Author: Tom McCaughtry

Date: 3/23/07

Standard Operating Procedure

Pertussis Toxin Treatment of mice

Hazards:
Pertussis Toxin (PT) is a potent toxin in humans. The LD50 in mice is 18 ?g/kg i.p. There is no LD50 information for humans. The toxin is harmful by inhalation, contact with the skin, or swallowed. However, PT is degraded by the low pH in the gut and is not absorbed. If swallowing occurs, induce vomiting. If skin pricking should occur, induce bleeding and flush with copious amounts of water. If i.v. or i.m. injection should occur, consult a physician. Attempt to obtain hyperimmune globulin to pertussis from the CDC. In an adult immunized against whooping cough, no long term ill effects are likely to result.

Protection:
Individuals working with this toxin should wear suitable protective clothing including lab coat, gloves, eye/face protection. Work in a well-ventilated area and avoid inhalation. Avoid contact with open wounds. Wash thoroughly any area of the body that comes into contact with the toxin.

Waste:
Contaminated pipettes, lab ware, etc. should be disposed of as hazardous waste (i.e. put in an appropriately labeled red biohazard bag and autoclaved).

Spill clean-up:
Remove any contaminated clothing and put in red autoclavable biohazard bag. Notify other workers in the area of the spill and control traffic though area. Wear shoe covers if spill is on floor. Put on gloves and cover spill area with paper towels. Pour 10% bleach over towels from edges of spill to center, be careful not to splatter. Decontaminate all objects in spill area. Allow 20-30 minutes of contact time. Pick up any sharps, including broken glass, with forceps and place in sharps container. Wipe area with disinfectant and clean towels, mop if spill on floor. Remove gloves and foot covers before leaving area of spill, put in biohazard bag, and wash hands. Autoclave waste.

Procedure:
Preparation of toxin: work in the hood in 6-258 NHH. Reconstitute a single vial of pertussis Toxin powder (List Labs #180) with 0.5 ml of sterile ddH2O. Use a needle and syringe to puncture the septum of the vial, to avoid potential generation of aerosols. Do not recap needle, dispose of directly into sharps container. Each vial, when reconstituted contains 100 ?g/ml pertussis toxin in 0.01 M sodium phosphate buffer, pH 7.0, with 0.05 M sodium chloride. Store toxin at 4oC in 10 ?l aliquots. Label box well “Pertussis Toxin – HAZARDOUS”.

Preparation of mice for injection: Label cage cards “Pertussis Toxin – HAZARDOUS”. If working in a procedure room in MCB, be sure to bring appropriate protective equipment and biohazard bags with you.
Treatment of mice: Thaw a single aliquot and dilute 1:50-1:5000 for a final concentration of 2 ?g/ml-20 ng/ml. Load an insulin syringe and inject 100 ?l i.p. into mice (2 – 200 ng). Do not recap needle, dispose of directly into sharps container. There is minimal risk to animal caretakers as the amount of unmetabolized PT excreted by the injected mice is likely to be extremely low. Bedding and carcasses can be disposed of as usual.