out at Eli Lilly and Company to determine if flu virus can be

isolated and recovered with this prototype rotor. Success of these preliminary

experiments with the K-I rotor will indicate the efficacy of producing the K-II rotor

for use as a production rotor.

17

E. The K-II Rotor.

I. Objectives

The success of the preliminary studies with the K-I rotor indicates that zonal

rotors which combined the functions of continuous-flow with isopycnic banding can be

built to operate in a variety of centrifuges. The size of the K-I rotor limits its use

for large-scale virus isolation. It was of considerable interest, therefore, to design

a centrifuge rotor which would effectively do the work of several K-I rotors, be

simple in design and operation, low in cost, and work on either electric or turbine

drives. This large-volume K-II rotor is the first of several rotor systems which are

being designed to do specific tasks. It is primarily designed for large viruses of low

density of the myxovirus type. As the need arises, other production-type rotors

can be designed to solve specific separation problems.

II. Materials and Methods

In order to evaluate the various possible types of continuous-flow rotors which

could fulfill the goals of this machine, the comprehensive design curves shown in

figure 8 have been prepared. These design curves are based upon the chosen data

in Table 2. The 100% removal of virus particles from the flow-through zone is

desirable particularly for viruses which grow to relatively low titers. The density

of 1. 193 is the observed banding density of flu virus in sucrose density gradients.

18

TABLE 2. — Selected data on which the K-II rotor is designed

Cleanout from sample zone 100%

Flow rate 10 liters/hr

Sample zone viscosity 0.0152 poises

Sample zone density 1.05 g/cc

Virus diameter 8.2 X 10 cm

Banding density in sucrose 1.193 g/cc

The choice of a flow rate of 10 liters per hour is arbitrary; however, a machine

of this capacity is already required. Also, the design curves may be scaled for any

desired flow rate by means of the relationship:

Q * Lr2N2 (1)e s

where

L ~ effective rotor length

rs ~ out-board sample radius

N ~ speed ~rpm

III. Results

There are five limitations that must be imposed upon the design of this machine:

(1) The rotor must operate below its fundamental flexural critical speed; (2) it must

be stable; (3) it must operate at some reasonable stress level; (4) the rotor weight must

be such that one person can easily handle it and disassemble it for routine maintenance;

and (5) the rotor temperature must be controlled. The first three limitations are shown

(Fig. 8) as areas in which a design is determined by the considerations of stress, rotor

19

speed, and rotor dimensions. The diagonal dashed lines indicate configurations which

are not practical. The unstable region (Fig. 8) is not a region in which the rotor is

truly unstable, but a region in which it is more difficult to design rotors for smooth

operation due to the demands upon the damper and suspension systems.

The rotor must be contained in a vacuum jacket to avoid overheating the rotor by

wind friction. This friction ("windage") becomes apparent when the horsepower lost

(shown in Fig. 9) is applied to the area of interest indicated in Fig. 8. Approximately

3-1/2 horsepower would be lost due to windage for any rotor in this region at

atmospheric pressure —a condition that would be intolerable both for the drive and

the rotor contents. It will therefore be necessary to operate the rotor in vacuum. It

is proposed that a central pumping station be set up which will be sized to permit

efficient evacuation of the machine facility. It is anticipated that only mechanical

roughing pumps will be necessary. Cooling of the vacuum jacket will be avoided to

reduce the cost of the machine. However, some cooling could be obtained cheaply

by circulating tap water through copper tubing soldered onto the vacuum jacket.

Where required, the jacket can be refrigerated.

Another practical limitation that should be considered carefully is speed. Although

the maximum operating speed shown in Fig. 8 is well within the "state of the art,"

there are not as many commercially available drives in this speed range as there are

at lower speed ranges. One result of this is that at higher rotor speeds one must pay

a higher capital investment and at the same time lose some design flexibility.

Therefore, the rotor system should be designed to operate at as low a speed that

would be consistent with the other design limitations.

20

A secondary but important consideration with regard to the design flexibility is

the matter of overspeed capability and its effect upon the machine capacity. Note

that the speed represented in Fig. 8 is not the maximum possible operating speed, but

the speed required to permit 100% cleanout of flu-type virus at 10 liters per hour flow

rate at the indicated rotor length and radius.

With these limitations in mind, the design of the rotor becomes relatively fixed.

The rotor will have a sample radius between 5 and 6.5 cm and will operate between

25,000 and 30,000 rpm with overspeed capabilities to approximately 40,000 rpm,

and it will not weigh more than 75 pounds.

There are four types of drives available that will operate under the speed and load

conditions of the K-II rotor. These are air turbine, oil turbine, low-speed electric

motor with speed increaser, and a high-speed electric motor. All of these methods

have been successful in driving high-speed centrifuges, and at this time no decision

has been made as to the best type of drive. It is most probable, however, that either

direct-drive electric motor or an air turbine will be the most economical means of

driving the machine. The choice between these two will depend primarily upon the

number of machines anticipated at a facility and the availability of compressed air.

If a good supply of compressed air is already available, then an air turbine would

clearly be the most attractive choice.

21

F. A Simplified Gradient Engine.

I. Objectives

Since the advent of the zonal ultracentrifuge there has been a demand for a device

which would deliver density gradients of 1000-ml to 1500-ml volume, having a

reproducible shape or steepness. In addition, there has been demand for a simple,

easily operable machine which could be easily sterilized.

II. Materials and Methods

These problems have been solved in large part with a new and simplified

gradient-forming device (engine). This gradient engine uses solutions which are

packaged in plastic blood storage bags which can be filled with sterile solutions under

sterile conditions. The gradient is formed by squeezing two bags containing two

different density solutions at differential rates. One bag contains the least dense

material for the lightest part of the gradient and the other bag contains dense material

for the heavier part of the gradient. Mixing of the two solutions takes place at a

simple "Y" connecting the two bags. Bags containing the sterile solutions can be

sealed off and used at a later date without losing sterility. Power for squeezing the

solutions from the bags is provided by a dropping 15-pound weight that turns a simple

cam shaft.

III. Results

Preliminary experiments indicate that gradients produced are quite similar to

gradients calculated on the basis of the shape of the cams, the difference between

the two being due to overcoming the viscosity of the heavier sucrose solutions. Such

effects are not as pronounced with density materials having a low viscosity. These

22

gradient machines will soon be put into routine operation in this laboratory for

extensive testing of function and reliability.

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12

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• -TOP END CAP

O-BOTTOM END CAP

NOTE - BOTTOM BEARING HAS

4 mil DIAMETRAL CLEARANCE

-O-

40 50

Fig. 5. Approximate per-revolution runout of titanium K-I rotor.

K3

ORNL DWG. 66-2936

ROTOR RADIUS, in.

700.7 0.8 0.9

1 1 ~T

60 —

-

50BAND WIDTH

t////rf?///A _LU

1/1

0

W 40to

—

PERCENTGO

o

—

20 —

10

n

o/

1 1 1 1 1 1 1 1 1 1 120 40 60 80 100 120 140

GRADIENT VOLUME, ml

160 180 200 220

Fig. 6. Continuous-flow, isopycnic banding of ragweed pollen in rotor K-I (42,000 rpm).

240

CO

29

1 2 3

FLOW RATE (I iters/hr)

ORKL DWG. 66-2937

Fig. 7. Type 3 bacteriophage removal from flow-through stream in the K-Izonal rotor as a function of flow rate. The dashed and solid lines represent twoexperiments. The results of the plaque assay of the T3 is estimated to be atleast 15%. The solid line represents one experiment in which the starting timeof T3 phage was 7X10' plaques per ml. The dotted line is a second experimentin which the titer was 1X1 0y per ml.

1<T

oz

-1 ]02

z

10

30

ORNL DWG 66-2938

—

—

LVD.-^-lO 9 8 7 6 5 4 3

—

CRITICAL SPEED

REGION

f %//"/// /—

o *£>

K % "%

o o% -o /x/

/X// ' ' / / /

<v/x/ / #/Y//Y/ -v/

—~%°- \ JO'A'

-s, 3 X/x / A, A / / STRESS CUTOFF

—

//'\\ \V A—^^^/^

"

"——^ziT£^2~—"——

1.0 10

SAMPLE RADIUS, cm.

W

Fig. 8. Length, speed, sample zone radius, and weight of rotor required for100% cleanout of virus at 10 liters/hr flow rate.

1000

31

10,000

ROTOR SPEED, rpm

ORHL DWG. 66-2939

100,000

Fig. 9. Horsepower per cm of length required to spin rotor in standard air.

1. S. I. Auerbach

2. R. Baldock

3. R. E. Biggers4. A. L. Boch

5. D. S. Billington6. C. J. Borkowski

7. G. E. Boyd8-9. Clark E. Center (ORGDP)

10-11. D. D. Cowen

12. C. J. Collins

13. F. L. Culler

14. P. B. Dunaway15. J. L. Fowler

16. J. H Frye, Jr.17. R. F. Hibbs (Y-12)18. A. S. Householder

19. J. S. Johnson

20. R. V,'. Johnson

21. W. H. Jordan

22. K. A. Kraus

23. M. T. Kelley24. J. A. Lane

25-26. C. E. Larson

27. T. A. Lincoln28. R. S. Livingston29. K. Z. Morgan30. D. J. Nelson

31. M. L. Nelson

32. J. S. Olson

33. R. B. Parker34. M. E. Ramsey

35. R. M. Rush36. M. J. Skinner

37. E. H. Taylor38. A. M. Weinberg39. G. C. Williams

40. H. I. Adler41-60. N. G. Anderson

61. W. A. Arnold

62. R. H. Baum63. M. A Bender

64. S. F. Carson65. E. H. Y. Chu66. G. B. Cline

67. W. E Cohn68. C. C Congdon

33

INTERNAL DISTRIBUTION

69. F. J. de Serres

70. D. G. Doherty71. W. D. Fisher

72-74. C. J. Whitmire, Jr.75. A. H. Haber

76. A. Hollaender

77. S. Karasaki

78. M. A. Kastenbaum

79. R. F. Kimball

80. S. P. Leibo

81. D. L. Lindsley, Jr.82. T. Makinodan

83. P. Mazur

84. G. D. Novel Ii

85. T. T. Odell, Jr.86. G. M. Padilla

87. A. P. Pfuderer

88. M. L. Randolph89. W. L. Russell

90. R. B. Setlow

91. G. E. Stapleton92. A. C. Upton93. E. Volkin

94. R. C von Borstel

95. T. Yamada

96. E. F. Babelay97. E. J. Barber

98. J. C. Barton

99. A. S. Berman

100. H. A. Bernhardt

101. Barbara S. Bishop102. H. D. Culpepper103. J. T. Dalton

104-118. E. C. Evans

119. J. Farquharson120. R. L. Farrar, Jr.121. W. W. Harris

122-124. W. L. Harwell

125. E. T. Henson

126. A. P. Huber

127. J. L. Liverman

128. J. G. Green

129. H. G. MacPherson

130. G. R. Jamieson

131. E. C. Johnson

132. T. J. Lewis

AppendixORNL-3942

Special

34

133. L. L. McCauley 141-142. Biology Library134. C. E. Nunley 143-145. Central Research Library135. P. R. Vanstrum 146. Reactor Division Library136. D. A. Waters 147-148. 0RNL-Y-12 Technical Library137. C. W. Weber Document Reference Section

138. S. J. Wheatley 149-195. Laboratory Records Department139. R. H. Stevens 196. Laboratory Records, ORNL, R.C.140. W. J. Wilcox, Jr. 197. Laboratory Shift Supervisor

EXTERNAL DISTRIBUTION

198. D. J. Davis, NIAID (Director), Bethesda, Maryland199. R. W. McNamee, Union Carbide Corporation, New York200. G. A. Andrews, ORINS, ORAU201. C. Baker, National Cancer Institute, Bethesda, Maryland202. F. Baronowski, AEC Production Division, Washington, D.C.203. N. F. Barr, Atomic Energy Commission, Washington, D.C.204. N. Berlin, National Cancer Institute, Bethesda, Maryland205. H. E. Bond, U.S. Naval Radiological Defense Laboratory, San Francisco, California206. Myron Brakke, U.S. Department of Agriculture, Agriculture Research Service, Plant Physiol.

Department, University of Nebraska, Lincoln 3, Nebraska207. H. D. Bruner, Atomic Energy Commission, Washington, D.C.208. W. R. Bryan, National Cancer Institute, Bethesda, Maryland209. J. C. Bugher, Puerto Rico Nuclear Center, Rio Piedras, P.R.210. W. W. Burr, Jr., Atomic Energy Commission, Washington, D.C.211. R. M. Chanock, National Institute of Allergy and Infectious Diseases, Bethesda, Maryland212. R. A. Charpie, Union Carbide Corporation, New York213. M. A. Churchill, Chief, Stream Sanitation Staff, Edney BIdg., TVA, Chattanooga, Tennessee214. W. D. Claus, Atomic Energy Commission, Washington, D.C.215. Helen Coates, National Institute of Allergy and Infectious Diseases, Bethesda, Maryland216. B. T. Cole, Department of Zoology, University of South Carolina, Columbia, S.C.217. E. Dapper, U.S. Army Chemical Corps, Fort Detrick, Maryland218. C. M. Davidson, Environmental Hygiene Branch, Edney BIdg., TVA, Chattanooga, Tennessee219. Irving S. Johnson, Eli Lilly Co., Indianapolis, Indiana220. 0. M. Derryberry, Director of Health and Safety, Edney BIdg., TVA, Chattanooga, Tennessee221. Harold S. Diehl, Sr., Vice President for Research in Medical Affairs, American Cancer Society,

521 W. 57th St., New York 19, New York222. C. L. Dunham, Atomic Energy Commission, Washington, D.C.223. K. M. Endicott, National Cancer Institute, Washington, D.C.224. S. G. English, Assistant General Manager for Research and Development, AEC, Washington, D.C.225. J. L. Fahey, National Cancer Institute, Bethesda, Maryland226. E. Frei, National Cancer Institute, Bethesda, Maryland227. F. E. Gartrell, Assistant Director of Health and Safety, Edney BIdg., TVA, Chattanooga, Tennessee228. H. Bentley Glass, Johns Hopkins University, Baltimore, Maryland229. Donald A. Glaser, Virus Lab., U. of Calif., Berkeley 4, Calif.230. H. N. Glassman, U.S. Army Chemical Corps, Fort Detrick, Maryland231. E. Grabowski, AEC Production Division, Washington, D.C.232. S. T. Grey, Food and Drug Administration, Washington, D.C.

35

233. N. S. Hall, University of Tennessee, Knoxville234. John Harris, AEC Public Relations Division, 0R0235. Fred J. Hodges, University of Michigan, Medical Center, Ann Arbor236. E. C. Horn, Department of Zoology, Duke University, Durham, N.C.237. J. G. Horsfall, Connecticut Agricultural Experimental Station, New Haven238. R. D. Housewright, U.S. Army Chemical Corps, Fort Detrick, Maryland239. R. J. Huebner, National Cancer Institute, Bethesda, Maryland240. J. S. Kirby-Smith, Atomic Energy Commission, Washington, D.C.241. R. M. Kniseley, ORINS Medical Division, ORAU242. K. W. Kohn, National Cancer Institute, Bethesda, Maryland243. P. Kotin, National Cancer Institute, Bethesda, Maryland244. E. L. Kuff, National Cancer Institute, Bethesda, Maryland245. W. T. Lammers, Davidson College, Davidson, N.C.246. A. D. Langmuir, Chief, Epidemiology Branch, Communicable Disease Center, Atlanta, Georgia247. R. E. Learmouth, National Cancer Institute, Bethesda, Maryland248. R. F. Loeb, College of Physicians and Surgeons, Columbia University, New York249. W. H. Magruder, National Cancer Institute, Bethesda, Maryland250. W. E. McMahon, Atomic Energy Commission, ORO251. R. G. Meader, National Cancer Institute, Bethesda, Maryland252. J. L. Melnick, Department of Virology and Epidemiology, Baylor University, College of

Medicine, Houston, Texas253. G. R. Mider, National Cancer Institute, Bethesda, Maryland254. Carl V. Moore, Washington University, St. Louis, Missouri255. P. T. Mora, National Cancer Institute, Bethesda, Maryland256. J. E. Moynahan, National Institutes of Health, Bethesda, Maryland257. M. A. Mufson, National Institute of Allergy and Infectious Diseases, Bethesda, Maryland258. W. G. Pollard, ORINS, ORAU259. C. A. Price, Department of Plant Physiol., Rutgers-The State University, New Brunswick, N.J.260. R. Quimby, National Aeronautics and Space Administration, Washington, D.C.261. H. M. Roth, Research and Development Division, AEC, ORO262. W. P. Rowe, National Institute of Allergy and Infectious Diseases, Bethesda, Maryland263. P. S. Sarma, National Institute of Allergy and Infectious Diseases, Bethesda, Maryland264. John Schacter, Union Carbide Corporation, New York265. D. G. Sharp, University of North Carolina, Department of Biophysics, Chapel Hill266. Byron T. Shaw, Administrator, Agricultural Research Service, U.S. Department of Agriculture,

Washington, D.C.267. C. S. Shoup, Atomic Energy Commission, Oak Ridge268. R. G. Smith, Food and Drug Administration, Washington, D.C.269. H. A. Sober, National Cancer Institute, Bethesda, Maryland270. Sol Spiegleman, Department of Microbiology, University of Illinois, Urbana271. W. M. Stanley, Virus Laboratory, University of California, Berkeley272. S. R. Tipton, Department of Zoology, University of Tennessee, Knoxville273. John Totter, Atomic Energy Commission, Washington, D.C.274. Rodes Trautman, Plum Island Lab., USDA, Greenport, L.I., New York275. Shields Warren, New England Deaconess Hospital, Boston, Massachusetts276. A. G. Wedum, U.S. Army Chemical Corps, Fort Detrick, Maryland277. J. White, National Cancer Institute, Bethesda, Maryland278. H. G. Wood, Western Reserve University, Cleveland, Ohio279. M. Zelle, Argonne National Laboratory, Argonne, Illinois280. R. E. Stevenson, National Cancer Institute, Bethesda, Maryland281. A. B. Sabin, Children's Hospital Research Foundation, Cincinnati, Ohio

36

282. C. E. Carter, Western Reserve University, Cleveland, Ohio283. E. R. Stadtman, National Heart Institute, Bethesda, Maryland

284-288. C. G. Zubrod, National Cancer Institute, Bethesda, Maryland289. Atomic Energy Commission, Washington, D.C.290. General Electric Company, Richland, Washington291. New York Operations Office292. AEC Patent Branch, Washington, D.C.

293-297. Division of Technical Information Extension, Oak Ridge298. UCLA Medical Research Laboratory299. Erwin Chargoff, Columbia University, New York, N.Y.300. R. D. Hotchkiss, The Rockefeller Institute, New York301. Curt Stern, University of California, Berkeley302. M. M. Wintrobe, University of Utah, Salt Lake City303. Arne Tiselius, University of Uppsala, Uppsala, Sweden304. Peter Alexander, Chester Beatty Cancer Institute, London, England305. Wayne Rundles, Medical School, Duke University306. J. A. Swartout, Union Carbide Corporation, New York, N.Y.307. Biomedical Library, University of California, Center for the Health Sciences, Los Angeles,

California

308-382. Daniel I. Mullally, Chief, Vaccine Development Branch, National Institute of Allergy andInfectious Diseases, Bethesda, Maryland