Jacobs:Protocol Safety

CMBL Guidelines

The primary and overriding rule for working in the CMBL is that you must clean up after yourself. The lab does not employ any technical assistants and no one is expected to clean or pickup after other people. Every time you do any work in the CMBL you are expected to leave all space, benchtops, supplies, and equipment ready for the next user (even if you did not find it in such conditions). Much of what follows is simply specific instances and examples.

Safety
Keeping the lab safe should be the top priority of everyone working in the CMBL. Some hazardous material require special training and techniques (e.g. radioactive materials) and it is the users responsibility to ensure that they have adequate training (e.g. radiation or animal training for the specific animal). No eating or drinking is allowed in the lab or culture room and food containers cannot be put in the trash in these rooms. Many reagents require that a lab coat, gloves be worn, and open-toed shoes be avoided, and is good lab practice in general. Wear goggles or face shield when handling liquid nitrogen or UV. Wear face masks when weighing toxic chemicals. Freshly cleaned lab coats are available for free from VA supply.

Glassware
You are responsible for cleaning any glassware that you use. It should be washed with tap water and soap and then rinsed thoroughly with dH20. It should then be placed on the drying rack and allowed to dry. If it was sterile to begin with it should be sterilized again. To sterilize glassware place it in a metal autoclave basket. When they fill up the baskets are brought to the autoclave all at once. Beakers, flasks and so forth should have their mouths covered with tin foil and have a piece of autoclave tape placed on them (note: autoclave tape is how you tell if something is sterile or not because it changes to have a black cross-hatched pattern on it once it is sterilized). It is important that glassware for sterilization be totally dry prior to being place on the cart. If not, water stains will be baked onto the glass that are hard or impossible to totally remove.

Metalware
The rules above also apply to use and cleaning of metalware (metal weighing spatulas, forceps, blade holders, etc). Metalware should be cleaned, thoroughly dried, totally enclosed in foil and placed in the sterilization basket and they will be baked rather than autoclaved.

Plasticware
Do not reuse disposable plasticware. Be sure to change pipets or pipet tips such that sterile material is not contaminated and samples are not cross-contaminated. For example if you are pipetting media from a sterile container into several dishes, it is OK to reuse the pipet as long as it only touches the inside of the media bottle. You must change to a new pipet if it touches a dish, the benchtop, the mouth or the outside of the bottle.

Pipet tips
If you open a box of sterile pipet tips outside of the hood, it is no longer considered sterile. It is very important the you mark the rack as unsterile with a pen or by removing the autoclave tape before you return the rack to the general supply. Alternatively, you may want to keep a rack or two of your own so you can be sure of sterility. If we are running low on pre-racked tips, please order some more and be sure to order RNase-free or sterile racks as needed.

Pipetting
Our pipettors have two stops, the first is a measured volume that you set with the dial, and the second is the max volume for the particular pipet. In normal use you depress to the first stop, immerse the tip, and release. At this point the measured volume is in the tip, so drops on the tip are to be avoided. To dispense, depress to the second stop. Note that going to the second stop to dispense ensures that all the fluid is ejected, however some air will also be ejected (known as blow through). This may introduce bubbles in the sample. If bubbles will be a problem in the technique you are doing, you may consider only going to the first stop or just slightly beyond to eliminate bubbles, but you may not eject all the fluid. For very thick or viscous fluids, you may need to operate the pipettor in “reverse mode”. To do this you go to the second stop before immersing the tip. This loads the tip with the max volume of fluid. You then go the first stop to dispense the measured volume. In “reverse mode” drops on the tip are part of the measured volume and should not be discarded. Note that some eppendorf pipettors have a third stop that ejects the tip. It is also important when using a pipet aid with a serulogical pipet that no fluid is sucked into the pipet aid. If this occurs you will need to check the pipet aid filter and possible change it. If needed find someone to show you how, but do not return it to service without checking the filter.

Reagents
When possible it is preferable for each experimenter to have their own reagents. If you order reagents, be sure to put initials on them and be responsible for storing them properly. Also, be sure to discard them once they expire or you no longer have a use for them. In many cases it is not practical for each investigator to have his/her own supply of certain reagent either because so many people need it, or because of cost, so in many cases we have a group supply. Remember to always use a clean spatula for measuring from common stock. That is, if you use a spatula to measure out a reagent from stock, you must clean and dry it before you use it to measure the next reagent. When measuring discard any excess you have removed from the bottle, do not return it to the common supply. If you use up the last of a reagent or we are low on a reagent notify someone so that it can be reordered, or reorder it yourself.

RNase free reagents
These reagents require special handling and storage because they are used in RNA work and RNase enzymes are very stable. RNase-free (and only RNase free reagents) are stored in a special marked RNase-free cabinet. It is very important that you wear gloves when you touch the reagents in this cabinet since your skin is the biggest source of RNase’s. Sterile spatulas must be used to measure out RNase-free reagents. If you do mistakenly contaminate RNase-free material, do not replace it in the RNase-free cabinet, but tell someone in the lab so that we can take appropriate action. Also, you should use pre-racked pipet tips that are certified from the manufacturer as RNase-free. This is because, the autoclave is not 100% totally effective at destroying RNase’s. Also, note that pre-racked pipet tips are can be ordered RNase-free or sterile and these are not exactly the same thing. To keep a rack RNase-free it should only be opened with gloves, but it need not only be opened in the hood. In contrast, a sterile rack can only be opened in the hood and you cannot touch the tips with or without gloves.

Flammable Materials
These must be stored in a flame proof cabinet. The large stock bottles of things like ETOH and acetone should only be removed to dispense the substance and the stock returned promptly to the cabinet. Avoid leaving large bottles of flammable materials unattended on the counters or on the side of the sink.

Water
There are three types of water in the CMBL. Tap water is used for washing. Deionized water is used for rinsing and comes from the special tap. We purchase sterile and RNase free water used in cell culture and RNA/DNA work.

Balances
Always use weighing paper or a weighboat. Be sure that all traces of you chemical are removed from the balance area when you are finished.

Media, serum, and other culture supplies
When mixing growth media for cell and tissue culture, when you open a bottle of media or a tube of serum, do not replace it to be used by others. In this way, we can isolate and reduce the chances of cross-contamination. If you don’t use a whole bottle or tube, mark it with you initials and save it for the next time you need to mix up media. Note, that we buy large bottles of serum from the same lot, and it is allequoted it into smaller centrifuge tubes that are stored frozen. Check media prior to use to ensure that it is not expired. Expired media may be used for short washing steps, but should not be used for culture, growth, or experiments.

Tissue & Cell Culture Room
The tissue and cell culture facilities are for culture work only. Other experiments should be conducted elsewhere. Please keep this room as clean and tidy as possible to reduce chances of infection. Check the temperature, CO2, and water levels of the incubators periodically and if you notice a problem notify someone. All dishes in the incubator should be properly labeled with the contents, investigator, and date. It is very important that any dishes you do not use are removed, made safe, and disposed of. Specifically, if you request dishes to be ready on a certain day and you do not used them, it is your job to dispose of them. After each use of the hood clean it with 70% ETOH (not 100% or 90%), and switch on the UV light. Remember the hood fan needs to run for 30min initially to ensure sterility in the hood. Never close the hood shield completely because it damages the fan. Also note that the water in the heater bath should be changed every two months.

Freezers
Try to minimize time that the freezer doors are open since this causes ice buildup. Material in the freezers should be marked with contents, investigators, and date. This helps with periodic inventories when freezer space is short. It is a big help if any samples or reagents that are no longer useful are removed promptly since freezer space is at a premium. Also, remember that space in the –80C freezer is assigned by shelf.

Cold Room
It is very important that the cold room stay clean, tidy and organized. This room is for storage of experimental material, equipment, reagents, and samples. Almost all of the culture media stored in the cold room is light-sensitive so it is critical that you turn off the light whenever you leave the room.

Other equipment
Note that instructions for use of many pieces of equipment are found taped to the equipment or on the wall nearby.

Fluorescent Microscope
If the UV light is used multiple times a day, keep the light on until it is done for the day. Frequently switching UV light will shorten the life of the bulb. The UV bulb needs to be change periodically before it burns out because it can explode when it get too old. So, check the hour meter to ensure that its not time to change the bulb. Please remember to put dust cover on the microscope when the bulb cools off.

Biohazard
Any material that has come into contact with or is intended to come into contact with living tissue or cells is considered a biohazard (and of course actual living cells). These should not be disposed of in the normal trash cans of the lab. Rather, they need to go in the red bag-lined trash cans. If you are working at the bench sometimes it is helpful to use a small jar or bag for pipet tips and pipets and later dump them in the biohazard trash. Bacteria (E coli or bacterial infections) need to treated with bleach prior to disposal. Also, certain toxic chemicals such as ethidium bromide for gel staining should be stored in a specific marked container and disposed of in a specified fashion.

Sharps
Needles, blades and broken glass should be disposed of in the sharps containers. Once full, these should be sealed with wide sticky packing tape or duct tape.

Fume Hood
The function of the fume hood is to protect you from dangerous substances (contrast with the culture hoods which are to keep sterile conditions and vent to the room and not the outside). Certain chemicals (eg  Mercaptoethanol) need to be used and kept in the fume hood. Additionally, any strong-smelling substances should be handled in the hood. To work properly, the front barrier needs to be lowered enough to keep the substance from escaping. Note, if you can smell the fumes it may not be low enough. Also, once you expose something with a fume hood material (e.g. a pipet tip used to measure some  Mercaptoethanol) it should stay in the hood and kept there until the substance evaporates.

Notebooks
All CMBL scientists are expected to keep detailed and organized lab notebooks. They should be kept in such a way that someone else can read and understand what you are doing. You may elect to take notes during an experiment that you later transcribe to you notebook to keep the notebook more organized. Also, note that upon departure from the CMBL, federal regulations require that you leave your original notebook on file with the PI, just in case we are audited. You may retain a copy.

Misc
If you use a blue absorbent pad on the bench to protect from spills, please throw it out when you are finished. Always use a vacuum collection bottle when using the lab vacuum source. Do not allow the bottle to overfill and make sure it is trapping all fluid and that no fluid is going into the vacuum nozzle. Make sure that all materials that contact liquid nitrogen are approved for cryogenic temperatures. Always use freezing vials, not eppendorf tubes, when storing samples in liquid nitrogen since normal glass may become brittle and explode.