As it says at the beginning of Lab 12 in your manual, the material covered in this lab assumes that you know what’s in Appendix 4 of your manual. You need to understand what’s covered in Appendix 4 on exponents, logarithms, and functions (especially exponential growth) to “do” Lab 12. And experience tells us that many of you need to review how to plot points on log-linear (semi-log) axes that’s covered at the very end of Appendix 4.
In Lab 11 you will be characterizing bacteria, and in Lab 12 you will be measuring bacterial growth. Knowing about the difference in the size of bacteria (prokaryotes) and eukaryotic cells is relevant to both of these labs. Prokaryotic cells are small when compared to eukaryotic cells; prokaryotes are typically about 0.5 – 3 μm in diameter, and eukaryotic cells are typically about 10 – 20 μm in diameter.
The small size of prokaryotes, as compared to eukaryote cells affects a number of biological functions. For example, the rate prokaryotes take up nutrients into their interiors, and remove waste products out into the external environment through their plasma membrane can greatly affect their cellular metabolic rates and growth rates. In general, the cellular metabolic rate and growth rate of prokaryotes is much higher than the rates for eukaryotic cells. This difference is because transport rates are in part a function of the plasma membrane surface area available relative to the interior volume of the cell. Therefore, small cells with small volumes have more surface area relative to their volume than do large cells. Let’s illustrate this surface area to volume relationship with small and large spheres.
Below are the formulas for the surface area of a sphere, and for the volume of a sphere written as a ratio of surface area to the volume. As you can see, the formulas for surface area to volume expressed as a ratio simplify to 3 divided by the radius.
So, a 2 μm diameter (1 μm radius) spherical prokaryote will have a surface area to volume ratio of 3, but a 12 μm diameter (6 μm radius) spherical eukaryotic cell will have a surface area to volume ratio of 0.5, i.e., the small prokaryotic cell has more surface area to its volume than a large eukaryotic cell. Prokaryotes having more surface area for their volume facilitates a higher metabolic rate, as well as a shorter generation time (the time it takes for one cell to divide into two cells) than large eukaryotic cells with less surface area for their interior volume. In Lab 12 you will see just how fast E. coli in liquid culture divide when they have lots of nutrients and oxygen available.
Remember, for Lab 4 you need to use the “Handout for Lab 4 Exercises” you received for your lab section during Lab 3 (along with your lab manual) to write your flow charts/diagrams with all their calculations on your Lab 4 notes pages. You need to have all these flow charts/diagrams with their calculations done before you come to your lab section next week.
Mitosis and meiosis simulations
You left lab 2 with a plastic bag with pipe cleaners and beads so you could practice simulating mitosis and meiosis before you come to lab 3. Remember, you need to bring your pipe cleaners and beads in their bag back with you to lab 3. For your information, we recycle these pipe cleaners and beads for use next semester.
Above is a diagram of mitosis (click on image to see it bigger). At the top is the key to identifying the maternal and paternal chromosomes of this somatic cell undergoing mitosis. At the left is the cell in interphase. As you know, during interphase the DNA of the chromosomes replicate. So when the cell enters prophase each chromosome has a two part structure consisting of sister chromatids joined by a centromere. At metaphase, the chromosomes randomly align at the metaphase plate perpendicular to the spindle. Also at metaphase the centromeres duplicate. At anaphase, the sister chromatids are pulled to opposite poles of the cell by the spindle fibers. The separated sets of chromosomes become enclosed in a nuclear membrane at telophase. Cytokinesis will complete the mitotic division. The two cells that are the result of this mitotic division are genetically identical to the original cell at the left.
Incidentally, your bag of pipe cleaners and beads has two different colored pipe cleaners to represent maternal and paternal chromosomes (and their sister chromatids), and it has extra beads so you can simulate the centromeres duplicating in both mitosis and meiosis.
Below is a stop motion animation of the mitosis simulation like you will do in lab 3.
(Direct link to YouTube video.)
The diagram above shows 1 of 2 alignments of homologous chromosomes at metaphase 1 of meiosis (click on image to see it bigger). Again, at the top is the color code for the maternal and paternal chromosomes of this germ cell undergoing meiosis. Please note that in this diagram of meiosis, there is no crossing over of sister chromatids. At the left is a germ cell in interphase, where it replicates its DNA, such that when this germ cell enters prophase 1, each chromosome has a two part structure consisting of sister chromatids joined by a centromere. At metaphase 1 of meiosis, homologous chromosomes pair and align at the metaphase plate in an alignment that determines the maternal and paternal chromosome complement of the resulting gametes. Following metaphase 1, is anaphase 1, and the maternal chromosomes go to one pole of the dividing germ cell, and the paternal chromosomes go to the other pole. Following telophase 1, the two meiotic products progress through prophase 2, metaphase 2, and anaphase 2. It is important to point out that there is no DNA replication between telophase 1, and prophase 2 of meiosis.
Carefully compare the alignment of maternal and paternal homologous chromosomes at metaphase 1, and the maternal and paternal chromosome complement of the resulting gametes. We call these resulting gametes, parental gametes. Below is a stop motion animation showing you how to simulate meiosis for “alignment 1″ and paternal gametes.
(Direct link to YouTube video.)
The diagram above illustrates alignment 2 of homologous chromosomes at metaphase 1 of meiosis, and recombinant gametes (click on image to see it bigger). At the left is the germ cell in interphase where it replicates its DNA, such that when this germ cell enters prophase 1, each chromosome has a two part structure consisting of sister chromatids joined by a centromere. Again, at metaphase 1 of meiosis, the homologous chromosomes pair, and align at the metaphase plate in an alignment that, again, determines the maternal and paternal chromosome complement of the resulting gametes. Be sure to note that this alignment 2 is different from the alignment 1.
Again, there is a correlation between the alignment of maternal and paternal homologous chromosomes at metaphase 1, and the maternal and paternal chromosome complement of the resulting gametes; we call these resulting gametes, recombinant gametes. Below is a stop motion animation showing you how to simulate meiosis for “alignment 2″ and recombinant gametes.
(Direct link to YouTube video.)
So why are these two different alignments at metaphase I of meiosis so important? The genetic makeup of the gametes formed depends in a large part on the alignment of maternal and paternal chromosomes at metaphase I of meiosis, and it’s random as to how the chromosomes align at metaphase I of meiosis. Furthermore, when two gametes come together at fertilization, the genetic makeup of an offspring is set, and the coming together of gametes is a random event as well.
Try to do t-tests with data collected in Lab 2 before come to Lab 3
Since you have your data from Lab 2, you can write out your flow chart for the t-tests in your Lab 3 notes before you come to Lab 3, then do (or attempt to do) your t-tests in your Lab 3 notes (after your t-test flow charts). Doing so saves you time during Lab 3, and gives you the opportunity to correct any mistakes you may have made, plus get out early or on time.
Preview of chi-squared tests you do in Lab 3
In Lab 3 you will count the phenotypes of the progeny from different parental corn monohybrid or dihybrid crosses. Once you have these counts, then you do χ2 tests to determine the genotypes of the parents of each cross.
You received a handout for Lab 3 during Lab 2 with all the genotypes and phenotypes of monohybrid and dihybrid corn crosses you will see in Lab 3. You count the frequency of phenotypes of offspring of these crosses. For example, there will be flats of corn seedlings from a monohybrid cross with a seedling height gene (the wild-type tall allele T, and the recessive dwarf t allele; T/T or T/t = tall height, and t/t = dwarf or stunted height). Here’s a labeled photo illustrating these two seedling phenotypes.
To illustrate how you do χ2 tests, let’s say for a monohybrid cross with the seedling height gene described above you count 46 wild-type tall seedlings, and 56 dwarf seedlings. The first thing you want to do is set up an observed and expected phenotype table like the one below.
As you can see, the “expected” cells in the table above are empty right now. This is because we have to first decide what genetic cross are we going to do a χ2 test. First, you want to note that there are offspring with two different phenotypes from this monohybrid cross, tall and dwarf. Now what are the genotypes of parents that would give tall and dwarf offspring, and what would be the phenotypic frequency of offspring? One of two crosses would be T/t × T/t ⇒ 3 tall : 1 dwarf, and the other would be T/t × t/t ⇒ 1 tall : 1 dwarf. (Note, while there are other parental crosses for this allele, these two are the only ones to give tall and dwarf offspring.) We need to do a χ2 test for each of these two possible parental crosses to be sure of our results. So, we test whether our offspring counts come from a parental cross of T/t × T/t. The first thing we need to do is to complete the table above with our expected frequency for a total of 104 offspring for this cross of T/t × T/t ⇒ 3 tall : 1 dwarf offspring, as shown below.
|Expected phenotype||76.5 (i.e., 76 or 77)||25.5 (i.e., 26 or 25)||102|
Now we state our H0 and Ha hypotheses. H0 is that our observed frequency does not differ from the expected frequency for a parental cross of T/t × T/t. Ha is that our observed frequency differs from the expected frequency for a parental cross of T/t × T/t. Next we use the equation for calculating χ2.
As stated in Lab 3 of your manual, we’ll be using an α = 0.05, and as described in Lab 3, the df = 1 for this χ2 test. So we use the Critical Values of the χ2 Distribution at the back of your manual, and we find that χ2calculated ≥ χ2critical. Thus we reject our H0 that our observed frequency does not differ from the expected frequency, i.e., it’s unlikely that the parental cross was T/t × T/t.
Next we test whether our offspring counts come from a parental cross of T/t × t/t ⇒ 1 tall : 1 dwarf, going through the same steps as above.
Our H0 and Ha hypotheses for this χ2 test are: H0 is that our observed frequency does not differ from the expected frequency for a parental cross of T/t × t/t. Ha is that our observed frequency differs from the expected frequency for a parental cross of T/t × t/t. Here is the equation for calculating this χ2.
Here we find that χ2calculated < χ2critical. Thus we accept our H0 that our observed frequency does not differ from the expected frequency, i.e., there’s a 95% probability that the parental cross was T/t × t/t.
Here are some labeled photos to show you the phenotypes of corn kernels and other corn seedlings from monohybrid and dihybrid crosses you will see in Lab 3. Plus there are counts you can use to practice doing more χ2 tests.
Sample corn kernel/seed counts
Monohybrid cross counts (observed numbers)
purple = 300 kernels
white = 98 kernels
Dihybrid cross counts (observed numbers)
purple flint = 123 kernels
purple sweet = 41 kernels
white flint = 30 kernels
white sweet = 12
Sample corn seedling counts
Another monohybrid cross counts (observed numbers)
tall = 82 seedlings
dwarf = 28 seedlings
Dihybrid cross counts
tall green = 61 seedlings
dwarf green = 19 seedlings
tall albino = 16 seedlings
dwarf albino = 6 seedlings
In Lab 2 you use a Vernier caliper to measure the length of objects. Most of you have never used a Vernier caliper, so this blog post has more images illustrating how to read the Vernier scale than what’s shown in your lab manual.
Below are photos of Vernier calipers (like you’ll use in Lab 2) being used to measure the length and/or width of different objects. Additionally, each photo includes a metric ruler so you can get an approximate linear measurement of what you are measuring with a Vernier caliper, i.e., so you use the correct “movable” OUTSIDE or INSIDE scale. Note, all the measurements illustrated below are external measurements (like you will do in lab), and they all use the movable OUTSIDE scale. We will tell you in lab about when you would use the INSIDE scale to make internal measurements.
As of around noon today, when you log into our Blackboard class site you will see a traffic signal icon showing a green, yellow, or red light, and a text description below indicating how you are doing in BIOL 13500, plus a link to your Bb grade book. Next Friday around noon you will see your “MyTotal” + “MyPercent” through Lab 2, as well as your “LastPercent” (to compare with your current “MyPercent” with your preceding week’s percent). From now on, your total, percent, and signal alert will be updated after the class completes each lab remaining in this class. The purpose of our signal alert system is to let you know how you are doing throughout the semester so you can earn the grade you want. Incidentally, I will send out a class tweet and FB post letting you know when I’ve updated our class Bb grade book, beginning next Friday.
Below is a recap of our first week’s suggestions for you to succeed in BIOL 13500, First-Year Biology Lab.
What you want to do before each lab:
- Read lab in manual. Pay close attention to “Information that you will need for this lab” section because many lab quiz questions & lab prep questions come from this section.
- Answer/solve all LON-CAPA lab prep problems/questions for each lab.
- Write all flow charts & needed calculations on that lab’s notes pages in your manual. Writing flow charts before you come to lab begins with Lab 2.
- Be very conscientious as to what exercises you will be doing in a lab, and have an idea of how you will do them so you get the most out of the demonstrations your TAs will do for you.
- Study for the in-lab quiz. There are sample quiz questions on our class Bb website.
What you will do during each lab:
- Take the in-lab quiz.
- Complete all lab exercises, and in-lab assessments.
- Keep neat and thorough lab notes, recording important information from that lab’s exercises. Keeping lab notes begins with Lab 2.
What you want to do after each lab:
- Go over the questions missed on previous week’s in-lab quiz, and figure out what did wrong. If you don’t understand what you did wrong, see the Grad TA who grades your section’s in-lab quizzes (see below).
- Check your points in the Bb grade book to see how you are performing.
- Check the correct answers to the LON-CAPA lab prep questions/problems you missed and figure out what did wrong. The correct answers to a lab’s LON-CAPA questions/problems you miss will be revealed to you a week after the class completes that lab.
How to get help in BIOL 13500
- Stay after lab to get help from one of your lab TAs
- Go to the BRC, LILY B-401, Mondays-Thursdays 9:00-5:00 PM, Fridays 9:00-1:00 PM
- Go to office hours
- Grad TA office hours in BRC, LILY B-401
- Yifan Yang (grades odd numbered lab section’s in-lab quizzes): Mondays 11:30 AM-12:30 PM; Thursdays 3:30-4:30 PM
- Rui Yan (grades even numbered lab section’s in-lab quizzes): Wednesdays 12:00-1:00 PM; Thursdays 3:30-4:30 PM
- Dr. Iten’s office hours in LILY B-101
- Tuesdays & Thursdays 11:30-12:30 PM
- If none of these office hour times fit into your schedule, email BCBLab@purdue.edu for an appointment
BCB Lab—Boot Camp for Biology Lab