I OF L.
ORNL P
115
.
9931a
.
.
-
-
MICROCOPY RESOLUTION TEST CHART
NATIONAL MUME MU OR STANDANOS-10
*
TESTS
.
.
.
.
2
L
.
<
te.
This paper was submitted for publication
in the open literature at least 6 months
prior to the issuance date of this Micro-
card. Since the U.S.A.E.C. has no evi-
dence that it has been published, the pa-
per is being distributed in Microcard
form as a preprint.
.
.
-
ik
-
:
.
111
.
.
:
.
.
.
17

1
SA
WM
12
J'
Me 1
"
T
.
AL
wLX
I
(1
:
:
.
1/2
-
&
.
-
.
.
1
1
.
1
-
L
.
-
$
.
-
7
-
LEGAL NOTICE
This report was prepared as 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 representa-
tion, expressed or implied, with respect to
the accuracy, completeness, or usefulness
of the information contained in this report,
or that the use of any information, appa-
ratus, 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 in the above, person acting on
behalf of the Commission" includes any em-
ployee 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 employ-
ment or contract with the Commission, or
his employment with such contractor.
".
.
.
.
NY
IS
A
.
1.
.
.
117
.
.
.
1
.....
-
IA
L
-
.
-
-
-
0
.
.
1
X
UN
i
'
1
1
.
.
.
.
1.
PN
G
.
WWW
Why
IN
IN
UN
".
.
.
/
PIA
EN
TA
I
VI.
L
"
NS
T
AI
WM
i
1
.
UN
.
*
.
TY.
..
.
-
ORAL P-115
-
Submitted to:
Science
DTIE

WL 161964
.
MASTERP
.
Y
.
'.
.
.
all esya
>
Natural Water Iraotionatian.
:
.
7
i
1532
>
Bulk Isolation of Virus Partioles ♡
.
.
COLN
m
ala t William T. Lamers Ve
id of Division of Health and Safety,
couhllite Tennessee Valley Authority
Biology Division, Oak Ridge National faboratory
Butach Pielgo, Tennessee
.
.
.
CA
Bore
→
.
.
W
.
24
14
.,
SWI
*prosoat addross:Biology Dopartment
Davidson Collogo
Davidson ,N.C.
The paper wa mbaattted for poblication
in the open utaratar at least aonths
prior to the innance date of the Micro-
aard. Since the 1.8.A.B.C. has no evi-
dince that it has been published, the pa.
per is being distribited to Microcard
form as a propriat.
A
.
.
LEGAL NOTICE -
The man weapon www Overl ord worth Honor test
tor, wou , memoria ya
Contactos
A. Manymore
,
er d
et ut more
me,
a
dunutulmurah
om
, at det, men detoed women w e niet tegen
Now I wona, pero no Who
mayor online daten
a contrato de Outen,
er d
et man
wentowe, w Wola
ortor.
I
'
-
-
1
.
i
.
.
7
sumimin
3
Regolamento del control time to time commission
o Present address: Biology department, Dawidaan College, Davidlam
.
.
!
-
.
Natural Viator Fraotionation
"
SOM
-
Bulk Isolation of Virus Partioles
William T. Lamers
Division of Health and Safety
Tennessee Valley Authority
IN
2
-
-
=
A
.
2
AK
9
22
FA
LI
R
.
Y
.
72
ev
10
:
.
*present address:Biology Department
Davidson College
Davidson ,N.C.
3
I
22
?
pe
FE
C
YE
.
*
IR
.
.
"
T. LAMMERS
*
*
.
11K
liatura) 776?
r^cticraticn
MEANZA
Bulk Isolation on Virus Particles
:
;
.
Resmirisiminen niin
.
Abstract:
articl... ci viral dimor. sic!!S:27. bor i sciotod from the
rajativa)y "Cloar. stroam"conditions of stts Bar Dam tailraco on
th? ???ns ses river. a narticl?shvo barr. charactrized by
derity, a scrution at 260 mi, ara .ectron microscopy.
TAS
. LU
-
5
-..
-
.
the isolation and characterization or subrnicroscopic particles
*!
't
s
'
suspended in larsa volumes of llouid arz of interest, in saritary
, .,
*-
:
7
2
A2
.............Sterft'a'ir su..ASDX
z
-
-
1
*
-
=
=
--
-
9"
TEA
.......
$
ita
ET
'
TA
WK
LS
engineering and public health(3,7,9,18,19,23), nesa? i.nization of
water(5), ce}] physiology(1.,2,6), and virology(16,17,22,26). "ho
i solation of microscopic particles irom large volumes of natural
water has been described in earlier parers(12,13,13). It is kr.cun
that co.3.?cida? sized particles, including virus narticles, cecur in
natural water(7,11., 23), and some work has been cionn on their rome:v:1).
(1,12,13,14,15), occurance(8,19,20), and activity(10,2+). Sor:o human
diseas? Soutbreaks have been attribued to virus particios carried in
water supplies(3,7,21), the investications roportod so far have
been concerned with the virus particles in rather concentrated
situations such as sewage(4,19,20), wasiing pools(9), or in situations
where the particles have been deliberately introduced for study
purpo se s(2+,25). The general knowledge of virus particles in potabje
water has been revieved recently(7).
In many areas of the natural water mass, especially relatively
uncontaminated stroans and lakes, little is known of the numbers,
types, or stability of virus particles. For wator that will be used
ultimately for human consumption, it is of nore than academic
interest to invastigate these factors. Water examination by the
1
TRS
.7.
M741
!
!
AR
-
S-
-
-
WA
I.
is
07::sicni lions ci virolcky, such as the plaqur assay, will 119
0....1: ort raitis ci to cocuror: arc stability of the specific,
kacent types (partici3: saroho! iur, but they vill y191d no
Incration on other types of virus particles occurring in the same
$11:31. m:mor oi virus partic??s in sest rolatively uncontaminated
3?e? .?" Yton 3523 is too 900 for "ho troet application or
bicohysic.l tacniques such as ultravic) et absorption or electron
microsco?y. liowavor, in orior to investigate tha total virus
polation or a natural watər is sig tha se biophysical methods would
seo... to ba tie only approach sircea widescribed or rare virus
particles, which mey be present, volila bo unceasured by the classical
cet!:ods. In aliition, knowr. virus particios have generally been
discoverod by their a ssociation with disease in other organisins,
but there is no reason to suppose that all virus particles cause
obvious patrological symptoms in their hosts' so that if the less
specii'ic bicpiysical methods can be applied, new types of virus
partic233 may be discovered.
Thus,for the investigation ci virus particies in natural water,
two thirris must ba accor!plished 32-сre the biophysical methods can
24
.
Y
-
particles 523. encies in the water. The second is separation of the
organic virus particles from the inorganic colloidal clay particles
whici will be cor.contrated with the virus particias. After these
steps have been accomplished the isolated particlos may be
exa inod by various bicphy sical. tzonniquas to obtain norphclogical
characteristics.
In tho investigations reported here, two 26 liter water sanpl.es
till
.

-
t
.
..
3
.
-
wore tain from the tail raco of its ar Pan on the Tennessee.
River sy rathods rescribed ej sor"?n?(12,13,14). The first sample
vas tayon or: 6/19/63 and the second on 1/2+/62. The investigation.
of the first simple was completel on 2/21/63 and that of the sacond
on 9/14/63. Both samples were hanized in the same manner excent
that in the case of the second sample only electron microscopy was
used ror exarination and a shorter tine elapsed from samplir:ā until
_-
.
.
final anal.y si s.
-
th
5
3
.
is
.
*.
As a first step, noncolloid:) particulate material was removed
from the water sample by continucus flow centrifugation at 50C (28 5
X%, Rmax:7cr:,1].ow rate 50 m3/min). The colloidal sized particles in
the supernatant water from this centrifugation were concentrated by
recentrifugation in batch lots of 940 m] in an angja head rotor
(21 K RPM for 2 hours, Rmax 12cm, Amin:6 cm). Particles with
sedimentatior rates as low as 200 S are sedimented in this
centrifugal field.
In the first water sample the coljoidal sized particles from
approximately 16 liters of the super natant water were collected by
centrifugation and the pellets saveri. By the time five liters had
been centrižuged a pell at was visible in each tube and by the time
six liters had been centrifuged the pellets appeared to be somewhat
gelatinous and tan in color. The pallets from this series of batch
centrifugations were combined in a single tube, resuspended in a
10 ml volume of the supernatant water and divided into four
aliquots ( one I nl and three 3 m2). The one m2 aliquot was sterile
filtered through a 0.45 u membrane filter to remove clumped and
aggregated material and the filtrate examined by electron microscopy
AL
N
.
SRR
-
-
HI
.
.
(1::: 2:21.194 results :10p" pactro jori: sosy is less that
01.::95:779 1 :atriy 7.5 7. virile Rüc?93 ir ki? on in
76 2107" ar ?. ?? ?03 3 ::? :":10:s !!"99, 10 :t: ... 07.97apmis
978 :39 221 dar si ti" TO :, 002r.sity 1.6,"28 3..3, 15.
1.?).. 0 1:lecl ir: ar a:'?'"?? ruter l'or + hours + 3" K2
(...24:. j C:.1::3.. 0:0). min 31 : nni extrifugation s?rvadi
to 3021269 11:9 (2.Pric virus :7*1*17*:!0: a accorrir: clay
partici?:37. ti coricartrata to vi:43 cortic]:: ir i sc in sit;' sorus.
bor. m ib? equilibrium was raathi, trio cartrifare was scoped
arii tartibga srafiert vos ni 13.1 throun an ultraviolet analytic)
syst
(1). 10 rzesriini; ci the ultraviclet
absorbion 20 260 g from the 41-ang: drzity prodierit:??? cor.sity
1..3) is siior ir. :1-re 3. Dis 26. 27 thit thora !?
virus garcinias 9295rt with a wide ranza carion siti?s..
ti
apana
22:
10 K5 litara
öots outi:3093 of virus partic.?re extranolations from ":
original 1022 Comorts of the sa?? 773 üuc vc?93 to tra criinn?
16 lito simile.
. ???9 results or ...ectron 10:0500n or the second water sample
gave an astiate or the nu:vor o virus particles in the original
Sple ci l. : 307 in 20 litors.
The differeres in tho wount or virus marticles batween the ti:0
00:103 rosorted hora is prona: reg?, an doror: zurates that with
the is0i' ti.o cortrirucal tochnians 19scribed the virus particias
i: alry water mass may be isolata ad pilysically investigated.
O If the cor.cer.tration of the virus particles in a givan water mass
0
..
Is joss char the cases prosinte:! ere, a largor water sample way
bu ta!:2r. so that a sufficiont rubar o virus particies ara
obtnin?;} icr investigatiori, mithi: rnea song any arouri ci wät ar con
bez processed by this meth.ca.
Deciric virus tyris cannot be positively inartifint :10??y 0.
the jasis of morphological charactari:"ios. ??curvor, t??:2 27 octron
microcraghs !0 show some morpholorical types of virus particias wich.
are similar, or even identical, in appagrancto know virus types.
Positive identification can only be made by using the classical
nothods of virology in conjunctior with morphology'until enough
irfcrmation is obtained to identify the particias on the basis of
morphology. ince it is now possihle to concentrate and isolata
virus articles from the alluta si sper sicris occurring in matura]
water, the ciassical methois may be applied to isolated æ? lovots
i'or positive identification of certain particles. sicwever, at
presort, the new unknown virus tynas can only be characterize: by
biophysical techniques. Ivariu. Dena usiniho
colloct
i tuł wita I.
PSG minotia with
فتنتمممتععمممممقحطغيط دم مth
واحط ج معت
in the contrition vi tire particles
I thank Drs.li.G. Ander an and W.:)..'i sher biology Division ORNI for
encourageuent and loan of facilitins. I thank Mr.Te: Dartlett,?echrical
Division, ORGDP for preparing the electron micrographs mit Procerry
Vaughan, CRIN 3 for technical assistance.
?:iter ::. ?
?....rarson, ána 1.Elochedi. 4, 265-263 (1962).
2.:... rakorch.Blochen.Bionhys. ,?75-200(1953).
3.0.C.ro:ngimor.jour.Pub,!!ealth 39, 75!4.771 (1949).
14011....019.8., 2.. Cavon son, J.,C.hing and P.%.Kabler, Ansr.jur. Fub.
10:7th 51,11.1E-1129(1.961.).
5.0....0.152..ber. ii. 47,776-513(156).
6.7...'izier,6.3.Cline, and N.G.Anderson, The hy siologist 6,179(1.953).
7.2.1..12b13r, in innov.licrobiol. 16,C...C] Irton ind.(Annual Reviews
Inc.?'a] o 13.to, alif.1962), p 127.
8. 5.1!...elly, wer.jour.Pub.realth 1-3,1532-1538 (1.953).
ge g erd it.. Sanderson, Fuw.tealth Repts.75,199-200 (1961.).
10.... Kristen son and K. Scaduauge, Acta.?'athol.et :icrobiol. Scard.
1+0,21.6-225(1957).
11.2.1. Trundiegel,Jour. Amer.later !!orks Assn.36,83.-25(1944).
32.%..Jammers, Lim:rol.and Occarorr. 7,224-229 (1962).
13.
cience 139, 1298-1298 (1963).
I'mo
paper presented at the XV International Congress of
Linnology, adi.son vis. 1962.
15. S.G.Jensen, H.hian, R. Stebbiris,i.C.Bacuis, and C... Peterson,
Amer.d cur.Pub.i!ealth 39,11.20-112(1949).
16.11. w Loring and 7...G.Wyckoff gour.3101.Chei. 121,225-230(1937).
17.0.cIntosh,7.1. elbic, Brit.Jour. xt1.Path. 21,153-160(1940).
18.1. c.cinney, :icrobiology for sanitary Engineers(IcCrawt!111.,11..,
1962), 142-143 and 1.64.165.
19.3.1..Helnici,J.com:ons,j.l.Coffey,and H... School, Amer.Jour.!!ycana
59,164-1.84(.1954).
20._ ,..Opton, and , Ibid. 59,185–195(1954).
0
/
-
-
-
-
..
1.415-11:24 (1661).
22...C.Orice and Rov.Gonyokoff, Nature 141, 685-686 (1938).
23.6.1!. Spies, Health Bull.Gregon State Ed. Health 21, 3-4 (1943).
24.1. Squori, A.Ioli, and A. Garrani, Ann.Sclavo. 3, 404-4 14 (1961).
25.2...@sielewski,S.Carlson, K.!!. Husna nn, and G.Cristofordis, Aron.
Hulu Eakt. 145, 184-189 (1961).
26.2.1.C.ilyokoff, Jour.Biol. Chem. 121, 219-223 (1937).
10unds
"igura 1 and 2:
21 actrcı: ::1.0: ranh3 cm Viru S.11':-? "artic?03 i soleted from a water
sas: 719 taker: ot intts Bar Par. tall.r:Ce on the Tanna sseo 1 Vor(80, onox).
164r3:
0
Absorption as 260 m in a CsCl din:1. ty yradient by viru 13-11%
particles. Ciorbriturari bor hours sui 30 31.7., an X: 8,1 cm,!imin:3.8 cm.
تستهل
مهمة
. م
مسلم:. م
:
.
... مدم. .. ... ... دسنس. سندسه.. ... ...
...
... سهام ، من سنصه.. ...
-
.
ر
ا
را
در
انجام
.
-
1
-
-.
.
-
.
Figh
|
. '
4
.
.
E
|
.
.
.
.
.
·······
.
… .
-
-
Fig 3
Refractive
Index, CsCI
my
Optical Density , 260

Floating
Material
Base
Peak
Bottom
Material
1-31
204
133 137
138
133
134
133
Density
of CSCI

en relation
t
remo
e
more
TT
: 0.
in
.
12.
!!
I
"
I
.
"
..
N
VLT
..
1.
---
R
**.
.
i
..
.
.
.
Wi.
*
:
-
i
1. 12
12
MY
-
"
-
DATE FILMED
7 / 22 /65
M
I
.
.
.
END
1.
-