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

202 Manual of basic techniques for a health laboratoryMicroscopic examinationFirst examine the slide using the ¥ 40 objective to see how the smear is distributedand then use the ¥ 100 oil-immersion objective.Corynebacterium diphtheriae appears as green rods (Fig. 5.16) containing green–black volutin granules. The rods may be arranged in rows (a) or in V-formation (b),or joined at angles, giving the appearance of Chinese characters (c). The presenceof slender rods containing volutin granules is sufficient evidence for starting treatmentfor diphtheria.If diphtheria is suspected, a specimen should be sent to the bacteriology laboratoryfor culture (see section 5.4.4).5.3.3 Staining with Ziehl–Neelsen stain (for the detection ofacid-fast bacilli)Ziehl–Neelsen stain is used to identify mycobacteria and oocysts of Cryptosporidiumspp. (see section 4.3.2, page 123).PrincipleWhen mycobacteria and oocysts of Cryptosporidium spp. are stained with a hotstrong solution of carbol fuchsin, they resist decolorization with a solution of acidor acid–ethanol and stain red. Tissues and other organisms are decolorized by theacid–ethanol solution and are demonstrated by a counterstain such as methyleneblue, which stains them blue.Mycobacterium leprae and oocysts of Cryptosporidium spp. only resist decolorizationwith weak solutions of acid or acid–ethanol. They are demonstrated using the modifiedZiehl–Neelsen technique (Table 5.1).Mycobacterium spp. and oocysts of Cryptosporidium spp. are referred to as “acidfast”due to their resistance to decolorization with acid solution. They do not stainwell with Gram stain or simple stains such as methylene blue.Materials and reagents● Microscope● Spirit lamp or Bunsen burner● Slide rack● Forceps● Carbol fuchsin solution for Ziehl–Neelsen stain (reagent no. 16) (filtered beforeuse)Table 5.1 Organisms stained by Ziehl–Neelsen stainSampleSputumSkinUrineStoolGastric lavageOrganismM. tuberculosisM. bovisM. lepraeM. ulceransM. tuberculosisM. bovisCryptosporidium spp.M. tuberculosisM. bovis