Nocardia under Fluorescence Microscope: Introduction, Staining Procedur

Nocardia under Fluorescence Microscope: Introduction, Staining Procedure, Result Interpretation

Nocardia under Fluorescence Microscope

Nocardia under Fluorescence Microscope as shown above image. Nocardia species is in auramine -rhodamine stain showing bright yellow intertwining and branching threads i.e. positive as shown above image. Initially, the clinician advised the patient for the AFB test of sputum due to suspecting tuberculosis but during the AFB stained smear examination, we found acid-fast structures resembling branching threads. From the same sputum, specimen smear prepared and performed modified auramine -rhodamine stain. After observation, we got this type of positive structure. Later organism confirmed as Nocardia, growing on culture media and using biochemical tests.

Introduction of auramine -rhodamine stain

It is a fluorochrome stain and used to visualize acid-fast structures of various microorganisms especially Mycobacterium tuberculosis and in modified form for Mycobacterium leprae, Nocardia species, Cryptosporidium parvum, Cyclospora cayetanensis , Isospora belli, and fungal spores. Ziehl-Neelsen (hot), Kinyoun (cold) are still widely used methods to detect acid-fast structures in these organisms in developing countries but sensitivity is high of fluorochrome stain. The acid fastness of Mycobacterium tuberculosis is due to having a thick cell wall composed of waxes and lipids that has a high content of mycolic acid.

Principle of Auramine -rhodamine stain

Auramine-rhodamine is the fluorochrome dye that forms a complex with mycolic acids found in the acid-fast cell wall of organisms that resist decolorization by acid-alcohol. Potassium permanganate, counterstain renders tissue and its debris non-fluorescent, therefore reducing the possibility of artifacts. The cellular structures visualized under U-V appear bright yellow or reddish-orange.

Test Requirements

Auramine Rhodamine stain (Primary Stain)
0.5% Acid alcohol ( Decolorizer)
0.5% Pottassium Permanganate
Test specimen (e.g. sputum )
Slide ( clean and grease-free)
Pencil ( diamond if possible)
Slide racks
Bunsen burner
Inoculating loop or sterile bamboo stick

Staining procedure of Auramine -rhodamine

Smear preparation

Take a purulent part of the sputum to a slide and make a thin smear using an inoculating loop or bamboo stick. Cover an area of approximately 2 cm square and spread the smear using circular movements. Allow it to air dry. Finally, perform heat-fixing passing the dried slide, smear facing upward, 2 to 3 times through the blue cone of a burner flame.

Staining Procedure

Put the fixed smear on a staining rack and flood the smear with rhodamine-auramine for 15 minutes. Do not let smear dry.
Wash off the stain with clean water.
Decolorize the smear by covering it with acid-alcohol for 2-3 minutes. ( But here in this case of Nocardia, we used 0.025 % acid alcohol i.e. modified form of auramine -rhodamine stain.)
Wash off the acid alcohol with clean water.
Now cover the smear with potassium permanganate for 3-4 minutes. Do not allow smear to dry.
Rinse thoroughly with distilled water and air dry.
Examine the smear by fluorescence microscope and use the 10X objective to focus the smear. Finally, observe the smear using the 40 X objective for acid-fast structures or acid-fast bacilli (AFB).

Result Interpretation

Test Positive: Acid-fast organisms fluoresce bright yellow or reddish-orange against a dark background.
Negative Test: Non-acid-fast organisms will not fluoresce

Precautions

Take precautions during handling the following reagents due to the following reasons-

Auramine-rhodamine stain is a possible carcinogen.
Acid alcohol is flammable and Corrosive.
Potassium permanganate is also corrosive.

Advantages over Z-N stain

Nearly 10% more sensitive than Z-N stain
It does not require the use of oil immersion fields and thus no need for cedarwood oil.
Heat is not required for auramine-rhodamine staining.

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