Tryptose Phosphate Broth as Supplementary Factor for Maintenance of HeLa Cell Tissue Cultures.

Adenovirus-infected tissue culture cells have been investigated by a variety of methods, including light and electron microscopy and cytochemical and fluorescent antibody techniques (1-7). The results of these studies have shown that adenoviruses produce characteristic sequential alterations in host-cell nuclei, and have demonstrated that certain of the structures which develop contain virus-like particles, deoxyribonucleic acid (DNA), and type-specific viral antigen. This evidence indicates that the intranuclear alterations described are manifestations of viral synthesis. The information provided by these studies is incomplete with respect to the role that sequential changes and various inclusions play in viral development because the techniques employed do not differentiate between infectious and non-infectious viral materials. Consequently, little is known of the infectivity of the particles and inclusions present in infected cells. Experiments were therefore designed to correlate the stages of adenovirus-induced cytologic change with production of infectious virus in HeLa cells. Adenoviruses types 5 and 7 were selected for investigation as being representative of two major subdivisions of the adenovirus group (7). The resuits of these studies form the basis for this report. Materials and MethodsViruses.--The prototype strains of the type 5 and 7 adenoviruses employed were furnished by Dr. R. J. Huebner.Tissue Culture.--Details of the propagation of the HeLa cell line and the preparation, growth, and maintenance of the stationary tube cultures have been described elsewhere (8,9).

“…Tissue Culture.--Details of the propagation of the HeLa cell line and the preparation, growth, and maintenance of the stationary tube cultures have been described elsewhere (8,9).…”

Section: Methodsmentioning

confidence: 99%

CORRELATION OF PRODUCTION OF INFECTIOUS VIRUS WITH SEQUENTIAL STAGES OF CYTOLOGIC ALTERATION IN HELA CELLS INFECTED WITH ADENOVIRUSES TYPES 5 AND 7

Boyer¹,

Denny²,

Miller³

et al. 1960

“…The washed cell sheets were then overlaid with 40 ml. of maintenance mixture composed of 67.5 per cent of Scherer's amino acid-vitamin solution (5), 25 per cent tryptose phosphate broth, and 7£ per cent chicken serum (8). Cultures were inoculated with I0 *.° to I@.° 50 per cent infectious doses of virus (IDa) and incubated at 35°C.…”

Section: Methodsmentioning

confidence: 99%

Characteristics of the Adenoviruses

Ginsberg

1958

Investigation of the early period of viral multiplication permits observation of certain discernible stages in the reproductive cycle. Detailed analysis of these steps ((a) combination of virus with susceptible cells, (b) synthesis of new viral particles, and (c) dissociation or release of virus) yields data concerning basic characteristics of the agent. This information is pertinent for purposes of classification, and to stimulate hypotheses concerning the nature of the reactions of a virus with susceptible host cells under conditions of natural infections. To obtain evidence concerning the characteristics of multiplication of the adenoviruses (1-3), investigation of 4 prototype viruses of this group was undertaken. A preliminary report of these studies has been made (4); detailed presentation of the experimental results forms the basis of this paper. These data indicate that the types 1 to 4 adenoviruses studied adsorb at a relatively slow rate to the host cells, strain HeLa cells; that the latent or eclipse period of the multiplication cycle is relatively long and is longer for types 1 and 2 than for types 3 and 4; that the incremental period of viral propagation is relatively short; and that only a very small amount of the maximum virus detectable is dissociated from the host cells during and after the incremental phase of the multiplication cycle. Materials and Methods Tissue Cu]#ure.--A strain of HeLa cells was employed throughout this investigation (5)"For the majority of experiments, a growth fluid composed of 40 per cent human serum and Hanks's balanced salt solution (BSS) was used for propagation of cells in serial cultures by methods described in detail previously (6). In the latter part of this investigation cells were cultivated in Eagle's basal medium (7) to which was added human serum in a final concentration of I0 per cent. These cells, as compared with those previously employed, were equally susceptible to adenovirus infection. Cen cultures were initiated by inoculation of 30 ml of a *

“…The first e.rperiments utilized tube cultures and fluids obtained from Microbiological Associates, Inc.; 3 the remaining experiments were done with cells grown in bottles in our laboratory and distributed to tubes or slides as needed. The additional tissue culture fluids were maintenance solutions containing tryptose phosphate (19) or yeast extract and glucose (20), and a nutrient solution consisting of equal parts of human serum, balanced salt solution, and tryptose phosphate. designated ARD (3) had been isolated originally as RI-67 (21) and will be called type 4.…”

Section: Methodsmentioning

confidence: 99%

Electron Microscopy of Hela Cells Infected With Adenoviruses

Harford

Hamlin

Parker

et al. 1956

HeLa cells were infected with adenoviruses (types 1–4) and sectioned for electron microscopy after intervals of 20 to 48 hours. Clusters of virus-like particles were found within the nuclei of infected cultures but not in those of uninfected controls. The particles were often arranged in rows as if in crystalline formation. Maximal diameter of particles was approximately 65 mµ, and internal bodies were demonstrated. Lesions of infected cells included target-like structures of the nuclear membrane, large nuclear vacuoles (type 2), and increased numbers of large irregular electron-dense granules in the cytoplasm 48 hours after infection. Examination of infected cultures by light microscopy, using the Feulgen reaction, showed intranuclear inclusion bodies and a cytopathogenic effect consisting of clumping of cells without pyknosis of nuclei. A lipide stain showed numerous cytoplasmic granules that were not identical with the large, irregular, electron-dense granules of the cytoplasm. Practically all the cells showed the viral cytopathogenic effect, but only a minority of cells were found to contain virus-like particles or intranuclear inclusion bodies.