Manual of basic techniques for a health laboratory - libdoc.who.int

5. Bacteriology 1995.2.4 Fixation of smearsWhen the smear has dried completely, fix it by covering theslide with a few drops of 70% methanol for 2 minutes or byquickly passing the back of the slide through the flame threetimes (Fig. 5.6).The fixed smear can be stained as described in section 5.3.It is sometimes useful to draw a circle around the smearwith a grease pencil, so that it can be seen more easily.5.3 Staining techniques5.3.1 Gram stainingFig. 5.6 Fixing a smear over a flameGram stain will enable the smear to be examined by microscopy for the presence ofbacteria, pus cells, Vincent’s bacilli and Candida albicans. Commensal bacteria, whichare always present, are not important. They do not need to be considered for furtherexamination or reported.Principle● Crystal violet stains all bacteria deep violet (Fig. 5.7).● Iodine solution fixes the violet colour more or less strongly in the bacteria(Fig. 5.8).● 95% Ethanol:— decolorizes certain bacteria when the crystal violet is not strongly fixed byiodine solution (Fig. 5.9 (a));— does not decolorize other bacteria when the crystal violet is strongly fixed byiodine solution (Fig. 5.9 (b)).● Carbol fuchsin, neutral red or safranine solution (pink):— re-stains (pink) the bacteria discoloured by ethanol (Fig. 5.10 (a))— has no effect on the other bacteria, which remain dark violet (Fig. 5.10 (b)).(a)(b)(a)(b)Fig. 5.7 Gram staining reaction:staining with crystal violeta: Gram-negative bacteria;b: Gram-positive bacteria.Fig. 5.8 Gram staining reaction:fixation using iodinea: Gram-negative bacteria;b: Gram-positive bacteria.(a)(b)(a)(b)Fig. 5.9 Gram staining reaction:decolorization with ethanola: Gram-negative bacteria;b: Gram-positive bacteria.Fig. 5.10 Gram staining reaction: restainingwith carbolfuchsin, neutral red orsafranine solutiona: Gram-negative bacteria;b: Gram-positive bacteria.