[Microbiology Laboratory] MYCOBACTERIA

MYCOBACTERIUM
Unique structure: it contains Mycolic acid
It is poorly stained by Gram’s stain
If stained: appear as weakly G+ staining bacilli
Acid-fast bacilli Do chest radiograph: if suggestive of tuberculosis
“acid-fastness” is due to presence of mycolic acid
Strict, obligate aerobes (inhabits apical portions of the
lungs in MTB)
Fastidious organisms Collect 3 sputum specimens for acid fast bacilli smear
Non-encapsulated, non motile, non spore forming for microscopy
Contain MUCH GRANULES giving it a beaded appearance (2 out of 3 positive smears = positive)

2 major pathogens Note: do culture and nucleic acid amplification (NAA) if

necessary
Mycobacterium tuberculosis: TB
Mycobacterium leprae: Leprosy

With increasing incidence of HIV, there is also an increased

incidence of atypical mycobacterium LABORATORY PROCEDURES
Ex. mycobacterium avium intracellullare complex, this Specimens
will proliferate more than the 2 major pathogens URT specimens: sputum – most common
Bronchial washings, broncho-alveolar lavage, bronchial
biopsies
Urine
MYCOBACTERIUM TUBERCULOSIS Blood
Common name: Koch’s bacillus Feces
Human Tubercle Bacilli
Tissue biopsies
Morphology
Needle aspirations
Acid fast bacilli
Long, slender, straight or curved rods Ideal sputum specimen
Chinese letter arrangement <10 squamous epithelial cells
Tend to grow in strands/filaments >25 leukocytes (neutrophils, alveolar macrophages)
due to “serpentine cord” formation Macrophages would mean that you obtained
Virulent strains: CORD factor respiratory specimen
If >10 squam ep cells and >25 leukocytes: salivary
specimen
When to suspect TB?
Need proper instruction upon expectoration
May use mucolytics: N-acetylcysteine
 Historic Features
DIGESTION AND CONCENTRATION TECHNIQUES
Exposure to a person with infectious tuberculosis (TB)
Purpose:
(+) result for M. tuberculosis infection
To dissolve fats and mucus
Presence of risk factors (HIV, DM)
To kill organisms other than mycobacterium
Diagnosis of CAP, unimproved after 7 days of treatment
Agents:
 Signs and Symptoms Typical of TB 4% NaOH
Prolonged coughing (>/= 2-3 weeks) +/- hemoptysis 4% H2SO4
Non-constitutional s/sx N-acetyl-Lcysteine + 2% NaOH
Pleuritic chest pain, chills, fever, night sweats 6% Oxalic acid
Non specific - loss of appetite, weight loss, weakness or 20% Na hypochlorite
easy fatigability, malaise

 Chest Radiograph: MICROSCOPY

Immunocompetent patients Differentiates acid-fast organisms from non-acid fast organisms
Upper lobe opacities Acid fast organisms – mycobacterium spp
+/- evidence of contraction fibrosis and cavitations Slightly acid fast – Nocardia
HIV patients Techniques
Lower lobe and multilobar opacities, hilar adenopathy, Carbol fuchsin
or interstitial opacities Ziehl-Neelsen (Hot method)
Kinyoun (Cold method)
Fluorochromes

Any patient with:

 Cough of equal or more than 2-3 weeks’ duration ZIEHL-NEELSEN METHOD
 HIV infection and unexplained cough or fever Hot method: employs heat and steaming process to enhance the
 Unexplained illness equal or more than 2-3 weeks primary stain
duration
 Diagnosis of CAP with no improvement Solutions
 High risk for TB CXR findings suggestive of TB even if
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 [Microbiology Laboratory] MYCOBACTERIA

Carbol fuchsin: Primary stain least 50 fields

Acid alcohol: decolorizer >10/field or <1/LPF +3 Positive
Methylene blue: counter stain Based on WHO, Center for Disease Control

Results
Acid fast organisms: red FLUOROCHROMES
Non acid fast organisms: blue Uses fluorescent dyes
Auramine O
Rhodamine B

Result
Yellow fluorescence against a dark green background

SMEAR PREPARATION
3x2 cm
Smear coiling procedure
Air dried then stained

Take note the red areas, bacilli are long and filamentous, this is a
characteristic of M. TB

KINYOUN METHOD
Cold method
Solutions
Carbol fuchsin: primary stain AFS PROCEDURE
Acid alcohol: decolorizer Flood slide with CARBOL FUCHSIN solution.
Malachite green: counterstain Apply heat for 4 – 5 minutes using a Bunsen flame. Avoid boiling,
Results evaporating or steaming.
Acid fast organisms: red Cool the slide for a few seconds then wash with water
Non acid fast organisms: bluish green Decolorize smear with ACID ALCOHOL until no further stain can be
rinsed off.
Rinse briefly in tap water, then counterstain with METHYLENE
BLUE solution for 20-45 seconds.
Rinse, drain and dry by blotting.
Examine the stained smear under the oil immersion objective.

CULTURE
Complex media which requires longer incubation
Egg-based media: egg albumin, Malachite green (to
enhance staining)
Lowenstein-Jensen
Petragnani
American thoracic Society (ATS)
Dorset

Non-egg based media: other vitamins, malachite green

Middlebrook 7H10
Middlebrook 7H11
METHOD OF REPORTING
No. of bacilli Report Interpretation
M. TUBERCULOSIS
0 0 = no AFB seen in 300 Negative
slow growth, 10-20 days at 37°C
visual fields
Colonies: appear as “cauliflower”
1-9/100 fields N+ Warrants follow-up
small, dry, scaly or warty
10-99/100 fields +1 Positive
1-10/OIF (oil +2 Positive slight yellow in color
immersion field) in at Eugonic growth” : luxuriant growth

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 [Microbiology Laboratory] MYCOBACTERIA

enhanced by glycerol

OTHER MYCOBACTERIA:
Dysgonic growth: scanty, smooth growth

NEW METHODS: Rapid

Results within 7 – 10 days MYCOBACTERIUM LEPRAE
Uses liquid/broth medium Common name: Hansen’s bacillus
Morphology:
Acid-fast bacilli found inside mononuclear and epitheloid
OTHER TESTS FOR MTB structure known as Lepra cells
Niacin accumulation test Found in palisade or parallel formation: Cigar packet or Picket-
Nitrate reduction fence arrangement
Growth inhibition by thiophene-2- carbocylic acid hydrazide (TH2) Thru slipping division
Serologic tests: RIA, ELISA, LA, PCR (most common) Cultivation: footpads of mouse/armadillo
Tuberculin test (+) acid fast bacilli in tissue samples? Assignment
CXR
If the lesion is at the site of the nostrils or ear lobes, we cut a small
 TB microscopy is still the cost effective diagnostic tool for MTB under part of that tissue (incision biopsy) then it is subjected for acid fast
TB-DOTS staining
So the type of reporting that we use for MTB is different from M.
leprae as long as we see acid fast bacilli, in keeping with the
TUBERCULIN TEST presence of M. leprae
Skin test for MTB the problem with lepromatous leprosy  involvement of nerves 
Based on the reactivity to indradermal injection of mycobacterial desensitization of that patient, so whatever pain to that patient will
antigen not feel
Just like BCG, whatever is used in tuberculin comes from M. bovis
Most common method: Mantoux test
Intradermal/ intracutaneous injection SPECIFIC AFS STAINING
Usually performed at the volar aspect of the arm
FITE’S METHOD
Types: Used to stain M. leprae
Old tuberculin (no longer used) Same solutions used with similar results like the Ziehl-Neelsen
Filtrate of glycerol broth culture by evaporation on waterbath method
Used only for tissue specimens
Purified protein derivative M. leprae usually shorter rods but the same staining reactions
Prepared from culture filtrates of bacilli grown on
synthetic medium and purified by precipitation with
Trichloroacetic acid (TCA)
Commonly used is PPD nowadays

(+) Result of skin test

10mm or > area of induration that occurs 48 hours to 72 hours
after ID injection
Significance:
Children: active infection
Adults – does not necessarily mean active infection
Previous infection
Immunization iwht BCG
Close contact with (+) TB case

 BCG is given 2x: at birth and on the 7th year

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