JOURNAL OF VIROLOGY, Jan. 1968, p. 21-25 Vol. 2, No.

I
Copyright e 1968 American Society for Microbiology Printed in U.S.A.

Efficient Filtration and Sizing of Viruses with

Membrane Filters
BINIE A. VER, JOSEPH L. MELNICK, AND CRAIG WALLIS
Department of Virology and Epidemiology, Baylor University College of Medicine,
Houston, Texas 70025
Received for publication 4 October 1967

Untreated membrane filters retain viruses by adsorption, as well as by physical

restriction which occurs when the pore diameter of the filter is smaller than that of
the virus particle. As originally recommended by Elford, membranes had to be
pretreated with proteinaceous material to preclude virus adsorption. However, coat-
ing materials that prevent adsorption of certain viruses do not necessarily prevent
adsorption of other viruses. In contrast to proteins, salts enhance virus adsorption.
Viruses treated with sodium lauryl sulfate to reduce the surface tension, or purified
viruses in distilled water, are not adsorbed to membranes. A procedure is recom-
mended by which viruses may be passed through membranes with a porosity twice
the diameter of the virus. Such filtrates, which contain 50 to 100% of the initial virus
concentration, should be used for sizing viruses by subsequent filtration through
smaller pores. The determination of virus size would then be based on the major
population of particles in the virus suspension. In the past, as little as 0.1 to 0.001%
of the initial virus population was the basis for size determination, because more than
99.9% of the virus was often lost by adsorption to membranes during the clarifying
procedures.

To prevent virus adsorption to collodion mem- MATERIALS AND METHODS

branes, Elford, in 1931 (la), recommended that
membranes be treated with fluids containing
proteinaceous materials. Nevertheless, investiga-
tors (3, 4) still filter and size viruses with un-
treated membranes that may adsorb the major
portion of the virus population. For example,
rubella virus has been reported as larger than
100 m, when untreated membranes were used
(4). Givan et al. (2) showed that the virus passed
100-m,u membranes if pretreated with infusion
broth.
In the studies cited above, only small amounts
of virus could be detected in clarified filtrates;
upon subsequent filtration, no infectivity could
be detected in the samples. When such clarified
filtrates are used for sizing a virus, they often
contain only a minute fraction of the initial virus
population. Since virus can be adsorbed to mem-
branes having a porosity several times greater in
diameter than the virus under test, erroneous sizes
of the virus may be reported. The current study is
concerned with the efficient filtration of viruses
through membranes, so that the major population
of virions is used in the size determination.
21
Viruses and their assays. Type 1 poliovirus
(Mahoney), vaccinia virus (WR), herpesvirus (JES),
and rubella virus (WER) were used in these studies.
Monkey kidney (MK) cells. Kidneys from immature
green monkeys were trypsinized and grown in Mel-
nick's medium A and maintained with medium B.
Since the medium in which viruses are suspended plays
an important role in the adsorption of viruses to
membranes, all viruses used in this study were grown
in MK cells maintained with medium B (lactalbumin
hydrolysate in Earle's salt solution). Viruses were
assayed by counting plaque-forming units (PFU).
Overlay medium consisted of Earle's salt solution,
0.4% NaHCOs, 1:60,000 neutral red, 0.1% skim
milk, and 1.5% Bacto-agar (Difco). Additives used in
overlays for plaque enhancement of poliovirus were
25 mm MgCls (10) and, for herpesvirus, 400 ,ug/ml of
protamine (7). Rubella virus was assayed by the
interference method using Newcastle disease virus
(NDV) as a challenge. After challenge with NDV,
sheep erythrocytes were added to cultures, and clear
plaques, to which erythrocytes did not adsorb, were
counted (5).
Membrane filters. Membrane filters (Millipore
Corp., Bedford, Mass.) used in this study were 25 mm
in diameter, and were contained in syringe adapters.
22 VER, MELNICK, AND WALLIS J. VIROL.

Membrane-coating agents. Materials used to treat TABLE 1. Coating agents used for pretreatmenit
membranes were prepared as follows. (i) A 100-ml of membranie filters with porosity twice the
amount of 10% fetal calf serum in distilled water was virus diameter
filtered at 25 psi through a 90-mm AP 20 clarifying
pad and then through 0.3-sI, 0.22-,, O.1-,u, and 0.05-Iu Percentage of virus recovered' in
membranes, in series. To the final filtrate, lOX Earle's HAIem- filtrate from membranes treated with
'brane
saline was added in sufficient volume to restore iso- Virus Po-
tonicity. This filtrate was then used to treat membranes rosity brs 1 0% iVeal
Virusrt Tris Fetal Infu- Bovine Cell
prior to virus filtration. It was capable of coating but buffer calf sion albumin extract
not clogging membranes subsequently used for virus serum Broth
filtration. (ii) Veal Infusion Broth (Difco) was con-
stituted as described by the manufacturer and used Poliovirus 50 0 100 100 100 100
without dilution. It was clarified as described above. Vaccinia 650 0 40-50 20 40-50 20
Herpes 300 0 100 30 60 0
(iii) Bovine Plasma Albumin (Armour) was made up Rubella 100 0 100 25 100 10
to a 1% suspension in tris(hydroxymethyl)amino-
methane (Tris) buffer and used after clarification as a Approximately 100 plaque-forming units of virus were
described above. (iv) Normal monkey kidney cultures hltered for each test.
were frozen and thawed to simulate a virus harvest, bTris = tris(hydroxymethyl)aminomethane.
and the material was clarified as above.
Sodium lauryl sulfate (SLS). A 10%/o suspension crease in titer was undoubtedly due to aggregated
of SLS was made in distilled water and adjusted to
pH 8.0 with HCl. The stock was then clarified through virus, as shown by the work of Sharp and his
a GS membrane to remove insoluble particles. associates (6).
Filtration of enteroviruses. In the above section
RESULTS we dealt with filtration conditions for viruses
present in low concentrations, only 103 PFU/ml.
Effect of different coating agents. In orientation We next examined the filtration conditions for
experiments, we found that different coating more concentrated virus. A 5-ml sample of un-
materials did not always effectively prevent virus diluted poliovirus was passed through a single
adsorption. Therefore, the following type of ex- filter holder containing a series of untreated mem-
periment was performed to determine the most branes (450 m,u, 300 my, 220 m,, 100 m,u, and
effective membrane-coating materials for the four 50 m,u, respectively). A sample of the filtrate was
types of virus used in this study. removed for assay; the remainder of the 50-
Viruses were diluted to contain 100 PFU/0.1 m, filtrate was passed through a 20-m,u mem-
ml in Tris buffer, which renders the virus sensitive brane, and this filtrate was also assayed. Simulta-
to adsorption on untreated membranes (8). The neously, 5 ml of undiluted poliovirus was filtered
diluted stocks were filtered through treated and as described through a series of membranes that
untreated membranes with a porosity size twice had been pretreated with 10%0 serum to preclude
the diameter of the virus under test. Membranes virus adsorption. A third 5-ml sample of virus
were first treated with the 5-ml volumes of the was treated with 1 %N SLS, at pH 7.5, to reduce the
reagents shown in Table 1, and were then washed surface tension of the suspension, and the sample
with 5 ml of Tris buffer to remove the residual was then filtered through a series of untreated
fluids used for treatment. Samples of each virus membranes. A fourth sample of virus was diluted
(5 ml) were then filtered through 25-mm mem- 100-fold in distilled water to reduce the salt con-
branes with 1 psi. Unfiltered virus and virus centration and surface tension, and 5 ml of this
filtrates were then assayed simultaneously and sample was also filtered through a series of un-
yielded the results shown in Table 1. treated membranes. The results of this test are
No detectable virus was found in filtrates of shown in Table 2.
untreated membranes or of membranes treated The untreated virus which was filtered through
with Tris buffer. Poliovirus was effectively passed the untreated membranes yielded a 50-m,u mem-
through all treated membranes without decrease brane filtrate of 3.0 log1o virus. Thus, 99.99% of
in titer. The results were not as uniform, however, the virus had been adsorbed to the membrane.
in the case of the other viruses tested. Vaccinia These results are similar to those obtained by
virus, herpesvirus, and rubella virus were not Hsiung (3), who determined the size of poliovirus
significantly adsorbed to membranes treated with based on holding back only 0.001% of the initial
serum or albumin. Thus, the sites that bind these virus with 10-m,u membranes, because only this
viruses are possibly those coated with albumin or tiny fraction of the original virus was present in
other serum proteins. the 50-m, membrane filtrates. As shown in Table
It is noteworthy that vaccinia virus could not be 2, samples of poliovirus filtered through mem-
quantitatively recovered in the filtrates. The de- branes pretreated with serum yielded a 50-mju
VOL. 2, 1968 FILTRATION AND SIZING OF VIRUSES 23
TABLE 2. Filtrationt of poliovirus branes with serum avoids the adsorption of
viruses. This led us to a procedure for analyzing
Log virus titer (PFUa/ml)
the number of aggregated units in a virus prepara-
Mem- tion. Viruses were passed through pretreated
Virus treatment treante Unfil- Filtrates membranes with a porosity twice the diameter of
ment tered 5-A 2-n the virus under test. The decrease in infectivity of
control mOems 20-em- the filtrate represented aggregated viral units.
brane brane
The quantitation of aggregated virus was per-
None None 8.1 3.0 0.0
formed by using vaccinia virus as a model. Un-
Serum 8.0 7.9 0.0 diluted virus was filtered serially through four
1% Sodium lauryl sul- None 8.1 8.0 0.0 650-m,u membranes pretreated with serum as
fate
Diluted 100-fold in None 8.0 8.0 0.0
distilled water TABLE 3. Limiting filtration of viruses indicating
percentage of virus population used to size virus
PFU = plaque-forming units.
Percentage of virus
Mem- passed Mem- Pevrcenag
membrane filtrate which contained virtually 100% Virusesa brane
filterb' brane
filterb pofae
vru
pasdi
of the initial virus. Subsequent filtration through (mA) Earlier Present (mA) present
study
studies study
a 20-m,. membrane failed to reveal any infectivity.
In the present experiment, in which 100% of the Polio 50 0.1-10 90-100 20 0
virus population was used, poliovirus was sized Vaccinia 450 0.01 40-50 220 0
as > 20 mA and < 50 mu. Similar results were Herpes 300 10 100 100 0
obtained when surface reducing agents were used, Rubella 100 0 90-100 50 0
or when the virus was suspended in distilled a Before being used for above experiments, each virus was
water. diluted to contain about 100 plaque-forming units per 0.1
A number of the enteroviruses were filtered ml in tris(hydroxymethyl)aminomethane buffer.
through a series of membranes as described above bJust before virus filtration, each membrane was treated
with and without added sodium lauryl sulfate, with 10% fetal calf serum as described in text.
with essentially the same results reported above
7
for poliovirus. TREATED MEMBRANES
Detergent-sensitive lipophilic viruses could be
passed through untreated membranes if they were
suspended in distilled water.
6
Limitingfiltration of viruses. When viruses were
filtered through untreated membranes, their diam-
eters were reported on the basis of a very small
percentage of virus present in the clarified filtrate enE
U) ~-
5
(for example, see references 3 and 4). Sometimes 0\
\
A
the filtrate was subsequently passed through an > _L ,Ol UNTREATED MEMBAES
additional membrane, which, regardless of size,
left no detectable infectivity, not because of 1-0
U 4
limiting pore size, as reported, but because of 4
>L
virus adsorption. 0
-J
Table 3 shows a comparison of the present
results with those of earlier studies in which un-
treated membranes were employed. Under proper 3 k 0 \\
conditions, the virus population should pass al-
most completely through a membrane of twice the
diameter of the virion. However, to cite the case \v T
2
of vaccinia (3), the determination of the porosity ST 2ND 3RD 4TH
of the membrane retaining the virus was based on UNFILTERED 650m# FILTRATES
only 10 PFU out of 100,000 PFU that had passed
membranes of larger porosity. Although the 10 FIG. 1. Quantitation of aggregated vaccinia virus. A
PFU were retained by the 100-m, membrane, it is 5-mi sample of undiluted vaccinia virus was filtered
likely that these few virions would have been ad- serially through four serum-treated 650-m, membranes.
sorbed to a membrane of any porosity. Samples of each filtrate were obtained for assay before
passing the filtrate through the next membrane. A
Quantitation of aggregated vaccinia virus. As duplicate 5-ml sample of vaccinia virus was filtered
originally described by Elford, treatment of mem- throughl four untreated 650-m,u membranes.
24 VER, MELNICK, AND WALLIS J. VIROL.

described above. A duplicate sample was filtered 4.1 MCC

through four 650-m,u untreated membranes. The V IRUS -,.
results of this experiment are shown in Fig. 1. p;WATER ;, *e
S* ALTS
Vaccinia virus filtered through the first pretreated
membrane was decreased threefold in titer. When
this first filtrate was filtered through the second
pretreated membrane, no further decrease in titer
was evident. Similarly, the third and fourth fil-
trates were equal in titer to the first filtrate. There- 450m# -;- -
fore, it is evident that the first filtration removed
virus aggregates which were too large to pass the
pores of the membrane. The mono-dispersed virus
in the first filtrate could then be serially filtered
without further loss of infectivity. On the other
hand, vaccinia virus filtered through untreated
membranes was dramatically adsorbed to these
membranes, with linear decrease in infectivity on 450m#--
serial filtration.
DISCUSSION
To determine virion size by filtration, the viral
suspensions must first be filtered through a series
of membranes for clarification, to preclude the
clogging of membranes (la). In the process of 450m#
filtration of a virus suspension through a series of
untreated membranes with pore diameters at
least twice the diameter of the virus, membrane- .i4f4.-
coating components adsorb to the membrane
filter (8). Membrane-coating components con-
tained in virus harvests are made up of organic,
cell-associated materials and serum. When they
occupy sites on membranes, they block virus FIG. 2. Schematic diagrams illustrating the effect of
adsorption. When membrane-coating compo- salts on virus adsorption. Serial filtration of virus
nents are added to a membrane containing ad- suspended in salts resulted in adsorption of membrane-
sorbed virus, the virions are rapidly eluted and coating components (MCC) to membranes. As MCC is
removed, the virus becomes amenable to adsorption by
replaced by membrane-coating components. subsequent membranes (right series). However, in
Virions suspended in salt solutions free of organic hypotonic solutions, neither virus nor MCC are ad-
material adsorb to membranes, whereas, in sorbed to membranes (left series).
distilled water, the virions pass through the mem-
brane pores and are found in the filtrate. If entero-
viruses adsorb to membranes during the early moved by adsorption to the clarifying membranes.
phase of a filtration procedure, membrane-coating Thus, the clarified virus is usually filtered through
components in the suspension will subsequently the fine porosity membranes in a medium con-
elute the virus into the filtrate, and the adsorption taining little or no membrane-coating component.
phase may go unnoticed. However, if suspensions The virions at this point are suspended in pre-
of a lipophilic virus are filtered through a mem- dominantly salt solution; this enhances the ad-
brane, and the virions are adsorbed, membrane- sorption of virus to membranes, regardless of their
coating components cannot elute these viruses pore diameter. Such filtrates contain only a small
(8). Thus, filtration of lipophilic viruses (even fraction of the initial virus population and should
through large-pore membranes), under condi- not be used for sizing a virus.
tions in which the virions adsorb to membranes In this report we have shown that pretreatment
before they are coated with membrane-coating of membranes with coating agents, as originally
components, yields filtrates with reduced infectiv- recommended by Elford in 1931 (la), or reduction
ity. of surface tension of virus suspensions (1, 6a), al-
As a virus harvest is serially filtered through lows virtually 100% of the virus population to be
membrane filters, as shown diagrammatically in found in the filtrate. Subsequent filtration through
Fig. 2, membrane-coating components are re- sizing membranes holds back virus on the basis of
VOL. 2, 1968 FILTRATION AND SIZING OF VIRUSES
size alone. Such filtrates are free from the non-
neutralizable, persistent fraction of viruses (9) and
are preferred for kinetic studies on the inactiva-
tion of viruses.
It has been emphasized that efficient filtration
of viruses requires that membranes be pretreated
with a proteinaceous diluent that will adsorb to
membranes and preclude virus adsorption. How-
ever, in the case of viruses which are resistant to
the effects of detergents, the virus suspension can
be treated with SLS and can be filtered through
untreated membranes without significant virus
adsorption to the membrane. Viruses can also be
diluted in distilled water in order to lower the salt
concentration to below 0.002 M, and then filtered
through untreated membranes without any virus
adsorbing to them.
ACKNOWLEDGMENTS
This investigation was supported by Public Health
Service grants A105382 and 5 Ti AI74 from the
National Institute of Allergy and Infectious Diseases.
The technical assistance of Lillian Brinkman is
gratefully acknowledged.
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