1. Faculty of Medicine and Health Sciences
Microbiology Lab
Experiment 5
Acid-Fast Staining
First Semester

2. ((Acid-Fast staining (Ziehl-Neelsen Staining
The Ziehl–Neelsen staining procedure was first described by two German doctors: the Bacteriologist Franz Ziehl (1859–1926) and the Pathologist Friedrich Neelsen (1854–1898).
This staining procedure is a differential staining method that is used to identify acid-fast bacteria, which include two main genera, Mycobacteria and Nocardia
Examples of acid-fast bacteria:
Mycobacterium tuberculosis : the causative agent of Tuberculosis
Nocardia asteroids: causes Nocardiasis , which is a Tuberculosis-like illness that mainly affects immunocompromised people.

3. According to their cell wall structure, acid-fast bacteria (Mycobacteria and Nocardia), are classified as Gram-positive bacteria because species that belong to these genera have a thick peptidoglycan layer in their cell walls.
However, the peptidoglycan of acid-fast bacteria contains large amounts of fatty acids, mainly Mycolic acid, and a wax-like material known as wax D.  These substances make their cell walls extremely hydrophobic.
Accordingly, acid-fast bacteria are poorly strained during Gram-staining procedure, since the stains used in Gram-staining, Crystal violet and Safranin, are both prepared as aqueous solutions. Thus, these stains will not be able to penetrate the extremely hydrophobic cell walls of acid-fast bacteria.
In conclusion, the hydrophobic nature of the cells walls of acid-fast bacteria requires a special staining technique.

4. Principle of Acid-Fast Staining:
1- Ziehl-Neelson Staining Method (Hot Method):
Ziehl-Neelson developed a special staining procedure to stain acid-fast bacteria. Their method includes the utilization of:
A- A stain with lipid-soluble properties (Cabolfuchsin) prepared in a solution that contains an organic solvent (Phenol)
B- Heating
By utilizing the above mentioned steps, the stain is forced to penetrate the cells wall of these types of bacteria.
During Ziehl-Neelson staining procedure, a heat-fixed smear is covered with acid-fast stain (Cabolfuchsin in 5% phenol) for minutes. Meanwhile, heat must applied gently. After that, the stained smear is washed with water.
After washing , the smear is then rinsed with a very strong decolorizer (Acid-Alcohol), which has the capability to destain (remove the the stain) from all other types of bacteria (non-acid-fast bacteria). However this strong decolorizer is does not remove the stain from acid fast-bacteria (hence the name acid-fast bacteria).

5. Note: The term (Fast) means stable, accordingly, acid fast bacteria means the bacteria that its is stably stained so the stain can not be removed even if a strong decolorizer was used.

6. The strong decolorizer used in acid-fast staining is acid-alcohol, which consist of 97% Ethanol and 3% HCl.
After washing with water to remove the decolorizer, the smear is stained methylene blue as a counter stain.
By the end of the staining procedure, acid-fast bacteria remains pink-red while all other types of bacteria are stained blue (regardless , whether these Gram-positive or Gram-negative bacteria).
Note: remember that Methylene blue is a basic stain. It stains all other types of bacteria in blue (see experiment 2)

7. 2- Kinyoun’s Staining Method (Cold Method)
In 1915, Kinyoun developed a modified method to stain acid-fast bacteria that has become known as the “cold staining” method. It was given this name because the heating step in Ziehl-Neelson staining procedure (Hot Method) was excluded and instead, a higher concentration of Cabolfuchsin was used.

8. Staining Procedure: 1- Ziehl-Neelson method (Hot Method)
Prepare a heat-fixed bacterial smear as describes before.
Cover the smear with acid-fast staining solution (Cabolfuchsin s in 5% Phenol )
Heat the slide gently for min. (Keep the smear covered with staining solution while heating the slide and do not allow it to dry. Once you see that the staining solution that covers the smear is about to dry, add more of it).
Allow the slide to cool down
Rinse (wash) the slide gently with water.
Decolorize the slide with acid-alcohol for 30 second.
Rinse the slide gently with water.
Counter stain with Methylene blue for 1-2 minutes.
Blot-dry the slide carefully with tissue paper.
Examine the slide under the microscope using oil immersion lens.

11. 2- Called Method (Cold Method:
Prepare a heat-fixed bacterial smear as discribed before
Cover the smear with Kinyoun Cabolfuchsin staining solution for min.
Rinse (wash) the slide gently with water.
Decolorize the slide with acid-alcohol for 30 second.
Rinse the slide gently with water.
Counter stain with Methylene blue for 1-2 minutes.
Blot-dry the slide carefully with tissue paper.
Examine the slide under the microscope using oil immersion lens.

12. Acid-fast staining of the sputum is a frequently used to test patients suspected to have Tuberculosis. The detection of acid-fast bacteria in the sputum can be considered as an important initial evidence the diagnosis process for patients suspected to have Tuberculosis.

13. The impermeable hydrophobic nature of the cell wall of Mycobacterium tuberculosis gives this bacteria special the following aspects:
1- Upon infection, Mycobacterium tuberculosis is phagocytozed by lung macrophages, but the bacteria resist killing by these phagocytes
The bacterium inhibits fusion of the phagosome with the lysosome.
Moreover, this hydrophobic cell wall gives the bacterium some protective features
against lysosomal enzymes in case fusion of the phagosome containing the bacterium
with the lysosome takes place.
2- In general, acid-fast bacteria resist killing by complement
3- Acid-fast bacteria resist many antibiotics because many of them cannot penetrate the hydrophobic cell wall of these bacteria.
4- The cell wall of acid -fast bacteria makes them able to resistant to dehydration. This implies that once Mycobacterium tuberculosis in contaminated aerosols from a Tuberculosis patient upon coughing , the bacteria can remain viable for along time once the released aerosols get dry. This will increase the possibility of transmitting the infection to surrounding people.

14. Healthy people with a normal immune system infected with Mycobacterium tuberculosis:
In most cases, once a healthy individual is infected with Mycobacterium tuberculosis , their immune defenses succeed to eliminate Mycobacterium tuberculosis from their lungs completely. .
2- However, in some cases, the immune defenses of some healthy individuals fail to eliminate Mycobacterium tuberculosis from their lungs after infection. In this case, the immune system takes an alternative protective procedure, which includes surrounding infected macrophages with T helper cell that keep activating infected macrophages. Activated macrophages restrict the replication of Mycobacterium tuberculosis inside them. In addition to T helper cells, infected cells are also surrounded by fibroblast that deposit fibrous tissue that prevent spreading of the bacteria in lungs. This kind of immune response is known as Granulomatous inflammation that leads for the formation of a kind of shield (( or quarantine)) so as to speak, to stop bacterial replication as well as to prevent its spread in lungs of infected persons. This protective shield is known as Granuloma, within which , Mycobacterium tuberculosis remains dormant.
3- In future, in case the immune system collapse for a reason or another such as Cancer, AIDS, the Granuloma disintegrate and Mycobacterium tuberculosis becomes active again and Tuberculosis develops ( thus is known as secondary Tuberculosis)

17. 10 Facts About Tuberculosis:
Tuberculosis (TB) is contagious and spreads through the air. If not treated, each person with active TB can infect on average 10 to 15 people a year.
Fact 2
More than two billion people, equal to one third of the world’s total population, are infected with TB bacilli, the microbes that cause TB. One in every 10 of those people will become sick with active TB in his or her lifetime. People living with HIV are at a much greater risk.

18. Fact 3
A total of 1.7 million people died from TB in 2009 (including people with HIV), equal to about 4700 deaths a day. TB is a disease of poverty, affecting mostly young adults in their most productive years. The vast majority of TB deaths are in the developing world, with more than half occurring in Asia
Fact 4
TB is a leading killer among people living with HIV, who have weakened immune systems.
Fact 5
There were 9.4 million new TB cases in 2009, of which 80% were in just 22 countries. Per capita, the global TB incidence rate is falling, but the rate of decline is very slow - less than 1%.
Fact 6
TB is a worldwide pandemic. Among the 15 countries with the highest estimated TB incidence rates, 13 are in Africa, while a third of all new cases are in India and China

19. Fact 7
Multidrug-resistant TB (MDR-TB) is a form of TB that does not respond to the standard treatments using first-line drugs. MDR-TB is present in virtually all countries surveyed by WHO and its partners.
Fact 8
There were an estimated new MDR-TB cases in 2008 with three countries accounting for over 50% of all cases globally: China, India and the Russian Federation. Extensively drug-resistant TB (XDR-TB) occurs when resistance to second-line drugs develops. It is extremely difficult to treat and cases have been confirmed in more than 58 countries.
Fact 9
The world is on track to achieve two TB targets set for 2015:
A-the Millennium Development Goal, which aims to halt and reverse global incidence (in comparison with 1990); and
B-the Stop TB Partnership target of halving deaths from TB (also in comparison with 1990).

20. Fact 10
41 million TB patients have been successfully treated and up to 6 million lives saved since million more lives could be saved between now and 2015 by fully funding and implementing The Global Plan to Stop TB