Chelex Extraction of Hairs Contaminated with Myobacterium Bovis

Chelex extraction of hairs contaminated with Mycobacterium bovis from Petra Carpenter

Worker should be vaccinated with BCG (+ve Heeff test) and sign COSHH forms

Find the samples Remove samples from fridge and place in a large tray on a clean bench. Have a flask of 80% ethanol as well as Presept and tissues and a disposable list of samples required. Wear a mask (type 3M-8812), goggles and double gloves.

Do not perform in a fume hood due to the creation of aerosols.

Sort through samples to find those required for extraction and place in a large plastic bag. Any loose hairs to be placed in 80% ethanol – decontaminated after 16 hours. Do not touch any other surface with potentially contaminated gloves. Once the samples have been sorted wipe the outside of the large bag with Presept. Soak the sorting tray with Presept, including gloves. Rinse off Presept with copious amounts of water after 1 hour. Gloves, alcohol hairs, tissue etc. can then be placed in bins for incineration.

Decontaminate the samples

Will need: 80% ethanol, 1.5 ml eppendorfs, scissors and tweezers, presept, tissue.

1. Take sealed bag containing badger hairs into a Class 3 laboratory in a plastic box 2. Sort 20 guard hairs with roots 3. Cut hairs 2cm from root 4. Place hair pieces root down into a microtube containing 80% ethanol. 5. Reseal hair bag and replace in box. 6. Wipe outside of box with Precept – return to C2 lab (alternative – just transfer roughly half, sort after decontam with alcohol – place 20ml in 50mL tube?)

Mycobacterium will be inactive after 16 hours at 4 degrees Extract the samples

Will need: 56°C rotating oven, possibly 37°C heatblock, 95°C heatblock, quality water, 5% chelex, tape, 1.5mL eppendorfs. Use 10% bleach to sterilise working surface and pippettman before commencing extraction. Include an extraction negative (can contain hair without root).

1. Remove ethanol from sample 2. Add 1ml of water to the hair and incubate 30 mins at room temp. 3. Spin 3 mins at 13000 g, then remove all but 30ul of the supernatant. 4. Add 200ul of 5% Chelex (made in low TE and autoclaved) and mix well 5. Incubate at 56°C for 45 mins mixing occasionally 6. Vortex for 10-15 sec, pierce lid, and boil at 95°C for 8 mins (check hair is completely immersed before boiling). Vortex again. 7. Stick clear tape on to seal the hole and spin 3 mins at 13000g (skip tape/pierce?) 8. Remove supernatant into a sterile tube.

Blood alternative test Add 50 to 100ul of blood to 500ul of 5% Chelex – then as above