View the PDF document

202 Manual of basic techniques for a health laboratory
Microscopic examination
First examine the slide using the ¥ 40 objective to see how the smear is distributed
and 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 presence
of slender rods containing volutin granules is sufficient evidence for starting treatment
for diphtheria.
If diphtheria is suspected, a specimen should be sent to the bacteriology laboratory
for culture (see section 5.4.4).
5.3.3 Staining with Ziehl–Neelsen stain (for the detection of
acid-fast bacilli)
Ziehl–Neelsen stain is used to identify mycobacteria and oocysts of Cryptosporidium
spp. (see section 4.3.2, page 123).
Principle
When mycobacteria and oocysts of Cryptosporidium spp. are stained with a hot
strong solution of carbol fuchsin, they resist decolorization with a solution of acid
or acid–ethanol and stain red. Tissues and other organisms are decolorized by the
acid–ethanol solution and are demonstrated by a counterstain such as methylene
blue, which stains them blue.
Mycobacterium leprae and oocysts of Cryptosporidium spp. only resist decolorization
with weak solutions of acid or acid–ethanol. They are demonstrated using the modified
Ziehl–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 stain
well 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 before
use)
Table 5.1 Organisms stained by Ziehl–
Neelsen stain
Sample
Sputum
Skin
Urine
Stool
Gastric lavage
Organism
M. tuberculosis
M. bovis
M. leprae
M. ulcerans
M. tuberculosis
M. bovis
Cryptosporidium spp.
M. tuberculosis
M. bovis