practical for mbbs students

1. Practical:
Ziehl-Neelsen staining
• Collection of sputum sample
• Preparation of smear
• Staining
• Reading ZN stained smear
1

2. Collection of sputum samples
• Two sputum specimens are considered as an
optimum means of identifying infectious cases
of tuberculosis.
(A morning sample on two consecutive days)
• With this approach, approximately 80% of
patients will be positive on the first specimen.
• an additional 20% on the second.
2

3. Macroscopic examination of sputum
• Salivary
• Mucous
• purulent/blood stained
• Mucopurulent/blood stained
Young children & patients with HIV/AIDS mayn’t
be able to produce sputum.
• In children: gastric washings
• In HIV/AIDS patients, induced sputum samples
can be collected. Induced sputum will be watery.
3

4. Collection of sputum
• Sputum must be expectorated from the
bronchi by deep forceful coughing.
• The recovery of sputum is best in the
early morning.
• The patient should rinse his or her mouth
by gargling with water prior to coughing
• Taking deep breaths and lowering the
head helps bring up the sputum. Sputum
must not be held in the mouth but one
should immediately spit into a sterile
container.
4

5. • If the ratio of squamous epithelial cells to leukocytes
exceeds 1 to 10, then the likelihood of a salivary
specimen increases.
Squamous epithelial cells are easily seen and recognized under
low power magnification.
5

6. Preparation & size of smear
• Use thin disposable bamboo applicator
sticks to make smears.
• Size of the smear should be 20mm by
10mm.
• Smears shouldn’t be made too thick.
6

7. Thickness of the smear
The thickness of a smear should be such
that a newspaper can be read through the
smear if held under the slide and the smear
should be evenly spread out on the slide. A
stained smear should show a light blue color
from methylene blue. If the smear is dark
blue it usually indicates that it is too thick.
7

8. Fixing the smear
• Fix the smear by gently heating the back
of the slide 3-4 times by flame.
8

9. Ziehl- Neelsen Staining
Principle:
• Acid fastness is due to the high content of
lipids, fatty acids and higher alcohols
found in the cell wall of mycobacterium.
• Mycolic acid (a wax), acid fast in the free
state, is found in all acid-fast bacteria.
9

10. Ziehl- Neelsen Staining
• Direct or concentrated smears of sputum
are examined.
• Sputum microscopy is the most reliable
single method in the diagnosis and control
of tuberculosis.
• New slides should be used for smears &
shouldn’t be reused.
• Smears should be prepared from the thick
purulent part of the sputum.
10

11. Procedure:
1. Place the slide over the staining rack with smear side
facing upwards.
2. Pour the filtered concentrated Carbol fuchsin (basic
fuchsin + phenol) over the slide to cover the entire
smear.
3. Heat the slide underneath until vapours start rising.
4. Don’t allow Carbol fuchsin to boil or slide to dry.
5. Continue intermittent heating for 5 minutes.
6. Wash both sides of the slide with tap water.
7. Cover the slide with 20% Sulphuric acid for 1 minute.
11

12. 8. Wash the slide with water & pour more acid. Repeat
this process several times until the decolourisation is
finished.
9. Counterstain with Loffler's methylene blue for 30
seconds.
10. Wash the slide with water & allow the slide to dry.
11. Put a drop of oil on stained area.
12. Now observe under 100x lens.
12

13. Reading ZN stained smear
• A smear may contain only a few AFB, a
minimum of 300 fields must be examined.
• This is equivalent to three long passes &
nine short passes on a slide.
• It takes approximately 15 minutes.
• AFB, if seen should be enumerated ( e.g.
five AFB/10 fields).
13

14. Three long passes
14

15. Nine short passes
15

16. Mycobacterium tuberculosis: Ziehl-Neelsen stain 16

17. Magenta ping Acid fast bacilli in singles, pairs & groups,
beaded & slightly. 4+ 17

18. Mycobacterium tuberculosis (Magenta pink) in tissue (blue).
Acid-fastness refers to resistance to decolorization
by acids during staining procedures
The most common staining technique is Ziehl-Neelsen stain,
in which the bacteria are stained dark pink (magenta pink).
Mycobacterium tuberculosis (magenta pink) in sputum
!
18

19. Observation
Acid fast bacilli, 1-4 x 0.2-0.8μm appears magenta pink, beaded
slightly curved rods in pairs or singles against bluish background of
pus cells, squamous epithelial cells and elastic fibers. Non-acid fast
organisms will stain blue.
Mycobacterium bovis will stain as acid fast bacilli, straighter,
shorter & stouter.
Uses:
• It is useful in detection of acid fast bacteria.
• Number of bacilli in smear may be counted and correlated with
infectiousness of person.
• This is useful in following patient’s response to treatment.
19

20. Grading of Ziehl-Neelsen(ZN)
smear
Number of AFB seen in oil
immersion fields
Report
0/300 fields AFB not seen
1-2/300 fields Doubtful, repeat the smear
1-9/100 fields 1+
1-9/10 fields 2+
1-9/field 3+
10 or more/field 4+
20

21. Concentration of Sulphuric acid
• 20% for Mycobacterium tuberculosis &
M. bovis
• 20% for Atypical Mycobacteria
• 5% for M. leprae.
• 0.5-1% for Nocardia.
21

22. 22

23. 23

24. 24