I. [MT 6320 LAB] Differential Staining: Acid-Fast Staining

This is employed for the detection of tubercle bacilli in sputum specimens.

Acid Fast Staining

Acid Fast Staining is employed for the detection of ___________________ in sputum specimens.

tubercle bacilli

This is the known causative agent of a disease of poverty called TUBERCULOSIS.

Tubercle bacilli (Mycobacterium tuberculosis)

What are the two types of tuberculosis? (2)

- Pulmonary Tuberculosis (PTB)
- Extra-pulmonary Tuberculosis (EPTB)

Acid-fast bacilli since these resist decolorization with acid-alcohol and appear red on a blue or green background after decolorization.

Tubercle bacilli

This is one of the recommended primary diagnostic and monitoring tool for TB by the International Standards of TB Care and WHO.

Direct Sputum Smear Microscopy (DSSM)

T/F: Nucleic Acid Amplification tests such as GeneXpert are now also used for TB screening.

True

What are the materials used for Acid-Fast Staining?

● Sputum specimen
● Clean, grease free slides
● Applicator sticks or wire loop
● Alcohol lamp
● Filter paper strips (same size of a glass slide)
● Yellow-colored waste disposal container
● Pencil
● Ziehl-Neelsen Staining Solutions
● 2 in x 3 in sputum smear template
● Staining rack
● Lysol solution
● PPEs

The specimen for Acid Fast Staining is preferably collected when?

Early morning

The specimen for Acid Fast must be collected in a ______________________ area.

well-ventilated

How much sputum is collected?

5-10mL

A ________________ specimen is thick, cloudy, and sticky

sputum

What are the main procedures in Acid-Fast Staining?

1. Specimen Collection
2. Sample Preparation Procedure
3. Staining Procedure
4. Microscopic Examination

Label properly the slide using a pencil or marker on the ________ portion.

left

The smear formed on the slide must have what dimensions?

2x3 inches

To make sure the sputum is spread properly, use an applicator stick by making ____________________ patterns.

concentric or coil like

What is used to disinfect the inoculating loop? (2)

1. dip in alcohol washed sand bottle
2. flame sterilize the loop

Place the smear 4-5cm from a newsprint, if the letters cannot be read it is ________________.

too thick

To see if the smear is too thick, place the smear _____________ from a newsprint.

4-5cm

T/F: Allow the smear to be completely dry at room temperature prior to heat fixation.

False

For acid fast staining, the slide must be passed over the flame ________ times for about ________ seconds each time.

2-3 times; 2-3 seconds

The smears of Acid Fast must be flooded individually with ________________________.

0.3% Zhiel's Carbol Fuschin

For acid-fast, heat slides underneath using an alcohol lamp until the steam starts to rise and a _____________________ appear/

metallic sheen

After heating, let the smears stand for how many minutes?

10 mins

What is used as a decolorizer for acid-fast staining?

acid alcohol

Decolorize until the __________ color disappears.

red

Decolorization lasts for how many minutes for acid-fast?

3 minutes

After draining excess water, what is used to flood the smear of acid-fast staining? and how long?

0.3% methylene blue or malachite green for not more than 1 minute

Acid Fast Smears are systematically read from ________________ to ______________.

left to right

What are the structures that may be present in an Acid-Fast Smear? (5)

a. Squamous Cells
b. Leukocytes
c. Mucus Threads
d. Bacterial cells aside from AFB (these bacterial cells will appear blue or green)
e. AFB = appear red or pink bacilli that appear thin or filamentous or beaded.

The total number of OIF to be read should be ______________ before reporting as negative for AFB

300

IUATLD stands for...

International Union Against Tuberculosis and Lung Disease

What is the scale of the description:

No AFB seen in 300 oil immersion (OIF)

0

What is the scale of the description:

1-9 AFB seen in 100 OIF

+n

What is the scale of the description:

10-99 AFB seen in 100 OIF

1+

What is the scale of the description:

1-10 AFB/OIF in at least 50 fields

2+

What is the scale of the description:

>10 AFB/OIF in at least 20 fields

3+