J. gen. Virol. (I978), 39, 377-380
377
Printed in Great Britain
Chromic-Acid Formaldehyde Fixation of Nucleic Acids of
Bacteriophage ¢6 and Infectious Bovine Rhinotracheitis Virus
(Accepted 7 December I977)
SUMMARY
Bacteriophage ¢6 nucleic acid was present as a torus after chromic acid-formaldehyde-OsO~ fixation and acetone and propylene oxide dehydration. A herpes
virus, infectious bovine rhinotracheitis virus, had its DNA mostly as a torus,
collapsed in the centre, or as a network, after glutaraldehyde-OsO4 fixation, but
in an uncollapsed torus or network formation after chromic acid-formaldehydeOSO4. This fixative stabilized nucleic acids, allowing acetone dehydration and
plastic embedding without collapse of nucleic acid to the centre of the virion.
Gonzalez et al. 0977) have shown that the double stranded RNA of bacteriophage ¢6
is present as a torus in the virion after glutaraldehyde-OsO4 fixation when dehydrated with
a water-miscible plastic but not when dehydrated with acetone. A toroidal conformation
of nucleic acid has been shown for only two viruses, bacteriophage ¢6 and a herpes virus.
The toroidal nature of herpes DNA in the virion was discovered after glutaraldehyde
fixation, ethanol dehydration and epoxy plastic embedding (Furlong et al. I972). Glutaraldehyde does not react with nucleic acids except after prolonged contact at elevated temperatures (Hopwood, I973) and even then no crosslinking occurs. Nucleic acids in glutaraldehyde-OsO4 fixed tissues are thus essentially unfixed. Chromic acid, on the other hand,
reacts with nucleic acids as well as with proteins (Johansen, I94o).
This note reports that a chromic acid-formaldehyde fixative stabilizes nucleic acids of
phage ¢6 and infectious bovine rhinotracheitis virus (IBRV) and preserves their integrity
during acetone dehydration followed by epoxy plastic embedding. A preliminary report has
appeared (Langenberg & Sharpee, I977).
Purified ¢6 preparations were gifts of C. F. Gonzalez and J. E. Partridge of this department. The bacteriophage was precipitated from solution by adding a few drops of 0"3
Alcian Blue (Polysciences, Warrington, Pa.) and centrifuging for I min at looo rev/min
to form a pellet for subsequent fixation.
Infectious bovine rhinotracheitis virus was propagated in cultures of a foetal bovine
kidney cell line (NL-BK-3). This cell line was used at the 37th to 39th passage levels.
Growth medium consisted of Earle's r99 medium (E, 11 ; Grand Island Biological Company,
N.Y.) and 0. 5 ~ lactalbumin hydrolysate (LAH; National Biochemicals Corp., Cleveland,
Ohio) supplemented with lo ~ foetal calf serum (tissue culture grade, Cat. no. 3000;
Kansas City Biological, Inc., Lenexa, Kansas) and antibiotics 0oo units penicillin G and
Ioo/~g of streptomycin sulphate/ml).
The Colorado I strain of IBRV was used at a constant passage level and was propagated
either in confluent cell monolayer cultures grown at 36 °C or on carbon-coated coverslips
(Langenberg et al. I972). Before inoculation, growth medium was removed and the cultures
were infected by adding maintenance medium (growth medium without serum) containing
Io 55 TCIDs0/ml IBRV. Incubation was continued at 36 °C until visible cytopathic change
occurred, usually x8 to 24 h after inoculation when the virus was harvested. Cells were
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378
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2
Glutaraldehyde-OsO4
(d/)
CrO3-formaldehyde-OsO4
Dureupart
Acetone
Acetone
01106
9/34
0/272
92/106
0/34
0/272
3/106
•3/34
86/272
0/106
0/34
13/272
1/106
1/34
12/272
41106
1/34
62/272
6/106
10/34
99/272
:??~;
(b)
(0)
. . . . . . 2;.....
(d) .... ~: ;
:
(e)
(f)
150 am
(g)
Fig. I. Toroidal configuration of the ds-RNA of bacteriophage $6 (large arrows) preserved after
CrOa-formaldehyde-OsO4 fixation and acetone dehydration. Torus sectioned in a plane perpendicular to the ring (small arrows).
Fig. 2. Infectious bovine rhinotracheitis virus DNA configuration after fixation and dehydration
as for ~6: (a) collapsed in centre of virion; (b) collapsed to one side; (c) toroidal ring; (d) sectioned in
plane of ring; (e) across plane of ring; (f) as 2 to 3 parallel lines; (g) as a network. Figures refer to
the frequency with which the different structural forms have been encountered after the indicated
fixation and dehydration method. Only extranuclear virions were tabulated.
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379
scraped off culture flasks with a rubber policeman and pelleted by low speed centrifugation
IOOOO rev/min for IO min in a RC-2B Sorvall centrifuge with an SS34 rotor. IBRV in the
supernatant was pelleted by centrifuging at 28 ooo rev/min for 45 min in a Spinco Model
LI ultracentrifuge with a type 3o fixed angle rotor. Pellets of IBRV, or buffer-rinsed coverslips with attached cells, were chilled on crushed ice and flooded with chilled 2 ~ glutaraldehyde (Polysciences) in o-I M-K~HPO~ and adjusted to pH 7"3 with solid citric acid. This
bufferwas used throughout. After fixing for o'5 h the glutaraldehyde fixative was replaced without rinsing by buffered o'5 ~ OsO~ at o °C for I5 min. At this point, samples were divided.
One half was washed twice in cold buffer, 15 min/change, dehydrated with a graded Durcupan A series (Polysciertces) to lOO ~ and embedded in complete Durcupan according to
the manufacturer's recommendation, or Durcupan A was gradually replaced with complete
Epon 81 z. The other half was dehydrated with a cold graded acetone series without rinsing,
brought to room temperature in 95 ~ acetone, and embedded in Epon 812 via propylene
oxide.
Bacteriophage 46, IBRV and IBRV-infected cells were also fixed in o.I ~ chromic acid
in o.i M-K2HPO4, the pH having been adjusted to 7"3 with solid citric acid. The chromic
acid-phosphate citrate was diluted from a stable IO x strength stock solution stored at
3 °C. Just before use, freshly prepared, neutralized paraformaldehyde was added to a concentration of I ~ from IO ~ solution. Virus and tissues were fixed overnight, I6 to ~8 h, at 3 °C,
rinsed four times for I5 rain each in cold phosphate-citrate buffer and post-fixed with
cold, buffered 0"5 ~ OsO4. Chromic acid-formaldehyde fixed samples were all dehydrated
with a cold acetone series, brought to room temperature at 95 ~ acetone and embedded in
Epon 812 via propylene oxide.
Thin sections were double-stained with uranyl acetate and lead citrate and viewed in a
RCA-3G or Philips 2oi electron microscope.
Bacteriophage ~56 nucleic acid was present as a ring in the virion after conventional acetone
dehydration and embedding in plastic (Fig. I), showing that it had been stabilized by the
chromic acid-formaldehyde fixation. The toroidal nature of phage 46 ds-RNA (Gonzalez
et al. I977) is thus confirmed with a second fixative. IBRV-DNA was mostly present as
dark staining material towards one side of the virion after glutaraldehyde fixation and
Durcupan dehydration but also in other configurations (Fig. 2). Little difference was found
between samples dehydrated and embedded in Durcupan or dehydrated in Durcupan but
embedded in Epon 812. After initial tests, the latter method was preferred for ease of
sectioning. After glutaraldehyde fixation and acetone dehydration, D N A in IBRV virions
was seen as being collapsed in the centre and also in other configurations (Fig. z), including the toroidal ring described by Furlong et al. (I97Z). Chromic acid-formaldehyde fixed
samples were not dehydrated in Durcupan but only in acetone and propylene oxide. No
IBRV-DNA was found collapsed or without some structure (Fig. z) using this fixative.
Approximately 40 ~ of 272 virions examined after chromic acid-formaldehyde fixation
had their nucleic acid in configurations conforming to a ring (c, d, e, Fig. 2). However,
approx. 6o ~ had their nucleic acid fixed in a manner resembling various sections through
a structure which could have been made up of more than one ring of D N A ( f to g, Fig. 2).
These rings may be closely appressed (c, d, e) or be in different planes (f, g). It must be
borne in mind that the various configurations shown in Fig. a were selected as typical forms.
Many intermediate forms were present and frequently a choice had to be made to assign a
particular section to one form or another.
The fine structure of virions was well preserved after chromic acid-formaldehyde fixation
as could be observed by projections extending from the outer surface of IBR virions.
~5
V l R 39
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380
C h r o m i c a c i d - f o r m a l d e h y d e m a y be useful as a control, or s e c o n d a r y fixative, in ultrastructural investigations. This contrasts with g l u t a r a l d e h y d e which does n o t react with
nucleic acids at t e m p e r a t u r e s o r d i n a r i l y used in electron m i c r o s c o p i c investigations ( H o p w o o d , 1973).
C o o p e r a t i v e investigation o f the U S D A , A g r i c u l t u r a l R e s e a r c h Service, N o r d e n L a b o r a tories, a n d the N e b r a s k a A g r i c u l t u r a l E x p e r i m e n t Station. R e s e a r c h c o n d u c t e d u n d e r P r o j e c t no. 2 I - I 2 . P u b l i s h e d as J o u r n a l Series, P a p e r no. 538t, N e b r a s k a A g r i c u l t u r a l
E x p e r i m e n t Station.
U.S. Department of Agriculture
Agricultural Research Service
Department of Plant Pathology
University of Nebraska
Lincoln, Nebraska. 68583 and
Norden Laboratories Inc.
Lincoln Nebraska 68528 , U.S.A.
W . G . LANGENBERG
R . L . SrlARPEE
REFERENCES
FURLONC,D., SWIFT,H. & ROIZMAY,B. (1972). Arrangement of herpesvirus deoxyribonucleic acid in the core.
Journal of Virology xo, Io71-IO74 .
GONZALEZ, C. E., LANGENBERG, W. G., VAN ETTEN, J. L & VIDAVER, A. K. (I977). Ultrastructure of bacteriophage 46: arrangement of the double-stranded RNA and envelope. Journal of General Virology 35,
353-359.
HOPWOOD, n. (I973). Recent advances in fixation of tissues. In Electron Microscopy and Cytochemistry,
pp. 367-38i. Edited by E. Wisse, W. Th. Daems, I. Molenaar and P. van Duijn. Amsterdam: NorthHolland Publishing Co.
JOHANSEN,D. A. (I940). In Plant Microtechnique, pp. 523. New York: McGraw-Hill Book Co., Inc.
LANGENBERG,W . G.. & SHARPEE,R. L. (1977). Stabilization of bacteriophage 46 and infectious bovine
rhinotracheitis virus nucleic acids by fixation x~ith buffered'chromic acid-formaldehyde. Annual Proceedings of the American Phytopathological Society, pp. 15o-i 5 I.
LANGENBERG, W . G. SCHROEDER, H. F., WELCH, A. B. & COOK, G. E. (I972). Epoxy film separable from glass
surfaces for selective hght and electron microscopy of tissue and in situ grown cells. Stain Technology,
47, 3o3-3o8.
(Received 27 September I977)
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