Instructions:

 Read and analyze each item carefully.

 Answer the given items by providing the necessary information asked for.
 Submit the file thru Moodle Online Learning System in PDF format.

Introduction
Tuberculosis is caused by Mycobacterium tuberculosis. The bacteria usually
attack the lungs, but TB bacteria can attack any part of the body such as the kidney,
spine, and brain. Laboratory methods are used to diagnose tuberculosis. One of these
methods is the Direct Sputum Smear Microscopy which is considered as the gold
standard for diagnosing TB.

QUESTIONS

1. Explain the reason why mycobacterial infections have to be treated for 6 months
or longer and the need to use multiple drugs when treating M. tuberculosis
infections.
Mycobacterial infections have to be treated for 6 months or longer and the need
to use multiple drugs when treating M. tuberculosis infections because there's a
risk of the infection becoming active. If treatment is not continued for a long
enough
duration, the surviving bacteria may cause the patient to become ill and
infectious again, potentially with drug-resistant disease.

2. Differentiate the following staining methods used in the detection of

mycobacteria.
a. Auramine-rhodamine
Although fluorescence microscopy is required for the visualisation of
mycobacteria, auramine-rhodamine stains do not use an antibody and are
 based on the acid-fast properties of mycobacteria. Fluorescent stains are
more sensitive to mycobacteria detection, especially in direct specimens,
because the organisms stain brightly and can be distinguished from
background material.
b. Kinyoun’s
Kinyoun stains mycobacteria red with carbolfuchsin. The Ziehl-Neelsen
stain requires heating and has been largely replaced in many laboratories
by the Kinyoun stain, which is an acid-fast stain that can be used "cold."
c. Ziehl-Neelsen
The Ziehl-Neelsen staining technique was developed by Ziehl and later
modified by Neelsen. Neelsen used carbol-fuschin from Ziehl's
experiment, with heat, and added a decolorizing agent using acid-alcohol
and a counterstain using methylene blue dye, thus developing the Ziehl-
Neelsen staining technique.

3. Complete the Ziehl-Neelsen Staining procedure.

Air dry the slide then fixed with heat.

STAIN PURPOSE TIME COLOR RESULT
a. Carbol Fuchsin Primary Stain 10 minutes b.Red
(heat the smear
 until vapor begins
to rise, do not
overheat/boil)

Rinse with tap water.

Acid Alcohol c. Decolorizer Until cloud of dye None
disappears
d. Methylene Blue e. Counterstain 30 seconds f. Red
Air dry the Slide.

4. Interpret the image.

Result: 0 ∴ No AFB seen

Result: 1+ ∴ 10-99/100 fields

Result: 0 ∴ No AFB seen

5. Complete the reporting guidelines for AFB Stain.

Result Interpretation
a.0 No AFB/300 visual fields.
b. +n 1-9 AFB/100 visual fields
c. 1+ 10-99 AFB/100 visual fields
d. 2+ 1-10 AFB/visual field in at least 50 fields
 e. 3+ More than 10 AFB/visual field in at least
20 fields

6. Identify the method used in the smear.

METHOD: Coiling is the technique used in the smear. This method is commonly used for
sputum specimens. The sputum must be dried and stained before being examined under a
microscope.