Tube Cell Culture Research Articles

Rapid 24-well plate centrifugation assay for detection of influenza A virus in clinical specimens

Two methods for detection of influenza virus in 234 clinical respiratory specimens were compared: (i) a 24-well plate-centrifugation assay using Madin Darby canine kidney (MDCK) cells and staining with monoclonal antibody pools to influenza A and B (Centers for Disease Control, Atlanta, GA) after incubation for 16 h and 40 h, and (ii) conventional tube cell culture using MDCK cells and primary rhesus monkey kidney cells. Influenza A was identified in 23 specimens (10%). No influenza B was recovered. The rapid centrifugation and tissue culture methods were positive for influenza A in 21 (91%) and 16 (70%) of the 23 specimens, respectively. Fourteen specimens were positive by both methods, 2 were positive by tissue culture alone, and 7 were positive by rapid centrifugation only. Of the 21 specimens positive by rapid centrifugation, 16 (76%) were detected after overnight incubation, and 5 (24%) were positive only after incubation for 40 h. Cytopathic effect was observed in 13 (81%) of the 16 isolates identified by tissue culture after an average of 6 days, and 3 (19%) were identified only by hemadsorption and staining with monoclonal antibodies at day 10. Compared with conventional tissue culture, the 24-well plate centrifugation assay is a more rapid and more sensitive method for detecting influenza virus in clinical specimens.

Factors affecting virus plaque confirmation procedures

Direct plaque counts obtained by using the monolayer cell culture assay technique reliably confirmed the number of viruses isolated. Analysis revealed some significant differences in the false-positive rate, depending on the test method used or virus samples evaluated. Plaques from laboratory stock viruses showed a higher confirmation rate than sewage plaque isolates. Test results with laboratory stock viruses suggested that confirmation rates may be affected by virus types present in the sample. Plaques picked by the stab and scrape method and immediately passed into cell culture tubes produced the most reliable counts as compared to those picked by the stab only method or those stored at −70°C in Earle's balanced salt solution with or without fetal calf serum. Plaque confirmation using this method was 90% or better. Although the term ‘false positive plaque’ has been applied to a particular plaque that was not confirmed, five of ten plaques picked by the stab and scrape method in one series of experiments were confirmed when repicked by the same method from original plaque bottles, indicating that a substantial number of unconfirmed plaques may be caused by plaque transfer techniques.

Detection of herpes simplex virus in clinical specimens using a DNA probe after centrifugal inoculation of A549 cells

Two methods for detection of herpes simplex virus (HSV) in 216 clinical specimens were compared: (a) 24-well plate centrifugation using A-549 cells followed by nucleic acid hybridization (Ortho Diagnostic Systems, Inc., Raritan, NJ) after incubation for 16 to 18 h, and (b) conventional tube cell culture using A-549 cells. HSV was identified by conventional tube cell culture in 44 of 216 specimens (20%) and in 36 specimens (17%) by the centrifugation-hybridization method ( P < 0.01). HSV was recovered by tissue culture from all specimens positive by centrifugation-hybridization. The sensitivity, specificity, and positive and negative predictive values of the centrifugation-hybridization technique for detection of HSV in clinical specimens were 82, 100, 100, and 96%, respectively. Centrifugal inoculation of A549 cells in 24-well plates followed by nucleic acid hybridization after overnight incubation should not replace conventional tube cell culture for detection of HSV in clinical specimens.

Early diagnosis of cytomegalovirus infection in renal transplant and dialysis patients by DNA‐DNA hybridisation assay

One hundred forty-eight urine specimens were collected from 47 renal transplant and dialysis patients and screened for the detection of cytomegalovirus (CMV). Diagnosis of CMV infection was suggested in 17 out of 47 patients (36.2%) by more than one of the five methods used. DNA hybridisation assay (DNA HA) using 32P-labelled probe detected CMV DNA in 15 (31.9%) of 47 patients, whereas virus isolation on conventional tube cell cultures (CTC), immunofluorescence incorporating monoclonal antibodies on centrifugation vial cultures (IF), complement fixation test (CFT), and electron microscopy (EM) yielded positive results in only nine (19.2%), 12 (25.2%), 11 (23.4%), and one (2.1%) of 47 patients, respectively. The significance of these results obtained by DNA HA lies not only in the apparent increase in number of patients diagnosed, but also in both early and rapid detection of CMV DNA. More importantly, the DNA HA is highly specific in that it correlates accurately with clinical and laboratory data characteristic of CMV disease. In respect of clinically manifest CMV disease, the specificity of DNA HA, CTC, IF, CFT, and EM was 87.5, 43.7, 56.3, 43.7, and 6.3%, respectively. These advantages of DNA HA make it the test of choice for early diagnosis of CMV infections in immunosuppressed patients.

Addition of magnetic field capability to existing extremely-low-frequency electric field exposure systems.

Magnetic field systems were added to existing electric field exposure apparatuses for exposing cell suspensions in vitro and small animals in vivo. Two horizontally oriented, rectangular coils, stacked one directly above the other, have opposite electric currents. This configuration minimizes leakage fields and allows sham- and field-exposure systems to be placed in the same room or incubator. For the in vitro system, copper plates formed the loop-pair, with up to 900 A supplied by a 180:1 transformer. Electric fields were supplied via electrodes at the ends of cell-culture tubes, eight of which can be accommodated by each exposure system. Two complete systems are situated in an incubator to allow simultaneous sham and field exposure up to 1 mT. For the in vivo system, four pairs of 0.8 x 2.7-m coils made of copper bus bar are employed. This arrangement is energized from the power grid via a 30:1 transformer; horizontal magnetic flux densities up to 1 mT can be generated. Pairs of electrode plates spaced 30.5 cm apart provide electric field exposure of up to 130 kV/m. Four systems with a capacity of 48 rats each are located in one room. For both the in vitro and in vivo systems, magnetic exposure fields are uniform to within +/- 2.5%, and sham levels are at least 2,500-fold lower than exposure levels. Potential confounding factors, such as heating and vibration, were examined and found to be minimal.

Detection of cytomegalovirus from blood leukocytes separated by sepracell-MN and Ficoll-Paque/Macrodex methods.

Two density gradient separation techniques for separation of blood leukocytes were compared for the laboratory diagnosis of cytomegalovirus (CMV) viremia. Of 510 blood specimens processed by both methods, 76 (14.9%) yielded CMV. Of the 76 positive specimens, 66 (87%) and 65 (86%) were processed by the Ficoll-Paque/Macrodex (F-P/M; Macrodex is dextran 70 in normal saline; Pharmacia, Pisataway, N.J.) and Sepracell-MN methods, respectively. Of the 76 CMV-positive blood specimens, 72 (95%) were detected in shell vial cell cultures, whereas only 42 (55%) were detected in conventional tube cell cultures. The time for recognition of specific cytopathic effects due to CMV in tube cell cultures (8.0 versus 7.1 days), the number of fluorescent foci in each positive shell vial culture (19.3 versus 20.1), and the costs of the reagents ($3.50 versus $2.80) were similar and independent of the leukocyte separation method (F-P/M versus Sepracell-MN). Recovery of CMV from heparinized blood (F-P/M method) was similar to that from EDTA-anticoagulated blood (Sepracell-MN method). The Sepracell-MN method is a rapid and sensitive method for detection of CMV from blood specimens and is recommended as a replacement for the more tedious and time-consuming F-P/M procedure.

Combined use of sonication and monoclonal antibodies for the detection of early and late cytomegalovirus antigens in centrifugation cultures

A total of 157 clinical specimens was inoculated into shell vials and conventional tube cell cultures containing confluent monolayers of human embryonic lung fibroblasts (HELF). Of 31 clinical cytomegalovirus (CMV) isolates, 30 specimens (96.8%) were positive by the immunofluorescence method on centrifugation vial cultures (CVC-IF), whereas the cytopathic effects (CPE) of CMV were detected in only 14 specimens (45.2%) in conventional tube cell cultures (CCC), P < 0.001 and in 22 specimens (70.9%) in centrifugation vial cultures (CVC-P), P < 0.1. Significantly more fluorescent foci were detected in centrifugation cultures inoculated with sonicated urine samples ( P < 0.001). CVC-P is more sensitive than CCC for the diagnosis of CMV ( P < 0.05), and a highly significant difference was observed when we compared the mean day to initial detection of CPE ( P < 0.001). For optimal detection of CMV, both CVC-IF and CVC-P should be used for the laboratory diagnosis of this virus infection.

Indirect Immunofluorescence Assay for Rapid Diagnosis of Influenza Virus

Monoclonal antibodies were used to detect influenza A and B in clinical specimens by an indirect immunofluorescence assay using Madden-Darby canine kidney monolayers in shell vials. Twenty-nine influenza virus isolates, including 23 type A and six type B, were detected in conventional tube cell culture at an average time of 4.2 days. Of these isolates, 18 (62%) were detected by indirect immunofluorescence 24 hours after inoculation, and 24 isolates (83%) were detected at 48 hours. Of 30 culture-negative specimens tested, influenza A was detected in one specimen by immunofluorescence. The shell vial assay is useful for rapid detection of influenza types A and B in clinical specimens, and the sensitivity is increased by staining at 48 hours in addition to 24 hours.

New and sensitive standard cell culture technique for the detection of cytomegalovirus in clinical specimens

Conventional tube cell culture has been recognised as the most sensitive technique available for human cytomegalovirus (HCMV) detection. Low-speed centrifugation of specimen inocula onto cell culture monolayers has been shown to increase the efficiency of infection with the AD 169 strain of HCMV. Therefore a centrifugal force of 900g for 1 hour at 37 degrees C was used to enhance the detection of HCMV cytopathic effect (CPE) in shell vials that contained circular coverslips with a monolayer of human embryonic lung (HEL) fibroblasts. Of 195 specimens, HCMV CPE was detected in 18 specimens (9.02%) on shell vial culture assay, whereas conventional tube cell culture was positive in only 13 specimens (6.6%). The shell vial culture assay was significantly more sensitive (P less than 0.05). Furthermore the development of the cytopathic effect on shell vial culture assay was significantly earlier (P less than 0.01) and more extensive. Urine samples were sonicated and the results obtained with immunofluorescence using human immune serum demonstrated that sonication increased both the intensity of fluorescence and number of fluorescent foci of HCMV-infected cells and also decreased the non-specific fluorescence of the background.

Dynamic cell culture system: a new cell cultivation instrument for biological experiments in space

The prototype of a miniaturized cell cultivation instrument for animal cell culture experiments aboard Spacelab is presented (Dynamic cell culture system: DCCS). The cell chamber is completely filled and has a working volume of 200 μl. Medium exchange is achieved with a self-powered osmotic pump (flowrate 1 μl h −1). The reservoir volume of culture medium is 230 μl. The system is neither mechanically stirred nor equipped with sensors. Hamster kidney (Hak) cells growing on Cytodex 3 microcarriers were used to test the biological performance of the DCCS. Growth characteristics in the DCCS, as judged by maximal cell density, glucose consumption, lactic acid secretion and pH, were similar to those in cell culture tubes.

Detection of cytomegalovirus by 24-well plate centrifugation assay using a monoclonal antibody to an early nuclear antigen and by conventional cell culture

During a 12-month period, two methods for detection of cytomegalovirus (CMV) in 1624 clinical specimens were compared: (1) centrifugal inoculation of MRC-5 cells on coverslips in 24-well plates and staining with a monoclonal antibody to CMV early nuclear antigen after incubation for 40 h (EA assay), and (2) conventional tube cell culture. CMV was identified in 183 (11.3%) specimens from 113 different patients. The EA assay was positive for CMV in 144 183 specimens (79%), and CMV was detected by recognition of specific cytopathic effect (CPE) in conventional cell culture in 143 183 (78%). Both methods yielded CMV in 56% of the specimens ( 104 183 ). CMV was detected by EA assay alone in 22% ( 40 183 ) and only by CPE in 21% ( 39 183 ) of the positive specimens. When all specimen types were considered, there was no significant difference in the detection of CMV between the two methods. However, bronchoalveolar lavage (BAL) fluids yielded CMV more frequently by EA assay than by CPE (58 compared to 48 of 574, p = 0.0178), and CMV was detected in blood specimens more often by CPE than by EA assay (20 compared to one of 149, p < 0.0001). In addition to CMV, other viruses were recovered by conventional tube cell culture, including herpes simplex virus (HSV) type 1 from 17 BAL fluids (two of which were positive for CMV by EA assay) and one liver biopsy and adenovirus serotype 4 from four separate urine specimens and three gastrointestinal tract biopsies from one patient. Our data strongly suggest that all specimens submitted for isolation of CMV should be processed for both EA assay and conventional cell culture to provide optimal recovery of CMV. This would also aid in the detection of other viruses, primarily HSV, that may be present in the specimen and play a role in the patient's disease.

The effect of centrifugation on the rapid detection of adenovirus in shell vials.

Monoclonal antibody, specific for the adenovirus group-reactive hexon antigen, was used for the detection of this agent by immunofluorescence 24 and 48 hours after inoculation of HEp-2 cell monolayers in 1-dram shell vials after low-speed centrifugation (700 X g, 30 minutes). Of 31 adenovirus isolates, 16 (sensitivity, 52%) and 30 (sensitivity, 97%) were detected after incubation for 24 hours and 48 hours, respectively. All adenovirus strains were detected in conventional tube cell cultures but required an average of four days incubation. The shell vial assay is rapid, sensitive, and specific and can be easily implemented in laboratories with cell culture experience.

Rapid detection of cytomegalovirus in bronchoalveolar lavage specimens by a monoclonal antibody method.

Cytomegalovirus (CMV), a common cause of pneumonia in immunocompromised subjects, is conventionally diagnosed in the laboratory by tube cell culture assays or by detection of characteristic inclusions in histologic sections. Of 160 immunocompromised patients, CMV infection was diagnosed in 19 subjects by bronchoalveolar lavage (BAL), using a monoclonal antibody directed against an early nuclear antigen of the virus. Cytospin preparations from BAL and MRC-5 cell cultures inoculated with the BAL specimens yielded positive results for 6 (31.6%) and 18 (94%) of the 19 subjects, respectively, within hours of the bronchoscopic procedure, whereas conventional tube cell cultures were positive for 11 of the 19 subjects (57.9%) only after an average of 9.3 days. The monoclonal antibody method permitted easy and rapid detection of CMV in BAL specimens.

Comparison of commercial sources of primary rabbit kidney and MRC-5 cell cultures for herpes simplex virus isolation

Two commercial sources of MRC-5 and primary rabbit kidney cell cultures were compared for sensitivity of herpes simplex virus isolation and cytopathic effect detection time. Forty-six of 78 previously positive and 50 of 234 fresh clinical specimens were positive and could be evaluated. Herpes simplex virus recovery from frozen specimens positive in at least one cell culture ranged from 85% in MRC-5 cells (M.A. Bioproducts) to 89% in primary rabbit kidney cells (M.A. Bioproducts). Herpes simplex virus recovery from fresh clinical specimens ranged from 94% in primary rabbit kidney cells (Flow Laboratories), MRC-5 cells (Flow Laboratories), and MRC-5 (M.A. Bioproducts) cells to 100% in primary rabbit kidney (M.A. Bioproducts) cells. Among the 96 positive specimens evaluated, no significant difference was found between commercially prepared MRC-5 and primary rabbit kidney cell cultures in either sensitivity or time to first detectable cytopathic effect. Cells from M.A. Bioproducts and Flow Laboratories were equally sensitive. Herpes simplex virus detection was dependent upon inoculating at least two cell culture tubes rather than if the cells were MRC-5 cells or primary rabbit kidney cells.

Evaluation of a diagnostic antigen for the detection of aujeszky's disease virus-infected subunit-vaccinated pigs

An early virus protein complex that is found in the maintenance medium of Aujeszky's disease (AD) virus-infected cells was evaluated as a subunit diagnostic antigen (SUDA) in the enzyme-linked immunosorbent assay (ELISA). This antigen was found in purer form and in larger quantities for up to 12 post-infection in the maintenance medium of AD virus-infected MDBK cell cultures than in the maintenace medium of virus-infected porcine Fallopian tube (PFT) and PK1a cell cultures. The SUDA was shown to be compatible with a lectin-derived subunit vaccine by the absence of positive ELISA reactions for antibody to this antigen in 25 AD virus-free subunit-vaccinated pigs. Following virus challenge, all of 24 surviving vaccinated pigs seroconvertd to SUDA within 10 days. Compatibility with the vaccine was further demonstrated by the absence of positive ELISA reactions for antibody to SUDA in 12 pigs that recieved five or six consecutive vaccine doses at 3-wk intervals. The sensitivity of the ELISA with SUDA was demonstrated by the detection of antibody in virus-infected vaccinated and non-vaccinated pigs for at least 15 and 22 weeks, respectively, following exposure to virus. The SUDA was also economical: it was calculated that 8000–14 000 test could be run with the antigen present in the maintenance medium of one 850 cm 2 plastic tissue culture roller bottle of virus-infected MDBK cells.

Detection and serotyping of herpes simplex virus in MRC-5 cells by use of centrifugation and monoclonal antibodies 16 h postinoculation.

Monoclonal antibodies (Syva Co., Palo Alto, Calif.) were used for the detection and serotyping of herpes simplex virus (HSV) isolates by immunofluorescence 16 h after inoculation of MRC-5 monolayers in 3.7-ml shell vials and after low-speed centrifugation. A total of 119 specimens were inoculated into conventional tube cell cultures and shell vials. Of 98 specimens inoculated on the same day of receipt in the laboratory (fresh specimens), all 23 (23.5%) HSV-positive specimens were identified by serotype in 16 h in shell vials by immunofluorescence, whereas only 8 of 23 HSV-positive specimens (34.8%) produced cytopathic effects in conventional tube cell cultures in this time period. Similarly, of 21 original specimen extracts previously determined to be culture positive for HSV (stored specimens), all were detected and serotyped by the immunofluorescence test with monoclonal antibodies 16 h postinoculation compared with the recognition of only 8 of these isolates (38.1%) by cytopathic effect that soon. This technique of centrifugal inoculation of HSV in shell vials containing MRC-5 cells permitted detection of this virus in all positive specimens with serotype determination within 16 h postinoculation.

Studies on Equine Viral Arteritis : I. Characterization of the Virus and Trial Survey on Antibody with Vero Cell Cultures

Two strains, the Bibuna and the Bucyrus, of equine arteritis virus (EAV) were tested for propagation in various cell cultures. They were grown in primary cell cultures of horse, rabbit, hamster, cat and pig kidney, and in established cell cultures of baby hamster kidney (BHK-21/13), hamster lung (HmLu), African green monkey kidney (Vero) and Cynomolgus monkey kidney (JINET). Cytopathic changes were comparatively common to all cells, and characterized by rounding and vacuolation of the cells and by increased density of the cytoplasm. Most affected cells were detached from the glass wall within 7 days after inoculation. The infectivity titers of the primary cell culture fluids were 104 to 106 TCID50/0.2 ml, and those of the established cell culture fluids 106 to 108 TCID50/0.2 ml. Both strains were able to replicate in the presence or absence of 5-iodo-2-deoxyuridine (IUDR). They were not stabilized by cation at 50°C, but were highly sensitive to ether and sodium deoxycholate, and moderately sensitive to heat and acid. The Bibuna strain was more sensitive to a high concentration of trypsin than the Bucyrus strain. Each virus formed distinct piadues 2 to, 3 mm in diameter on the monolayer of Vero cells. Serum samples were collected from 107 horses in Japan in 1972 and 1978 and tested for EAV antibody by neutralization tests in monolayer cell culture tubes or plaque reduction neutralization tests in Vero cells. No antibody, however, was detected from any sample so far.

Titrations of viruses on cell cultures. Pharmacology or single particle hypothesis.

Titration of foot-and-mouth disease and Poliomyelitis viruses on pig and monkey kidney cells have been analysed by means of the single-particle (Poisson distribution) and the pharmacology (Gauss distribution) hypothesis. These titrations, carried out on a large number of cell culture tubes, have shown: that the dosage-effect relationship for the cytopathogenic effect of the virus is a Poisson distribution; that the Poisson distribution can be disturbed by some factors such as pipetting errors and aggregation of virus particles in the fluids.

Laboratory diagnosis of mumps by immunofluorescence on experimentally inoculated cell cultures.

AbstractWith immunofluorescence, hemadsorption, hemagglutination and reading for cytopathic effect as indicators of mumps infection in roller tube cell cultures, the relationship was observed between inoculated virus doses and time before the appearance of the various indicators. The preparation of specimens for immunofluorescent staining was facilitated by the introduction of a simple procedure for obtaining samples of routine monolayer cultures directly from infected roller tubes.

Rhinovirus Inhibition by 3-Substituted Triazinoindoles

SummarySix compounds in a series of 3-substituted triazinoindoles were tested for inhibitory activity against a large number of rhinovirus strains. All of the compounds produced some inhibition of rhinovirus growth as measured by the suppression of viral CPE in WI-38 cell culture tubes. Two of the compounds, 2,2-dimethyl-3-[(5-methyl-5H-as-triazino [5,6-b | indole-3-yl)amino]-1-propanol (SK&F 28938) and 2-methyl-4-| (5-methyl-5 H-as-triazino [5,6-b] indole-3-yl)amino|-2-butanol (SK&F 30097), with paired methyl groups in the side chains were the most active. In gradient plate plaque reduction experiments using HeLa cells, SK&F 30097 had a minimum inhibitory capacity for rhino-viruses ranging from 21 to 32 μM (6–9 μg/ml). HeLa cell morphology was not altered by SK&F 30097 at a concentration of 350 μM (100 μg/ml). The mechanism of the antiviral action of the 3-substituted triazinoindoles is currently unknown.