support for microbiology laboratories - to be used by medical students in the 2nd year of study

1. Laboratory diagnosis in infections produced by
germs of the Genus Mycobacterium

2. Genus Mycobacterium
General characters:
• filamentous, non-motile, non-sporulated bacilli;
• obligate aerobes
• Bacterial wall – high lipid content: mycolic acid (gives
the name of the genus) - 2 types of effects:
• 1. resistance to antiseptics
• 2. staining only possible with heating BUT after that,
discoloration with acids & alcohols is not possible →
“acid-alcohol resistant” / “acid fast” bacilli
– Gram staining not possible
– Other staining techniques required e.g. Ziehl-Neelsen

3. Ziehl-Neelsen Staining
• Mycobacteria – impermeable to dyes due to high
lipid and wax content of cell wall – usual staining
techniques (e.g. Gram) cannot be used
• heat and phenol (carbolic acid) help penetration
of dye inside mycobacterial cells
• gold standard for diagnosis of tuberculosis and
leprosy
• + Nocardia, Cryptosporidium

4. Ziehl-Neelsen Staining
• used for Mycobacterium tuberculosis and Mycobacterium
leprae = acid fast bacilli: stain with carbol fuschin (red
dye) and retain the dye when treated with acid (due to
lipids i.e. mycolic acid in cell wall)
Reagents
• Carbol fuchsin (basic dye) - red
• Mordant (heat)
• 20% sulphuric acid (decolorizer) – acid fast bacilli retain
the basic (red) dye
• Methylene blue (counter stain) – the other elements of
the smear, including the background will be blue

5. Mycobacterium avium – Ziehl Neelsen
staining

6. Genus Mycobacterium
- Clinical significance -
• 41 species of which over 20 isolated in human infections
• Tuberculosis (pulomonary & extrapulmonary) -
M.tuberculsis, M.bovis
• Other (similar) lung infections - M.avium
• Skin infections (possible dissemination) - M.ulcerans
• Leprosy - M.leprae

7. Robert Koch (1843-1910)
• German physician,
pioneer microbiologist
• “founder of modern
bacteriology”
• Identification of causative
agent of tuberculosis
(Mycobacterium
tuberculosis a.k.a. Koch´s
bacillus)

8. Mycobacterium tuberculosis
- Collection of specimens -
• Sputum:
– Challenging! – avoid contamination with saliva and
secretions from upper air ways
– Optimal moment: in the morning (higher amount of
sputum secreted during the night and stagnant in
lower air ways)
– Indirect method:
• Patient energically rinses mouth with saline solution
• Coughs and expectorates in sterile container (Petri dish)
– Direct method:
• Bronchoscopy / tracheal punctioning

9. Mycobacterium tuberculosis
- Collection of specimens - continued
• Pleural fluid: pleural puncture
(thoracentesis/ pleural tap)

10. Mycobacterium tuberculosis
- Collection of specimens - continued
Urine:
- first morning miction
- clean uro-genital area
- eliminate first flow
- collect middle flow in sterile
container
- Send to lab immediately or
store at 2-8°C
- Repeat in 2-3 days
(intermittent elimination of
bacilli)

11. Mycobacterium tuberculosis
- Collection of specimens - continued
• CSF: lumbar puncture (spinal
tap)

12. Mycobacterium tuberculosis
- Collection of specimens - continued
• Bone marrow aspiration:
suspected bone tuberculosis
• Ascites (peritoneal fluid):
paracentesis (peritoneal
puncture)

13. Mycobacterium tuberculosis
- Microscopy -
• Preliminary treatment of specimen:
– Homogenization with 4% NaOH (also destroys associated flora)
– 30 min at 37°C
– Centrifugation: 10-15 min, 3000 rpm → sediment used for
further examination
• Ziehl-Neelsen stained smear:
– examined for at least 10 min (minimum 100 microscopic fields)
– thin red, encurved bacilli, arranged in groups or in angulated
pairs or isolated;
– positive result expressed as “presence of acid fast bacilli”

14. Mycobacterium tuberculosis - Ziehl-Neelsen
Staining

15. Mycobacterium tuberculosis
- Isolation & Identification-
• The previously homogenized (NaOH – destruction of
associated flora) and concentrated (centrifuged)
specimen – inoculated on solid media
• Lowenstein-Jensen (egg, glycerin, asparagin) – slant in
tube
• Incubation at 37°C for 2 - 4 weeks (slow growing germs)
• Identification:
• Colonial characters: prominent, rough, irregular colonies,
slightly yellowish
• Further identification – Ziehl-Neelsen stained smear from
colonies

16. Mycobacterium tuberculosis – Ziehl Neelsen
staining from culture

17. Mycobacterium tuberculosis
- Isolation & Identification- continued
• Niacin test:
– production of niacin by M.tuberculosis in egg-containing medium →
canary yellow colour (test is negative for other mycobacteria)
– Paper strips impregnated with reagents for detection of niacin
produced by mycobacteria
• Peroxidase test: M.tuberculosis, M. bovis – positive test (other,
atypical mycobacteria – negative test)
• Catalase test: M.tuberculosis and M.bovis - weakly positive test
(atypical mycobacteria - strongly positive test)
• Tuberculostatic susceptibility tests
• Oxygen requirement:
– M.tuberculosis – aerobic
– M.bovis is microaerophilic

18. Mycobacterium tuberculosis
- Antimicrobial susceptibility -
• Multidrug resistant (MDR) strains – major public health
problem
• Treatment of tuberculosis is standardized – association
of antiTB drugs adjusted according to response to
treatment regimen (1st
→2nd
→ 3rd
line regimens)
(follow link below)
• http://www.cdc.gov/tb/publications/guidelines/Treatment.htm

19. Mycobacterium tuberculosis
- Immunization-
Bacille de Calmette et Guérin (BCG): attenuated strain
of M.bovis (lower virulence; no virulence in humans)
Vaccination policies adjusted to morbidity in various regions
(see link below)
http://www.bcgatlas.org/
In Romania BCG (TB vaccine) given to all newborns
(during the 1st
week of life)
Guidelines for TB vaccination:
• http://www.cdc.gov/tb/publications/guidelines/vaccines.htm

20. Mycobacterium leprae
• Clinical significance: Leprosy –
chronic infection (Hansen´s
disease, named after Gerhard
Hansen, 1841-1912 –
Norwegian physician)

21. Leprosy
• slowly progressing bacterial infection affecting:
– skin
– peripheral sensitive nerves in the hands and feet
– mucous membranes (nose, throat, eyes)
– Internal organs, bones
• Transmission via infectious droplets (infectivity is low):
– acquisition of disease requires close, longtime contact with
patient with active infection, not treated; after 2-3 days of
treatment – patient is not contagious any more
• 2 clinical forms:
– lepromatous
– tuberculoid

22. Leprosy - continued
• Lepromatous leprosy (multibacillary):
– low immune response
– spongy tumor like swellings on the face and body
– degenerative lesions of internal organs and bones
– deformity & sensitivity loss in extremities (loss of fingers, toes,
nose tip, lips)
– long evolution (20-30 years)
• Tuberculoid leprosy (paucibacillary):
– isolated, asymetrical skin lesions
– not contagious

23. Leprosy lesions

24. Tuberculoid leprosy

25. Mycobacterium leprae
- Laboratory diagnosis -
• Collection of specimens:
– skin lesion biopsy, skin
scrapping
– Nasal exudate
• Microscopy:
– Ziehl-Neelsen stained
smear: acid fast bacilli,
accumulated in
intracellular, encapsulated
globular masses - “leprosy
globi” - in lepromatous
leprosy
• (Cultivation – not applicable)

26. Prevention and treatment of leprosy
• Hygiene (washing hands), disinfection, monitoring close
contacts of leprosy diagnosed cases
• Early diagnosis – very important in order to initiate
treatment and control further transmission (infectivity of
leprosy patient tends to zero after first 2-3 days of
treatment)
• Multidrug therapy (MDT): combination of 2-3
antimicrobials: Dapsone + Rifampicin + Clofazimin – 6
months – 2 years
• + Corrective surgery