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ZIEHL NEELSEN STAINING

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Title: ZIEHL NEELSEN STAINING


1
ZIEHL NEELSEN STAINING
  • Dr Maliha Sumbul

2
  • Over the last century, tuberculosis (TB) has
    killed more than 100 million people and this has
    continued relatively unchanged over the last 50
    years, despite the development of effective
    antituberculous drugs. This chapter summarizes
    the current status of the epidemiology,
    pathogenesis, diagnosis, treatment, and control
    of pulmonary tuberculosis. We have excluded
    nontuberculous mycobacterial disorders and the
    various forms of extrapulmonary disease, except
    pleural TB.

3
Historical overview
  • Egyptian mummies with severe skeletal deformities
    suggest that TB has existed since antiquity
    (Pott's disease).
  • After the plague devastated Europe during the
    Middle Ages, TB (the White Plague) began to
    take its heavy toll.
  • TB affected famous kings and political figures
    (e.g., King Edward VI, King Louis VIII of France,
    John Calvin, Cardinal Richelieu, Napoleon II).
  • TB, also called writer's or artist's disease,
    killed, among others, Nicolo Paganini, Robert
    Louis Stevenson, Franz Kafka, George Orwell, all
    five Brontë sisters, Thomas Mann, Albert Camus,
    and Igor Stravinsky.
  • The Nobel Prize for Medicine for TB-related work
    was given to the following
  • Dr. Robert Koch for the discovery of TB bacillus
    (1905)
  • Dr. Gerhard Domagk for the discovery of the first
    antibacterial drug (Prontosil) also pioneered
    anti-TB drug development (1947)
  • Dr. Selman Waksman for the development of
    streptomycin as an anti-TB drug (1952)
  • In 1993, the World Health Organization (WHO)
    declared TB a global emergency, the only disease
    ever so designated. In 2003, WHO reported a
    continued TB pandemic

4
  • Dr. Robert Koch

5
  • Also known as the acid-fast stain
  • First described by two German doctors Franz
    Ziehl (1859 to 1926), a bacteriologist and
    Friedrich Neelsen (1854 to 1894), a pathologist.
  • It is a special bacteriological stain used to
    identify acid-fast organisms, mainly
    Mycobacteria.
  • Mycobacterium tuberculosis is the most important
    of this group, as it is responsible for the
    disease called tuberculosis (TB) along with some
    others of this genus. It is helpful in diagnosing
    Mycobacterium tuberculosis since its lipid rich
    cell wall makes it resistant to Gram stain.
  • It can also be used to stain few other bacteria
    like Nocardia. The reagents used are
    Ziehl-Neelsen carbol fuchsin (basic dye), acid
    alcohol and methylene blue / malachite green.
    Acid fast bacilli will be bright red after
    staining

6
Ziehl-Neelsen Staining Procedure
  • STEP 1 Flame slides to heat fix
  • ALCOHOL FIX IS BETTER 1 to 2 drops of 70
    v/v ethanol or methanol for 2-3 min

7
  • STEP 2 Flood the entire slide with Carbol
    Fuchsin
  • Ensure enough stain is added to keep the slides
    covered throughout the entire staining step.

8
Do not overheat
  • STEP 3 Using a Bunsen burner, heat the slides
    slowly until they are steaming.  Maintain
    steaming for 5 minutes by using low or
    intermittent heat (i.e. by occasionally   passing
    the flame from the Bunsen burner over the slides)
     
  • Caution Using too much flame or heat can cause
    the slide to break

9
  • STEP 4 Rinse the slide with water

10
  • STEP 5 Flood the slide with 3 acid-alcohol and
    allow to decolorize for 5 minutes.
  • Throughout the 5 minutes, continue to flood the
    slides with 3 acid-alcohol until the slides are
    clear of stain visible to the naked eye. 
  • To the right are examples of slides
    insufficiently and sufficiently flooded with 3
    acid-alcohol.

11
  • STEP 6 Rinse the slide thoroughly with water and
    then drain any excess from the slides.

12
Malachite green
  • STEP 7
  • Flood the slide with the counterstain, Methylene
    blue.
  • Keep the counterstain on the slides for 1-2
    minutes.

13
  • STEP 8 Rinse the slide thoroughly with water

14
  • If all steps are performed correctly you should
    have a slide that looks like this

15
Reporting
  • Negative X
  • No AFB seen

16
Differences b/w ZN stain methods I and II
  • Method I for M.tuberculosis and M. ulcerans
    strongly acid fast, 3 v/v acid solution is used
    to decolorize the smears
  • Method II for M. leprae only weakly acid fast,
    1 acid solution is used to decolorize

17
Hot and cold ZN methods
18
  • Quality Control Parameters
  • A positive and negative control slide should be
    included with each run of stains.  This will
    verify the correct performance of the procedure
    as well as the staining intensity of the
    acid-fast organisms.
  • Control slides should be reviewed before patient
    smears are read to confirm that the mycobacteria
    stain acid-fast.  If the results of the QC slides
    are acceptable, go on to the patient smears.  If,
    however, the control slide(s) are unacceptable,
    review procedures and reagent preparations.  When
    the problem has been identified and corrected,
    remake and stain all of the patient's slides from
    the problem run along with a new set of controls.

19
NEGATIVE QC
POSITIVE QC
  • ACCEPTABLE RESULTS QC
  • Only blue background
  • UNACCEPTABLE RESULTS QC
  • -Remains red after decolorization
  • -Background is not properly decolorized
  • ACCEPTABLE QC
  • Red bacilli against a blue background
  • UNACCEPTABLE QC
  • -Bacilli are not stained red
  • -Background is not properly decolorized

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23
An acid fast stain is used to diagnose the presence of mycobacteria in tissue and cytologic preparations. Note the thin red rod-like organisms
24
What are Mycobacteria?
  • Tuberculosis complex organisms are
  • Obligate aerobes growing most successfully in
    tissues with a high oxygen content, such as the
    lungs.
  • Facultative intracellular pathogens usually
    infecting mononuclear phagocytes (e.g.
    macrophages).
  • Slow-growing with a generation time of 12 to 18
    hours (c.f. 20-30 minutes for Escherichia coli).
  • Hydrophobic with a high lipid content in the cell
    wall. Because the cells are hydrophobic and tend
    to clump together, they are impermeable to the
    usual stains, e.g. Gram's stain.
  • Known as "acid-fast bacilli" because of their
    lipid-rich cell walls, which are relatively
    impermeable to various basic dyes unless the dyes
    are combined with phenol. Once stained, the cells
    resist decolorization with acidified organic
    solvents and are therefore called "acid-fast".
    (Other bacteria which also contain mycolic acids,
    such as Nocardia, can also exhibit this feature.)

25
More sensitive than an acid fast stain, this auramine stain requires fluorescence microscopy. Note the yellow-orange rod-like mycobacteria. Of course, the most sensitive method for detection of mycobacteria is microbiologic culture
26
  • Centers for Disease Control and Prevention (CDC)
  • Case Definition of Tuberculosis (Laboratory
    Criteria)
  • Isolation of M. tuberculosis from a clinical
    specimen or (when culture not obtained)
  • Demonstration of acid-fast bacilli in a clinical
    specimen
  • Clinical Elements (All of these are needed when
    not confirmed by laboratory test results.)
  • A positive skin tuberculin test
  • Signs and symptoms compatible with tuberculosis
    or an abnormal chest radiograph
  • Treatment with two or more antituberculous drugs
  • A complete diagnostic evaluation with exclusion
    of other, alternative diagnoses

27
DIAGNOSIS OF TB
  • CLINICAL FEATURES
  • MT TEST
  • LAB TESTS
  • (AFB STAIN, AFB C/S)
  • RADIOLOGY
  • (X-RAYS)

Symptoms of tuberculosis include Fever
Night-time sweating Loss of weight Persistent
cough Constant tiredness Loss of appetite
28
A TB skin test is typically performed on the
forearm. After sterilizing the skin, a measured
amount of PPD is injected intracutaneously
29
After the PPD is injected intracutaneously, a
small wheal appears on the forearm. The patient
should be instructed not to scratch the wheal
(but it tends to itch).
30
Within 48 to 72 hours, a positive TB skin test is
marked by an area of reddish induration greater
than 10 mm. It is the induration (firm bump) that
is gently palpated that determines the size, not
the area of redness. This reaction is slightly
larger than the average positive test 17 mm in
size. The positive reaction shown here was
obtained with a TB skin test performed 20 years
after the initial infection.
31
Man must want to achieve more than he is able to
achieve If we do not reach for the impossible,
we shall never reach far enough to discover the
possible. Our wishes should be boundless
Dr. Gerhard Domagk (1947 Nobel Prize in Medicine
for the discovery of the first antimicrobial
drug)
32
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