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Plastic chambers for the cultivation of cells on microscope slides and their application in virology

Article in Canadian Journal of Microbiology · September 1967

DOI: 10.1139/m67-147 · Source: PubMed

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PLASTIC CHAMBERS FOR TI-IE CULTIVATION OF CELLS ON MICROSCOPE SLIDES AND THEIR APPLICATION IN VIROLOGY

During our studies of virus quantitation in tissue culture using immuno- fluorescence, glass coverslip cultures proved highly unsatisfactory. Owing to their fragility, the handling of coverslips in large numbers was cumbersome;

it was also difficult to be sure, throughout the staining and mounting pro-

cedures, which side of the coverslip carried the cell monolayer. To overcome these limitations, it was decided to adapt standard glass slicles for cell culture, as these could then be treated in much the same way as tissue sections on

• slides. To achieve this, simple plastic (Plexiglas, Rohm & I-Iaas Co.) chambers

(Fig. 1) were designed and constructed in our laboratory. The present corn- munication describes the use and handling of these chambers as applied to the study of viruses in tissue culture. The tissue culture charnber, as shown in Fig. 2, co~lsists

• f a plastic ring,

a nontoxic rubber (Ronthor Reiss Corp., Little Falls, New Jersey, U.S.A.) gasket, and two stainless steel clips; the plastic ring with the rubber gasket is fastened to a standard microscope slide with the help of the clips. Three of these assembled chambers can easily be acco~n~nodated in a 100 X 100 X 15 mm square disposable plastic petri dish (Falcon Plastics). It has also been found possible to place two of these chambers on the same slide, so that of the two resulting cell sheets, one can be infected and the other left as a con- trol, and when ready, both of these receive identical treatment throughout the washing, fixing, and staining procedures. After asse~nbly, the chambers can be sterilized by exposing them to ultra- violet light (Westinghouse Sterilan~p G36T6EI) for 15-20 mill a t a distance

• f 8-10 in. from the larn~.

Infected cultures can be soaked in disinfectant solution overnight and boiled in tap water for about 30 min. They are thor-

• ughly washed in running tap water before rinsing in three changes of deion-

ized water. Each chamber can be seeded with approximately 120 X lo3 to 130 X lo3 cells in 1.0 ml of medium and incubated in an atmosphere of CO?

• air mixture

a t 36 "C. Confluent cell monolayers, with an average of 100 X lo3 cells per cha~nber, are ready for use within 10-12 h after seeding. Inoculation of these cultures with viruses and the maintenance of them are carried out in essen- tially the same way as that employed for handling cells grown in petri dishes. The cells can be examined directly under the low power objective of a micro-

• scope. When ready, they are put through the washing, fixing, and staining

steps after removing the chambers from the slides; the frosted ends of the slides can be marked with an ordinary lead pencil.

'Colombo Plan Scholar, on lcave from the University of Karachi, Pakistan.

Canadian Journal of hlicrohiology. Volurne 13 (1967)

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CANADIAN JOURNAL OF M

I C R O B I O L O G Y .

• VOL. 13. 1967

FIG.

• 2. Diaqrammatic representation of the parts of a chamber. (a) Top view of the

plastic ring; (b) side view of the plastic ring; (c) silicone rubber gasket; (d) stainless steel clip.

These chambers have now been successfully used for the cultivation of a variety of cells including HeLa, BS-C-1, I-IEp-2, and second generation Cercopithec~~s aethiops kidney cells. Cultivation of cells in this f as h' ion over- comes inany of the disadvantages inherent in working with coverslip cultures, without significantly affecting the quantities of media and reagents involved. Cell sheets grown on a known area of the slide can be put through the required manipulations without disruption; this is virtually in~possible when large numbers of coverslip cultures have to be handled a t the same time. Deinhardt and Dedmon (1965) have reported the construction of a coverslip holder which may render batch handling of slides easier, but it does not safeguard against damage to the cell sheets. Stainless steel rings, fastened to glass coverslips with a mixture of Vaseline and paraffin, have been used in the cultivation of cells for virus study (LVildy et al. 1961). Similarly, Bergman et al. (1963) en~ployed glass rings fixed on slides with Vaseline. Use of rubber gaskets, as reported here, obviates the necessity of having to remove Vaseline and paraffin from the cultures before

• Can. J. Microbiol. Downloaded from www.nrcresearchpress.com by China University of Science and Technology on 06/03/13

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NOTES

1109

• staining. More recently, use of "Perspex" rings has also been reported (Mc-

Leod and Blackburn 1966). Here the rings were fixed in annuli drilled in the slides, making this procedure technically involved and cumbersonie for routine application. The chambers reported here are cheap and easy to construct. Since all parts of the chamber can withstand commonly used disinfectants and boiling for prolonged periods of time, they can be reused. No special skill is required in assembling and handling them. They can be employed for a variety of purposes, including virus macroplaques and microplaques (with or without agar or methyl cellulose overlays), plaque purification, cloning of cells, cyto- chemistry, autoradiography, and chroniosome studies.

Acknowledgment

The authors are indebted to Mr. J. Robillard of the Medical Faculty Workshop for technical assistance and advice throughout this study.

BERGMAN, S., STENRAM, I., and STENRAM,

• U. 1963. Trials with a rapid method for quantita-

tive ancl qualitative virus determination by ~nicroscopic examination of the indivi- dual cells in virus inoculated monolavers on slides. Acta Pathol. Microbial. Scand.

58, 141-151.

DEINHAR~.~,

• F. and DEDMON,
• R. E. 1965. Simplified method for fluorescent antibody staining
• f tissue cultures. Nature. 205. 1122-1123.

RICLEOD,

• D. H. and BLACKBURN,'
• N. K. 1966. A slide-ring preparation for cell culture and

virological research. Lab. Pract. 15, 190-191. WILDY, P., SMITH, C., NEWTON,

• A. A., and DEKDY.
• P. 1961. Quantitative cytological studies
• n HeLa cells infected with herpes virus. Virology, 15, 486-500.

RIBONUCLEASE AS AN ANTIMICROBIAL AGENT

The small molecular size of ribonuclease, the presence of its substrate, ribonucleic acid, in ~iiernbrane-bound ribosomes, and the role of soluble ribonucleic acid in cell wall formation and in protein synthesis give ribonu- clease anlple opportunity to act as an antimicrobial enzyme. During the 19501s, its lethal action was demonstrated against Rhizobizlm (5), Escherichia

coli (4), and Bacillus megateriz~m

(2, 3). After a period of several years in which no work was done in this area, interest was revived, with several publications on the killing of yeasts (6, 7) aiid of theriilophilic bacilli (9). In these publications, no mention was made of the above-inentioned earlier studies; Tl~onipson and Shively (9), in fact, claiiiied that "lethality of ribonu- clease for viable bacterial cells has not previously been demonstrated". Furthermore, the impression has been given in the literature that the results

• btained with B. rnegaterium (2, 3) were due to the use of a "special strain"

Canadian

Journal o f

• Microbiology. Volume 13 (1967)
• Can. J. Microbiol. Downloaded from www.nrcresearchpress.com by China University of Science and Technology on 06/03/13

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