key: cord-1028902-mljknov4 authors: Skilling, D. E.; Barlough, J. E.; Berry, E. S.; Smith, A. W. title: A simple, rapid method for preparation of virus isolates from cell culture for electron microscopy date: 1986 journal: J Tissue Cult Methods DOI: 10.1007/bf01665758 sha: e2d81a83c9934aba1111d0b8539c95cb85b93fd2 doc_id: 1028902 cord_uid: mljknov4 A simple procedure for the rapid preparation of virus isolates from cell culture for negative-contrast electron microscopy was devised. Using only conventional centrifugation steps (i.e. without ultracentrifugation), the procedure produced consistent, fine-quality preparations of a variety of virus types differing in size/shape and buoyant density. Negative-contrast electron microscopy (NCEM) is a simple and effieaeious means of visualizing viruses present in eell eulture supernatants and in elinieal speeimens of various kinds (1, 2, 7, 12, 21) . Several NCEM approaehes for improved results involving eoneentration of samples by centrifugation have been described (7, 8, 13, 17, 22, 23) . Conventional methods have utilized preparatory low-speed centrifugation (clarification) to remove unwanted debris, followed by ultracentrifugation to pellet virus partieles (6) (7) (8) 11, 21, 23) . We report here a simple and rapid proeedure for the preparation of virus particles from cell culture material for NCEM that eliminates the need for an ultracentrifugation step. This technique is used routinely in our laboratory and has proven to be extremely useful tor the rapid detection of viruses present in small quantities of eell eulture material. until 4 + eytopathie effeet is observed. 3. Harvest eultures t)y ()ne or more freeze-thaw cyeles I -70 ° C). {room temperature) in a eonventional eentrifuge. 5. Transfer supernatants to polypropylene miero tubes and centrifuge at 2500 × g for 10 min (room temperature) in a C E N T R A l -4 bench-top centrifuge equipped with a model IEC 817 fixed-angle rotor, 6. Transfer supernatants to fresh micro tubes and respin at 8850 × g for 20 rain (room temperature) in the CENTRAR-4. 7. Resuspend pellets in 10 Ixl sterile-filtered distilled water. 8. Transfer one drop of each sample to Parafilm t/. 9. Float individual Formvar-eoated copper grids for 2 min on each drop, then blot dry with filter paper. 10. Touch each grid to a drop of sterile-filtered distilled water, then blot dry with filter paper. 11. Float each grid for 1 min on a drop of 1.5% phosphotungstic acid (pH 7.0), then remove excess stain with filter paper. 12. Leave all grids under ultraviolet light for at least 15 to 20 min. 13. Examine grids with an electron microscope at an accelerating voltage of 80 kV. Viruses of infected cell cultures identified by NCEM have included members both of DNA (Adenoviridae, Herpesviridae, Poxviridae) and RNA (Picornaviridae, Calieiviridae, Coronaviridae, Reoviridae, Paramyxoviridae) virus families (Fig. 1) . This technique has permitted detection of a wide variety of viral agents differing in size/shape and buoyant density. In our experience it has shown itself to be an especially reliable procedure for the rapid morphological identification of new virus isolates from cell culture. Only small volumes of material are required; with the development of cytopathic effect in a single culture tube, enough material is provided not only to passage the isolate onto fresh monolayers but also to examine it by NCEM at the same time. In many cases we used this technique in conjunction with immune electron microscopy for preliminary serologic identification of isolates. It is useful for the identification both of eytopathic and noncytopathic isolates, and we have obtained successful results by direct examination of clinical specimens, including feces and skin serapings (Fig. 1 Ck The success of the procedure lies in part in the intermediate eentrifugation step (2500 × g for 10 min), which etk fectively cleans the preparation by removing most of the nonviral, particulate (cellular) debris remaining after preliminary clarification. Of corresponding significance is the final, moderate-speed centrifugation step (8850 x g for 20 mini which we have tbund to be most effective for pelleting many viruses and a desirable alternative to ultracentrifugation. The resuhing pellets have produced clear, even spreading of virus particles on grids, with minimal amounts of unwanted virion aggregation ("chunping"), background staining, and suhcellular debris (a frequent contaminant of ultracentrifuged preparations). Importantly, the ultrastructural integrity of most virus particles has been well preserved. The major advantages of NCEM that have been cited traditionally are its speed, simplicity, and capability of identifying viruses on a morphological basis without the need for intermediate, specific reagents (1, 2, 6, 7, 12) . Its major disadvantage is a relative insensitivity when compared to certain other techniques. such as enzyme immunoassay, radioimmunoassay, or virus isolation (1, 2, 22) . Because of this, various preparatory procedures for concentrating virions fiom sample material have been described. These have included ultraccntrifugation 13.7,8,19,20) , pseudoreplication (10,161, density gradient centrifugafion (4, 9) , immune electron microscopy ( 1, 6, 14, 19, 21 ,23k ammonium sulfate precipitation (5k and polyaerylamide hydrogcl absorption (22) . Some have concentrated viruses by simple low-speed centrifugation (13, 17) , although the validity of this type of procedure has been questioned (18) . Of all these methods, uhracentrifi.1gation has reeeived without doubt the widest acclaim and acceptance for rapid viral diagnosis {1,6-8, 11,171. However, ultracentritugation is not without its own disadvantages (sedimentation time. disruption of virion morphology, eosedimentation of subcellular debris, requirement tot ready access to an uhraeentri-{uge) (9, 15, 20, 22) . With the procedure described in this report, the need for a relatively prolonged ( 1 or 2 h) ultracentrifugation step has been eliminated, decreasing sample manipulation and resuhing in eve,* greater simplification of the process. Importantly, the time required for the preparation of samples has been reduced considerably. Search time tor the location and identification of isolates with the electron mieroscope has been minimized (usually averaging less than 1 rain, frequently much less) by the fine, even quality of the final preparations. Although some degree of virion loss is inevitable in the two lower-speed centrifugation steps, this had had little effect on our ability to rapidly locate and identify isolates from cell culture supernatants. This procedure will probably prove useful for the detection and identification of a broad range of virus isolates in addition to those described in the present communication. V. I>raelieal aspects of diagnostic eleeu'on microscopy Rapid viral diagnosis by electron microscopy. Stand Two iridovirus isolates from Carassius auratus Ig.l A model tor xesieular exanthema ,.irus. the prololype of Ihe ealici'.;irus group An improved method for the rouline identification of feea] viruses using arnmonium sulihle precipihdion E. Negative contrast electron microscopic techniques lor diagnosis of viruses of velerinar?, importance. ('orne Diagnoslic electron microscopy c,f [aeces. 1. The viral t]ora c,f Ihe theees as seen b; electron microscopy mpro'~ed deicelion of viruses b'~ electron mim'oSCOl) > after direct uhl'aeentrituge preparation of specimens Feline infectious peritonitis "titus. Zentralbl. Veteritmrmed Slagno. 5. Rapid diagnosis of cyh;'mega-h+,Srus intectkm in in(ants by electron micr,'+seop Comparison of the features of astr(n,'irus+!s and caliciviruses seen in samples o| |e,:'es b'.< eleelron microseop The application o1 negalive contrast electron tnicrc, scop? at. =A, low-speed eentrJhlgatJo~l leehnb{ple for the preparation of grids [br direct virus exarnhtalion b~ eleetltltl microscop3 Enterovirus typing b, immune electron mieroscop~, using low-speed centrifugation approaches to ultracentrifugation Jle idetitificalion of reo'.irus-like agent in diarrheal stools Phillips. A. 1.1. Rapid preparation of faecal specimens f,r deteetitm of ,iral particles b+ electron microscopy Dete«tion of rotavirus particles fi'om patients with gastmenteritis Immunoele«tron mi«roscopic eomparisons of «aliciviruses Experimema[ shnlies of a coronavirus and coronavirus-like agent in a bar|'ier-mair~tained feline breediv, g «olony Ah]erson, C. The use of negative «onlrast eleemm mieroseopy (NCEMI for diagnosis of viral infe