LABORATORY DIAGNOSIS OF TUBERCOLOSIS

HEALTHCARE LESSON 5.3

DIAGNOSTIC METHOCS FOR TUBERCOLOSIS
1. Staining
 Bright-field microscopy
 Fluorescent microscopy
2. Skin Testing
 Purified Protein Derivative

STAINING
 Artificial coloring of microbes using dyes

DYE
 A colored organic compound that has the ability to
combine with certain substances and to impart color
to them.
 Two Components:
o Chromophore- gives the specific color, Greek
"color bearers"
▪ Responsible for the COLORING property
o Auxochrome- responsible for transferring the
color of a dye to the material upon which it
acts. Greek "increases"
▪ Responsible for the DYEING property

MORDANT
 Refers to any substance that will fix the stain so that
the material will retain the stain.

TYPES OF STAINING
1. DIRECT- the microbe is the one that is stained.
a. Simple- general study of microbes
b. Differential- used to contrast two or more
organisms for the basis of differentiation.
c. Selective- staining of certain cell structures.

2. INDIRECT- the background is the one that is stained.

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 LABORATORY DIAGNOSIS OF TUBERCOLOSIS
HEALTHCARE LESSON 5.3

ACID-FAST STAINING
 Purposes:
1. Early diagnosis of mycobacterial infections
2. Monitor patients with ongoing
antimycobacterial therapy (PRISE)
 ACID-FASTNESS: The ability to resist decolorization
with acid-alcohol
 Types:
1. Ziehl-Neelsen (Hot Method)
▪ Routine
2. Kinyoun’s (Cold Method)
▪ Demonstration of acid-fast bacilli in tissues
3. Fite-Faraco’s
▪ Counterstain: Hematoxylin instead of
methylene blue
4. Auramine-Rhodamine stain (Truant’s)
▪ Flourescent organisms on black
background
▪ (+) MTB: appear as yellow fluorescent
bacilli
5. Spengler’s: for blind individuals
▪ (+) Black
6. Pappenheims
▪ MTB: red
▪ M. smegmatis: blue
7. Baumgarten’s
▪ MTB: blue
▪ M. leprae: red

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 LABORATORY DIAGNOSIS OF TUBERCOLOSIS
HEALTHCARE LESSON 5.3
SPECIMENS FOR AFB STAINING
 Specimens:
1. Sputum
▪ Early morning deep expectorated cough
(Sputum)
▪ The patient is requested to submit sputum
for three consecutive days
▪ Removal of adherent sputum: washed
sands or small glass beads with 90-95%
spirit or 5% cresol
▪ Use BARTLETT'S classification to determine
if sputum sample is acceptable:
• <10 epithelial cells/lpf (if increased,
saliva)
• >25 PMN's/lpf
2. Secretions obtained by bronchoscopy
3. Blood
4. Urine
5. CSF
▪ Pellicle formation (web-like structures)
upon refrigeration
6. 3P’s: Pleural, peritoneal, pericardial
▪ Tubercular effusion: increased adenosine
deaminase

DIGESTION AND DECONTAMINATION

 Methods for decontamination and digestion of
mycobacteria:
1. N-acetyl-L-cysteine (NALC) and 2-4% NaOH
▪ NALC: digesting/mucolytic agent/liquefies
mucus
▪ NaOH: decontaminating agent
2. Trisodium phosphate and benzalkonium
chloride (Zephiran)
3. Dithiothreitol and NaOH
4. QUATS: quaternary ammonium compounds
▪ Inactivated by organic substance

SKIN TEST FOR TUBERCOLOSIS

 Purified Protein Derivative (PPD)
o Determines exposure to M. tuberculosis
o Organism is killed by heat and ammonium
sulfate, precipitated and injected intradermally
▪ (+) redness after 48 hours
MYCOBACTERIUM SPECIES ▪ Positive skin test indicates previous
 The cell wall of Mycobacterium spp. contains large exposure to the bacteria but not
amounts of lipids and waxes primarily MYCOLIC ACID necessarily an active disease.
which are stored in MUCH GRANULES. o Mantoux: Intracutaneous
 Mycolic acid renders Mycobacterium resistant to o Von Pirquet: Scratch
decolorization during acid-fast staining, hence the o Vollmer: Patch
name acid-fast bacillus.
 Mycolic acid also prevent the digestion of
Mycobacteria during phagocytosis
 ONLY the GENUS MYCOBACTERIA are acid-fast.
1. Partially acid-fast organisms: Nocardia
asteroides, Rhodococcus spp, Legionella
micdadei, Isospora spp, Crytosporidium spp

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