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

200 Manual of basic techniques for a health laboratoryMaterials and reagents●●MicroscopeSlide rack● Crystal violet, modified Hucker (reagent no. 18)● Lugol iodine, 0.1% solution (reagent no. 36)● Acetone–ethanol decolorizer (reagent no. 4)● Carbol fuchsin solution for Ziehl–Neelsen stain (reagent no. 16) (diluted 10-fold with 95% ethanol), neutral red, 0.1% solution (reagent no. 40) or safraninesolution (reagent no. 47).Method1. Fix the smear as described in section 5.2.4.2. Cover the smear with crystal violet stain for 60 seconds.3. Wash off the stain with clean water. Drain the slide and cover the smear withiodine for 60 seconds.4. Wash off the iodine with clean water. Decolorize rapidly with acetone–ethanol.Only 2–3 seconds are needed.5. Cover the smear with carbol fuchsin for 2 minutes.6. Wash off the stain with clean water and place the slide upright in a slide rack todrain and air-dry.Microscopic examinationFirst examine the slide using the ¥ 40 objective to see how the smear is distributedand then use the ¥ 100 oil-immersion objective.Gram-positive organismsGram-positive organisms appear dark purple (Fig. 5.11) (e.g. staphylococci, streptococci,micrococci, pneumococci, enterococci, diphtheria bacilli, anthrax bacilli).Fig. 5.11 Gram-positive organismsFig. 5.12 Gram-negative organismsGram-negative organismsGram-negative organisms appear red (Fig. 5.12) (e.g. gonococci, meningococci,coliform bacilli, shigellae, salmonellae, cholera vibrios).Fig. 5.13 Candida albicansIdentification of specific organismsCandida albicans appears as large (2–4mm in diameter) oval or round Grampositivespores (Fig 5.13(a)) with mycelium-like filaments of varying lengthwith rounded ends (Fig. 5.13(b)).“Actinomycetes” are seen as large granules, sometimes visible to the naked eye(white to yellow colour). The centre is Gram-negative, the periphery Grampositive(Fig. 5.14). They are found in pus from skin, sputum, etc.