Staining with semichon's acetocarmine

Primary Fixation

Fix specimens or tissues and affix them to slides or coverslips. AFA is the preferred fixative. Note that Semichon's is itself a fixative originally designed to simultaneously fix and stain wet smears. We move our coverslips from columbia jars to ceramic staining racks for processing. All stains and solutions are kept in screw top jars. We remove the lids as needed and move the racks from jar to jar and then tighten down the lids for storage.

Fixative Rinse

Rinse out excess fixative in 70% Ethanol for 3 minutes. Specimens that have been stored in 70% ethanol can go directly to staining.


Semichon's acetocarmine is a regressive stain, that is, specimens are overstained and then destained to remove excess stain and differentiate structures.Stain in Semichon's Acid Carmine for 2-3 minutes. Large gregarines may need to stain for up to 25 minutes.


Rinse out excess stain in 70% Ethanol for 3 minutes. Destain in 1% Acid Alcohol. Time varies from 5 seconds to 5 minutes depending on species and size. Remove excess background stain by dipping in 5% Acid Alcohol for 1-2 seconds. Rinse in Alkaline Alcohol for 3 minutes to neutralize destain acids and set the final stain tone and color.


Dehydrate in a graded ethanol series: 95% (5 minutes); 100% (3 changes, 3-5 minutes each).


Clear in a graded xylene series: 50% xylene/50% Absolute alcohol (5 minutes); 100% xylene (3 changes, 3-5 minutes each).


Mount in warm (37-40 C) Damar Balsam.


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    Coors coverslip staining rack (left), Columbia coverslip jar (right), coverslip storage box (rear).

    Acetocarmine staining series in screw top jars.

    Acetocarmine stained gregarine, phase contrast microscopy.

Acid Carmine Stain

Glacial Acetic Acid
250 ml
Distilled water
250 ml
Carmine (Alum Lake)
ca. 3-5 g
70% Ethanol
ca. 500 ml

Combine water and acid in a flask and add carmine until no more will go into solution. Bring contents of the flak 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.

1% Acid Alcohol Destain

1% vol/vol HCl in 70% ethanol.

5% Acid Alcohol Destain

5% vol/vol HCl in 70% ethanol.

Alkaline Alcohol

A saturated solution of Sodium Bicarbonate in 70% Ethanol. Dilute 1:1 with 70% EtOH before use.