Use of CFA


In order to ensure appropriate use of Complete Freund’s Adjuvant (CFA), the UConn Health’s Institutional Animal Care and Use Committee (IACUC) has implemented a policy on the use of CFA.


CFA is an oil-in-water emulsion containing mycobacterial cell wall components. It is used as an agent to potentiate antibody response to injected immunogens by eliciting a local inflammatory response. CFA should be used only when absolutely necessary (e.g., weak antigens, induction of auto-immune response) and may only be used for the initial immunization. Incomplete Freund’s Adjuvant (IFA) or other adjuvants that produce less intensive inflammatory responses (e.g., RIBI® or TiterMax®) should be used whenever possible. Improper use of Freund’s adjuvant can cause undesirable and painful side effects such as excessive inflammation and swelling, abscess formation, ulceration, and tissue necrosis.

Any proposed deviation from these guidelines must be fully explained and justified to the Animal Care Committee in the research proposal.

Occupational Health and Safety

Due to the fact that CFA contains killed mycobacteria, accidental inoculation into personnel handling this agent can result in subsequent positive tuberculin titers and/or local inflammatory lesions, which have poor response to antibiotic therapy. These lesions can be severe and result in granulomas, abscesses, and/or tissue necrosis. Extreme caution should be taken when using CFA.

Personnel Protective Equipment (PPE), especially gloves and eye protection, must be worn when handling CFA. Needles should not be recapped, Luer lock syringes should be used, and anesthesia or sedation of animals being injected is recommended to minimize the risk of accidental exposures to personnel.



CFA may be used only for the first (priming) immunization. IFA should be used for any subsequent, booster immunizations. Inoculum should be free of extraneous microbial or chemical contamination. Filtration of the antigen before mixing with adjuvant is recommended.

Sites of administration of the CFA should be chosen so as to avoid anatomic sites used for handling or restraint of the animals being inoculated. Intravenous administration of CFA is prohibited. Intramuscular, intradermal, and footpad administrations (rodents only, one footpad only) are permitted only with scientific justification and approval of the IACUC.

Injection sites should be clean and free of debris as contamination is likely to result in local infections. Clipping of hair and surgical preparation of the injection sites will reduce the potential for the development of infection and/or abscess formation. Sterile Luer lock syringes and needles should be utilized. Needles should be 25 to 31 gauge in size. The number of injection sites should be limited to the minimum necessary to accommodate the volume of material being administered. Injection sites should be sufficiently separated to prevent coalescing of any inflammatory responses from different injection sites.

Animals may receive booster immunizations of IFA or another adjuvant, as required, to develop the desired immune response as long as no adverse effects from prior immunizations are present. There should be a minimum interval of two weeks between initial and subsequent immunizations.

The following is a list of acceptable routes of administration and volumes of CFA for the indicated species:

Species Subcutaneous IM SubQ via Footpad ID IP
Mouse 0.1 ml/site

4 sites maximum

NR (Not Recommended) Requires Scientific Justification 0.05 ml/site

4 sites maximum

0.25 ml maximum
Rat 0.2 ml/site

4 sites maximum

NR Requires Scientific Justification 0.05 ml/site

4 sites maximum

0.5 ml maximum
Rabbits 0.25 ml/site

4 sites maximum

0.25 ml/site

4 sites maximum

Requires Scientific Justification

NR 0.05 ml/site

1.0 ml total

Not permitted

Animals must be closely monitored for complications following CFA administration. Daily observation of injection sites must be documented in written records for the initial 2 days post immunization and a minimum of once weekly thereafter. Injection sites should be closely monitored for the development of severe lesions. When the adjuvant has been administered intraperitoneally in rodents, the animals must be monitored closely for excessive abdominal distension.


1. ARAC Guidelines: Recommendations for Consideration in the Research Use of Inflammatory Agents. NIH Office of animal Care and Use. May, 1996.

2. Jackson, LR and Fox, JG. Institutional Policies and Guidelines on Adjuvants and Antibody Production. ILAR Journal, Vol. 37(3): 141-152, 1995.

Effective Dates:     December 3, 2020 through December 31, 2023