Arielle Cruz
Acid-Alcohol is a mixture compounded from an acid and an alcohol, often used in bleaching or decolourising samples that have been previously stained. To prepare 1% acid alcohol, you can mix 1 part acid to 99 parts 70% ethanol. For example, you can mix 1ml of acid with 99ml of alcohol. This method is commonly described in histology texts such as Carson's Histotechnology: A Self-Instructional Text.
How to Prepare 1% Acid Alcohol for Staining
| Characteristics | Values |
|---|---|
| Acid-Alcohol Use | Bleaching or decolourising samples that have been previously stained |
| Ingredients | 70% ethanol and 0.1N (0.1M) hydrochloric acid |
| Preparation Method | Mix 1 part acid to 99 parts 70% ethanol (1 mL acid to 99 mL alcohol) |
| Example Protocol | Dip sample in 1% Acid Alcohol twice |
| Related Protocols | Re-hydrate with 95% alcohol, rinse with water, stain with Hematoxylin Solution, counterstain with Eosin Solution, dehydrate with 100% alcohol, clear with xylene, add Cytoseal, and apply a coverslip |
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What You'll Learn
Mix 1ml acid with 99ml 70% alcohol
Acid-Alcohol is a mixture of acid and alcohol used for bleaching or decolourising samples that have been previously stained. It is commonly used in histology, the study of the microscopic anatomy of cells and tissues.
To prepare 1% acid alcohol, you will need to use 70% ethanol and hydrochloric acid (HCl). The protocol for this is outlined in histology texts such as Carson's Histotechnology: A Self-Instructional Text.
Firstly, you should use a concentrated HCl stock and consider it as 100% HCl, which is approximately 38% in reality. Then, mix 1ml of this acid with 99ml of 70% ethanol. This mixture will give you a 1% acid alcohol solution, which is commonly used for staining in histology.
It is important to follow safety protocols when handling chemicals and to wear appropriate protective equipment, such as gloves and safety goggles. Always ensure that you are in a well-ventilated area when working with chemicals.
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Rehydrate with 95% alcohol
When preparing slides for staining, rehydration is a critical step. The purpose of rehydration is to reintroduce water to the tissue sample, which has been dehydrated during the staining process. This step is necessary to ensure effective staining and preservation of the tissue.
During the rehydration process, it is essential to use a graduated series of alcohol solutions, starting with a lower percentage and gradually increasing the concentration. This gradual approach helps to gently reintroduce water to the tissue, preventing any potential damage that could occur from abrupt rehydration.
One common protocol for rehydration involves using 95% ethanol. This concentration of ethanol is crucial because it serves as a "bridge" between the non-polar solvent, xylene, and the more hydrophilic tissue environment. Xylene, as an organic solvent, is immiscible with water, so transitioning directly to a lower concentration of ethanol can cause issues.
By using 95% ethanol, you can effectively remove residual xylene and other hydrophobic substances from the tissue. This step ensures that the tissue can gradually adapt to the presence of water, preventing the formation of "schlieren" (flow marks) and other disturbances that could impact the staining quality.
Additionally, 95% ethanol plays a role in removing excess water from the slides after rinsing. This step is crucial before applying certain stains, such as alcoholic eosin, where the presence of too much water can result in poor staining. The 95% ethanol step helps differentiate and remove excess stain, ensuring a more precise and controlled staining process.
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Add glacial acetic acid
Acid-Alcohol is a mixture compounded from an acid and an alcohol, commonly used for bleaching or decolourising stained samples. Glacial acetic acid, a name for water-free (anhydrous) acetic acid, is a mild acid used for mixing acid rinse solutions in histological staining. It is a much weaker base than water and has been used to treat cancer since the 1800s. In the context of staining, glacial acetic acid is useful for several reasons:
Increasing Tissue Penetration Time
Glacial acetic acid helps increase tissue penetration time, which is beneficial when working with challenging tissue samples that require extended processing. This property ensures that the stain can effectively penetrate the tissue, resulting in more accurate and reliable results.
Reducing Shrinkage
Glacial acetic acid also plays a crucial role in reducing shrinkage when combined with dehydrating tissue fixatives. This feature is especially important in maintaining the structural integrity of the tissue being stained, as shrinkage can distort the sample and compromise the accuracy of the staining procedure.
Compatibility with Dehydrating Fixatives
The compatibility of glacial acetic acid with dehydrating tissue fixatives is essential for effective staining. By combining glacial acetic acid with these fixatives, the tissue is adequately prepared for the staining process, ensuring optimal results.
Synthesis of Other Compounds
Additionally, glacial acetic acid is valuable for synthesising other compounds used in staining procedures. It is utilised in the synthesis of acetic anhydride, cellulose acetate, and acetic esters. These compounds play various roles in the staining process, including enhancing stain penetration, improving colour development, or providing a compatible medium for specific stains. For instance, ether acetates are used as solvents for nitrocellulose, acrylic lacquers, varnish removers, and wood stains.
Safety Considerations
It is important to note that glacial acetic acid is a corrosive liquid with a vinegar-like odour. Therefore, it should be handled with caution and used only for professional purposes, adhering to safety guidelines and protocols specific to your laboratory or institution.
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Heat alum and add alcohol
To prepare 1% acid alcohol for staining, you'll need to heat alum and add alcohol. Here's a step-by-step guide:
Start by heating 50 ml of 10% alcoholic hematoxylin solution. Bring it to a boil and maintain the temperature for one minute. This step ensures the hematoxylin is properly dissolved. Once dissolved, remove the solution from the heat source.
At this stage, you'll need to add mercuric oxide (red). Slowly add 2.5 grams of mercuric oxide to the heated solution. The solution will gradually change colour. Continue heating until the solution turns dark purple. This colour change is a crucial indicator that the mixture is ready for the next step.
Now, it's time to cool the solution. Place the container with the solution in a cold water bath. This will help bring the temperature down gradually and uniformly. Do not add ice or cold liquid directly to the solution, as it may cause an uneven temperature change.
Once the solution has cooled sufficiently, add the alcohol. Measure out 20 ml of glacial acetic acid (concentrated) and slowly incorporate it into the mixture. Stir gently to ensure even distribution.
Finally, before using the solution for staining, remember to filter it. Filtering ensures that any impurities or undissolved particles are removed, resulting in a smooth and consistent mixture. This step is essential for obtaining accurate and reliable results during the staining process.
By following these steps, you will have successfully heated alum and added alcohol to create a crucial component of the H&E staining protocol. This technique is commonly used in histology and pathology to examine tissue samples under a microscope.
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Dip in 1% acid alcohol
To prepare a 1% acid alcohol solution for staining, you will need to mix 1 part acid with 99 parts 70% ethanol. For example, you could mix 1 mL of acid with 99 mL of alcohol. This solution can then be used for dipping.
When performing an H&E stain, the slide should be dipped in 1% acid alcohol twice after the initial stain and wash steps. First, stain the slide in Mayer's Hematoxylin Solution for 30 seconds. Then, rinse the slide in running tap water for 2 minutes. Next comes the acid alcohol step: dip the slide in 1% acid alcohol twice. After this, wash the slide again with running tap water for 2 minutes.
The slide can then be counterstained. Rinse it quickly in 95% alcohol (20 dips x 2) and then dehydrate it through 100% alcohol (10 dips x 2) until it is dry. Finally, clear the slide in xylene for 2 minutes, twice.
It is important to note that the concentrations and timings provided here are specific to the H&E staining protocol and may vary depending on the specific application and desired results.
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Frequently asked questions
1% acid alcohol is used for staining and is also known as a bleaching or decolourising agent for samples that have been previously stained.
Mix 1 mL of acid with 99 mL of 70% ethanol.
- Rehydrate with 95% alcohol for 2 minutes, twice.
- Rinse in running tap water for 2 minutes.
- Stain in Mayer’s Hematoxylin Solution for 30 seconds.
- Rinse in running tap water for 2 minutes.
- Dip in 1% Acid Alcohol twice.
- Wash with running tap water for 2 minutes.
- Counterstain in Eosin Solution (1 mL Eosin Solution + 2 mL Acetic Acid Glacial) for 2-3 minutes.
- Rinse with 3-4 washes of tap water.
- Rinse quickly in 95% alcohol (20 dips x 2) and dehydrate through 100% alcohol (10 dips x 2) until dry.
- Clear in xylene for 2 minutes, twice.
- Add 2-3 small drops of Cytoseal and slowly apply a coverslip, avoiding air bubbles.
