Laboratory diagnosis of
Tuberculosis
Case scenario
A 35-year-old man presented to the OPD with a history of cough for 2
weeks, evening rise in temperature, loss of appetite, and weight loss.
(PTB)
• Name the most possible clinical condition
• Name the causative agent
• Tests for identification of the organism
• Sample collection-
• Sputum collection in pulmonary TB cases
• Two sputum samples are collected: one spot sample and other early
morning sample.
• Quantity should be 2-5 ml and preferably mucopurulent.
• Sterile wide-mouthed screw capped container.
• Other samples in PTB:
o Induced sputum
o BAL
o Gastric lavage in children.
Ziehl-Neelsen Staining
• Principle:
Acid fastness is due to the presence of mycolic acid in the cell wall of
mycobacteria.
Mycolic acid is a complex hydroxylated branched-chain fatty acid
that prevents ordinary dyes from entering the cell.
Applying heat helps the penetration of strong carbol fuchsin and
phenol penetrate the tuberculous bacillus's cell wall and kills the
bacteria
Once the bacilli are stained, the stain cannot be easily removed,
hence called acid-fast bacilli.
• Method:
Select a new slide and label the slide with the Laboratory number with a
diamond marking pencil.
Make a smear from a yellow purulent portion of the sputum using an
applicator stick.
Air dry for 15–30 minutes.
Fix the smear by passing it over a flame 3–5 times for 3–4 seconds each
time.
Pour 1% filtered heated carbol fuchsin to cover the entire slide.
OR
Gently heat the slide with carbol fuchsin on it until vapours rise. Do not
boil.
Leave carbol fuchsin on the slide for 5 minutes.
Gently rinse the slide with tap water until all free carbol fuchsin stain is washed
away. At this point, the smear on the slide looks red in colour.
Pour 20-25% sulphuric acid onto the slide x 2–4 minutes (till dark pink colour
disappears).
Rinse gently with tap water. Tilt the slide to drain off the water.
A properly decolourised slide will appear light pink in color. If the slide is still red,
reapply sulphuric acid for 1–3 minutes and rinse gently with tap water. Wipe the
back of the slide clean with a swab dipped in sulphuric acid.
Pour 0.1% methylene blue onto the slide x 30 seconds.
Rinse gently with tap water.
Allow the slide to dry.
Examine the slide under the microscope using 10x objective lens to select the
suitable area and then examine under 100x lens using a drop of immersion oil.
• Observations- bright red/ pink coloured, thin slender bacilli, irregularly
stained or beaded appearance in green/blue background
• Modifications:
o M. leprae: 5% Sul acid
o Nocardia: 1% Sul acid
o Coccidian parasites: Kinyun stain (cold) 1% Sul acid
{Now NTEP}
Fluorescence staining
• Faster screening of smears than with ZN
• ~10% more sensitive than ZN
• Does not require the use of oil immersion
• Primary Stains - Auramine O, Auramine O-Rhodamine B
• Counter Stains – Potassium Permanganate
• Observations: M tuberculosis bacilli appear as bright
yellow rods against a dark background
• Examples of other Acid-fast bacilli
Culture of MTB
• Lowenstein Jensen media
( Demonstrate L J media with and without growth (sealed LJ bottle))
• Composition of LJ medium-
Coagulated hen’s eggs
Malachite green (inhibits most other bacteria)
Glycerol serves as a carbon source enhances the growth of
Mycobacterium tuberculosis. For cultivation of M. bovis, glycerol is
omitted and sodium pyruvate is added.
L-Asparagine and Potato Flour are sources of nitrogen and vitamins
• Type of media: Egg based
• Sterilization method: Inspissation: describe the reason (already
taught) and method of Inspissation for MTB, 80-85°C , 3 consecutive
days
• MTB growth characteristics: Rough, Buff & Tough
• Generation Time: 20 hrs
• Time taken for growth: 6-8 week
Liquid media
