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

5. Bacteriology 2192. Remove the supernatant, resuspend the deposit and use an inoculating loop(sterilized by flaming) to prepare three smears. Spread the fluid thinly over eachslide (see section 5.2.3).3. Allow the smears to air-dry and fix with methanol.4. Stain the slides with:— Gram stain (see section 5.3.1)— Ziehl–Neelsen stain (see section 5.3.3)— Giemsa stain (see section 9.10.3).Bubo aspirates1. Prepare a smear from the aspirated fluid as described in section 5.2.3.2. Fix the smear in methanol for 2 minutes and stain with Wayson stain (see section5.3.4).5.10.3 Microscopic examinationAspirated cavity fluidExamine each slide using the ¥ 40 objective and the ¥ 100 oil-immersionobjective.Look for any bacteria present on the slide stained with Gram stain.Look for acid-fast bacilli (mycobacteria) on the slide stained with Ziehl–Neelsenstain.When examining the slide stained with Giemsa stain, identify the predominanttype of blood cell present — leukocytes, lymphocytes or mesothelial cells (from thelining of the cavity) and any atypical cells which may suggest cancer cells.If there are more than a few cells present or if the fluid is bloodstained, send theslide to a bacteriology laboratory for culture.Bubo aspiratesFirst examine the slide using the ¥ 40 objective to check the distribution of thematerial and then use the ¥ 100 oil-immersion objective to look for Yersinia pestis.Yersinia pestis is seen as bipolar organisms which stain blue with pink ends.5.11 Examination of pus for Bacillus anthracisBacillus anthracis is a pathogen of several types of animal. It is responsible for cutaneousanthrax where it shows in its early form as a blister on the skin often called amalignant pustule.5.11.1 Materials and reagents●●●●Protective clothingGlovesMicroscopeMicroscope slides● Inoculating loop or sterile cotton wool swabs (see section 5.4.1)● Loeffler methylene blue (reagent no. 35)● Potassium permanganate, 4% solution (reagent no. 46).