Transcript Acid-Fast staining (Ziehl

Faculty of Medicine and Health Sciences
Microbiology Lab
Experiment 5
Acid-Fast Staining
First Semester 2016-2017
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.
 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.
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 10-15 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).
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.
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 Grampositive 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)
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.
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 10-15 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.
Rinse the slide gently with water.
Blot-dry the slide carefully with tissue paper.
Examine the slide under the microscope using oil immersion lens.
2- Called Method (Cold Method:
Prepare a heat-fixed bacterial smear as discribed before
Cover the smear with Kinyoun Cabolfuchsin staining solution
for 10-15 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.
Rinse the slide gently with water.
Blot-dry the slide carefully with tissue paper.
Examine the slide under the microscope using oil immersion lens.
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.
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.
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)
10 Facts About Tuberculosis:
Fact 1:
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.
Fact 3
A total of 1.7 million people died from TB in 2009 (including 380 000 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
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 440 000 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).
Fact 10
41 million TB patients have been successfully treated and up to 6
million lives saved since 1995. 5 million more lives could be saved
between now and 2015 by fully funding and implementing The
Global Plan to Stop TB 2011-2015