Microbiology

EXERCISE 9: ACID FAST STAINING

December 11, 2013
Pamantasan ng Lungsod ng Maynila
College of Medicine
ACID FAST STAINING 4. For the diagnosis of TB, the following specimens can be used for
o A differential staining procedure used to identify acid fast organisms acid fast staining:
such as Myocardia and Nocardia o Sputum
o Acid-fastness: Property of bacteria that refers to their innate o pleural biopsy
resistance of decolorization by acid o broncho-alveolar washings
o Acid-fast organisms are difficult to stain via standard gram staining o fiberoptic bronchoscopy
procedures o biopsy specimen of lung
o The high mycolic acid content in the cell wall of acid-fast organisms o CSF
are the one responsible for the staining pattern o gastric washing
o biopsy from other anatomical site
PROTOCOL
1. Prepare a mycobacterium smear and heat-fix it. 5. The following steps are to be followed in collecting a sputum
2. Flood the slide with carbol fuchsin for 2 minutes. sample for a patient with TB
3. Ash with tap water. Add decolorizer via the acid-alcohol solution until o Sputum should be collected for:
the smear is colorless. all patients (adults and older children) suspected of having
4. Wash with tap water. pulmonary or laryngeal tuberculosis
5. Counterstain with methylene blue solution for 5-15 seconds patients diagnosed with extra-pulmonary TB who are coughing or
depending on the thickness of the smear. who have an abnormal chest x-ray
6. Wash with tap water and allow to dry. patients being evaluated for a positive TB skin test who are
7. Examine under oil immersion. coughing or who have an abnormal chest x-ray
o Collect samples 8 - 24 hours apart
RESULT prior to the initiation of chemotherapy; then
o A positive acid fast stain will yield an organism that is stained pink or at least monthly until direct smears are negative for acid-fast
red against blue background bacilli (AFB);
weekly for patients who need to be released from isolation
until direct smears are negative for AFB; then
continue to collect at least monthly and at the end of the 8-week
initial treatment phase until cultures become negative. It is very
important that culture status be documented after 2 months of
treatment, then
continue to collect monthly (especially for patients who are not on
directly observed therapy) as long as the patient is able to cough
and produce a suitable specimen;
and at the end of treatment

o Notes:
Specimens should be collected in either a well-ventilated area or
a sputum collection booth.
ANSWERS TO QUESTIONS Collection of early morning specimens is preferred because of the
1. The components of the acid-fast stain protocol are: carbol fuchsin, overnight accumulation of secretions; however, you may collect
acid-alcohol solution and methylene blue specimens at any time for patients who have a deep cough that is
a. Carbol fuchsin: primary stain, lipid soluble and contains phenol. The readily productive.
phenol assists the stain to penetrate the cell wall. Collect sputum in a sterile container for processing and
b. Acid-alcohol: decolorizer, removes the primary stain except for acid- examination.
fast organisms. Instruct the patient to breathe deeply and cough from deep down
c. Methylene blue: counterstain, occupies the microbe once primary in the lungs. Instruct them that saliva and upper respiratory
stain is removed. secretions are not sputum and are not acceptable specimens. For
patients unable to bring up sputum, deep coughing may be
2. The hot method of acid fast staining is called the Ziehl-Neelsen induced by inhalation of an aerosol of warm, hypertonic (5%-15%)
method due to the usage of heat to help drive the primary stain into the saline.
waxy cell wall. The cold acid fast staining is known as Kinyoun
method which eliminated the usage of heat and was replaced by using 6. To determine if the sample is sputum rather than saliva, one has
a higher concentration of phenol. to first conduct gram staining of the sample.
o A microbial culture must be positive for pathogenic bacteria and
ADVANTAGES not just normal flora inhabiting the mouth and the upper airways.
HOT COLD o Another test is to identify if the sample contains many white blood
cells that indicates the bodys response to an infection.
smear is examined under a
o Microscopy of the specimen revealing 25 squamous cell is saliva.
lower magnification
relatively cheap due to usage
fluorescent stain binds more
of ordinary light microscope 7. The definitive test for TB is still a culture.
avidly as compared to carbol
as opposed to fluorescent o The sputum is placed on a petri dish with nutrients and allowed to
fuchsin with mycolic acids
ones grow for three weeks. A sample positive for TB will yield a colony
(more bacilli are stained)
of bacteria, of the genus Mycobacteria.
No Need for heating, saves
o However, due to the time constraint of such test, several tests can
time
be employed for the diagnosis of TB: skin test, blood test or a
chest x-ray.
DISADVANTAGES o A skin test employs injection of tuberculin in the lower art of the
HOT COLD arm.
o A positive result correlates with an induration at the site of
organisms apparently dead, injection 48 to 72 hours after.
smear is not examined under o Blood tests employ the measurement of your WBC when it is
or rendered non-cultivable by
a lower magnification, thus, it mixed with tuberculosis antigens.
chemotherapy may still be
does not allow the
fluoresce positive
examination of a larger area 8. All Mycobacteria as well as Nocardia and Rhodococcus are acid
artifacts may be mistaken for
in a shorter time fast. The head of the sperm as well as spores of some coccidians are
acid-fast bacilli
also acid fast.

3. The mycolic acid and waxes found in the cell wall of Mycobacteria
is the one responsible for the acid-fastness of the organism.