u/ScoopJr

I'm having trouble figuring out how to properly do the different staining. Here's how I performed the different stains and my results in an image. The bacteria is Mycobacterium Smegmatis

Gram Staining

1. Flood slide with Crystal Violet for 1 minute, water rinse

2. Flood slide with Iodine (mordant), 1 minute, water rinse

3. Acetone Alcohol drop by drop at an angle until color stops running for 3-5 seconds, water rinse

4. Counterstain flood slide with Safranin, 1 minute, water rinse, blot dry

Acid Fast Stain

1. Flood slide with Carbolfuschin for 10 minutes (cold method), water rinse

2. Apply 5-6 drops of alcohol acid on slide at angle until no more color runs off, water rinse

3. Counterstain with Methylene Blue for 1 minute, water rinse, blot dry bulbous paper

Endospore Stain

1. Flood slide with Malachite Green and steam with blowtorch method for 5 minutes (ensure fumes and stain doesn't dry out), water rinse both sides until no more Malachite

2. Counterstain with 0.5% Safranin for 1 minute, water rinse, blot dry

Problem

The problem I seem to have is for Acid Fast Stain (A), whether I flood the slide with alcohol acid or slowly do it drop by drop at an angle, I always seem to get rid of all the carbolfuschin and I just see the methylene blue under the microscope. The B picture is one that my classmate got for the same bacteria. My staining (A) makes it difficult to identify the bacteria because it looks blue instead of the pink/purple to identify the Mycobacterium.

Endospore, I seem to get spots of green and the rest is pink, which is confusing since this bacteria doesn't have endospores. I should only see pink here confirming the Endospore neg.

Any tips or advice?? Thank you for taking the time to help and read my post :)

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