642 MED ICAL MICROBIO LO GY
RE FE R E N CE S
ANDERSON, E. A., ARMSTRONG, J. A. & NIVEN, J. S. F. (1959). Fluorescence micro -
scopy; observations of virus growth with aminoacridines. Symposium Society
General Microbiology No. IX, p. 224. Cambridge Univ. Press.
BARER, R. (1959). Lecture Notes on the Use of the Microscope. 2nd ed. Oxford:
Blackwell.
BRADLEY, D. E. (1961). The preparation of specimen support films. Chap. 3 in
Techniques for Electron Microscopy. Ed. Kay, D. Oxford: Blackwell.
BRENNER, S. & HORNE, R. W. (1959). A negative staining method for high resolution
electron microscopy of viruses. Biochim biophys. Acta (Amst.), 34, 103.
DAVIES, H. G., WILKINS, M. F. H., CHAYEN, J. & LA COUR, L. F. (1954). The use of the
interference microscope to determine dry mass in living cells and as a quan titative
cytochemical method. Quart. J. micr. Sci., 95, 271.
GLAUERT, A. M. (1961). The fixation, embedding, and staining of biological specimens.
Chap. 8 in Techniques for Electron Microscopy. Ed. Kay, D. (Oxford: Blackwell.
GLAUERT, A. M. & PHILLIPS, R. (1961). The preparation of thin sections. Ibid.,
Chap. 9.
HALE, A. J. (1958). The Interference Microscope. Edinburgh : Livingstone.
HORNE, R. W. & NAGINTON, J. (1959). Electron microscope studies of the develop-
ment of and structure of poliomyelitis virus. y. molec. Biol., 1, 333.
HORNE, R. W. & WILDY, P. (1963). Virus structure revealed by negative staining.
Advanc. Virus Res., 10, 101.
KELLENBERGER, E., RYTER, A. & StcRAuD, J. (1958). Electron microscope study of
DNA containing plasms. y. biophys. biochem. Cytol., 4, 671.
MCCARTNEY, J. E. (1951). An improved microscope lamp. J. din. Path., 4, 234.
NAIRN, R. C. (1964). 2nd ed. Fluorescent ProteinjTracing. Edinburgh: Livingstone.
NEEDHAM, G. H. (1958). The Practical Use of the Microscope. Springfield: Thomas.
PALADE, G. E. (1952). A study of fixation for electron microscopy. y. exp. Med.,
95, 285.
PEASE, D. C. (1960). Histological Techniques for Electron Microscopy. New York &
London: Academic Press.
VALENTINE, R. C. (1961). Contrast enhancement in the electron microscopy of
viruses. Advanc. Virus Res., 8, 287.
WREDDEN, J. H. (1947). The Microscope. London: Churchill.
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CHAPTER 45
- STAINING METHODS-
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43
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As bacteria consist of clear pro plasmic mattes, erAg out s sightly in
ihkw - refractke ilex from the medium in which they are rowm , it is difficult with
etfirciMi the ordtilg microscope, except when special Mafia& o illumination are
used, them in the unstained condition. Staining, therefore, is of
primary importance for the recognition of bacteria.
The use and general principles of bacterial staining have been discussed in
Chapter 2.
METHODS OF MAKING FILM OR SMEAR
PREPARATIONS
Before describing the various staining processes, details of the methods
employed in making films must be considered.
Film preparations are made either on coverslips or on the ordinary 3 x 1
in. glass slides, usually the latter. It is essential that the coverslips or slides
should be perfectly clean and free from grease, otherwise uneven films will
result.
Coverslips.-These should be or in. square, and of No. 1 thickness, i.e.
0.1 mm. thick. (Thicker coverslips-No. 2-may prevent the oil-immersion
objective from coming near enough for the specimen to be focussed.) They
are cleaned by placing them in a mixture of nitric acid, 6 parts; potassium
bichromate, 6 parts; water, 100 parts. They should be dropped one by one
into the fluid. The solution is contained in an evaporating dish and boiled.
Alternatively, they may be cleaned in the dichromate-sulphuric acid solution
(p. 661) by the method described for slides on p. 862. The coverslips are then
well washed, first in tap water and later in distilled water, and stored in a
stoppered jar in 50 per cent. alcohol. Before use they are dried with a soft
clean cloth, such as an old handkerchief. For routine use, the coverslips may
be sufficiently clean as supplied by the maker and require only to be wiped
free from grit and dust with a clean dry cloth.
Slides.-These may be treated in a manner similar to coverslips. A
quicker and quite satisfactory method for ordinary routine use is to wipe the
slide with a clean dry cotton cloth and then, holding its end with forceps, roast
it free from grease by passing it 6-12 times through a blue Bunsen flame. The
heating should be as strong as is possible without cracking the slide. Cracking
is rendered less likely by allowing the slide to cool somewhat before laying
down, or by laying it on a warmed metal rack. Another method of cleaning is
to moisten the finger with water, rub it on the surface of some fine sand soap,
and then smear the surface of the slide. After removing the soapy film with a
clean cloth the surface is clean and free from grease. For special purposes,
such as the staining of flagella, slides are cleaned with hot dichromate-
sulphuric acid solution followed by flaming, as described on p. 661. If the
slide is perfectly clean.,a drop of water can be spread over its surface in a thin
even film; otherwise die water collects into small drops and a film cannot be
made.
After the films have been made and examined the slides should be dis-
carded. They should not be cleaned and used again, since it is difficult to ensure
that all organisms are removed.