Auramine-Rhodamine Stain Test Cost & Procedure

Nocardia in a rhodamine-auramine stain

As seen in the image above, the Nocardia species exhibits vivid yellow intertwining and branching threads in an auramine-rhodamine stain, indicating that it is positive. The doctor initially suggested the patient do the sputum.

AFB test because they suspected tuberculosis. However, when we examined the AFB stained smear, we saw acid-fast structures that resembled branching threads. The same sputum was used to create a modified auramine-rhodamine stain and a specimen smear. In the following observation, we obtained this kind of good structure. Using biochemical testing and an organism that was growing on culture media, the latter was identified as Nocardia.

Auramine-rhodamine staining principle:

The fluorochrome dye auramine-rhodamine combines with the mycolic acids in the acid-fast cell walls of organisms to prevent color loss from acid-alcohol by making tissue and its byproducts non-fluorescent, potassium permanganate counter stain minimizes the risk of artifacts. Under U-V, the cellular structures are shown as bright yellow or reddish-orange.

Test specifications:

• Auramine dye rhodamine (Primary Stain)
• Acid alcohol, 0.5% ( Decolorizer)
• Potassium Permanganate 0.5%
• test subject (e.g. sputum )
• Slide ( clean and grease-free) ( clean and grease-free)
• Pencil ( diamond if possible) ( diamond if possible)
• Moveable racks
• Bunsen stove
• Vaccination loop or clean bamboo stick

Auramine-rhodamine staining method

Preparation for smears

Smear a tiny layer of purulent sputum onto a slide using an inoculating loop or bamboo stick. Spread the spot in circular motions over an area roughly 2 cm square. Let it dry naturally. Finally, perform heat-fixing by passing the dried slide through the blue cone of a burner flame 2 to 3 times with the smear facing upward.

Staining

• Placing the fixed smear on a staining rack, cover it for 15 minutes with rhodamine-auramine. Never allow the smear to dry.
• Make use of clean water to remove the discoloration.
• Cover the smear with acid-alcohol for two to three minutes to remove color. (However, in this instance, we utilized 0.025% acid alcohol,e.g., an altered version of the auramine-rhodamine stain.)
• With clean water, rinse the acid alcohol away.
• Potassium permanganate should be applied over the smear for 3–4 minutes. The smear must not be allowed to dry.
• Thoroughly rinse with distilled water, then let air dry.
• Use a fluorescent microscope to examine the smear, focusing it with the 10X objective. Lastly, use the 40 X objective to scan the smear for bacilli or structures that can withstand acid (AFB).