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THE-ts

This is to certifg that the

thesis entitled

Sedimentation Characteristics of the
T—3 Bacteriophage of Escherichia 0011 B

presented bg

Richard James DeLong

has been accepted towards fulfillment
of the requirements for

Doctor of Philosophy d - Microbiology and
agree m_T—Pub ic Health

MM‘mM/tg.

Major professor

Date m

0-169

SEDIMENTATION CHARACTERISTICS OF THE T3 BACTERIOPHAGE
OF ESCHERICHIA COLI B

BY
RICHARD Jf‘l'KDE LONG

A THESIS

Submitted to the School for Advanced Graduate Studies of
Michigan State University of Agriculture and Applied
Science in partial fulfillment of the
requirements for the degree of

DOCTOR OF PHILOSOPHY

Department of Microbiology and Public Health

Year 1956

 

 

ACKNOWLEDGEMENTS

The author expresses his sincere thanks and
deep appreciation to Dr. Walter N; mack for the guid-
ance and advice he has so generously given during the
course of this study and to the Research Division of
the united States Army; Fort Detrick, Frederick,
Maryland for financial assistance in the form of a

fellowship.

 

 

 

TABLE OF

INTRODUCTION . . . . . .
MATERIALS AND METHODS .
RESULTS . . .
DISCUSSION . . .. . ..
SUMMARY'.

BIBLIOGRAPHX'.

CONTENTS

INTRODUCTION

Characterization of viruses, both.p1ant and.animal,
has shown considerable individual differences in physical,
chemical andzmorphological preperties. Among the bact-
erial viruses, very little physical and chemical infern-
ation characterizing this group has been obtained. Most
‘work on bacteriophages has been performed with.the
familiar "T" strains of Escherichia coli B. Even.members
of this system.have been investigated.only sparsely in
relation to their particle constants.

A search of the literature reveals work on purifi-
cation and concentration of bacteriophages for
comparatively few strains. Thus, physical and chemical
analyses have been executed for relatively few bacterial
viruses.

For example, sedimentation characteristics have
been collected for only T2, T6 and T7 of the "T" system
and for the staphylococcus bacteriophage of Northrop
(Sharp 93 gr. 1911.6, Kerby 93; 3;. 19m, Northrop 1937 and
Putnam 22.§;. l9h9). Diffusion data have been compiled

for the Th, T6, T3 and.the staphylococcus bacteriophage

(Nbrthrop 1937, Polson and Shepard l9h9, Goldwasser
and Putnam 1951). The staphylococcus virus and T2,

T6 and T7 have been analyzed for nitrogen content
(Northrop 1937, Hbok 33 21- l9h6, Putnamu2§.gl. 19h9,
and.Kerby g§.gl. l9h9). Specific volumes have been
determined for T2, T6, T7 and NorthrOp's staphylococcus
bacteriophage. The most popular approach to investi-
gating the bacteriophages has been electron.microscopy.
In this respect, all the T-phages have been photographed
by various workers (Williams and Fraser 1953, Anderson
1952, and Fraser and Williams 1953).

The work accomplished by various investigators on
purified bacteriOphages has been.compiled.by Evans
(1952) in tabular form (figure 1).

Responsible, in part at least, for an aversion to
physical and chemical inquiry of these entities, is the
difficulty of purification and concentration.

In the past, most workers have labored with.proced-
ures for purification and concentration which are in-
herently time-consuming and laborious. Any method which
'would yield a high degree of purity and concentration,
yet, which could be simply and quickly performed, would
lend an impetus not only to bacteriOphage research.but
also to the utilization of bacterial viruses as research

tools.

 

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The determination of the particle constants of
bacterioPhages allows not only bases for comparison
between these viruses but also permits these constants
to be used as criteria in other experiments with each
particular strain of bacteriophage. For example, if
the sedimentation constant of a particular bacterio—
phage is known at neutral pH, then this constant can be
used as a basis in showing the effect of pH on its
sedimentation properties. Experiments of this type,
of course, can be extended to include the effect of any
physical, chemical or biological agent on the sedimen-
tation characteristics.

Of the T group of bacterial viruses, one of the
least investigated has been the T3 strain. With the
exception of electron microsc0pic studies (Williams and
Fraser 1953, Fraser and Williams 1953) and diffusion
results (Polson and Shepard l9h9), no other chemical or
physical information has been revealed for this bacterio-
phage.

Although.T3 is more difficult to concentrate than
the even-numbered bacteriophages, it should be.more ideal
for the study of physical behavior because of its more
Spherical shape.

It has been the purpose of the present study to

 

 

 

augment the characterization of the T3 bacteriophage
by securing data on some of its physical and chemical
properties and, especially, on its sedimentation

characteristics.

MATERIALS AND METHODS

Medium

F medium was used for cultivation of E. coli B.

Its components were:

NHflCl

mgsou

KHZPOh
NaZHPOu
Lactic acid
Distilled H20

Agar

1 gram
0.1 gram
1.5 grams
3.5 grams
9 grams

1 liter

20 grams

pH adjusted to 6.9

Buffer Systems

The buffer systems and their respectiveij values

used in these experiments were as follows:

Pl!
5
6

10
ll

Buffer System

 

M CHBCOOH + M Na02H302
IE4 NaZHroLL + 15; NatHZPOLL

MKHPO +MNaHPO
3' 21+ '5' 2 it

MNaHZPO‘I'MNaHPO
‘5’ 2 1* 3 2 L“

M NH3 + M NH1+Cl
M NH3 + M NHuCl
% NaZHI’OLL + % Na3POv4

Preparation of T3 Bacteriophage Stock Suspensions

Throughout the course of these experiments, care
was exercised.in manipulating the T3 bacteriophage so
that the least amount of inactivation of the bacterio-
phage might occur.

Petri dishes, containing standard "F" agar medium
adjusted to pH 6.9, were inoculated with.one drOp of a
suspension from an 18 - 2h hour slant culture of

E. coli 2. The drOps were spread evenly over the entire

 

surface of the agar, using a glass spreader. These
plates were incubated for approximately 18 hours at 37° C.
At this time, 0.5 ml. of T3 bacteriophage was added to
the agar surface of each plate and thoroughly mixed with
the bacteria by means of a glass Spreader. The plates
were again incubated at 370 C. for about 18 hours.

Upon the completion of this incubation period, 5 m1.

of buffer (pH 7) was added to each.plate. Once more,
the mixture of bacteria and bacteriOphage was agitated
with a glass spreader and the suspension from each plate
was decanted into an Erlenmeyer flask. Four to six
batches of crude lysate were prepared in this manner and
pooled.

The pooled lysate, obtained from the agar plates, was

centrifuged at 3,000 rpm. for 10 minutes in an Inter-
national centrifuge (Model PR-l). The supernatant
fluid was decanted and filtered through a bacterial-
sterilizing, Seitz filter. The filtrate was placed in
plastic tubes, transferred to rotor (B) and centrifuged
at u2,0h0 rpm. (llh,610 g) for one hour in a Spinco
model E ultracentrifuge.

The supernatant fluid was withdrawn by means of a
syringe and needle and the sediment was redispersed in
buffer (pH 7) in an amount to accomplish about a ten-
fold.increase in bacteriophage concentration. The re-
dispersed bacteriOphage was centrifuged at 6,000 rpm.
for 10 minutes in an International centrifuge (Model
PR-l). The supernatant fluid was withdrawn carefully
with a flat-tipped.needle and a syringe, placed in
plastic tubes and transferred to rotor (J). This sus—
pension was centrifuged again at h2,0h0 rpm. (110,660 g)
for 30 minutes in the ultracentrifuge. After this second
cycle of ultracentrifugation, the supernatant fluid was
withdrawn and the sediment was rediSpersed in buffer (pH 7)
in a volume about 1/5 that of the immediately preceding
suspension. Hence, an approximately fifty-fold concen—
tration of bacteriOphage was achieved by volume reduction.

The concentrated bacteriophage suspension was

centrifuged.once more in an International centrifuge
(model PR—l) at 6,000 rpm. for 10 minutes and the
supernatant fluid was withdrawn very carefully. This
supernatant fluid was used as stock material and the
sediment was discarded.

The volumes of crude lysate, at the beginning of
the concentration procedure, usually amounted to about
hOO-SOO ml. The final volumes, after concentration,
were usually about 6—10 ml. The mean infective titer
of the T3 bacteriophage increased from 7.7 x 1010
plaque-forming particles per ml. in the Seitz filtrates
to 3.h x 1012 plaque-forming particles per.ml. in the
concentrated stock suspension.

Concentrations of the T3 bacteriophage were best
expressed as weights per unit of volume when physical
constants were being determined. In order to obtain
concentrations expressed in these units and, yet, prevent
the bacteriOphage from desiccation, the following pro-
cedure was utilized. A clean, tared pycnometer was
filled with the buffer (pH 7) used to suspend the T3
bacteriophage. This was transferred to an analytical
balance and weighed. The pycnometer was then thoroughly
cleaned.and dried. Using this pycnometer, the same
volume of buffer (pH 7) plus T3 bacteriOphage suspended

in it was weighed. The difference in tho two weights

10

‘was considered the weight of the T3 bacteriOphage. The
result of the weight of the bacteriOphage divided by
the volume used, gave the concentration as weight per
unit of volume. The pycnometer and its contents were
always allowed to reach the temperature of the weighing
room.before weights were determined.

An important factor in preparing the stock bacterio-
phage suspensions for this study was the preparation of
a.material without loss of biological activity. In order
to determine the loss of activity resulting from the
process of preparation, the volume from the Seitz filtrate
was measured before processing, after which.an assay of
the infective activity of the T3 bacteriOphage was per-
formed by the standard agar plate method (Adams 1950).

At the end of the processing, the preparation's volume
was measured and another assay for infectivity was made.

From.this information, an estimation of the active
T3 particles that should be present in the final stock
suspension, assuming no loss of activity whatsoever,
could.be computed. For instance, the volume of the
filtrate from preparation B was 2h0 ml. and its infective
titer was 8.7 x 1010 plaque-forming units per ml. The
final volume of preparation B was 6 ml. The reduction in

volume of the suspending medium.was forty-fold. 0n

ll

multiplying the infective titer of the filtrate by to,
the calculated titer for active T3 particles in the
concentrated stock was 3.5 x 1012 plaque-forming units
per ml. The actual, infective titer of the concen-
trated preparation was 3.6 x 1012 plaque-forming units
per ml. Similarly, the calculated titer of preparation
A demanded 3.3 x 1012 plaque—forming units per ml. and
the actual titer was 3.1 x 1012 plaque—forming units per
ml. Preparation C was not tested for infective titer.
Such close agreement between the calculated and
observed titers of the concentrated preparations indicates
very little loss in activity of the T3 particles when

processed by the method.employed in the present study.

Biological Assay;pf_lpfectiyehBacteripphage Partiples

 

A 0.5 ml. amount from.a sample to be assayed was
pipetted into hdS ml. of nutrient broth.and diluted
serially. The dilutions selected for assay (usually
three successive dilutions) were transferred in 0.1 ml.
amounts to tubes containing 2.5 ml. of 0.2 per cent agar
and 0.5 ml. of a bacterial suspension, which was washed
from a 12—18 hour slant culture of E. coli 8, The soft
agar had been melted previously and cooled to hS“ 0.

The bacteriua—bacteriophage mixture was poured onto the

 

12

surface of nutrient agar plates and Spread evenly over
the entire surface of the nutrient agar. After harden-
ing of the soft agar layer, the plates were incubated
for 18 hours at 370 C. and the plaques which formed on
the plates were counted. Each dilution that was assayed
had been plated in triplicate and the average number of
plaques that had been formed on the three plates was
used as the plaque-count for a particular dilution. The
plaque-count from the dilution which yielded the statis—
tically best value was utilized to calculate the concen—
tration of bacteriOphage in the original sample. The
concentration was expressed as "plaque-forming units per
ml."

Titer computations were performed_in the following
manner:
plaque—forming units = plaque-count x.reciprocal of

per ml. dilution x 10

Nitrogen Analyses

Nitrogen determinations were performed by a micro-
Kjeldahl method. Because of the small amounts of
nitrogen expected, a colorimetric method was adOpted.
Digestion and nesslerization were accomplished by the
method of Folin and Denis (1916). Nessler's solution

was made according to that prescribed by Koch and

13

McMeekin (1921;). The transmittancy readings were per-
formed on a Bausch and Lomb Monochromatic Colorimeter

at a wave length of 505 mu.

-.—-.-.

Electron micrographs of the T3 particles were
prepared in order to have another means of determining
the purity of the preparations and to measure the
particle sizes directly.

Samples of the final stock suspension were prepared.
The samples were placed on collodion membranes. One set
of the samples was shadowed with palladium and photo—
graphed, while the other set was photographed unshadowed.
The electron microscOpe's magnification was calibrated

from a ruled diffraction grating.
Density Determinations

The densities of the T3 particle and the medium in
'which it was suspended were determined in an identical
manner. A tared pycnometer, which was calibrated to
contain 5 m1. of distilled water at 20° 0., was employed
for both determinations of density. The pycnometer was
made of Pyrex glass, having a low coefficient of thermal

expansion. Before filling, the empty pycnometer was

placed in a constant temperature water bath at 2L1..0° C.
(i 0.10 C.) for 5 minutes. It was then filled and
reimmersed in the water bath for another 5 minutes. If
any change in volume occurred, it was corrected to
volume in the bath. The pycnometer's surface, after
removal from the water bath, was thoroughly dried with
an absorbent, lintless paper and immediately weighed on
an analytical balance. The balance weights employed in
all of the weighings conducted in the present study were
unlacquered metal. Weights of specimens were measured
accurately to 10-h place. The calibrating liquid was
distilled water.

The teznperature of the pycnometer and its contents
was equilibrated to the average temperature of the
ultracentrifugal experiments .

Three weighings were performed for each sample and
the values obtained were averaged.

Calculations of density were made using the formula
given by Bauer (19h?) which yields results accurate
within about 0.1 per cent.

The formula prescribed. by Bauer is:

w d l
d=fio)° ()

15

where,

'W = uncorrected weight of sample

05:
u

uncorrected weight of calibrating liquid

9.:
H

density of sample at experimental
temperature

dO density of calibrating liquid at

experimental temperature

This equation does not correct for the effect of air

buoyancy of the samples on the balance pans.

Viscosity Detenminations

 

An 0stwald capillary viscometer, within the range
of pure water, was used to determine viscosities. All
viscosities were conducted in a constant temperature
water bath at 2h.0O C. (it 0.10 0.). The timing mech-
anism was a mechanical stOp watch (1' 0.2 seconds).

Apparatus was arranged, by means of clamps, in such
a.manner, that the viscometer was always in the same
vertical position within the constant temperature bath.

After filling the viscometer, it was placed in the
constant temperature bath and the temperature was
allowed to equilibrate by waiting for a period of 5
minutes before starting any determinations.

Viscosities were performed in the usual manner.

That is, distilled water's known viscosity and density

were used to determine
viscometer used.

cos ity was determined .

16

a constant C in relation to the

Then, knowing C, the sample's vis-

The relation may be expressed

mathematically as follows:

nk

where ,

0
II II II

then ,

and,

rf’

where ,

a”

Sedimentation Constant

0 Pk tk (2)
viscosity of water

a constant

density of water

time of flow of water

nk

pktk

(3)

4——

C pu tu (LL)

viscosity of sample
density of sample

time of flow of sample

Analyses

 

It is well known,

that in the determination of

sedimentation constants, the sedimentation rate is

17

dependent upon concentration of the solute. Therefore,
it is customary to measure the sedimentation rate at
different concentrations and obtain the sedimentation
constant of a freely sedimenting particle at infinite
dilution by extrapolating to zero concentration.

The same procedure was employed for the sedimen-
tation determinations reported within the present study.
Varying concentrations of the T3 particles were

made by taking a.measured volume (usually 2 m1.) of the
stock material and placing it in an open glass vessel.
The open glass vessel was transferred to a desiccator
containing anhydrous CaSOu. The desiccator was placed

in a 37° C. incubator and the liquid was allowed to
evaporate. It was found, by trial, that about 0.03 ml.
of liquid evaporated per hour under the conditions des-
cribed. When enough evaporation had occurred to give the
desired concentration (determined by accurately measuring
the remaining volume) the material was removed and centri-
fuged at 2,000 rpm. for 5 minutes in an International
centrifuge (No. 2). The supernatant fluid was carefully
withdrawn and transferred to the analytical cell of the
ultracentrifuge. Two cells of 3 mm. and 12 mm. light-
path.diameters were used according to the concentration

of the solute being centrifuged.

18

Calibration of the free thermocouple against the
reference thermocouple was performed before the
analytical sedimentation experiments were started.

All analytical experiments were performed at
12,590 rpm. (11,518 g) and.photographs were taken at
eight.minute intervals after the centrifugal velocity
had become constant.

The temperature of the analytical rotor was
determined immediately preceding and following an ex-
periment. This was accomplished with the free thermo-
couple which measures the temperature of the rotor. The
mean temperature rise of all the sedimentation rate
experiments was 0.30 C. The range of variance of the
temperature was 0.00 C. - 0.90 C.

The measurements of the boundary displacements were
made on the photographic images after the photographic
plates were deve10ped. At first, measurements were made
only after the photographic images were enlarged, but it
was decided to test the necessity of this extra manipu-
lation. Both direct measurements and.enlarged.measure-
ments were conducted on the same photographic image. The
mean deviation between the two methods amounted to

0.006 cm. Since sedimentation rates, even under the

best conditions, were reported reproducible to within

19

only about 1 per cent (Pickels 1952, Svedberg and
Pedersen 1911-0) , it appeared that the enlargement pro-
cedure could be obviated because the difference found
between the two sets of measurements would mean a
difference in the sedimentation rate of only about 0.5
Per cent. A perusal of the literature revealed that
the direct method of measurement is commonly employed
(Svedberg and Pedersen 3.91m, Pickels 191m.)-

For each sedimentation experiment, measurements
were made of all the boundary positions, except the last,
and these values were averaged. Five boundary positions
in each experiment were photographed. The first photo—
graph was made immediately after the ultracentrifugal
velocity had become constant and the four succeeding
PhOtOgraphs were exposed at 8 minute intervals there-
after. The migration of the boundaries of the first
four photographs was measured. Each migration of the
boundary from the first four photographs was calculated
for the sedimentation constant and these three values
were averaged, yielding the sedimentation constant for
a particular experiment.

Calculations of the sedimentation rates were made
according to the equation of Svedberg.

The Svedberg expression states that by measuring

20

the position of the boundary which demarcates the
supernatant fluid and sedimenting solute, at known
intervals, the mean value for the sedimentation con—

stant is obtained. Svedberg's equation is,

1n X2 "' 1n Xl
w? (t2 - t1) (5)

s:

 

where,

sedimentation constant

x = distance of boundary fram axis of rotation

angular velocity

time

Sedimentation experiments are conducted at differ-
ent temperatures in various laboratories and so, for
the sake of camparison, it is customary to correct
measured values of the sedimentation constant to some
standard condition. Svedberg has standardized conditions
not only in relation to temperature but also to the
density and viscosity of the suspending medium. This
sedimentation constant, symbolized.sw20, is the rate a
material would have in.a.medium.having the viscosity
and density of water at 200 C. Svedberg's equation for
SW20 is:

.- S n (O - pWZO) (6)
3we " 111,20 (0 - p7

 

21

where,
n = viscosity of medium at experimental temperature
0 3 density of solute
p = density of medium
nw20 = viscosity of H20 at 200 C.
pwgo 3 density of H20 at 200 C.

Units of sedimentation are usually given in
Svedberg units, symbolized S, which.may be defined as
centimeters p33 second 35 £912. In other words, one
Svedberg is 10"13 centimeter per second.

From.the sedimentation and diffusion data, the
particle weight may be computed. Svedberg has derived
an expression utilizing these data. This formula is as

follows:
RTSWO. _ _ __ (7)

where,

particle weight
gas constant

absolute temperature

U H3 :0 :3
u

diffusion constant

SW20 3 sedimentation constant

0
I

' density of particle

density of medium

'6
u

22

‘Whan calculating the T3 particle weight, the value
for D in this equation was taken fram the data of Polson
and.Shepard (l9h9). These authors reported the value
for D as 1.19 x 10"7 em.2 per second. The diffusion
canstant given by these authors was measured at various
concentrations and extrapolated to zero concentration.

Not only particle weight can be estimated from
SW20 but also particle size. The apparent or equivalent
diameter of a particle may be defined as the diameter of
an unhydrated sphere of the same mass and volume as the
actual particle under scrutiny and, therefore, does not
correspond necessarily to the over—all dimensions of the
particle.

Apparent diameters were estimated, assuming
spherical particles and no hydration, fram a formula

based on Stokes' Law,

2 _ 9ngWZ‘O
r .-
2 (pv - pg) (8)

 

Here,
r = radius of particle
n = viscosity of H20 at 200 0.

density of particle

’U
<3
ll

- density of solvent

'6
m
I

23

It is possible to estimate the particle size from
infectivity and nitrogen data also. If the infectivity
is determined, assuming that each plaque formed is the
result of one phage particle (Luria, Backus and Williams
1951), and the nitrogen measured for a purified prepar-
ation (assuming here that no extraneous nitrogen is
present), it becomes possible to approximate apparent
particle sizes.

A relation expressed by Putnam (1950) is:

weight of particle in grams = LLITr3 pv (9)
'3'

 

 

 

hence, 1— T 1/3
W‘
I, , <10)
EJT'Pv
3 L
L. 4
where,
w 3 weight of a single infectious unit in grams
r = radius of particle
pv = density of particle

w' may be obtained fram the following relation:

N

2
ll

grams of nitrogen

' concentration of phage from.infectivity
measurements

0
I

.nd
ll

nitrogen ratio to concentration weight

2h

In these equations it is assumed that the bacterio-
phage is camposed of all nitrogen-containing compounds.
Such an assumption is, perhaps, reasonably justified
since most bacteriophages, which have been chemically
analyzed, are composed of approximately 100 per cent
protein and.nucleic acids (Taylor l9h6, Kozloff and
Putnam 1950, Hook et a1. 19h6 and Evans 1952).

In addition, particle weights may be computed from

infectivity measurements. Using Avogadros' number,

M Z'WN (l2)

in'which,

{Z
l

' particle weight

’ weight of a single infectious unit

2
:

Avogadros' number

All the assumptions made for the particle size
expression from.infectivity measurements must be con—
sidered also for this relation. Values obtained from it
may be regarded as maximum values, since they depend on
the efficiency of the biological assay and the purity of

the bacteriOphage preparation.

;pH and Sedimentation Rate

Samples of stock bacteriophage of known concentration

25

(mg. per:m1.) were sedimented for 30 minutes in the
ultracentrifuge using rotor J at M2,0h0 rpm“ (110,660 g).
The pellets were resuSpended in distilled water and
centrifuged again for 30 minutes at the same velocity.
The supernatant fluid was removed and the pellets were
redispersed in buffer solutions at different pH values.
These samples were analyzed for sedimentation prOperties.
Each.sample possessed the same concentration (mg. per ml.)
of bacteriOphage.

At pH 5 and pH 11, after ultracentrifuging at the
operating velocity employed for the T3 particles in
neutral pH, the rotor was accelerated to 54,000 rpm.
(211,906 g) to determine the presence or absence of any

other boundaries.
Precision of Techniques

It is important to know the precision of a set of
measurements or of a laboratory procedure in an experi-
ment. A statistical measure of dispersion or variance
is often useful as an index of precision. Two of the
most familiar parameters of variance are the standard
deviation and the coefficient of variation. The standard

deviation is expressed as:

26

NIH

N
o .-. E (x1 - u)2 (13)
i = 1

N

o = standard deviation

8. measurement

N
u

u = arithmetic mean

number of measurements

The coefficient of variation is defined as:

100 o

C.V. = "jE-‘ (1h)

This parameter is expressed in per cent while the
standard deviation is given in the same units as those
measured.

The coefficient of variation for viscosity deter-
minations was 0.6 per cent. The same parameter for
density measurements of the T3 particle was 15 per cent
while that of the medium was 0.5 per cent. Nitrogen
analyses gave a value of 23 per cent for the coefficient
of variation. The sedimentation constants resulted in
a value of 1.3 per cent for the same parameter.

In the determinations of viscosity and density of
the suSpending medium, the coefficient of variation

applies to the method employed, since portions of the

27

same sample were used. For the remaining, probably most
of the variance is due to differences between sample
materials and not to the.methed or technique, since
different samples of the same type of material were
analyzed. It is known, for example, from repeated
nitrogen determinations on preparations of bacteriOphage
produced in this laboratory, that analyses performed on
portions of the same preparation are precise within less
than 1 per cent.

The coefficient of variation for particle densities,
nitrogen analyses and sedimentation constants gives an
estimate of the degree of dispersion that may be en-
countered between different preparations of the T3
bacteriophage when measured under the conditions described

in the present study.

28

RESULTS

All values reported in this study are average
values of all experiments completed for each particular
item. For example, the density values are the average
values of three determinations, viscosity results of
five, nitrogen content of three and sedimentation
constant of three.

The density of the suSpending medium was 1.01 grams
per m1. at 2h.0O C. The T3 particle density was 1.43
grams per ml. at the same temperature. Its specific
volume, which is the reciprocal of the density, was
0.70 ml. per gram.

The nitrogen content of the T3 bacteriOphage was
found to be 9.3 per cent. On a gram.weight basis, the
nitrogen content was 0.3 x 10~16 gram of nitrogen per
infective bacteriophage particle.

The sedimentation of T3, at neutrality, is depicted
in figure 2. A single, sharp, symmetrical boundary
migrating at a constant rate is observed. It is noted
that the light-absorbing region, due to Opalescence of
the bacteriophage, sediments with this boundary.

Sedimentation constants of three preparations of
T3 suspended in buffer (pH 7) are given in figures 3, h

and.5. Figure 6 is a plot of mean values for these

29

v
53
CL
F—I

O
,\

L'\

(\1

   

u n... mnr“. .
. mmmfimmfifl r. H I! OOHOM, Hume. 9,34. .wfi u._r
AR... was moment aw “unmeamnmdm manna)...

... C

 

no he..- . cu... at

 

 

 

433-3

30

PREPARATIOS A

’1

‘9

 

“-—-4—-5—+—+—§—-’.

canoe-traud
(~.Ih.’ .
Puma-o . Sodinants» = went-.8- W at u Ger-can
War-33hr. of 22‘. - :3! 7 be?!” an GB “in

.041-..

 

 

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1

Figure.h.

tn; oruturn

medium.

31

Patrmnon a

JL

M

3 . L g ‘

3?:‘12‘. catration
(mg.fll.)

30'11.‘.t3:.t -_-, “'

-.‘.: state. ;,.=s-romod at an "we; '

"f ';,.-u::; ~ .2. the “spending

 

..-

"¢‘rl_1r.H-Iil. .I n

1:50

hoo ,

353

300

32

PBEPARATIOH C

 

 

 

i——1 L 1 2t 41—4

Concentratim
(mg . /nl . )

a". so Mgntstjg, wt,“ 2 “$13551;th na-r-‘Pmod ‘t m avgrge :1
tflfieramg or 25'1 '5' , ; ’2’ enter as the suspending
no u,

 

 

° W

33

l i I
l 2 3

 

snacmmticn
1 xiv/m. a 2

Figure 6. Kean value 3:3...“ 2;? _, _<.~».e:¥%::~as A, B and 5..

 

m

 

 

31L

constants, derived from the graphic method of Askovitz
(1955). It is seen that the extrapolated values from
the mean plots (LL83S, LL73S and h67S) do not change sig—
nificantly from the direct extrapolations (h82S, h77S
and M678).

From.these three preparations the sw20 was computed,
yielding a value of h69S.

Within the range of pH’é - pH 10 only a single
boundary was observed.

Figure 7 is a plot of pH versus sedimentation con-
stant. The small differences observed within the range
of pH 6 - pH lOInay be due to the use of different
buffer systems. This same phenomenon is reported for
T7 (Kerby g£_al. 19h9) and these authors suggest that
it may be due to differences in hydration of the bacterio-
Phage particle in different buffer systems. They state
that although corrections for differences in viscosity
and density of the buffer systems were made the slight
variations in the sedimentation rate remained.

At pH 11 a multiple boundary, which was rather
diffuse, appeared (figure 8), indicating degradation of
the T3 particle.

At pH 5 the sedimentation rate increased to such an

extent that the lowest, automatical1y-controlled, centrifugal

500

 

 

P
_ A = 1V; neurone. n/Snnzpoh
E3 = a 333+ 11 IBhCl
0 = 14/5 3.23%.. 14/5 Iago),
_ Jo H/S K3301, + III/5 nazsrch
l I l J I
5 7 8 9 10 11
pH

Vigure 7. Sediaentatian expel-incite perfumed at a
concentration of 1. 5 mg per ml. and an average taperature
Of 2.1.OOC.

36

 

 

 

 

Figure 8. Asymmetrical, diffuse boundary of T3
sedimenting in a buffer adjusted to pH 11.

37

velocity on the ultracentrifuge proved to be too great
to obtain a sedimentation pattern. A boundary accumu-
lated at the meniscus at about 5,000 rpmt which, on
further application of centrifugal force, became very
diffuse and in a short time disappeared completely.

A possible explanation for such sedimentation
behavior at pH 5 might be the occurrence of heterogen-
eous aggregation of the particles. That is, at this pH
the T3 pOpulation might be composed of aggregates con-
taining different numbers of particles.

At pH‘5 and pH 11, after termination of the usual
observation period, the centrifugal force was increased
to 54,000 rpm. (211,906 g) to determine if boundaries
other than those observed at the centrifugal force
employed for sedimenting T3 particles were present. None
were observed.

Figure 9 demonstrates the absence of boundaries at
54,000 rpm.

USing the sedimentation data, the apparent diameter
of the T3 particle was calculated from equation (8),
resulting in a value of 45 mu, while that calculated from
equation (10), utilizing biological data, was 76 mu. The
particle weight of T3 was cemputed from equations (7) and

(12). Equation (7), employing sedimentation information,

38

 

 

Figure 9. Photograph showing the absence of any
boundaries in buffer (pH 11} at a centrifugal force
of 54,000 rpm (211,906 g).

 

 

 

 

39

6

gave a value of 28 x 10 and equation (12), which
utilizes biological data, gave a value of 199 x 106.

Figure 10 is an electron micrograph of a prepar-
ation of the T3 bacteriophage. The preparation is quite
homogeneous in relation to size and morphology of the
particles and little extraneous material is present.

The T3 particle diSplays the shape of an almost Spherical
structure which, upon close observation, is seen to have
somewhat of a hexagonally-shaped periphery. Also irreg-
ularities appear on the surfaces of many of the particles.
These irregularities are illustrated better in figure 11.

In figure 12 it can be observed that some of the
particles display definite, short processes extending
from their main.structures.

Uhshadowed preparations had been eXpected to reveal
some internal structure of the T3 particle. This was
not demonstrated (figure 13) because of the low absorb-
ing power for electrons by the T3 particle.

Measurements of the particles were made from shadowed
and unshadowed air-dried Specimens. The average diameter
of the particle in the shadowed photographs measured 50 mu,
while in the unshadowed, the average diameter measured

38 mu. These two values differed by 24 per cent.

 

Figure 10. An air—dried specimen of the T3 bacteria—-
phage stock suspension. Magnification (25, 500 .2}.
... Palladium shadowed.

 

 

 

 

Figure 11. Photograph of T3 particles displaying
irregularities on the surfaces of the particles.
Magnification (58,500 x). Palladium shadowed.

 

Figure 12. Photograph showing "tails" of the T3
particles (circles). Magnification (Lfl,500 x).
Palladium shadowed.

 

 

1+3

I O ;’. . . '0 ‘ a o
n .. . ' .m o ‘. .50..
’: . ' O ... ', o. '. .h. ‘
a ; .‘5 I . 0. : ' ‘

a .. . , , . \

Figure 13. An unshadowed, airwdried Specimen of
the T3 bacteriophage stock suspension. Magnification
(h8,960 x).

 

DISCUSSION

A substance which sediments with a single, sharp,
symmetrical boundary is customarily designated an
"apparently molecularly homogeneous" substance. This
designation is meant to convey the understanding that
a substance is purified within the limits of an ultra-
centrifugal method. It is entirely possible that
impurities may be present in concentrations below that
of the sensitivity of the ultracentrifugal instrument.
Moreover, an "apparently molecularly homogeneous" sub-
stance need not be homogeneous in other respects; for
example, it may not be homogeneous in relation to its
electrOphoretic preperties. Thus, semantically, it
would be more correct, perhaps, to label such a substance
”apparently centrifugally homogeneous". Nevertheless,
a single sedimenting boundary of the T3 bacteriOphage
may be obtained.

The purity of a substance, as defined above, can
be judged qualitatively by inspection of the sharpness
and symmetry of the boundary. The sharper the boundary,
the:more homogeneous is the substance.

Sedimentation diagrams that have been observed in

the present investigation appear to be as sharp and

 

1L5

symmetrical as any that have been reported.in the
literature for any of the bacteriophages (Putnam gt_gl.
19149, Jesaitis and Goebel 1955, and Putnam 93; §_1_. 1952).

In order to make a comparison of the measured
particle constants of T3 with those of other purified
bacteriOphages more facile, figure 1 has been inserted
once more on page up but this time with.the values of
T3 tabulated. The strains, which are related morphologi-
cally and serologically, have been grouped together.

Since there is nothing in the literature on the
sedimentation preperties of T3, comparison cannot be
made with values obtained by other workers. However,
comparisons can be made between the morphologically
similar and serologically related T7 phage (Kerby gt_gl.
l9h9, Putnam.et.al. 1952).

From figure 1 it is seen that T3 and T7 are serologi—
cally related. Also, they are very similar in size and
shape when viewed with.the electron microscOpe. Possess—
ing such similarities, they might be expected to be
similar in some other respects. Their Sw20 values (MEGS
for T7 and h69S for T3) differ by approximately 2 per cent.
The specific volumes (0.68 for T7 and 0.70 for T3) agree
6

within 3 per cent and the particle weights (31 x 10 for

T7 and 28 x 106 for T3), derived from.sedimentation,

is

 

typo Ultramfintmuon coirio Intootivity hurtiolo
913m

 

 

 

 

 

rihzge Volta.
Sufi dog Mohoq an (svzolmu-
0" . 10’
too. nu «1% In 113/; Fla-g? Ill 106 106
no »
T1
T5
1.3 3
re 1000 59 0.66 39-16 107
700 50
Th 0.80 53

 

rs loco 61 0.1;; 91; 0.66 Lek 9a 167%

 

 

 

 

 

 

 

 

 

 

 

 

 

 

9?}? F1}; 10.
0.3 X
T3 {5:} ‘; a”, 1.19 36 0.79 10‘16 76 28 199
17 use 1.3 0.68 0.51: 804 nine
it 0.1 2 0.8 1.0
“ml 690 77 i 35 3 1°. 93 5” L

 

i’igm-o 1. hwticlo occwuntl at can purified untow-
(1.”: er swat-s, 195?).

1+7

agree within less than 10 per cent. Putnam.§tflal.

(1952) report 5 values for T7 which varied from

w20
hSOS to h9OS with a mean of h658.

Kerby gt El. (l9h9) reported that the T7 bacterio—
phage at very dilute concentrations (0.025 mg. per ml.)
exhibits a much lower sedimentation rate than at the
commonly employed concentration range (5 mg. to 0.5:mg.
per ml.). These authors stated that they performed the
sedimentation eXperiments on T7 by an absorption-method
which.permits measurements of bacteriOphages at dilute
concentrations. This was possible because of the bacterio-
phage's high absorbency for traviolet light. The
instrument used in the present investigation for the
sedimentation of T3, employed the refractive index method
and did not allow measurements of very dilute suSpensions.
Therefore, it was not possible to determine whether T3
displayed similar sedimentation behavior with the re-
fractive method. Sharp gt a1. (1950) reported that this
anomaly occurs with dilute suSpensions of polystyrene
latex particles also. These authors conclude that it is
due to instability of the boundary between the supernatant
fluid and the sedimenting solute.

Dual sedimentation has been reported for the

bacteriOphages T2 and T6. With T2 a single boundary of

 

us

about 1000 S is always found below pH 5.8 and a single
boundary of about 700 S is obtained above pH 5.8. An
immediate transition from the slow rate to the fast can
be accomplished by pH adjustment and the transition is
reversible. A similar effect of pH on sedimentation
behavior is observed with the T6 phage, except that
both forms frequently have been found together over a
wide range of pH. The fast component is seen singly at
leh.9 and has a value of about 1050 S while the slower

component has a value of about 800 S.

 

Taylor gt El: (1955) claimed that in their work on
the T2 phage they found, by indirect methods, that the
dual sedimentation behavior was not due to reversible
aggregation or hydration but to a reversible change in
shape of the particles.

The stability of the sedimentation rate for T7,
when subjected to changes in pH, covered a fairly wide
range (Kerby 23.31. 19u9). T7 sedimentation was found
to be constant from pH 5 through.pH 10. In comparison,
the stability for T3 ranged from pHC6 through.pH 10.

A peculiar behavior by T3 was observed when the pH
was adjusted to 5. At this pH, the sedimentation rate
increased greatly. At pH 11, T3 showed signs of degrad-
ation. This was evidenced by the appearance of a multiple
boundary. T7 also diSplayed another boundary at pH ll

(Kerby gt 3;. l9u9)

 

M9

In the pH region where sedimentation stability was
apparent, small variations occurred in the sedimentation
constants. Upon plotting the data, a correlation appeared
to exist between sedimentation constant and the type of
buffer system utilized. This same phenomenon was re-
ported for T7 (Kerby gt 3;. l9h9) and these workers
pointed out that the amall differences observed.may be
due to differences in hydration of the bacteriophage
particles.

The close agreement between SW20 values of differ-
ent preparations of T3 is satisfying. This indicates
that the method of preparation used in this study is
quite reproducible. Indeed, when one considers the ex—
treme variability that may be encountered in biological
materials, such agreement is almost amazing.

Electron micrographs of the T3 and T7 particles
reveal structures almost morphologically identical. The
irregularities observed in shadowed preparations and the
inability to depict defined, internal structures in the
unshadowed samples were reported also for T7 by Kerby
_e_’_c_ _a_l. (1919).

It had been mentioned before that electron micro-
graphs of the T3 particles displayed short processes

extending outward from the main structures of the particles.

 

50

These might be considered short "tails", analogous to
the "tails" of T1, T2, Th, T5 and T6. These "tails"
were visible in only a fraction of the population but,
probably, this could have been correlated with.the
plane of orientation of the "tails". T7 displayed the
same short, stubby "tail" (Kerby e§_§l, l9h9).

The difference in the two particle size measure-
ments, made from.shadowed and unshadowed preparations,
could be due to the high contrast accomplished by shadow-
casting, thereby rendering the outer edges of the
particles visible. Since the extent to which matter
scatters electrons depends on its density, it would
appear that the T3 bacteriophage is not homogeneously
dense but contains a less dense periphery circumscrib-
ing a more dense core.

Kerby et a1. (l9h9) reported particle sizes for
T7 measured from shadowed and unshadowed Specimens.

Their values were 73 mu for the shadowed and 51 mu.for
the unshadowed samples, resulting in a difference of

31 per cent between the two measurements. Electron
micrographs of T3 have been published by Williams and
Fraser (1953). These authors utilized frozen—dried prep—
arations. Their particle size measurements from shadowed

samples gave a value of h7 mu. They presented excellent

 

51

photographs which demonstrated the short tails and
hexagonal shapes. In another publication, Fraser and
Williams (1953) photographed T3 from.air-dried prepar—
ations. The particle measurements from these air-dried,
shadowed.samples were 50 mu. Earlier’measurements
(Anderson l9h6) gave a value of h5:mu.

Upon comparing the bacteriophages T3 and T7, it
becomes evident that they are closely related and very

similar. It appears that the only significant difference

 

between the two lies in the degree of their response to

homologous antisera.

52

SUMMARY

The T3 bacterioPhage has been investigated in
relation to some of its physical preperties and, par-
ticularly, its sedimentation behavior.

The 3w20 was found to be h69 S when the values from
three determinations were averaged. A single, sharp,
symmetrical boundary, migrating at a constant rate, was
observed.

Sedimentation stability extended through.pH 6 -
pH 10. At pH 11, degradation of the virus occurred.
Increased sedimentation rate was observed at pH 5.

The specific volume of the T3 particle, derived
from three measurements and averaged, was 0.70 ml. per
gram.

Micro—Kjeldahl analyses revealed 9.3 per cent
nitrogen for the T3 bacteriOphage and 0.3 x 10"16> gram
of nitrogen per infective particle. These values were
lower than those found for T7, and could be indicative
of less extraneous material in the T3 preparations.

Equivalent diameters, calculated from sedimentation

and infectivity data, gave values of h5 mu and 76 mug

reapectively, while particle weights, computed from

 

53

sedimentation and infectivity data, gave values of
28 x 106 and 199 x 106, respectively.

Particle sizes of T3 measured directly from
electron micrographs averaged 501ml.

The shape of the T3 particle in air-dried prepar-
ations was roughly spherical but, on closer observation,
it was noticed that the particles displayed hexagonally-
shaped structures and possessed a short, stubby "tail".

The particles were not of uniform density.

 

 

 

5h

BIBLIOGRAPHY

Adams, M. H., Methods in Medical Research.
J. H; Comroe, Jr. (ed.), 2, Yearbook Publishers,
Chicago, 1950.

Alber, H. K., Determination of Specific Gravity, Ind.
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Anderson, T. F., Nbrphology and chemical relations in
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Symp. Quant. Biol., ll:l-l3, l9h6.

- , Stereoscopic studies of cells and
viruses in the electron microsc0pe. Am. Naturalist,
.§6:91-100, 1952.

 

Askovitz, S. 1., Rapid method for determininngean values
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Bauer, N., Determination of density, Physical Methods of
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Beams, J. W., The production and.maintenance of high
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Cohen, S. S., and Anderson, T. F., Chemical studies on
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, and Arbogast, R., Chemical studies in host-
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55

Delbruck, M5, Bacterial viruses. Adv. in Enzymol.,
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<, Biochemical mutants of bacterial viruses.
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Demerec, M. and Fano, U., BacteriOphage-resistant
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Evans, E. A., Jr., Biochemical studies of bacterial
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Folin, 0., and Denis, W., Nitrogen determinations by
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Fraser, D., and Williams, R. 0., Details of frozen-dried
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Goldwasser, E. and.Putnam, F. W., Physicochemical
prOperties of bacteriOphages. J. Biol. Chom.,
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Herriott, R. M. and Barlow, J. L., Preparation, purifi-
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J. Gen. Physiol., 36:17-28, 1953.

Hook, A. E., Beard, D., Taylor, A. R., Sharp, D. G.
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Jesaitis, M. A. and Goebel, W. F., Lysis of Th.phage by
the Specific lipocarbohydrate of phase II
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Kabat, E. A., and Mayer, M. M., Experimental Immuno-
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Kerby, G. P., Gowdy, R. A., Dillon, E. S., Dillon, M. L.,
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56

Koch, F. C. and kabekin, T. L., A new direct nessleri-
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, Williams, R. C. and Backus, R. 0.,
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, Ultracentrifugation. Methods in Med.
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Pollard, E. G., The Physics of viruses. Academic Press
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SEDIMENTATION CHARACTERISTICS OF THE T3 BACTERIOPEAGE
OF ESCHERICHIA COLI B

By
RICHARD J. DE LONG

AN ABSTRACT

Submitted to the School for Advanced Graduate Studies of
Michigan State University of Agriculture and Applied
Science in partial fulfillment of the
requirements for the degree of

DOCTOR OF PHILOSOPHY

Department of Microbiology and Public Health

Year 1956

Approved by

 

 

Richard J. DeLong

ABSTRACT

Some of the physical prOperties of the T3 bacterio—

phage of Escherichia coli B were investigated and, in

 

particular, its sedimentation behavior. The specific
volume of the T3 particle was 0.70 ml. per gram. Nitro-
gen analyses of the T3 bacteriophage revealed 9.3 per cent
nitrogen. The nitrogen content of the infective T3
particle was 0.3 x 10-16 gram of nitrogen. The sediment-
ation constant was A69 Svedberg units. A single, sharp,
symmetrical boundary, migrating at a constant rate, was
observed. Sedimentation stability extended through pH 6 -
pH 10. At pH 11 degradation of the bacteriOphage occurred.
Increased sedimentation rate was observed at pH.5. Equiva-
lent diameters, calculated from sedimentation and infectivity
data, were H5 mu and 76 mu, respectively. Particle weights,
calculated from sedimentation and infectivity data, were

28 x 106 and 199 X 106, respectively. The particle size

of the T3 bacteriOphage, measured directly from electron
micrographs of air—dried specimens, was 50 mu. The T3
particle's morphology was roughly spherical in air—dried
preparations. Stubby ”tails” were observed on some of

the particles.

 

 

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