zACID FAST STAINING PROCEDUREDr. Sunarjati Sudigdo Adi, dr. MS., SpMK(K)

I. General objective After finishing skill practice of this session, the student will be able to perform Ziehl-

Neelsen staining procedure from the sputum specimen II. Specific objective

At the end of skill practice, student could interpret the result of Ziehl-Neelsen staining III. Methods

a. Presentation b. Demonstrationc. Coaching d. Self practice

IV. List of equipments, materials, and reagents Equipments & materials

Sputum specimen Sputum container Bunsen burner Lighter/ matches Cotton soak with 95% alcohol Glass slides Pencil glass/ marking pen Sputum smear preparation positive AFB (not stained) Inoculating loop Filter/ tissue paper Sands with 70% alcohol Methanol 96% (spiritus) Toothpicks Slide holder or box Microscope Iron stick with cotton

Reagents Carbol fuchsin (the Primary Stain) Decolorizer HCl 3%+alcohol 95% Methylene blue (the Counterstain) Water (preferably in a squirt bottle)/ tap water Oil emersion

LEARNING GUIDE

No S T E P 0 1 2 SMEAR PREPARATION

01. Clean glass slide by wiping it with wet cotton ball 95 % alcohol and/ or flame directly 2 – 3 times over the fire to make the slide fat free

02. Mark a target oval area about 2x3 cm below the glass slide with a marking pen/ pencil glass

03. Flame a bacteriological loop to get red-hot and the loop become sterile. Let it cool at room temperature WITHOUT touching anything.

04. Open the sputum container carefully and place the lid face up on the work surface

05. Dip the loop into a sputum sample, select the best portion of sputum, select the most purulent (the thickest part) of the sputum or bloodstained particles if present

06 Place the loop full of sample in the center of target area.

07 RESEAL the sputum container tightly 08 Dips the loop into the bottle contains sands with 70 % alcohol, moves the

loop up and down, and rotates in the sands. Reflame the loop and let cool and put it in the rack

09. Using a toothpick smear the specimen with coiling methods. Spread the specimen using a toothpick vertically and horizontally and smear the specimen in small circular motions to distribute the specimen evenly until about 2x3 cm in size.

10. DISCARD the applicator stick into a discard container containing sands and

suitable disinfectant (alcohol 70%) 11 ALLOW the smear to air dry completely at room temperature

12. Fixation: Pass bottom part of the slide through the flame of a Bunsen burner 3-4 times but don’t get burned the smear is now ready for staining procedure.

This process kills the bacteria and fixes them to the slide so they won't wash off during staining or rinsing.

Ziehl-Neelsen staining procedure01 STEP 1: After fixation of the slides, place the fixed slide on the staining rack

with the smeared side facing upwards 02. STEP 2: Flood the entire slide with Carbol Fuchsin Ensure enough stain is

added to keep the slides covered throughout the entire staining step

03. STEP 3: Using a Bunsen burner or iron stick with cotton, 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 under the slides), prevent the stain for boiling. Allow the slide to cool for 30-60 seconds Caution: Using too much flame or heat can cause the slide to break.

STEP 4: Gently rinse the slide with tap water to remove the excess carbol fuchsin stain. At this point, the smear on the slide looks red in color.

STEP 5: Decolorize the slide by dip it into 3% chloric acid-95% alcohol for few seconds until the slides are clear of stain visible to the naked eye.

To the right are examples of slides insufficiently and sufficiently flooded with acid-alcohol.

STEP 6: Rinse the slide thoroughly with water and then drain any excess from the slides

STEP 7: Flood the slide with the counterstain, Methylene Blue. Keep the counterstain on the slides for 1 minute.

STEP 8: Rinse the slide thoroughly with water

STEP 9: let it dry at room temperature or dry it by blotting with filter/ tissue paper (do not rub!!)

Interpretation Morphological Characteristics - Acid-fast bacilli range from 1 to 10 μm in length and 0.2 to 0.6 μm in width. - They typically appear as slender, rod-shaped bacilli, but they may appear curved or bent. - Individual bacteria may display heavily stained area referred to as beads and areas of alternating stain producing a banded appearance. - Some mycobacteria other than M. tuberculosis may appear pleomorphic, ranging in appearance from long slender rods to coccoid forms, with more uniform distribution of staining properties. Method of Examination Ziehl-Neelsen stained smears should be examined with a 100x oil immersion objective.

Procedure of microscopy: Switch the lamp of microscope on Open diaphragm maximally Adjust the condenser up maximally Put the slide on the stage Put 1 drop of emersion oil Use 100 x objective lens turn until it touch the slide Turn coarse adjustment knob until you find the field Turn fine adjustment knob to set the focus of your eyes Start to count the bacteriaReporting Results of Acid-Fast Bacilli Smears One reporting system recommended by the International Union against Tuberculosis and Lung Disease (IUATLD) when reporting ZN stained smears observed at 1000x is: 1. AFB not found in 100 HPF : negative 2. 1 – 9 AFB/ 100 HPF : report the number of bacteria 3. 10 – 99 AFB/ 100 HPF : + or +1 4. 1 – 10 AFB/ 1 HPF: ++ or +2 5. > 10 AFB / 1 HPF: +++ or +3 Note : if 1 – 3 AFB/ 100 HPF, repeat exam using new specimen, if still 1 – 3 report as neg, if 4 – 9 report as pos.

Note: 0 = not doing at all 1 = do the step partially 2 = do the step completely