Media+&+Autoclave

=__Media & Autoclave__=

Words of “wisdom”

 * Even though our autoclaves are not under intense use, attempt to use them as efficiently as possible
 * Do not mix dry items like glassware and liquids. The inevitable result will be heavy water condensation on the solid items
 * Keep lids loose
 * Set a timer and remove your items promptly. To do otherwise is rude.
 * If you discover that someone rudely left their things in an autoclave for an extended period of time, remove them and neatly place them on the cart to cool
 * Fill out the log

Liquids and media

 * Do not fill any vessel more than half-full with liquids
 * Always place liquids in a larger pan that can handle any boil-overs. It is no fun crawling into the beast to scrape out charred and nasty remains. Everyone will take a turn at this if this becomes a problem.
 * Here is a handy guide for normal volumes and appropriate sterilization times:
 * up to 1 liter, 20 minute liquid cycle
 * 1-2 liters, 30 minute liquid cycle
 * 2-4 liters, 60 minute liquid cycle

Solids

 * Place autoclave tape on items to help indicate when a "sterile temperature" has been achieved
 * Include a 15 minute dry cycle for all lab consumables to help minimize condensation
 * Place all items on a tray (avoid using bins) and leave space between them for the heat and steam to penetrate.
 * Avoid packing trays like a pile of bricks. The items at the center will likely not be sterile. You have been warned.

Waste

 * It is federal law that all foreign soils be autoclaved prior to disposal.
 * Place solid waste in a bag, and seal with autoclave tape.
 * Place plates in a clear autoclave bag, without a biohazard symbol and seal with autoclave tape. Slit the top of the bag to help with steam penetration (these seem silly, but they make a big difference!)
 * It is OK to mix solid and liquid waste for sterilization because you will be using a liquid cycle for microbial annhilation - the most stringent killing cycle.
 * Place all items in a metal bin and set the autoclave for a 60 minute liquid cycle.
 * If you used a bag with a biohazard symbol on it, deface the symbol after autoclaving because there are no longer any biologically hazardous materials.

Problems

 * Surprisingly enough, our autoclaves are very sophisticated. So, the machine will not allow generally unsafe conditions (i.e., elevated pressure or temperatures) to persist; nor will it deem a run finished if the temperature or pressure were out of acceptable ranges.
 * If something should occur, the safest thing to do is find the lab manager.
 * WARNING - DANGEROUS: If you have specially been shown where the main power switch is, it's OK to turn the main power off, but the switch is hidden and no one should EVER attempt this if they have not been trained.
 * __No one should EVER make adjustments to the valves inside the autoclave__. Doing so may cause damage to you, the instrument, and result in having the repair person called to fix your mistake.

Standard recipes

 * It is preferable to make a large volume of media, i.e., 2 liters, and divide it into 4 flasks, as opposed to weighing out the components several times.
 * Be very careful when weighing out all reagents. Some strains are ridiculously sensitive to minor changes in media composition.
 * Label media with the person who prepared it, instead of the person who pours. I recommend labeling the foil caps with your initials as you make the media.
 * If you think something is wrong with a batch of media, dump it out. Don’t simply assume that things will “work out” or try to correct a compositional problem.
 * If you are making selective plates, add a stir bar to the flask before autoclaving. Then add the antibiotics and gently stir them in on a hot plate. Voila! No bubbles!
 * Do not pour agar down the drain. When finished pouring a sleeve of plates, fill put the flask in a sink and fill the flask half-full of tap water to dilute any remaining agar and allow it to solidify before it is put into the drain (solidified agar can be put in the drain). The agar will harden quickly so the flasks can be rinsed thoroughly with deionized water a few hours after they are put in the sink.


 * **LB**: per liter
 * 10 gram Bacto-Tryptone
 * 5 g yeast extract
 * 10 g NaCl
 * adjust to pH 7.4 with NaOH (1 M soln)
 * for plates, add 15 g agar (the cheap technical grade, if available)
 * add antibiotics after autoclaving (and before pouring) from 1000X stock (Cm in 95% EtOH; Kn in dH2O, filter-sterilized) and kept in the freezer
 * Add a stir bar to flasks that will receive antibiotics after sterilization
 * **BHI** (brain-heart infusion): per liter
 * 37 g BHI powder
 * for plates, add 15 g agar (the cheap technical grade, if available)
 * Add a stir bar to flasks that will receive antibiotics after sterilization


 * **Adding antibiotics**: Selective agents should __never__ be added to hot media. Cool media intended for plates in the 60 ºC water bath for at least 45 minutes, then