Microbiology, Acid Fast Bacillus - Specialized staining technique for myobecterium tuberculae and other bacterias having complex cell wall.

1. Mycobecterium

2. Miss Hina Asif
Hassaan Bin Nasir
Mustaqeem
Pooja Ropeta

3. Acid Fast
It is the differential staining
techniques which was first
developed by Ziehl and later on
modified by Neelsen. So this
method is also called Ziehl-
Neelsen staining techniques.

4. History of Acid Fast Staining
• In 1882 ROBERT KOCH reported the
discovery of the tubercle bacillus and
described the appearance of the bacilli
resulting from a complex staining
procedure.
• During the same time period several
other researchers (Ehrlich, Ziehl,
Rindfleisch, and Neelsen), intending to
improve on Koch’s method, introduced
modifications to the reagents and the
procedure.

5. • Franz Ziehl was the first to use carbolic
acid (phenol) as the mordant. Friedrich
Neelsen kept Ziehl’s mordant, but changed
the primary stain to the basic fuchsin .
• This method became known as the Ziehl-
Neelsen method in the early to mid 1890s.
•

6. • Acid fast organisms like Mycobacterium
contain large amounts of lipid substances
within their cell walls called mycolic acids.
These acids resist staining by ordinary
methods such as a Gram stain.
– It can also be used to stain a few other bacteria, such as
Nocardia.

7. LIPID RICH CELL
WALL

8. • The stains used are the red colored Carbol
fuchsin that stains the bacteria and a
counter stain like Methylene blue or
Malachite green.

9. Procedure
ACID FAST STAINING

10. Basic Requirements
Carbolfuchsin (Red)
Acid Alcohol
Counterstain with
Methylene Blue
Acid - Fast Cells – Red
Non Acid – Fast – Blue

11. Acid-fast Stain Reaction Explained
• Primary stain: The hot pink appearance of Acid-fast cells is
caused by Carbol fuchsin, the primary (first) stain, which is
driven into acid-fast cells using the heat from a water bath.
– Carbol Fuchsin is a lipid soluble, phenolic compound,
which is able to penetrate the cell wall

12. • Secondary stain (counterstain): The methylene
blue counterstain imparts Blue color to the
colorless nonacid-fast bacteria, but doesn't
change the color of acid-fast cells.​

13. Application of Primary Stain
• 1. Carbol fuchsin primary stain of acid-fat stain;
2. Carbol fuchsin being applied to slide that had
been prepared with acid-fast controls and an
unknown bacteria. Blotting paper has been put
on top of the slide. Then the blotting paper is
saturated with stain and heated over water bath;
3. Clothes pins are useful for handling the slide;
4. Blotting paper is discarded and slide is rinsed.

15. Application of Decolorizer
• 1. Acid alcohol decolorizer for acid-fast stain; 2. Drizzle decolorizer down slide
for 10 - 15 seconds, while watching to see that stain is removed from negative
control; 3. Rinse

16. Application of Counterstain:
• 1. Secondary stain (counterstain), crystal violet;
• 2. Crystal violet is applied to slide and left for
one minute;
• 3. Rinse; 4. Stained acid fast slide,

18. Observation

20. Mycobacterium
Kingdom: Bacteria
Phylum: Actinobacteria
Class: Actinobacteria
Order: Actinomycetales
Suborder: Corynebacterineae
Family: Mycobacteriaceae
Genus: Mycobacterium
Species: M.tuberculosis

21. MYCOBACTERIUM-INTRODUCTION
• Mycobacteria are aerobic, Rod shaped and nonmotile bacteria (except
for Mycobacterium marinum, which shows motile within macrophages
• Cell wall –rich in lipids
• Very slow growing (15-20 Hr)
• They do not have capsules, and most do not form endospores.
• are characteristically acid fast.

22. Cell wall
• The distinguishing characteristic of
all Mycobacterium species is that the cell wall is thicker
than in many other bacteria, being hydrophobic, waxy,
and rich in mycolic acids or mycolates.
• contains a polypeptide layer, a peptidoglycan layer, and
free lipids.
• There are porins in the membrane to facilitate transport.
• Beneath the cell wall, there are layers of arabinogalactan
and peptidoglycan that lie just above the plasma
membrane.

23. Mycobacterial cell wall:
1-outer lipids, 2-mycolic acid, 3-polysaccharides (arabinogalactan),
4-peptidoglycan, 5-plasma membrane, 6-lipoarabinomannan (LAM),
7-phosphatidylinositol mannoside, 8-cell wall skeleton`

24. The high concentration of lipids in the cell wall of Mycobacterium
tuberculosis have been associated with these properties of the bacterium:
• Impermeability to stains and dyes
• Resistance to many antibiotics
• Resistance to killing by acidic and alkaline compounds
• Resistance to osmotic lysis via complement deposition
• Resistance to lethal oxidations and survival inside of
macrophages

26. Mycobacterium tuberculosis
complex
• M. tuberculosis
• M. bovis (subsp. bovis and caprae)
• vaccine strain M. bovis BCG (Bacille Calmette-Guérin)
• M. africanum
• M. canettii
• M. microti
• M. pinnipedii
These species are, with the exception of M. bovis BCG, considered to cause
tuberculosis (TB) in humans and animals. Despite their close genetic similarity,
these organisms differ considerably with regard to epidemiology, pathogenicity
and their host spectrum.

27. Habitate
• Mycobacteria are widespread organisms,
typically living in water (including tap
water treated with chlorine) and food
sources. Some, however, including the
tuberculosis and the leprosy organisms,
appear to be obligate parasites and are not
found as free-living members of the genus.

28. Myobacterium Division
• A natural division occurs between slowly– and rapidly–
growing species.
– Mycobacteria that form colonies clearly visible to the
naked eye within seven days on subculture are termed
rapid growers,
– - while those requiring longer periods are termed slow
growers

29. • Two media are used to grow MTB
1- Middlebrook's medium
-which is an agar based medium and
2- Lowenstein-Jensen medium which is an
egg based medium.

31. “Tuberculosis is defined as an infectious disease
caused by a bacterium; that most commonly
affects the lungs.”
It can also be a crippling and deadly disease, and
is on the rise in both developed and developing
worlds. Globally, it is the leading cause of deaths
resulting from a single infectious disease.
 Currently, it kills “three million people” a year
and could claim up to 30 million lives if not
controlled.
What is Tuberculosis?

32. Types of Tuberculosis
Mycobacterium which is carried by humans.
Mycobacterium T.B. can present it self in the
human body in different forms effecting any
where from “the intestines, bones, joints, skin,
and the genito urinary, lymphatic, and nervous
systems.”

34. The primary stage of the disease may
be symptom-free, or the individual may
experience a flu-like illness. This is
called the “inactive stage.”
Within the active stage of the disease,
there might be a slight fever, night
sweats, weight loss, fatigue.
The symptoms may vary depending
on what type of tuberculosis you
contract.
Symptoms of Tuberculosis

36. PULMONARY
TUBERCULOSIS

37. INTRODUCTION
Pulmonary Tuberculosis (TB) is an infectious
disease that mainly affect the lungs
parenchyma.
TB is a contagious bacterial (M. tuberculosis)
infection that mainly affects the lungs
parenchyma, but may spread to other organs.
TB is an ancient disease. Signs of skeletal TB
(pott disease) were evident in Europe from
Neolithic times, ancient Egypt, and in the pre-
Columbian New World.
Physicians in ancient Greece called this illness
as “phthisis” reflecting its wasting character.

38. TB has remained an enemy of human society for all age.
TB is not only a problem for the person suffering from it
or their families but a public health problem of the entire
world.

39. TB spread from person to person by airborne transmission.
Infected person release droplet nuclei (1-5 micro meter in
diameter) through,
Talking
Coughing
Sneezing
Laughing
Singing
If not treated properly, TB can be fatal.

40. Clinical presentation of
Pulmonary TB
• Chronic cough
• Weight loss
• Pyrexia of unknown origin
• Unresolved pneumonia
• Chest pain
• fatigue

41. SIGNIFICANT LAB TEST
Tuberculin skin test:
Injecting a small amount of protein from
tuberculosis bacteria between the derived
layer of the skin (usually forearm).
Sputum examination and Cultures;
Is examined under a microscope to look for
tuberculosis bacteria and used to grow the
bacteria in a culture.

42. Interferon-gamma Blood test;
A simple blood is mixed with synthetic proteins
similar to those produced by the tuberculosis
bacteria.
If people are infected with tuberculosis
bacteria, their white blood cells produce certain
substances (interferons) in response to the
synthetic proteins.
SIGNIFICANT LAB TEST

43. IMAGING CONSIDERATION
Chest CT Scan
Chest X-ray

45. Treatment
Anti TB drugs:
Duration 9 months:
Isoniazid along with pyridoxine (vit B6)
Rifampicin
Ethambutol
Pyrazinamide