SO NOW YOU'RE A LAB TECH FOR THE
BLOOM LAB
HERE'S WHAT YOU NEED TO KNOW.
A SURVIVAL GUIDE CREATED BY
MOLLY HAYES
AND
JULIE THIBODEAUX
Welcome
Welcome to the Bloom/Yeh labs. You are now an official lab tech and it is your duty to keep things running smoothly around here. Or at least to try.
Being a lab tech is essentially an easy job but does require some attention to detail and a good memory at first. You will be responsible for several tasks, most of which are outlined in this manual. Things may seem a bit daunting at first. You will probably want to take notes on the things as you are trained. This manual is designed to make your first few days a little less hectic and serve as a reference as you get more accustomed to being a lab tech but one of the most important things to remember is to never be afraid to ask for help. The other people in the lab will be the best references you can find. If you are uncertain about something, ask someone to explain it before you try it yourself.
Because the jobs you do directly affect the research done by other's in the lab, often if lab tech work is done wrong, it will throw off someone's experiment and rerunning experiments can take a long time. Of course everyone makes mistakes sometimes but if you ask questions you will probably make fewer.
You will be dealing with a lot of numbers as you work in the lab. There are grant numbers used in the store room, photocopy numbers, phone numbers etc. You may want to make a notecard with all of these numbers listed on it and have that card with you each time you work (until you memorize them from use of course). This card should include:
all grant numbers and where they can be used
all photocopy numbers and where they can be used
the lab phone numbers: 962-2363- lab 962-1182- Dr. Bloom
room numbers: 622 Fordham Hall
the home phone number of at least one person in lab
Physically getting into Fordham Hall can sometimes be a challenge, as you may have already figured out. The first floor doors are usually locked at all times. The second floor doors in the rear of the building near Wilson Hall are usually kept open. The third floor doors are usually locked as well. If this is a great hassle for you or if you feel another need to have keys to the lab, such as working late hours or doing a lot of photocopying on the first floor, you are permitted to have keys. You must make a $10 deposit to Billy York in the basement of Wilson Hall. Ask someone to take you to see him.
Working in the Bloom/Yeh labs is great once you learn the ropes. Don't get discouraged at first, you will catch on! You will have a lot of freedom with this job in that you do what needs to be done each day and the rest is up to you. If you are having problems keeping yourself busy, ask someone if they need anything done. Someone will almost always have some kind of project that you could do. But most of all, enjoy yourself, learn a lot and meet some great people. Good luck!
Mail/Fedex
Check the mail daily. The mailboxes for the Bloom Lab are located in 213 Coker Hall. Dr. Bloom's mail box is located on the far right hand wall of this room as is the Bloom Lab mail box. Elaine's mail box is located on the wall immediately to the right after entering the mail room. Be sure to check all three of these mail boxes daily. And if you are feeling generous, check the mail boxes of the graduate students located in the hallway of Coker Hall.
There are three outgoing mail boxes. Dr. Bloom will ask you to deliver mail; therefore, knowing which box to use is of great importance:
The U.S. and Campus mail boxes are located in the hallway in Coker near the door facing Wilson Hall. The other mail box is the un-metered mail box for unstamped letters. There is a slot for such mail located under Dr. Bloom's box in the mail room of Coker Hall.
You may also be asked to place items in other peoples' mail boxes. Most of this mail is for the accountant or other people who work in Coker Hall. These mail boxes are located on the left hand wall in the mail room of Coker Hall. The mail should be placed in the box above the name tag.
All FED EX
mail can be done in room 216F of Coker Hall.
Simply fill out the form found on the counter of this room. Be sure to bring your account numbers as
well as the address and phone numbers of both the person to whom you are
sending the mail and the Bloom Lab. All
this information is necessary to send FED EX mail. The secetary in charge of FED EX in this office is Julia. Ask her if you have any question.
Photocopying
You will inevitably be asked to do photocopying for the lab. There are several photocopy machines available for this purpose. One machine is located on the first floor of Fordham Hall across from the bathrooms. This room is accessible with a key only. If you do not have your own set of keys, ask someone if you may borrow theirs. This photocopy machine is very slow and breaks down easily. It is best to use this machine for simple photocopying of a few pages only.
There are two photocopy machines located in 217 Coker Hall. This room in accessible by a key kept in 216F Coker. It is kept on the counter of this room and is connected to a large piece of wood. Remember to return it once you are done photocopying. If you have keys to Fordham Hall you can use those instead. They open the photocopy copy room as well.
There is another photocopy machine located in Wilson Hall across from the Zoology Library. The door to this room should be unlocked at all times.
All of the photocopy machines require specific photocopy numbers. These are a kind of "password" number. Some machines take all of the numbers, some only take a few. Be sure to bring all of the photocopy numbers with you when you go to photocopy.
There are some quick tips to make photocopying more
efficient:
-Photocopy all books or magazines last page first, or backwards in other words. This way you don't have to sort the article when you are done.
-If you are making copies of an unbound article, place it in the slot on the left hand side of the cover of the copier. Press enter and the machine with copy the entire article quickly and efficiently and you don't even have to lift a finger.
-To make several copies of a multipaged (unbound) article, do the above, press the SORT button and the number of copies you want. This copies the articles as a whole but separates each article into its own slot. You can also staple these articles by using the staple button.
Libraries
Another responsibility of working in the Bloom lab is obtaining articles from research journals. Therefore, much of the photocopying you will do will be in UNC's libraries. There are three libraries you will use:
1) The Zoology Library in Wilson Hall on the second floor. The bound journals are on the second floor of the library; you will find a steel staircase in the back of the library. The new journals are in a small room off the right hand side of the library behind the circulation desk. Once you find the article(s) you need, check them out with your ONE card at the circulation desk. You can then take them across the hall to the copy machine. Be sure to return the journals to the circulation desk once you are finished copying.
2) The Botany Library is located on the second floor of Coker hall on the same end as the photocopy room. You will use this library less often than the Zoology library, but is still useful from time to time.
3) The Health Sciences Library (HSL) is the library that you will find the most useful. The easiest way to get to this building is to go down the back staircase of Fordham (take a left out of the Bloom lab, go outside, enter door to your left to get to staircase) to the third floor, go back inside Fordham, and then exit Fordham through the door to your immediate right. A ramp outside the door will lead you to the street which runs beside the nursing school. Find the brick path that runs in front of the nursing school. You will pass Berryhill and the Medical School buildings (on your left) before you come to the HSL (a 5 story, off-white building). Be sure to take the Health Sciences Photocopy Card. If the Photocopy Card is out of money, tell Dr. Bloom or Elaine and they will give you more money to put on it. The machine for this is in HSL on the first floor to the left when looking at the elevators. All journals are found on the 2nd through 4th floors:
2nd floor: new journals,
3rd floor: bound journals A-I,
4th floor: bound journals J-Z.
It is possible to determine where you will find a journal before you actually check all three libraries. Simply go to www.unc.edu on the internet, click on LIBRARIES , and then on-line card catalog. You can perform an author/title/subject search and it will tell you in which library the journal is housed.
Often you
will receive lists of articles to photocopy.
The titles of the journals will sometimes be abbreviated and sometimes
this can be confusing. As you become more
familiar with the journals identifying
them will become easier, but always remember: if you ever have questions,
simply ask!!
The Autoclave:
The autoclave is a wonderful yet dangerous and sometimes fickle machine. The purpose of autoclaving items is to sterilize them; therefore, there are many items that need to be autoclaved before use to keep from contaminating experiments.
There
are two basic types of autoclaving:
liquid
and dry:
Liquid: Since researchers in the Bloom lab work mainly with yeast and E coli, making sterile media is yet another duty of the lab techs. Media is generally made in one or 1.2 liter quantities. Because the media must be autoclaved, the 2 liter flask is generally used. This brings about a very important point about autoclaving: DO NOT fill any bottle or flask full or even to the last measurement indicated on the container...or else
(1) you run the risk of letting your liquid boil over
(2) the container may crack and break in the autoclave.
Cover the flask with aluminum foil once the ingredients of the media are mixed.
In addition to media, a researcher in the lab may request sterile water. This is simply a request for you to fill several 100 mL glass bottles to the 75 mL mark with double distilled water and to autoclave. Appropriate lids for the bottles should be found and tightened only enough so that they stay in place and no tighter. A tight lid could potentially cause the bottle to explode in the autoclave.
Once the liquid is prepared in the appropriate container, it should be placed in a tub with about 2 inches of water. Be sure to put autoclave tape on all items that go into the autoclave. Autoclave tape is white before autoclaving and marked with the word AUTOCLAVED or black bars afterward. This allows people to be able to tell what has been autoclaved and what has not.
Running the machine in liquid mode:
1. Place the tub in the autoclave and close the door.
2. Press the lock button.
3. Press the button with the flask on it.
4. Set the far left hand timer to 35-45 mins. Be sure the second timer is on 00:00.
5. Set the temperature to 120 degrees.
6. Press the start button. It's green with a triangle on it.
The liquid cycle is now in progress. It should take about an hour to completely finish. Be sure to remove all liquids when the cycle is done and tighten the lids after the liquid has cooled to prevent contamination. The items will be very hot when they come out, so be sure to protect your hands with the thick gloves/mittens found around the lab.
Dry: Blue and yellow pipetman tips, test tubes, cortex tubes, eppendorf (eppi) tubes, toothpicks, velvets, and glass pipettes are all items which need to be autoclaved before use in the lab. Pipetman tips should be placed in the appropriate boxes found on the shelf facing the sink. Once filled, a piece of autoclave tape should be placed on the boxes. Test tubes should be placed in test tube racks and topped (loosely) with lids. One long piece of autoclave tape can go across the top of the tubes. Eppis can be placed in small buckets and covered with foil. The foil lid should be marked with autoclave tape. Glass pipettes should be separated according to size into tin cylindrical containers. When the containers are full, they should be sealed with a lid and marked with autoclave tape. If a researcher requests sterile centrifuge tubes, be sure first to loosely fit the tubes with lids. The topped tubes can be put into a small tub and autoclaved.
When a load of dry goods are ready to be autoclaved, they can be placed into tubs or wire baskets. The dry cycle runs pretty much the same as the liquid cycle except for a few basic things:
1. The tub should not have water in it.
2. The dry cycle button has a pair of scissors on it.
3. The first timer should be set at 30 mins.
Everything else is pretty much the same. These cycles take the amount of time indicated on the timer and should be removed promptly. Condensation may develop on the items; this is a bad thing, as contamination may result. To prevent this from happening, the pipetman tips and glass pipettes are placed in the 70 degree oven in the media pouring room for a day to dry out.
Cleaning
Dishes. Dishes usually pile up in the sinks on a daily basis. When you are ready to take a load down to the wash room, be sure to rinse everything first. Test tubes and cortex tubes should be scrubbed with a brush in the soapy water. They should be collected along with their lids, stir bars (magnet-mixers) , and bottle tops into the white plastic baskets, as they are too small to be put loosely into the dish washers. If possible things in baskets should be placed facing down with should be open for optimal cleaning. Stack all of the dishes (from both the Bloom and the Yeh labs) onto a cart and be sure you have soap before you venture.
The dish washing room is on the first floor. Find or wait for an available washer. Load one dish per peg into the machine. On the top, right, front, inside corner of the machine, you will find a small "bucket". Pull this bucket off of the machine (be careful because it is filled with water). Dump the water and fill the bucket half way with the powdered soap. Replace the bucket and close the door to the machine. Be sure the machine is on the GLASS setting. Push the button that is marked A and the button marked 1. The machine should start automatically. You can leave the cart sitting in front of the machine. The cycle usually lasts about 35 minutes. When you return with the dishes, you can distribute them back to their places around the lab.
Glass Pipettes. Dispersed around the lab benches are several plastic cylinders. These are the containers into which glass pipettes go when they are dirty. Dirty pipettes fill up over the span of a couple of days, but they are relatively easy to clean:
Wearing latex gloves, collect all pipettes from both the Bloom and the Yeh labs. Put the pipettes into the huge cylinder located next to the sink on the media prep countertop. On the pipette washer you will see a large piece of tubing with a metal tightener on the end. Attach this tubing to the normal water faucet. Turn on the hot water. You will notice that the water fills and gushes out of the washer cyclically. Let this process continue for approximately one hour. After an hour, attach the tubing to the distilled water faucet. Allow the distilled water to run for about half an hour.
The pipettes are clean at this point, but they are not sterile. Remove the basket of pipettes from the washer and distribute the pipettes into the appropriately labeled (pipettes come in 1, 2, 5, 10, and 25 mL sizes) tin cylinders which you will find on the shelf beside the large Sorvall Centrifuge in the main lab. Be careful when carrying the tins or removing the lids because the pipets may spill out of the lids if they are removed without caution. If you fill the tin containers, attach a piece of autoclave tape such that it holds the top in place and autoclave the pipettes along with the next load of dry goods.
When the pipettes come out of the autoclave, be sure to put the tin containers into the 80 degree oven overnight. This gets rid of any condensation that may arise. The containers of sterile pipettes are kept on top of the 80 degree oven in the pouring room. Be careful not to let the containers roll off the oven.
Velvets. If you find small squares of velvet in the sink, they need to be scrubbed in the soapy water and laid out flat to dry on the countertop. Once dry, they can be bundled in stacks of 5-10 and wrapped in foil. The foil should be marked with autoclave tape, and the velvets should be autoclaved with the next load of dry goods.
Stocking Items
Another important aspect of the job is keeping the lab stocked with the equipment researchers use. On the chalk board in the main lab there is a "needs" board. Often you will need to buy the items on the board to keep them in stock. There are two main ways of restocking items: things can be bought directly from the biology storeroom or they can be ordered from catalogs.
The Storeroom
In the basement of Wilson Hall there is a biology storeroom. Here, many of the items used in lab (pipette tips, labeling tape etc.) are sold. Since you will not be acquainted with the storeroom initially, it may take some time to find the items for which you are looking. On the end of each aisle, a list of items on the shelves is posted. These are wonderful cheat sheet. All alcohol and other organics are located in the back of the storeroom in a closet with a yellow fire door. You must personally ask Amyy Butcher, the store attendant, for all office supplies and slide coverslips. These items are located in an adjoining room. Fill out one of the tickets on the table next to the door at each visit. Have to ticket checked over by Amy before leaving the storeroom.
Ordering Items
You will often be asked to stock items that the storeroom does not carry. These items include some media making materials, glass beads, and other lab equipment (gloves and autoclave tubs). To order items you must look up the item's order number in one of the many catalogs located over the desk with the telephone in the main room of the lab. Fisher is the distributor we use most. Items such as printing paper are ordered through the University Central Stores. We have a specific account number for each company, the Central Store Room number being our grant number and the Fisher account number is written inside the front cover of the catalog. These companies will ask for a check number which is can be found on a piece of paper in the top right hand desk drawer of the telephone desk. All of these numbers start with a K and are six digits long. Mark off the circle next to the number once it has been used. Be sure to have all of your lab information with you when ordering items because you will need to tell them the phone and room numbers. This is a complicated process so, as usual, if you have any questions, feel free to ask someone.
Media
Media is the stuff that the bacteria grows on or in. One of your jobs will be to prepare different kinds of media for people to use. There are many different kinds of media and they come in two different forms, plates or liquid. Plates are liquid media with an extra ingredient (agar) that makes them gel-like and they must be poured into petri dishes.
The basic premise of making any type of media is the following:
Hints: In order to avoid mixing ingredients within their containers, use a different scooper for each particular ingredient.
Because all media is autoclaved and because liquid expands in the autoclave be sure the container you use is considerably bigger than the amount you have made. Usually the container should only be half full.
In general if you are making liquid media you will prepare a large amount and then transfer it to 125 ml bottles (the ones with the black tops). Do not put more than 100ml in these bottles. Remember not to tighten the lids too tightly or they will explode in the autoclave.
Liquid media is generally placed, after it is autoclaved, in the small room on the shelf parallel to the windows. This shelf if labeled with a specific location for each type of media.
Plates
As mentioned, plates have agar in them. When assembling the ingredients for plates be sure to add the correct amount of agar to the mixture. When stirring the media on the stir plate the agar will not go into solution therefore you should simply stir the media for a few minutes. About as long as it takes liquid media to go into solution. Autoclave as a liquid.
To pour plates:
-Take the media out of the autoclave and place it in the hood in the supply room. The hood's blower and lights should be on.
-One liter of liquid transfers to about 25-30 plates. Take one bag of petri dishes from the box in the supply room. Open the bag in the hood, remove the plates (be sure they are right side up) and label them. Place the plastic bags in the bin to the right of the hood. they will be used again. The labeling key should be taped to the glass of the hood. Simply take the marker of the color denoted and run it in one line up the length of the plates so that each plate has a line (or lines) on it that denotes what the plates are. If you have prepared plates that are unusual or are not part of the labeling system write the type on the bottom of each petri dish in small writing.
- Allow the media to sit for 15-30 minutes. Media should be about 50-60 degrees or cool enough so you do not burn your hand when holding the flask. Cooling reduces the amount of condensation that forms on the lids of the plates. Be careful not to cool too much or the liquid will solidify in the flask.
- When ready to pour divide the plates into 5-6 high bunches. Turn on the Bunsen burner. Using the plastic grip glove to hold the flask, slowly pour the media into the plates. Plates should be about half full.
- Flame the top of the flask every 5-6 plates to maintain sterility.
- Sometimes if the media is poured quickly or has been shaken bubbles will form on the plates. These can be popped simply by passing the flame of the Bunsen burner over them. Be careful though because the plastic of the petri dish may melt and the top and bottom may be stuck together.
- When finished pouring clean any small spills from the table with a paper towel, push the plates slowly to the back of the hood (be sure not to splash the media around) and turn the UV light on.
-The next day you must rebag the plates. Be sure there has been no contamination. Take one of the bags from the bins next to the hood and place the plates inside upside down. Using a piece of label tape, secure the top of the bag tightly and write what the plates are, your initials and the date on the tape. There are three or four storage places for different types of plates. Ususally you can give the plates to the person who requested them otherwise be sure to ask someone where they go until you learn where the different types go.
Adding Trp
Tryptophan (trp) is a common ingredient in all media. It is diluted into a 1% solution in sterile conditions with water and is stored in the refrigerator. If your media calls for trp, after autoclaving add it to the media with a pipette. Be sure to do this in sterile conditions (flame the pipette, the trp container lid, and the lid of the media upon contact) or the media can easily become contaminated.
Confusing Media
There are several types of media that use the same recipe but have ingredients either added or omitted.
YNB
Will be asked for as YNB which is YNB (yeast nitrogen base) and dextrose. Any other form of this name means you need to add a specific amino acid according to the recipe card.
YCA- add only adenine
YCU- add only uracil
YCAU- add uracil and adenine (do not add trp)
YCAT- adenine and tryptophan (do not add ura)
anything with gal at the end means to substitute galactose for glucose (dextrose).
NOTE: YNB comes in two forms, with or without Ammonium Sulfate. Be sure to know whether the batch you are using contains Ammonium Sulfate or before you start. Not doing so could result in adding as much as six times the needed amount of Ammonim Sulfate.
SD (synthetic dextrose)
SD media has its own recipe which contains several amino acids. They are often called "dropout" medias and lack amino acids present in the casein amino acids. They will be asked for as SD minus something (SD- trp for example). For most "dropout" medias there are containers of the indregients premixed on the top shelf in the ingredient cabinet. Use the omission mix recipe and add these premixes as you would the ommission mix (unless otherwise stated on the bottle). If you are asked to make a "dropout" media that has more than one ingredient missing (SD- trp-his) use the ommission mix in the cabinet labeled minus 5. This mix has all of the amino acids except trp, ade, ura, leu, and his. Therefore, if you are asked to prepare something like SD- trp-his you would prepare the media with the Omission media recipe, using the minus 5 mix, but would add the other three amino acids.
Omission media for plates (with agar) must have the pH adjusted to 5.8. Add 150 microliters of NaOH (found in stock by the pipette washer sink). Be careful not to get 10N on your skin because it is very caustic.
The basic amount for amino acids are 45mg/500ml.
Leu x 2 (90mg/500ml)
Ade x 1/4 (11mg/500ml)