Carnoy Fixative Ⅱ: Preparation, Protocol & Applications

Carnoy Fixative Ⅱ is a rapid-acting, non-aqueous fixative composed of absolute ethanol, chloroform, and glacial acetic acid, widely used in histology, cytogenetics, and chromosome preparation for its excellent nucleic acid preservation and fast tissue penetration.

Composition

Absolute ethanol : chloroform : glacial acetic acid (6:3:1, v/v/v)

Primary Applications

Chromosome spreads, cytogenetics, lymph node and mucosal tissue fixation, nucleic acid preservation

Fixation Mechanism

Protein precipitation and dehydration; acetic acid counteracts tissue shrinkage caused by alcohol and chloroform

Penetration Speed

Very rapid; small tissue pieces fixed within 30–60 minutes at room temperature

Storage & Stability

Prepare fresh before use; store tightly sealed away from heat and ignition sources

Key Advantage

Excellent preservation of DNA and RNA; compatible with FISH and in situ hybridization workflows

Composition and Reagent Preparation

Carnoy Fixative Ⅱ is prepared by combining absolute ethanol, chloroform, and glacial acetic acid in a volumetric ratio of 6:3:1. For a standard 100 mL working volume, combine 60 mL absolute ethanol, 30 mL chloroform, and 10 mL glacial acetic acid in a glass container with a tight-fitting lid. Mix gently until homogeneous.

Critical notes:

• Always add reagents in the order listed to minimize exothermic reactions.
• Prepare fresh immediately before use; do not store mixed fixative for extended periods.
• Use only glass containers — chloroform degrades many plastics.
• All steps must be performed in a well-ventilated chemical fume hood due to the volatility and toxicity of chloroform.

Aladdin Scientific offers Carnoy Fixative Ⅱ as a ready-to-use formulation (SKU: C1373510), eliminating the need for in-house preparation and reducing exposure risk.

Step-by-Step Fixation Protocol

The following protocol is suitable for small tissue pieces and cell pellets intended for histological or cytogenetic analysis.

1. Sample preparation: Trim fresh tissue to pieces no larger than 3–5 mm in thickness to ensure complete and rapid penetration of the fixative.
2. Fixation: Immerse the sample in a minimum of 10× its volume of freshly prepared Carnoy Fixative Ⅱ. Fix for 30–60 minutes at room temperature for small pieces or cell pellets. Larger tissue fragments may require up to 2–4 hours. Avoid over-fixation, which can cause excessive tissue brittleness.
3. Intermediate washes (optional): For cytogenetic preparations, replace the fixative once or twice during the fixation period to ensure complete removal of lipids and cellular debris.
4. Dehydration / transfer: After fixation, transfer tissue directly into 70% ethanol or 100% ethanol for downstream paraffin embedding, or proceed with spreading onto slides for chromosome preparations.
5. Chromosome spreading (cytogenetics): Resuspend the fixed cell pellet in fresh fixative, drop the suspension onto pre-chilled clean glass slides, and allow to air-dry before staining.
6. Downstream processing: Fixed material is compatible with standard hematoxylin and eosin (H&E) staining, Feulgen staining, FISH, and in situ hybridization workflows.

Note: Carnoy Fixative Ⅱ is not recommended for immunohistochemistry (IHC) requiring antigen preservation, as protein precipitation may compromise epitope integrity.

Applications and Tissue Compatibility

Carnoy Fixative Ⅱ is particularly valued in the following research and diagnostic contexts:

• Cytogenetics and karyotyping: The standard fixative for chromosome spreads from cultured lymphocytes, bone marrow, and other dividing cells. Its non-aqueous composition preserves chromosome morphology and allows high-resolution banding.
• Nucleic acid studies: Compatible with DNA and RNA preservation, making it suitable upstream of FISH, in situ hybridization, and PCR-based analyses on tissue sections.
• Lymphoid and mucosal tissues: Particularly effective for lymph nodes, spleen, and intestinal mucosal biopsies where rapid fixation is required to preserve cellular architecture.
• Glycogen detection: Used in conjunction with PAS (Periodic Acid–Schiff) staining, as it preserves glycogen more reliably than aqueous fixatives.
• Plant and insect tissue: Widely used in botanical and entomological research for fixing small, dense specimens that require rapid penetration.

Carnoy Fixative Ⅱ is not recommended for routine formalin-equivalent fixation requiring long-term archival storage, thick tissue blocks, or IHC applications dependent on intact protein epitopes.

Safety, Handling and Waste Disposal

Carnoy Fixative Ⅱ contains chloroform, a suspected human carcinogen and CNS depressant, and glacial acetic acid, a corrosive irritant. Strict safety measures must be observed:

• Personal protective equipment (PPE): Wear nitrile gloves, splash-proof safety goggles, and a laboratory coat. Use a full-face shield when handling large volumes.
• Ventilation: Perform all handling in a certified chemical fume hood. Do not use near open flames — ethanol is highly flammable.
• Storage: Store tightly sealed in an explosion-proof refrigerator or a flammables cabinet, away from oxidizers and ignition sources.
• Spill response: Absorb spills with inert dry material (e.g., vermiculite or dry sand). Do not use paper towels for large spills. Ventilate the area immediately.
• Waste disposal: Collect all waste fixative in clearly labeled halogenated solvent waste containers. Dispose of according to local institutional and governmental regulations for halogenated organic waste. Do not pour down the drain.

Consult the Safety Data Sheet (SDS) for Carnoy Fixative Ⅱ (C1373510) available on the Aladdin Scientific website for complete hazard information.

Troubleshooting Common Issues

The following table summarizes common problems encountered when using Carnoy Fixative Ⅱ and recommended corrective actions:

• Poor chromosome spreading / clumping: Ensure fixative is freshly prepared; aged fixative loses efficacy. Adjust the fixative-to-cell ratio and drop height when making chromosome spreads. Humidity and slide temperature significantly affect spreading quality — optimize empirically.
• Tissue over-hardening or brittleness: Reduce fixation time. Carnoy Fixative Ⅱ acts rapidly; excessive exposure causes protein over-precipitation and brittleness that complicates sectioning.
• Incomplete fixation / soft tissue center: Reduce tissue block size to ≤3 mm thickness. Ensure sufficient fixative volume (≥10× tissue volume) and agitation during fixation.
• Weak or absent FISH signals: Verify that fixative was freshly prepared. Residual lipids or inadequate washing steps can quench hybridization signals; include additional fixative washes before slide preparation.
• Excessive tissue shrinkage: While some shrinkage is inherent to Carnoy fixation, excessive shrinkage may indicate insufficient acetic acid in the mixture. Verify the 6:3:1 ratio was correctly prepared.

Frequently asked questions

What is the difference between Carnoy Fixative I and Carnoy Fixative Ⅱ?

Carnoy Fixative I is composed of absolute ethanol and glacial acetic acid (3:1, v/v), while Carnoy Fixative Ⅱ adds chloroform to the formula (ethanol:chloroform:acetic acid, 6:3:1). The addition of chloroform in Fixative Ⅱ improves lipid dissolution and penetration speed, making it preferred for denser or more lipid-rich tissues and for cytogenetic applications.

Can Carnoy Fixative Ⅱ be used for immunohistochemistry (IHC)?

Carnoy Fixative Ⅱ is generally not recommended for standard IHC applications. Because it acts as a precipitative fixative, it can disrupt protein conformation and compromise antibody-epitope recognition. For IHC workflows requiring reliable antigen retrieval, neutral buffered formalin (NBF) fixation followed by appropriate antigen retrieval is the preferred approach.

How long can samples remain in Carnoy Fixative Ⅱ before processing?

For optimal results, samples should be processed promptly after the recommended fixation time (30 minutes to a few hours depending on tissue size). Prolonged storage in Carnoy Fixative Ⅱ is not recommended, as over-fixation leads to excessive tissue hardening and brittleness. If immediate processing is not possible, transfer samples to 70% ethanol for temporary storage.

Is Carnoy Fixative Ⅱ compatible with FISH (Fluorescence In Situ Hybridization)?

Yes, Carnoy Fixative Ⅱ is one of the most widely used fixatives for FISH applications, particularly for chromosome spreads prepared from cultured cells. Its ability to preserve nucleic acid integrity while removing lipid membranes facilitates efficient probe hybridization. It is considered a standard fixative in clinical and research cytogenetics laboratories for FISH-based chromosomal analysis.

What precautions are needed when disposing of Carnoy Fixative Ⅱ waste?

Carnoy Fixative Ⅱ waste must be collected in dedicated halogenated solvent waste containers due to the presence of chloroform. It should never be poured down the drain or mixed with non-halogenated waste streams. Disposal must comply with institutional chemical waste regulations and applicable local environmental legislation. Contact your institutional environmental health and safety (EHS) office for guidance.

References

1. Carnoy J. La biologie cellulaire. 1884. Original description of Carnoy fixative formulations.
2. Bancroft JD, Gamble M. Theory and Practice of Histological Techniques. 6th ed. Churchill Livingstone; 2008.
3. Rooney DE, Czepulkowski BH, eds. Human Cytogenetics: Constitutional Analysis. 3rd ed. Oxford University Press; 2001.
4. Kiernan JA. Histological and Histochemical Methods: Theory and Practice. 4th ed. Scion Publishing; 2008.

Products are supplied for research and development use only. Not for use in humans, animals, diagnosis, or therapy.