JOURNAL OF CLINICAL MICROBIOLOGY, Feb. 1975, p. 225-233 Copyright © 1975 American Society for Microbiology
Vol. 1, No. 2 Printed in U.S.A.
Improved Recognition and Quantitation Technique for Oncornaviruses R. STEPHENS,* K. TRAUL, D. WOOLF, G. LOWRY, AND J. LELEK Pfizer Inc., Maywood, New Jersey 07607
Received for publication 23 September 1974
A technique to recognize and quantitate oncornaviruses using perforated pointed BEEM capsules has been developed in our laboratory. Virus samples that presented problems in counting, or those which could not be evaluated at all by negative staining, could be clearly defined and counted using the thin-sectioning BEEM capsule technique. Perforation of the BEEM capsule allowed rapid infiltration of reagents into the tip of the virus pellet and made further manipulation and orientation unnecessary. The sensitivity of this technique, determined by making serial dilutions of viral concentrates, allows observation of as few as 5 x 105 virus particles per ml. Precision in counting by this technique varied only + 0.3 log in repeat aliquots of identical concentrated virus samples quantitated, making this a highly useful and reliable system.
Since 1949, several electron microscope virus quantitation methods have been described (5, 10, 12, 13, 19). Precision in the technique varied only slightly, but problems were encountered with recognition and/or loss of virus. With the introduction of negative staining, a procedure that defines surface morphology, a new era began in electron microscopy particle counting. Using the negative-staining procedure, Watson (17) and Watson et al. (18) added viruses and known concentrations of latex particles during the preparation procedure. This presented problems in accurate quantitation because latex particles and virus were lost in the preparation steps. A review by Sharp in 1965 (11) on virus quantitation stimulated new interest in developing more accurate methods to recognize and quantitate virus particles. The Monroe and Brandt procedure (8) was developed in our laboratories to quantitate oncornaviruses. Using the rapid semiquantiation (negative-staining) procedure, oncornaviruses present more problems in recognition and quantitation than most viruses because of the following conditions: (i) highly pleomorphic particles, (ii) occasionally, low numbers of virus particle, (iii) occasional mycoplasmal contamination, (iv) lack of virion surface specialization in some instances, and (v) presence of large amounts of cellular debris. Even with the most reliable method introduced in virus recognition and quantitation, there has always been a degree of uncertainty. Recently, attention was shifted to thin sectioning methods as a means of quantitation of viruses (2, 4, 7). This paper describes a sectioning method for virus quanti-
tation using pointed BEEM capsules, in which many of the problems of virus recognition and quantitation have been eliminated. MATERIALS AND METHODS
Several oncomaviruses and a herpes type virus were produced in vitro in our laboratories for this study: simian sarcoma virus (SSV-1), Mason-Pfizer monkey virus (M-PMV), gibbon ape lymphoma virus (GALV) all were produced from NC-37 cells (normal lymphoid), rhabdomyosarcoma (RD)-virus from RD114B cells, J-96, a Russian cell line (human leukemia, subsequently found to be infected with mycoplasma), and Epstein-Barr virus (EBV) from 6410-EBV, and P3HR-1 cultures. Purification of virus. The SSV-1, GALV, MPMV, J-96, and RD viruses were purified and concentrated from clarified culture medium by ultracentrifugation as previously described (15). EBV was obtained by direct high speed pelletization (78,000 x g) from the culture fluid. Virus concentrates were resuspended in 0.01 M tris(hydroxymethyl)aminomethane, 0.1 M NaCl and 0.001 M ethylenediaminetetraacetic acid (TNE) buffer, or spent medium. Both frozen, stored and fresh virus preparations were used. Preparation of virus for assay. For the BEEM capsule method (sectioning), aliquots (0.1 to 0.5 ml) of the virus concentrates were repelletized directly into the capsules using an SW50.1 rotor equipped with special Teflon adaptors for holding the BEEM capsules (Fig. 1). Samples were centrifuged for 30 min at 20,000 rpm (48,000 x g), 25,000 rpm (68,474 x g), and 30,000 rpm (110,000 x g) using a Beckman Model L-4 ultracentrifuge. After centrifugation the BEEM capsules were removed from the buckets and the supernatant fluid was carefully removed with a microliter pipette while the capsule was held at 450 angle. The pellets were
225
226
STEPHENS ET AL.
then fixed by carefully overlaying 2% glutaraldehyde in phosphate-buffered saline. Standard embedding procedure. The samples remained in glutaraldehyde for a maximum of 12 h followed by postfixation in Dalton chromeosmium fixative for 2 to 3 h. Pellets were then dehydrated in serial changes of ethanol, 50% (made with 2% uranyl acetate), then 70, 90, and 100%, and then through propylene oxide and into Epon 812 (prepared with 1 part component A to 2 parts component B). The samples were then allowed to polymerize at 35, 45, and 60 C for 24 h each, respectively. Rapid infiltration procedure. When necessary to accelerate penetration of fixatives, the part of the BEEM capsule containing the virus pellet was perforated with a fine pointed needle under a magnifying glass without disturbing the pellet (see Fig. 3). The capsules were suspended in small plastic containers in which fixation, dehydration, and embedding were performed allowing penetration of the reagents from the sides as well as from the tops of the pellets. Further acceleration of the process was obtained by microinjection of fixatives with a 0.5-ml syringe and a 27-gauge needle before suspending the capsule in the plastic containers. Samples were then polymerized in the manner previously described. Testing the sensitivity of the assay. Serial dilutions of the concentrated virus (SSV-1 and GALV) were made, then added to a predetermined amount of NC-37 cell debris to ensure that the resulting pellets would be of a measurable size. The cell debris was prepared by homogenizing NC-37 cells, clarifying the homogenate at 2000 x g for 10 min, removing the supernatant fluid and clarifying it at 50,000 x g for 30 min. The resultant pellets from this last step were then resuspended in TNE so that 0.2 ml of suspension was approximately 3.0 mm in height in a BEEM capsule. The virus samples were then processed as earlier described.
BEEM CAPSULE
J . CLIN . MICROBIOL .
Measurement of pellets and thin sections. To calculate the volume of the cone-shaped viral pellet, measurements were made using a (Zeiss) light microscope equipped with an eye-piece micrometer. Four measurements were made along the vertical axis to determine the height of the pellet, while two measurements were made on the horizontal axis to determine the diameter of the base. These numbers were averaged and substituted into the formula v = 7r/3 r2h, to calculate the volume of the cone where v = the volume to be determined, r = radius of the base, and h = height of pellet from tip to base. The size of the thin sections was kept constant by measuring the length and width (1 x w) of the trimmed face of the block with the micrometer. All sections were cut at 85.0 nm according to Peachey's continuous interference color index (9). Using the formula I x w x h (h = 85.0 nm), the volume of the section was then determined to be 4.6 x 10-6 mm3. The sections were stained with lead-citrate (16), and studied by electron microscopy (Siemens IA). Determination of virus counts using the BEEM capsules. Due to differential layering of virus and cell debris into the pellets during centrifugation, sections were cut randomly at several levels from the tip, through the middle and base with respect to the centrifugal axis. Counting the number of observable photoframes per grid square (lines printed on the fluorescent screen which are the same size as 6.5 x 9.0 cm photographic plates) at 10,530 magnifications, it was determined that there are 1,395 photoframes per section. Using this as a constant, an average number of virus particles per section was determined (10 sections were counted from each third). This number was then multiplied by the total possible number of sections in the cone and the number of virus particles per milliliter was then determined by relating this to the starting volume of virus sample used. Negative-staining procedure. The virus suspensions were prepared by diluting the samples 1:3 in 2% phosphotungstic acid with the aid of microdiluters (14). The diluted preparations were then placed on 0.6% Formvar, carbon-coated, freshly ionized 200mesh grids, and air-dried. The virus content of each specimen was determined by counting the number of virus particles per grid square (the average count in 20 or more squares of each 200-mesh grid) (1 virus particle/square = 3.4 x 107 virus particles/ml [8]). Virus particles plus latex. To optimize the BEEM capsule technique, latex particles (Dow Chemical Co., Midland, Mich.) were used. The concentration of the latex particles was determined using data provided by the manufacturer. We prepared a stock solution of latex particles, which contained 1013 per ml. This was diluted 1:100 for use and equal volumes of virus were mixed with equal volumes of latex, also latex particles alone were prepared. Serial dilutions were made and observed with the electron microscope by both negative staining and the BEEM capsule technique.
RESULTS Using the nonperforated BEEM capsule techFIG. 1. Cross sectional diagram of BEEM capsule nique, samples could be processed and observed in 5 to 7 days. However, the capsule perforation adaptor for SW 50.1 rotor with capsule in place.
TECHNIQUE FOR ONCORNAVIRUSES
VOL. 1, 1975
modification shortened the total time to 2 days. Optimal conditions of centrifugation were 25,000 rpm for 30 min; under these conditions, all virions were sedimented, and particle distribution was uniform throughout each region of the pellet. At 20,000 rpm for 30 min, the virions were not completely sedimented and the particle distribution was not uniform throughout each region of the pellet. At 30,000 rpm for 30 min, radial layering occurred with some of the virus samples affecting the precision of the system. (Similar steps would have to be taken to standardize the system for other microparticulates.) The virus pellet (Fig. 2) was 3 mm in height. For sectioning, the pellet was divided into volumetrically equivalent thirds in order to have the same number of thin sections per third of virus cone, and sections were taken at random from the tip, middle, and base. The pellet height was adjusted by varying the amount of input concentrated virus. The pointed BEEM capsules have a capacity of roughly 0.7 ml, and 0.1 to 0.5 ml of the concentrated virus suspensions were added. Perforation alone or microinjection of fixa-
227
tives into the BEEM capsule allowed rapid infiltration of reagents into all parts of the pellet, the technique was unique in itself because there was no need to manipulate the pellet for better preservation (Fig. 3). Perforations were made along the side of the capsule
.7ml
.3ml
.2ml
VIRUS PELLET
3mm
FIG. 2. Schematic diagram of BEEM capsule and virus pellet.
C FIG. 3. Illustrates perforation and infiltration procedure (A), microinjection procedure (B), rapid osmium infiltration into perforated capsule (C) compared to nonperforated capsule (D) after 20 min appearance before (E) and after (F) removal of the processed block from the capsule.
which was in contact with the pellet with only minimal amounts of disturbance. Infiltration of reagents was observed to proceed from the top and sides of the pellet. The rapid penetration of the sample enabled us to place it directly into a 60 C oven and it was polymerized overnight, eliminating the gradual hardening steps. Quantitation of identical samples of virus concentrates was performed by the negative staining and BEEM capsule techniques and results were compared. Consistent correlations were obtained on all virus samples studied (Fig. 4 and Table 1). Several thousand virus particles were counted from each sample by the two above techniques. The BEEM capsule technique showed a higher retrieval, in that 0.5 to 1.0 log more virus was observed per milliliter than in negative staining. Both systems showed a high degree of consistency in repeat counts of the same sample. When negatively stained samples were prepared using equal amounts of virus and latex particles, good correlation was obtained in quantitating virus. But, when the same virus samples were prepared using the BEEM cap-
12
J. CLIN. MICROBIOL.
STEPHENS ET AL.
228
SSVI
a- RD -VIRUS
b
c =GALV
d= MPMV
=
1
E
(n
> -j u
I IIII I?2.
ee= J-96
f
z
EBV
l0 I
9-
1 I 2
b
3
4 5 6 EXPFRIMENT
. I1.111 2 NUMBER
3
4
5
6
7
FIG. 4. (A-F). Comparison of virus particles/ml using Monroe and Brandt procedure (8) with aliquots of virus particles in BEEM capsules. Each bar represents an average count of 2 aliquots of each virus. Bars: solid indicates beem capsule; open indicates negative stain.
TABLE 1. A comparison of concentrated viral aliquots using negative staining and BEEM capsules techniques Virus particles/ml Virus Negative-stain BEEM capsule Virus count count"
RD SSV-1
GALV M-PMV J-96 EBV
9.5 1.2 1.3 1.3
x 1010
x 1011 x 1011 x 1011 4.4 X 108 3.3 x 108
1.1 2.2 1.4 1.1
x 1012 x 1012
1012 x 1012 6.2 x 109 4.7 x 109 x
value ~~~~~~~~Log change'
+1.15 +1.10 + 1.10 +0.98 +1.18 +1.40
a Virus counts were determined by the Monroe and Brandt (8) procedure using a modified negative stain method described by Stephens et al. (14). "Comparison of Fig. 4 with Table 1 shows the correlation between average virus count in each experimental group and the difference in log value between the two techniques.
sule procedure, erratic results were obtained. Expected numbers of latex particles were not recovered. Due to their buoyant density, they were found mainly in the base of the pellet. When centrifugation speed was increased to 40,000 rpm to sediment the latex particles, the problem of radial layering occurred with the virus. Radial layering presents a problem in quantitation of the virus particles due to uneven distribution on the horizontal axis of the pellet. Little or no radial layering was observed with the virus at 25,000 rpm. At 30,000 rpm the latex particles were uniform but some of the virus preparations exhibited radial layering. Another problem encountered when sectioning and observing latex particles was that much of the cell debris had a similar morphology to the latex particles. In parallel studies of the two techniques, the morphology of several oncornaviruses was compared. Figure 5 depicts a composite of six different oncornaviruses as observed by negative staining. Several factors affecting the accuracy in virus quantitation is negative staining can be identified (pleomorphic viruses, mycoplasma, and much cellular debris). However, as shown in Fig. 6, 7, 8 and 9, very clear differentiation of the viruses can be made using the BEEM capsule technique. When centrifuging concentrated viral aliquots into BEEM capsules (25,000 rpm), differential layering did not present a problem in quantitation as equivalent thirds were used in counting. Virus distribution was consistent throughout each given third of the pellet (example Table 2). To investigate the sensitivity of the BEEM capsule technique, serial dilutions were made from several lots of SSV-1 and GALV. Virus was
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WF
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Vol. 1, No. 2 Printed in U.S.A.
Improved Recognition and Quantitation Technique for Oncornaviruses R. STEPHENS,* K. TRAUL, D. WOOLF, G. LOWRY, AND J. LELEK Pfizer Inc., Maywood, New Jersey 07607
Received for publication 23 September 1974
A technique to recognize and quantitate oncornaviruses using perforated pointed BEEM capsules has been developed in our laboratory. Virus samples that presented problems in counting, or those which could not be evaluated at all by negative staining, could be clearly defined and counted using the thin-sectioning BEEM capsule technique. Perforation of the BEEM capsule allowed rapid infiltration of reagents into the tip of the virus pellet and made further manipulation and orientation unnecessary. The sensitivity of this technique, determined by making serial dilutions of viral concentrates, allows observation of as few as 5 x 105 virus particles per ml. Precision in counting by this technique varied only + 0.3 log in repeat aliquots of identical concentrated virus samples quantitated, making this a highly useful and reliable system.
Since 1949, several electron microscope virus quantitation methods have been described (5, 10, 12, 13, 19). Precision in the technique varied only slightly, but problems were encountered with recognition and/or loss of virus. With the introduction of negative staining, a procedure that defines surface morphology, a new era began in electron microscopy particle counting. Using the negative-staining procedure, Watson (17) and Watson et al. (18) added viruses and known concentrations of latex particles during the preparation procedure. This presented problems in accurate quantitation because latex particles and virus were lost in the preparation steps. A review by Sharp in 1965 (11) on virus quantitation stimulated new interest in developing more accurate methods to recognize and quantitate virus particles. The Monroe and Brandt procedure (8) was developed in our laboratories to quantitate oncornaviruses. Using the rapid semiquantiation (negative-staining) procedure, oncornaviruses present more problems in recognition and quantitation than most viruses because of the following conditions: (i) highly pleomorphic particles, (ii) occasionally, low numbers of virus particle, (iii) occasional mycoplasmal contamination, (iv) lack of virion surface specialization in some instances, and (v) presence of large amounts of cellular debris. Even with the most reliable method introduced in virus recognition and quantitation, there has always been a degree of uncertainty. Recently, attention was shifted to thin sectioning methods as a means of quantitation of viruses (2, 4, 7). This paper describes a sectioning method for virus quanti-
tation using pointed BEEM capsules, in which many of the problems of virus recognition and quantitation have been eliminated. MATERIALS AND METHODS
Several oncomaviruses and a herpes type virus were produced in vitro in our laboratories for this study: simian sarcoma virus (SSV-1), Mason-Pfizer monkey virus (M-PMV), gibbon ape lymphoma virus (GALV) all were produced from NC-37 cells (normal lymphoid), rhabdomyosarcoma (RD)-virus from RD114B cells, J-96, a Russian cell line (human leukemia, subsequently found to be infected with mycoplasma), and Epstein-Barr virus (EBV) from 6410-EBV, and P3HR-1 cultures. Purification of virus. The SSV-1, GALV, MPMV, J-96, and RD viruses were purified and concentrated from clarified culture medium by ultracentrifugation as previously described (15). EBV was obtained by direct high speed pelletization (78,000 x g) from the culture fluid. Virus concentrates were resuspended in 0.01 M tris(hydroxymethyl)aminomethane, 0.1 M NaCl and 0.001 M ethylenediaminetetraacetic acid (TNE) buffer, or spent medium. Both frozen, stored and fresh virus preparations were used. Preparation of virus for assay. For the BEEM capsule method (sectioning), aliquots (0.1 to 0.5 ml) of the virus concentrates were repelletized directly into the capsules using an SW50.1 rotor equipped with special Teflon adaptors for holding the BEEM capsules (Fig. 1). Samples were centrifuged for 30 min at 20,000 rpm (48,000 x g), 25,000 rpm (68,474 x g), and 30,000 rpm (110,000 x g) using a Beckman Model L-4 ultracentrifuge. After centrifugation the BEEM capsules were removed from the buckets and the supernatant fluid was carefully removed with a microliter pipette while the capsule was held at 450 angle. The pellets were
225
226
STEPHENS ET AL.
then fixed by carefully overlaying 2% glutaraldehyde in phosphate-buffered saline. Standard embedding procedure. The samples remained in glutaraldehyde for a maximum of 12 h followed by postfixation in Dalton chromeosmium fixative for 2 to 3 h. Pellets were then dehydrated in serial changes of ethanol, 50% (made with 2% uranyl acetate), then 70, 90, and 100%, and then through propylene oxide and into Epon 812 (prepared with 1 part component A to 2 parts component B). The samples were then allowed to polymerize at 35, 45, and 60 C for 24 h each, respectively. Rapid infiltration procedure. When necessary to accelerate penetration of fixatives, the part of the BEEM capsule containing the virus pellet was perforated with a fine pointed needle under a magnifying glass without disturbing the pellet (see Fig. 3). The capsules were suspended in small plastic containers in which fixation, dehydration, and embedding were performed allowing penetration of the reagents from the sides as well as from the tops of the pellets. Further acceleration of the process was obtained by microinjection of fixatives with a 0.5-ml syringe and a 27-gauge needle before suspending the capsule in the plastic containers. Samples were then polymerized in the manner previously described. Testing the sensitivity of the assay. Serial dilutions of the concentrated virus (SSV-1 and GALV) were made, then added to a predetermined amount of NC-37 cell debris to ensure that the resulting pellets would be of a measurable size. The cell debris was prepared by homogenizing NC-37 cells, clarifying the homogenate at 2000 x g for 10 min, removing the supernatant fluid and clarifying it at 50,000 x g for 30 min. The resultant pellets from this last step were then resuspended in TNE so that 0.2 ml of suspension was approximately 3.0 mm in height in a BEEM capsule. The virus samples were then processed as earlier described.
BEEM CAPSULE
J . CLIN . MICROBIOL .
Measurement of pellets and thin sections. To calculate the volume of the cone-shaped viral pellet, measurements were made using a (Zeiss) light microscope equipped with an eye-piece micrometer. Four measurements were made along the vertical axis to determine the height of the pellet, while two measurements were made on the horizontal axis to determine the diameter of the base. These numbers were averaged and substituted into the formula v = 7r/3 r2h, to calculate the volume of the cone where v = the volume to be determined, r = radius of the base, and h = height of pellet from tip to base. The size of the thin sections was kept constant by measuring the length and width (1 x w) of the trimmed face of the block with the micrometer. All sections were cut at 85.0 nm according to Peachey's continuous interference color index (9). Using the formula I x w x h (h = 85.0 nm), the volume of the section was then determined to be 4.6 x 10-6 mm3. The sections were stained with lead-citrate (16), and studied by electron microscopy (Siemens IA). Determination of virus counts using the BEEM capsules. Due to differential layering of virus and cell debris into the pellets during centrifugation, sections were cut randomly at several levels from the tip, through the middle and base with respect to the centrifugal axis. Counting the number of observable photoframes per grid square (lines printed on the fluorescent screen which are the same size as 6.5 x 9.0 cm photographic plates) at 10,530 magnifications, it was determined that there are 1,395 photoframes per section. Using this as a constant, an average number of virus particles per section was determined (10 sections were counted from each third). This number was then multiplied by the total possible number of sections in the cone and the number of virus particles per milliliter was then determined by relating this to the starting volume of virus sample used. Negative-staining procedure. The virus suspensions were prepared by diluting the samples 1:3 in 2% phosphotungstic acid with the aid of microdiluters (14). The diluted preparations were then placed on 0.6% Formvar, carbon-coated, freshly ionized 200mesh grids, and air-dried. The virus content of each specimen was determined by counting the number of virus particles per grid square (the average count in 20 or more squares of each 200-mesh grid) (1 virus particle/square = 3.4 x 107 virus particles/ml [8]). Virus particles plus latex. To optimize the BEEM capsule technique, latex particles (Dow Chemical Co., Midland, Mich.) were used. The concentration of the latex particles was determined using data provided by the manufacturer. We prepared a stock solution of latex particles, which contained 1013 per ml. This was diluted 1:100 for use and equal volumes of virus were mixed with equal volumes of latex, also latex particles alone were prepared. Serial dilutions were made and observed with the electron microscope by both negative staining and the BEEM capsule technique.
RESULTS Using the nonperforated BEEM capsule techFIG. 1. Cross sectional diagram of BEEM capsule nique, samples could be processed and observed in 5 to 7 days. However, the capsule perforation adaptor for SW 50.1 rotor with capsule in place.
TECHNIQUE FOR ONCORNAVIRUSES
VOL. 1, 1975
modification shortened the total time to 2 days. Optimal conditions of centrifugation were 25,000 rpm for 30 min; under these conditions, all virions were sedimented, and particle distribution was uniform throughout each region of the pellet. At 20,000 rpm for 30 min, the virions were not completely sedimented and the particle distribution was not uniform throughout each region of the pellet. At 30,000 rpm for 30 min, radial layering occurred with some of the virus samples affecting the precision of the system. (Similar steps would have to be taken to standardize the system for other microparticulates.) The virus pellet (Fig. 2) was 3 mm in height. For sectioning, the pellet was divided into volumetrically equivalent thirds in order to have the same number of thin sections per third of virus cone, and sections were taken at random from the tip, middle, and base. The pellet height was adjusted by varying the amount of input concentrated virus. The pointed BEEM capsules have a capacity of roughly 0.7 ml, and 0.1 to 0.5 ml of the concentrated virus suspensions were added. Perforation alone or microinjection of fixa-
227
tives into the BEEM capsule allowed rapid infiltration of reagents into all parts of the pellet, the technique was unique in itself because there was no need to manipulate the pellet for better preservation (Fig. 3). Perforations were made along the side of the capsule
.7ml
.3ml
.2ml
VIRUS PELLET
3mm
FIG. 2. Schematic diagram of BEEM capsule and virus pellet.
C FIG. 3. Illustrates perforation and infiltration procedure (A), microinjection procedure (B), rapid osmium infiltration into perforated capsule (C) compared to nonperforated capsule (D) after 20 min appearance before (E) and after (F) removal of the processed block from the capsule.
which was in contact with the pellet with only minimal amounts of disturbance. Infiltration of reagents was observed to proceed from the top and sides of the pellet. The rapid penetration of the sample enabled us to place it directly into a 60 C oven and it was polymerized overnight, eliminating the gradual hardening steps. Quantitation of identical samples of virus concentrates was performed by the negative staining and BEEM capsule techniques and results were compared. Consistent correlations were obtained on all virus samples studied (Fig. 4 and Table 1). Several thousand virus particles were counted from each sample by the two above techniques. The BEEM capsule technique showed a higher retrieval, in that 0.5 to 1.0 log more virus was observed per milliliter than in negative staining. Both systems showed a high degree of consistency in repeat counts of the same sample. When negatively stained samples were prepared using equal amounts of virus and latex particles, good correlation was obtained in quantitating virus. But, when the same virus samples were prepared using the BEEM cap-
12
J. CLIN. MICROBIOL.
STEPHENS ET AL.
228
SSVI
a- RD -VIRUS
b
c =GALV
d= MPMV
=
1
E
(n
> -j u
I IIII I?2.
ee= J-96
f
z
EBV
l0 I
9-
1 I 2
b
3
4 5 6 EXPFRIMENT
. I1.111 2 NUMBER
3
4
5
6
7
FIG. 4. (A-F). Comparison of virus particles/ml using Monroe and Brandt procedure (8) with aliquots of virus particles in BEEM capsules. Each bar represents an average count of 2 aliquots of each virus. Bars: solid indicates beem capsule; open indicates negative stain.
TABLE 1. A comparison of concentrated viral aliquots using negative staining and BEEM capsules techniques Virus particles/ml Virus Negative-stain BEEM capsule Virus count count"
RD SSV-1
GALV M-PMV J-96 EBV
9.5 1.2 1.3 1.3
x 1010
x 1011 x 1011 x 1011 4.4 X 108 3.3 x 108
1.1 2.2 1.4 1.1
x 1012 x 1012
1012 x 1012 6.2 x 109 4.7 x 109 x
value ~~~~~~~~Log change'
+1.15 +1.10 + 1.10 +0.98 +1.18 +1.40
a Virus counts were determined by the Monroe and Brandt (8) procedure using a modified negative stain method described by Stephens et al. (14). "Comparison of Fig. 4 with Table 1 shows the correlation between average virus count in each experimental group and the difference in log value between the two techniques.
sule procedure, erratic results were obtained. Expected numbers of latex particles were not recovered. Due to their buoyant density, they were found mainly in the base of the pellet. When centrifugation speed was increased to 40,000 rpm to sediment the latex particles, the problem of radial layering occurred with the virus. Radial layering presents a problem in quantitation of the virus particles due to uneven distribution on the horizontal axis of the pellet. Little or no radial layering was observed with the virus at 25,000 rpm. At 30,000 rpm the latex particles were uniform but some of the virus preparations exhibited radial layering. Another problem encountered when sectioning and observing latex particles was that much of the cell debris had a similar morphology to the latex particles. In parallel studies of the two techniques, the morphology of several oncornaviruses was compared. Figure 5 depicts a composite of six different oncornaviruses as observed by negative staining. Several factors affecting the accuracy in virus quantitation is negative staining can be identified (pleomorphic viruses, mycoplasma, and much cellular debris). However, as shown in Fig. 6, 7, 8 and 9, very clear differentiation of the viruses can be made using the BEEM capsule technique. When centrifuging concentrated viral aliquots into BEEM capsules (25,000 rpm), differential layering did not present a problem in quantitation as equivalent thirds were used in counting. Virus distribution was consistent throughout each given third of the pellet (example Table 2). To investigate the sensitivity of the BEEM capsule technique, serial dilutions were made from several lots of SSV-1 and GALV. Virus was
eAi;a97'IWX%4$.f->;w_,i|z. st Z . \E < :x ..-
i ts '9
VP
WF
s
.....
a
'4
3^
4
%& ,'t
"
a_
K
;:n 9+
.
ir_
't;R
\,t
1.
*.
._,
\l
_w
. =k '-: r
_ _i{ -r
4*.t.t
