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

204 Manual of basic techniques for a health laboratoryMethod1. Fix the smear with methanol for 2 minutes.2. Cover the smear with Wayson stain for 15 seconds.3. Wash the slide in clean water and place it upright in a slide rack to drain and airdry.Microscopic examinationFirst examine the slide using the ¥ 40 objective to check the distribution of thematerial and then use the ¥ 100 oil-immersion objective.Yersinia pestis appears as bipolar organisms which stain blue with pink ends.5.3.5 Staining with Loeffler methylene blue (for the detection ofBacillus anthracis)Loeffler methylene blue is used to stain Bacillus anthracis, which causes anthrax(see section 5.11).Note: Anthrax is a highly contagious disease. Gloves and protective clothing shouldtherefore be worn when specimens suspected of being infected with anthrax arehandled. The staining procedure should be carried out in a safety cabinet.Materials and reagents●●MicroscopeSlide rack● Potassium permanganate, 4% solution (reagent no. 46)● Loeffler methylene blue (reagent no. 35).Method1. Cover the slide with potassium permanganate for 10 minutes.2. Wash the slide in clean water and cover the smear with Loeffler methyleneblue for 1 minute.3. Wash off the stain with clean water and place the slide upright in aslide rack to drain and air-dry.Fig. 5.17 Bacillus anthracisMicroscopic examinationFirst examine the slide using the ¥ 40 objective and then use the ¥ 100oil-immersion objective.Bacillus anthracis appears as large blue rods surrounded by a mauve capsule; thebacilli appear in chains (Fig. 5.17).5.4 Examination of sputum specimens and throat swabsThe presence of pathogenic organisms is revealed by microscopic examination ofsputum specimens and throat swabs. The organisms include:● Bacteria: Gram-positive and Gram-negative acid-fast bacilli.● Fungi or yeasts: filaments of mycelium with or without pores. They may be pathogenicor saprophytes that have multiplied in the sample after collection (correctidentification by a specialized laboratory necessary).● Actinomycetes: granules, see page 200.