In order to store bacterial isolates long term, they need to be in a glycerol solution. These solutions can be prepared in the lab to ensure longterm storage of bacterial isolates.
- Turn on the Biological Safety Cabinet. This is done by lifting the sash to operating sash height, marked on the left side of the hood. This should turn on the blower, white light, and window alarm.
- Clean the cabinet with 70% ethanol, including the work surface, walls, and glass.
- Lower the sash and press the UV button. This will turn on the UV light for 15 minutes. It will turn off automatically.
Remember, before placing any objects in the hood, they must be sprayed with 70% ethanol.
- Plated pure cultures of well-formed colonies
- BHI Liquid Media
- Sterile Inoculation Loops
- 70% Ethanol
- 100% Glycerol
- Incubator (set at appropriate temperature for bacterial growth)
- Micropipettes (P1000)
- Pipette Tips (1000uL)
- 1.5mL Screw Cap Cryovials
- Microtube Rack
- Serological Pipette
- Serological Pipette Tips (5mL)
- Label tubes with corresponding numbers/labels.
- Use the serological pipette to add 5 mL of BHI broth into each of the tubes.
- Remove cover of petri dish with bacteria colonies and use sterile inoculation loop to pick an isolated colony.
- Aseptically transfer the colony from the stick to the broth. Swirl gently ~10-20 times in the broth to remove the colony from the loop.
- Repeat steps 3-4 for each isolate to be prepared.
- Incubate at the appropriate temperature overnight. (15-18 hours)
- Label sides of microtube with corresponding numbers/labels as well as the date and initials. Optional: Add cap inserts to vials for easy viewing.
- Microwave glycerol for ~10 seconds to make it easier to pipet. Do not overheat.
- Aliquot 200 uL of glycerol to each cryovial and replace cap.
- Vortex overnight culture briefly and add 1 mL to the appropriate cryovial.
- Invert until mixed.
- Store in -80C Freezer.