Carmine

Acid Carmine Staining Protocol
based on Semichon [1929].
Modified from various sources and mistakes

Reagents

Working Notes: Neutralizes destain and colors final preparation. Dilute 1:1 with 70% EtOH before use.

A saturated solution of sodium bicarbonate in 70% Ethanol

Alkaline Alcohol

Working Notes: Secondary (Bleaching) Destain. May also be used as a primary destain for very large gregarines.

Hydrochloric Acid
70% Ethanol

5 ml
100 ml

Strong Acid Alcohol Destain

Working Notes: Primary (Differentiating) Destain

Hydrochloric Acid
70% Ethanol

1 ml
100 ml

Working Notes: Combine water and acid in a flask and add carmine until no more will go into solution. Bring contents of the flask to ca. 95 C over a boiling water bath, but DO NOT BOIL. Cool and filter. (A double drip coffee filter works well.) Measure stain volume and add an equal volume of 70% EtOH. Store away from excessive heat and light. Keeps for years. Discard stock stain when stained cytoplasm begins to appear muddy or stained specimens appear bluish-purple.

Glacial Acetic Acid
Distilled water
Carmine Alum Lake
70% Ethanol

250 ml
250 ml
5 g
500 ml

Semichon's Acid Carmine

Weak Acid Alcohol Destain

Primary Fixation: Fix specimens or tissues and affix them to

Procedure

slides or coverslips. AFA is the preferred fixative. Note that Semichon’s is itself a fixative and may be used to simultaneously fix and stain wet smears.
Fixative Rinse: Rinse out excess fixative in 70% Ethanol for 3 minutes.
Stain: Stain in Semichon's Acid Carmine for 2 minutes. Large gregarines may need to stain for up to 5 minutes.
Stain Rinse: Rinse out excess stain in 70% Ethanol for 3 minutes
Primary Destain: Destain in 1% Acid Alcohol. Time varies from 5 seconds to 5 minutes depending on species and size. Larger species may be destained in 5% Acid Alcohol.
Secondary Destain: Remove excess background stain by dipping in 5% Acid Alcohol (1-2 seconds).
Neutralization: Rinse in Alkaline Alcohol for 3 minutes to neutralize destain acid.
Dehydration: Dehydrate in a graded ethanol series: 95% (5 minutes); 100% (3 changes, 3-5 minutes each).
Clearing: Clear in a graded xylene or toluene series: 50% xylene/50% Absolute alcohol (5 minutes); 100% xylene (3 changes, 3-5 minutes each).
Mounting: Mount in Damar Balsam (Damar Xylene).

Dr. R. E. Clopton