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

252 Manual of basic techniques for a health laboratory●Reagents needed for Gram staining (see section 5.3.1) and Ziehl–Neelsen staining(see section 5.3.3).MethodCollection of specimensThe genitals should be cleansed beforehand, using soap and water. Collect a midstreamspecimen (see section 7.1.1) in the sterile flask. Examine as quickly as possible.(Another way is to collect the urine in a conical tube rinsed only in boilingwater and to examine it immediately.)Preparation of slides1. Pour 10ml of fresh urine into a sterile centrifuge tube. Seal the tube with eithera screw-cap or a plug of sterile cotton wool fixed with gauze and string.2. Centrifuge the specimen at 1500g for 10 minutes. If tuberculosis is suspected,centrifuge a further 10-ml specimen at 5000g for 20 minutes.3. Pour off the supernatant from the two tubes (Fig. 7.42). Using an inoculatingloop (sterilized by flaming) (Fig. 7.43), mix the deposit with distilled water untilit forms a homogeneous suspension.Fig. 7.42 Pouring off thesupernatant fluidFig. 7.43 Mixing the urinary deposit4. Using an inoculating loop (sterilized by flaming), prepare asmear from each of the two suspensions (Fig. 7.44). Leavethe slides to air-dry.5. Fix the slides by flooding with ethanol and flaming or byheating.6. Stain the first slide with Gram stain (see section 5.3.1) andthe second with Ziehl–Neelsen stain (see section 5.3.3).Fig. 7.44 Preparation of smears from the urinarydepositMicroscopic examinationExamine the slides under the microscope using the ¥ 100objective.Examine the slide stained with Gram stain for the following (see section 5.3.1):●●●●●pus (many leukocytes stained red by Gram stain)Gram-negative bacilli (Fig. 7.45(a))Gram-positive cocci (Fig. 7.45(b))Gram-positive diphtheroid bacilli (Fig. 7.45(c))Gram-positive fungi (Fig. 7.45(d)).