Laboratory techniques for evolution experiment
Important note
Make sure that you write everything down in this lab. You will have a lab report due at the end of the lab, and you will need this information. Dont say to yourself I know I will remember this, because you wont (trust me, Ive had some bad experiences) Important things to keep track of: Procedure, measurements, observations, things that you messed up (it happens its called human error . Its better to report it than to have an incorrect lab report. You wont get points off for accidental errors. You will get points off for falsifying data) ***You will not have a copy of the procedure. The methods section of your lab report is a big part of your grade. Make sure to keep close track of what youre doing (down to the amount of sample on the spread plate)
Methods example
Incorrect: We took a sample from the test tube and put it on our plate.
Correct: Using a micropipette, we took 100L of 90% saline, 10% E. coli and inoculated our petri dish.
Methods example
Incorrect: We took a sample off of our plate and put it in a tube.
Correct: We chose a colony that survived UV irradiation and, using a toothpick, put the sample in an Eppendorf tube containing 1mL of LB broth
General outline of the lab
Why are we doing this lab?
The purpose of this experiment is to see evolution in action. Working in pairs, you will take a common bacterium (E. coli) and subject it to various selective pressures and see which bacterium survive and reproduce. At the end of the experiment, they will write a lab report in the same format as a professional scientist (abstract, introduction, methods, results, discussion).
How does this relate to evolution?
Organisms must survive various selective pressures. For example, animals in the desert must live in an extremely hot environment. We know that Earths temperature is increasing (global warming), so animals that can better handle this change will survive and leave offspring. Maybe the increase in temperature means less water. A camel that can better regulate its water usage will be more successful at surviving and reproducing. Over a long period of time, more camels in the desert will have this advantageous trait
In this experiment, we are subjecting the bacterium E. coli to two major selective pressures and seeing which ones survive and reproduce.
Why are we using E. coli?
We are using E. coli because it has a very fast replication rate
Think: It takes humans 9 months to create a baby, then another 15 years for that child to reach sexual maturity and have the ability to have children of his/her own. Additionally, most people wait until their 20s or 30s to have children. E. coli reproduces every 30 minutes in ideal conditions. So, after 24 hours of incubation, a single bacterium will have ~17,000,000 (17 million) descendants. In humans, this would take A MINIMUM of 500,000,000 years (half a billion years) for someone to have 17 million descendants
Selective pressures
The first selective pressure we will force upon our E. coli is UV radiation. UV radiation can be thought of as pure energy. It is the reason why you wear sunblock when its sunny out to prevent cancer. UV light causes mutations in E. coli, however some may be better able to survive this pressure than others.
We will let our E. coli grow overnight, then take a colony that survived the UV light and subject them to the second selective pressure (lambda) bacteriophage (also known as T4 bacteriophage). bacteriophage is a virus that attacks E. coli. Some E. coli will be better at resisting infection than others. Can you think of any reasons why?
(or T4) bacteriophage
Important materials
Materials we will be using
Petri dishes (also known as plates) with LB agar Micropipettes Micropipette tips Eppendorf tubes Spreaders Liquid LB dH2 O (distilled water, free of any impurities or contaminants)
Petri dishes with LB agar
LB agar: A jelly-like medium that provides a suitable environment for bacterial growth. LB agar has everything a bacterium needs to survive and reproduce. It is made from seaweed and other chemicals. It is roughly the consistency of Jell-o
Micropipette
Micropipette tips
Bad technique here: Make sure to keep boxes CLOSED
Eppendorf tubes
Spreaders
Note: The LB agar on the petri dishes is about the consistency of jell-o. With this in mind, try not to break the surface of the agar
Laboratory techniques
The aseptic technique
Purpose: We are working with bacteria (E. coli). Bacteria are everywhere, and we want to make sure that our sample does not become infected with unwanted bacteria
The aseptic technique
When you need to be aware of cross-contamination:
Keep you petri dishes and micropipette tip boxes closed. Only open them when needed, and be quick! Change micropipette tips after each use. For the purpose of this lab, each tip can only be used once. Keep spreaders in bleach solution until they are ready to be used.
Make sure to thoroughly rinse spreaders before spread plating or else you will kill your sample and not get any results.
Keeping track of plates
Purpose: Your petri dishes will be collected at the end of each class. You need to make sure it is properly labeled so that you know which plate is yours.
Procedure: Write your group number, date, period, and amount of E. coli solution plates around the perimeter (outside edge) on the lid.
Why might it be important to keep the center of the plate free of any writing?
Mixing a solution in an Eppendorf tube or test tube
Purpose: To make sure your sample is evenly distributed through out the test tube Mixing in an Eppendorf tube: Make sure the Eppendorf tube is capped. Gently invert the tube 5 times. Then, gently flick the base of the tube 5 times.
Mixing in a test tube: Gently flick the test tube near the water line at least 5 times. You should see your solution swirl around (like a tornado)
Micropipetting
Purpose: To measure out a small amount of liquid (microliters, L, see below for explanation) Equipment: Micropipettes, micropipette tips, lab bench trash can
How to use a micropipette
Usage: 1. Set the micropipette to the correct measurement. 2. Place a fresh tip on the micropipette. 3. Push down to the first natural stop. 4. Insert the micropipette into the solution, and slowly release the stop. 5. When ejecting your sample, slowly push down to the first natural stop, then push down to the second stop. You need to push down to the second stop to make sure all of your sample is ejected
Spread plating
Purpose: We spread plate our sample to ensure that we have a complete and equal distribution of our sample on the plate Procedure: To perform spread plating, you first need to place your sample in the petri dish using a micropipette. Then, take your spreader in your dominant hand and gently push the sample from front to back. With your nondominant hand, slowly rotate the petri dish to ensure equal distribution ***Warning: Be careful! You do NOT want to break the agar. You dont have to be rough with your sample!
Spread plating
Other important information
Naming
We are using the bacterium Eschericia coli (E. coli) Note how the name of this bacterium is written.
The name should be italicized If using the abbreviated name (E. coli), the E should be in uppercase and it should have a period. The coli should be in lowercase.
What is a colony?
A colony is an isolated dot on the petri dish Each colony is made up of the direct descendants from one E. coli Bacteria grow through a process called binary fission (basically mitosis). As such, all the descendants from one bacterium are assumed genetically identical (or, identical enough for our purposes)
Microliters (L)
We are using micropipettes to measure out liquids in microliters. A microliter (L) is 1/1,000 of a milliliter (mL)
Thinking another way- you can fit 1,000 microliters in one milliliter A milliliter is pretty small to begin with so a microliter is a tiny amount (thats why we have to use a special piece of equipment, the micropipette, to measure it out)
Visualizing a microliter
This red dot is exactly the size of a microliter in comparison to the milliliter shown on the left If youre viewing this slide on your computer, you can zoom in to see just how small the dot really is. (If youre interested, the milliliter is 5 inches by 2 inches. The microliter is 0.005 inches by 0.002 inches)
Milliliter (mL)
Microliter (L)