Biology 1AL - Lecture 1: Introduction

>>INSTRUCTOR: Good evening, we have overflow, we have 700 students, this holds 513 so we
have Evans set up. So fill in the middle and then if you want
a seat please go to 10 Evans, okay. Okay. Good evening. Let's get started please.
I love the energy in the room, that's great, so we'll get started here so this is Biology
1A, my name is Mike Meighan, I'm the lecturer for this course, I've been doing this for
20 years, in that time, I know that I lecture fast, if you need me to repeat something,
please raise your hand, do I like questions, I usually reward the first three people who
ask questions with the See's chocolate. Not this semester I haven't had time to get. So
let's get started here. A few things the syllabus please read it carefully. It has information
on there, now we do not have lab this week, Tuesday through Friday.
We have construction in the lab rooms, and we currently do not have sinks and faucets
and a bunch of other things it's supposed to be ready next Tuesday Septemberﾊ4th because
Monday Septemberﾊ3rd it's a holiday. So I know you haven't a chance to read things,
it's completely understandable. So book, Campbell, fall 2012, it's on bSpace it's also available
on replica copy it's probably much cheaper and fast tore go through to replica copy and
buy it, you can preorder it I posted the URL on bSpace it's bound there, I don't remember
the cost, it's 200 and some pages, so I recommend you get that. Fall 2012 only.
There's some changes relative to other semesters. There's a package of pre labs and worksheets
you need those pre labs and worksheets before you go to lab. Every lab, and I write this
on the board it's going to be on a handout slide as well, but I stress this for every
lab, including next week there's a pre lab and that's due at the start of lab, what will
happen is you go into the lab room, turn in your pre lab, at the start of lab there's
a 5 point quiz. So the pre lab has to be turned in for the quiz score to count. There's a
quiz, 5 points, tat start of lab. And then as you work through the lab, there's
a worksheet. And this worksheet is oftentimes due at the
end of the lab. But sometimes due the next lab period, depending on how much work you
have to do on that okay. So this is every lab.
[Instructor writing on board] There are 10 labs. So you'll have 50 points
worth of quizzes. We will drop the lowest quiz score so what
counts towards your grade will be 9 out of 5 of those.
So 45 points. That's what will count toward your grade.
You must turn in pre lab for this quiz score to count and then you turn in the worksheet
later. These pre labs and worksheets the way they'll be graded basically if it's average
work of two, if it's really good, a three, a pretty sloppy a 1 if it's admissible, 0.001,
not 0 at least I know you turned something in and didn't turn something in. But obviously
there was something turned in, so when I go to look at grades I know you did something
here asﾊopposed to nothing. Those do not count towards your grade, the
pre labs and worksheets do not count directly towards your grade, but at the end of the
semester I make a Histogram, and basically look the distribution of the students, and
I guarantee 90% or above is an A, and we do this thing just like with 1A we will have
ranges for various grades and we will look at these students here, who are close to that
next grade. And we consider them to be a potential bump.
And what happens is, I meet with the GSIs and we discuss individually every student
who is a potential bump. What the GSI does, well look at the pre labs and worksheets rah
yes they were active participants they helped out others, etc., so the bumps aren't guaranteed.
But usually about 75% of the bumps are done, but 25% of them aren't done and usually the
GSI has good reasons not to do the bumps so is that clear how that works? So pre labs
and worksheets not directly towards the grade but the quiz does so, the GSI is going to
be careful the grading the other thing going to count towards your actual grade is exam
1, is worth 100 points. [Instructor writing on board]
I think I have 4 examples of exam 1 in the lab exam later; it's probably the best 4 or
$5 you can spend in this class. When you work through these exams you're going
to say, wow this exam is pretty similar to this exam and this exam is pretty similar
to this. In fact as we get closer I'll hold extra office hours and the main emphasis,
in order to do this answer you need to know concept A B and C, then a student will say,
can you go over this problem and I will say the same concepts we used before will be applied
again and I'll show you how the concepts are applicable there, so the lab exam reader will
help you tremendously, this is in the evening, don't remember the date I have it on a slide.
Lab exam 2 is worth 64 points. And so in the end, you have 209 points that will count toward
your grade, okay. Now, I don't like giving out grades because
no one's really happen by with grades but we'll discuss it and I'll entertain your comments
and things with respect to the lab exams, lab exam 1 Wednesday night the 18th, all students
take the same lab exam 1. It's going to be about 12 to 15 pages long,
multiple choice, and we'll discuss that more, okay. But it's pretty challenging.
I'll be honest with you it's very challenging so work through these the exams in the exam
reader. Lab exam 2 is a new format this semester. Now, when I told students last semester about
the new format they all regretted they took it last semester, I'll tell you last semesters
and this semester format. All previous semesters we set up a lab practical where we set up
the sections, because the second half of the course will involve the sections, some various
invertebrates, lab. So that second lab involved setting up 30 stations with microscopes etc.
and then we timed students they had a 1 hour 45 minutes at the station and they had to
rotate to the next and the next and go through 1 an hour and 45 minutes, it's challenging
to the time frame people say it's too nerve wracking, educates, we spent about 400 to
500 hours setting up those exams, now that we have 3 classrooms even though one of them
is not ready to use, but now that we have 3 classrooms we don't have that option so
on this night, Thursday night you're all going to take the same lab exam 2. And that way
you don't have just a minute 45 a question, you divide how much time you want for a given
question, so that's the new format. Any questions a about that?
All right. So that's going to be lab exam 2, that is not Thanksgiving, Thanksgiving
is the week before, I've had several people e mail me about that.
I wouldn't want to be here either. Question? >>STUDENT: [Indiscernible]
>>INSTRUCTOR: So, this exam 1 cover the first 6 labs.
[Instructor writing on board] And this second exam will cover lab 7 through
10. The last 4 labs. So this is worth 60 points so it not comprehensive.
It's just the second half of the course. Other questions?
>>STUDENT: [Indiscernible] >>INSTRUCTOR: Do I have this backwards?
Does anyone have the syllabus it's correct on the syllabus. May I have a typo there.
Lab exam 1 isﾊ oh, sorry, Thursday the 18th and Wednesday the 28th, so I have the dateﾕs
wrongﾊ the days wrong but the dates are correct, okay. Thank you.
All right. So, for lab, there's a pre lab due at the
start of lab, the quiz start of lab, worksheets please bring your lab manual. I know that
seems obvious but for the first lab you're going to be looking at various organisms it's
extremely help to have the diagram with the organisms look like, so when you say it's
an amoeba, it looks like what's in your book and not the grain of sand on the slide. Aye
seen hundreds of grains of sand labeled amoeba, but these things look like the figures in
your lab manuals so bring them. We talked about the bump cases, now we'll deal with
some rumors, always have to deal with rumors. Let's talk about the grades, we talked about
the bumps things like that. So respectﾊto As, what do you think the ratio is of A grades
to Ds and Fs? So if I take a look through number of As,
so No. A grades given this class and that's some form of an A, A , A +, verses the Ds,
D +, Dﾊ ﾊand Fs, all put together, what's the ratio? Is it A? 1 to 1? Bﾊ so this is
the first one. 2 to 1, C 3 to 1, D 4 to 1, E 5 to 1? I would use the iClicker but I can't
get it to work right now. Those who think A? About 15 hands those who think B?
Getting to me more maybe 20. C?
Quite a bit more. Maybe 30. D.?
Quite a bit more. E?
I need a drink. It's E.
We give out 5ﾊ easily, in fact the ratio is closer to 10 times.
So we give about 10 times more As than we do Ds and Fs so this rumor that it's impossible
to pass 1AL are from the few students, 15 or so, every semester who we don't pass, okay.
But we give out 10 times more As, all right. So anyway that's what up with the grades,
we'll discuss that more. Now, what will happen here is you're actually, GSI quite these quizzes
and so there's a lot of them, so the level of difficulty with vary, I will adjust the
give levels. So assigned reading you have for each lab, the worksheet you'll be doing
as you go through the lab, lots of study resources, okay.
I said this class is hard, it is. But we're here to help you.
I'll have lots of extra office hours when we get closer to the exams there's lots of
information on our website, things like that. We'll have exam handouts, as we get closer
to the date telling where you'll take it. It will take and post study materials so please,
please take advantage of the resources that you have.
I have reviews that are webcast from the past, I'll also hold lots of extra office hours,
probably 15 extra office hours, the GSI, will hold extra office hours. Do the exam reader,
because you will see they're similar. We're giving great advice, take advantage of it.
E mail is a great thing, unfortunately if you e mail me and say, I don't quite get genetics.
Well I spent hours in lecture trying to do it I won't be able to do it via e mail. So
there are times I say come to office hours it's not fruitful to do it through e mail
or chat. So again, as we get closer to things, lab exam 2, we will have pictures of the ring
tank things like that so there's resources. Techniques to study.
I know most of you are juniors and seniors or sophomores and a lot of you have figured
out study skills and approaches, I think a amazing way to study for biology is to form
study groups. Interact with each other, ask each other questions, ask each other to explain
things, draw diagrams to summarize things it's a very useful way.
One semester I formed study groups for students, I gotﾊrooms had about 100 study groups,
so please, please take advantage of those things. Office hours are listed here; Monday
is 11 12, Monday 1 to 2. The favorite thing about teaching is office
hours it's a chance to interact with you, it's a chance to work with you, and a lot
of times the student will come and I can work backwards until we get to a common set of
knowledge and then we can work toward and then I will see students get genetics so please
come to office hours. The labs will be as follows: The first one
safety equipment cells and vibrio will spend 5 labs on this vibrio, I'll go over shortly
what it involves. We're going to take seawater, plate it out, look for bioluminescent bacteria,
we're going to have a purified colony of the bacteria and do PCR on it of two specific
genes, once we get the PCR product we'll send out or sequencing once we get the sequence
back, you'll blast it against the database you identify the species and then what we'll
do is build a tree that shows the relationship of the brief organizations that we found in
lab. So it's involving a lot of modern molecular biology so we'll be streaking it to get the
purified colony we'll have an enzyme lab because enzymes are what control reactions, catalyst,
especially biological systems, we'll get the purified colony it's critical that it's a
purified single strain. We'll do a lab of photosynthesis, so we'll study out light is
captured and how that light is energy is converted into chemical energy to form corn sugar and
things like that. We'll then do some experiments involving computation
which is a series of steps, very easy to do, and we're going to determine the number of
genes in a biochemical pathway so, the pathway we'll will booking at is a production of an
amino acid called histidine. And there are various steps involved in that
pathway. So by finding mutantsﾊ without even knowing which step they are, there's
a lot of mutants in here, so maybe 10,000 mutants and we do is start mating them and
we do mating so we make that first mutant which we'll call mutant No.ﾊ1, with mutant
No.ﾊ2, mutant No.ﾊ1 with No.ﾊ3 and we'll do 2 with 3.
2 with 4. And down the line. So we'll do matings like
this and from this technique which is quick to do, you'll see you can determine the number
of genes, and what order of steps those genetics they occur in the pathway.
So, if it's a very fast way to learn a lot of about biochemistry so it takes about 3
labs to do we'll have to do PCR on this bacteria that we isolated to find the genes to get
the gene product, so that we can then sender them out for sequencing. We'll send them out
or sequencing we'll have to make sure those PCR product work, get the right concentrations
for sequencing once we get the information out that's when we take and bioinformatics
to identify the species we'll look at mitosis and meiosis here, so cell duplication, whether
there's a change with meiosis, half the change or mitosis [Indiscernible]. And then basically
we'll do a lab genetics looking at fruit flies. Then we'll do entire lab in bioinformatics
and we'll continue this 1, 2 and 3 steps and then we have 2 exams and someone said this
is Wednesday, I had that backwards? But it's the 18th. That one's correct it's the 18th.
Okay. Lab exam 2.
Covers the anatomy lab, we do a rat dissection, the anatomy is extremely similar to yours
there's a few exceptions. So we'll do a rat anatomy lab and look at blood pressure measurements
we'll have an inverter, we will detect invertebrates, we'll do demos and then we have a lab on reproductions
development and then this lab exam on Novemberﾊ28th so those are the exams. Some themes that you
will learn through out 1A and 1AL. 1A, you wont have time to learn the things
in the lab class I have to go over techniques as well. So I rely on 1A to teach you some
things, next week we're looking at cells. Cells come from preexisting cells.
Biological organisms are congresswoman posed of cells. Those are two components of the
cell theory. We'll talk about hierarchy, I think everybody heard of this, molecules,
macromolecules. Organelles. Cells. Organ systems, various things like this. So we'll be talking
about that. The hierarchy. Evolution. How many of you had Bio 1B? A big part of
that is ordinary evolution. The changes in DNA given time with a species.
Classification we'll look at this second half when we're looking at the various organisms
the animals so there's 3 domains. Within the prokaryotes there are two domains, and there's
been a lot of confusion about our terms of bacteria.
I don't really like the term bacteria, that's because a few years ago we discovered bacteria
that we didn't know existed. So there are three domains. They're the eubacteria.
And there's the archae and eukarya. Those are the prokaryotes. I will use the
eubacteria for one domain and the third domain are the eukaryote, which are the eukaryotes.
[Instructor writing on board] And basically, these are considered to have
membrane bound organelles. In particular the nucleus.
So, within the prokaryotes there are two domains. Cladistics are a way for us to identify, express
relationships between individuals, whether they're species within a population, etc.,
we'll talk a lot more about that later. And then this one you've already started to see
a little bit in the 1A class. So Dr.ﾊPauly taught this morning about cellulose verses
starch. Now, you know that with potato starch, cornstarch,
no problem to eat it. But this is cellulose, this isﾊ you can eat it but you won't be
digesting it, that's fiber. So simple difference between an alpha and beta configuration in
the glycosidic bound make a huge difference in the structure and in its function you will
see this over and over again. A difficult concept in biology which I will introduce
now, I don't expect you to get the term now, but eventually you will need to get it and
I have to warn you, it took me 6 years to get the Campbell authors to do this right
in the book. Taken me years to get [Indiscernible] so ploidy
refers to the sets of chromosomes. Not the number, but the sets of chromosomes.
So to help you with the analogy, a deck of cards has 4 suits.
The ploidy of a deck of cards is 4 because there are 4 sets of cards.
The types of cards, 2, 3, 4, those are the types of cards, or types of chromosomes so
for a human, when we talk about 2N = 46, [Instructor writing on board], the number before the N
is the number of sets. So in a 2N cell there are 2 sets, consistent
of 46 chromosomes, so how many are in each set?
23. So 23 came from mom. And 23 came from dad.
All right. So, with expected life cycle we can illustrates
some aspects of that, I mention one of the themes is mitosis meiosis, so let's look at
this so within the individual here within the gonads, meiosis is going we produce eggs
and sperm so this is haploid, 1 haploid, when these fuse, that's one cell 2N 46 chromosomes.
And through mitosis we duplicate the DNA, and cell division. So no change in ploidy
so we have two cells and 4 and then 8, etc. so we'll talk a lot about the reproductions
of development lab. We'll go through the steps. So mitosis does not change the ploidy, meiosis
pass the ploidy. These are called gametes before they can under go a mitosis they must
have a fertilization event. So does anyone know the name of a cell, general category
that's produced by meiosis but can undergo mitosis?
>>STUDENT: [Indiscernible] >>INSTRUCTOR: Spore. So a spore is a cell
that was produced but meiosis typically and it can undergo mitosis. All right.
So safety, that's one aspect of the lab, it's very straight, common sense, so work through
that. As part of the requirement you to sign the safety sheet I'm not going to spend the
time in that. The GSI will point out the safety aspects. What I will point out because we've
had a few issues is when he do certain labs like the dissection lab or take blood pressure
measurement we ask you to be seated, several students faint. When it's a shorter distance
it's not a bad thing. We've had people catch their hair on fire,
so if you're in lab, and you'reﾊ everyone's patting they're head, it could be because
your hair is on fire. So when you work with flames tie back long hair I don't have that
issue anymore. But be careful of those things. Micropipetters so we can actually remove, dispense, add,
volume, as far as 20 I understood this is a 200 so from ranges to 20 to 200, so we have
a thousand, that we use for 200 to 2,000, and this is accurate piece of equipment, you
always have to use a tip with them, so I would like you to just repeat after me, you must
use a tip. One more time.
Third time. Excellent thank you.
You may ask why does he stress this? One lab we had 30 of these micropipetters out. 28
of them contaminated because people forgot to use tips. So we want you to use a sterile
technique, GSI, I try to teach you a good technique, I'm always correcting the GSI,
you can't see this so this is the autoclave, so these tips are sterile so we want to remove
these tips using a sterile technique. To do that, we're going to take a rotate the lid
and then I'm going go in and grab with the micropipetter and then close it and if I open
this like this, all the spores, bacteria in the room are contaminated so you want to rock
the lid open like this and grab that tip. Okay. If you look on this, it reads 100 here
you can't read this, I'll zoom. I know does that give you a headache?
Okay that's 100. So what that means is, atﾊthis plunger here
there's 2 points of resistance, 1, 2. I'll do that again.
This is set that 100, that would eject and there's [Indiscernible] so if you go and grab
solution, make sure it's set at the right volume and then press down to that point of
first resistance I go into my tube, and what I do is you put the tip in there and you will
see this in lab you slowly release that plunger if you let your finger off of it it will ricochet
up and that sucks too much solution in and that will contaminate this tip. And then to
dispense it... Onto this plate, which formally I would not
be doing. I press down on that. Now unfortunately this dye is viscus so I can't get that liquid
out. Normally the solutions you work aren't that viscus, this one is.
Then there's an ejector here and you will see this in lab. The ejector. So we have three
different micropipetters, and always remember the first point of resistance. GSI will do
this in lab, each of you will pick up one and each will go through it with them.
The way you will test your technique is if you had a P 200 here and you had pipe petted
a microliters that volume of water should be 1.00ﾊgrams, so what you can do is weigh
it so we'll have weighing dish here, so put the weigh dish here, put this to 0 and add
your sample to that weighing dish, measure the volume, the weight as I should say, measure
the weight, re zero it, add another example, measure it, add another sample, re zero it.
So what you're going to do a take repeated reading so see how accurate you are, you should
be within 5%. That means your value here should be .009ﾊ
0 pointﾊ to 0.15. So you do stuff like this. The other part of lab is using the microscope.
Now I have it here. Now there'sﾊ it's like me trying to tell you how to drive without
having a car and stuff. So when you take and do this in lab it will make more sense. So
let me go over a few things here. We have some knobs here.
This is the courseﾊ can we zoom in on the microscope please.
With this camera. Is that going to work?
All right. I'll spin it this way. Can you see that?
So there's a big focus and that's the course if you notice the stage is moving quickly
up and down, so when you do the core focus that's moving. You can't say the stage is
moving but it is. So the lenses are fixed in place. So when
I change the magnification, what I'm doing is changing the depth of focus. So I need
a volunteer to help illustrate this. Want to volunteer? Yeah.
So, a given position is in focus, okay. So, you hold out your hands like this.
That's the depth of focus. So remember looking down.
So, when I have that microscope here, my specimen is here, so when I have my specimen I have
to raise the stage or lower it depending on which way to get that into the focal point,
the focal point is fixed for the lenses, the way you set the focus of the microscope is
you position the stage of the specimen into this, the lower power the focus is big like
this as you change the magnification, that depth of focus gets smaller, okay. And what
happens there is we're going to optically [Indiscernible] the specimen. Does someone
have a water bottle? Transparent water bottle. That's fine. That's
better yet. Okay.
So this is transparent. So what would happen is if I hadﾊ so imagine this is what's in
focus if I raise the stage I can look at the top of this and if I do this it gets wider
and wider, so you will optically sections the specimen with the higher power your sections
are thinner and you can get more detail. Okay.
So that's the reason the microscope is to increase magnification and get the ability
to see the sections. So exercises you'll do in lab, you'll be looking at the nylon cloth,
the space between the fibers this leading edge to this leading sedge 100 micrometers,
the cloth is fixed it's always 100 micrometers and there's ruler marks we see, because in
one of the eye pieces when I have my specimen in focus and you'll go through the steps in
lab, when my specimen is in focus, and it really doesn't take long to do this, but when
my specimen is in focus I will see that measuring device superimpose on that image and I can
use that measuring device to measure some unknown organisms but we have to calibrate
it first. So aspect of lab is you need to calibrate the ruler. So the best way to sill
strait this, those who wear glasses imagine you have 100 edges on your glasses, and if
I held a piece of paper up close, I would see those etches, superimposed on the paper
right and if I hold it further away I would see them imposed if I grab binoculars I would
see them imposed. It's like pulling the thing closer so the distance they represent should
get smaller or bigger? Smaller. Okay. And there's a direct mathematical relationship
and you'll determine that. One exercise we have you do in lab is illustrate this section
is we have some slides made and on this slide is a solid piece of glass tubing and hollow.
So, just like thisﾊ if this was empty, and I could section with the middle of this I
would expect to see what an edge here, another edge very close by, all of the internal space
another edge another edge, right? Because it's hollow.
If this was solid I would see the edge and edge but I wouldn't see the second edge there
nor would I see that, so based upon that is this the hollow or the solid one?
Hollow because there's one edge there's the other edge there's the air space, edge, edge.
So by having position that specimen, such that the middle of that hollow tube is your
optical section you can tell that. So you'll be doing that a lot, it's not easy but you'll
work through it. Part of the microscopes the focus knobs this is the stage, this is how
you move the stages the parts of this of course, light source, it getsﾊ the light's produced
down below, LED there's a series of lenses, the condenser lenses focus on the light on
specimen here, and the objectives here, transfer the light to the oculars onto your eyes these
are the focus knobs, the big one is the course, the other one's the fine focus, this just
controls the rest state, this is on off the light source.
So the main aspect of this, lenses to focus the light onto your eyes and light source
condenser the light focus onto your specimen. And here's to list of all of the parts.
Will your GSI have a diagram the microscope and ask you to identify the parts? It's possible.
But truth of the matter is, it's not so critical that you be able to identify all of the parts,
the more important thing is do you know the function of the parts so that's what you should
know is the function. When we use these microscopes since these
are binoculars what you're going do is get the specimen focus with justﾊ close your
left eye it's nice clear focus, close your right and you can adjust the focus of this
eye piece relative this, this ask called after adjustment so you want to have these be in
proper focus for the eye. You see this in lab.
Students have some difficulty with this part. The real part they have the biggest difficult
with is the following: When I have thisﾊ these tubes spread very far apart I see two
circles of light, what you want to do is move them close together until you get one field
of view, I see one circle of light that means I use both eye that's the most difficult part
of the microscope. Rotate it it out so it's far apart. Two [Indiscernible] of light [Indiscernible].
And that varies with each person in here, the GSI will help you, and this just illustrates
what's going on there. You adjust those to match the two field of views so one uniform
round field of view. It's not that difficult, the key thing is if you're having difficulty,
go back and make sure the focus is set for each eyepiece independent of one another.
Okay. Because if you haven't done that, you're not
going to get the two circles to overlap. The other thing if you struggle too much we
can takeﾊ with a ruler, we can measure the distance between your pupils and try to set
it. Okay you guys are laughing but when you go in lab you will see that people struggle
with this. Anyway, it's nice to use 2 eyes because then you get a better sense of the
focus of it. Unfortunately the microscopes have inherent design flaw in them, I didn't
build them but we have to use them. Do not hold both the left and right focus
knobs at the same time. If you hold them both and you rotate one and maintain the other
one stationary there's a shaft in that you will break the shaft inside, these are about
$3,000 to replace, so bad design I didn't design them so when you focus, only use either
your right hand or left hand but don't use both at a the same time because you will break
that shaft it's a very bad design. The other very bad design of the microscope
and we haven't been able to correct it with these objectives you will use different ones
during lab, high power, low power, etc., the 40 X objective was designed poorly, so the
40 X objective is very difficult to clean. Only use lens paper, and use lens cleaner
with it. Okay.
And if you're having difficult ask the GSI. It's a bad design they recessed the lens a
lot, and as a result sometimes when crud gets on it it's hard to get off. So those are the
two bad design flaws of the microscope that we haven't been able to correct. So we'll
look at specimens in there, there are various ways to do this. What happens is the light
interacts with the specimen and we see that and then we see the image of that specimen
so there are ways to enhance that contrast you can add stains that enhances that contrast,
so this is a stain that's specific for DNA, so this is a nucleus here so it stains more.
What [Indiscernible] might be all of these small dots here, what other structure in here
might have DNA? Mitochondria? So those potentially could be mitochondria but this is the nucleus,
so you're going to do this in lab, you're going to stain your cheek cells so you'll
use a toothpick, scrape yourﾊ gently, scrape the inside of your cheek put saline with dye
in it, it will stain the nucleus and you will see that and it will look like this.
So this was unstained, this is stain so you can see contrast, there are ways focal microscopes,
florescence, these have exclusive resolution they use florescence to label things and then
they have a pinpoint source of light and they focus it.
Okay. In terms of use the microscope, the key thing
is resolution. So what we're really want to do is be able
to tell that these are two distinct points you tell it easily, but as we get closer it's
more difficult to tell these two distinct points. So resolution refers to this distance
D. is two distinct points. So the smaller the D value, the better the resolving power,
so this resolution and D power the better resolving power and there's a formula you
can use to solve that, and let's just go over some aspects of that formula here. So the
distance is 0.67 x the wavelength this is the wavelength times light used. You can use
electrons which have a really short wavelength so this D ain't much smaller value and this
N is a feature of the solution between your cases, but it's 1 but you can put oil between
there, how many used oil, so what you do with oil emergent lenses you take the M value which
makes it a higher value which means this is lower value. So oil is 1.3, it's smaller value.
Okay. And the siren alpha is an aspect of the design
we're not going to change. So here's the calibration. So right now if the distance here here to
here is 100. Now, we used different materials in the past so we're made all new slides this
time so I have to be careful it's completely different. So the distance between the fibers,
between the fibers the space the gap is 100 microns, always. So if that's the case at
this magnification, how does each ruler mark represent?
So this distanceﾊ known distance 100, how much does each represent? 2.5, 100 divided
by 40, if 10 had to fit there it would be 10 for each one. Remember that distance changes
within magnification, so that's one aspect of the lab you'll do you'll collaborate it.
You'll look at it to see if it's hollow, solid, which is which and which about what you're
doing there in terms of that optical sections. The cells you're looking at in terms of the
prokaryotes, the two domains are eubacteria and archaea, and we're going to be looking
at them, they're very small they tend to be about 1 to 5 microns at most they don't have
a lot of morphology to them, we'll talk about 3 different shapes, rod shaped, spiral, round
shape, different terms for that. Help to identify them we'll talk about gram staining and it's
a procedure that we use and it reflects the chemistry of the cell walls.
So, I want to go how the staining is done, and there's two broad categories I'm sure
that everyone's heard of gram positive and gram-negative bacteria. Raise your hand if
you haven't. Okay. So most of you have heard of it. So
let's start with a gram negative, and gram positive, okay.
So these are known bacteria. So we know what these are ahead of time. Okay.
So, let's start with gram positive. What we do is we make a slide and then I'll
put over there we take a slide, we put a solution bacteria on here, liquid, and we let it dry,
heat it up, now we treat with the stains and so the staining procedure is follows, draw
one of these gram positive bacteria they have a thick cell wall, and then there's the cell
membrane that's very thin and then the cytoplasm and they have DNA, and it has proteins with
it it's not naked DNA. But the first thing we do have fix it, we add crystal violet.
So it's a purple color dye. [Instructor writing on board]. And that's
retained by the cells we treated it with iodine as well. And then we decolor it we do these
for a specific length of time and that removes some of the violet.
Then we counter stain. Once we've done the rinse. We counter stain, so that's an additional
stain with safranin red, which is a red color. And then we detain again.
And this is for known periods of time, and then we'll re stain it. But in the end the
gram positive bacteria will be violet color. Very easy technique. It's antibiotics or certain
gram positive other where specific gram negative. If you have a gram-positive antibiotics and
it's gram negative it won't do you good. So gram negative, we're we'll go through the
same staining procedure but has a thin cell wall, the cell membrane, when we do the crystal
violet, and there's DNA in there and proteins and all of that other stuff. We do the destaining.
We do the counter stain. And in the end this appears red.
Very fast technique, it only takes a few minutes to do this and you can easily categorize this
gram positive and gram negative and this will tell you what kind of antibiotic to use. Here's
the gram positive bacteria, this thick cell wall it retains that crystal violet a lot
more, which is why in the end this is violet color and with the gram negative, they have
a thinner cell wall they also have a extra membrane outside of this, which is kind of
bizarre, it's usually the toxic part of gram negative bacteria, so in fact gram negative
bacteria tend to be more toxic to individuals than gram positive because of this outer membrane,
but the bottom line is thin cell wall when we do the staining at the end it's red and
not violet. Okay. And here to show you some slides, E.ﾊcoli,
these are rod shaped, so the bacillus shaped and they're red, gram negative.
This is just an electromyograph. Better resolution here and this is gram positive there. Round
oxide. So it shows you a sourdough starter. By the way you wereﾊ we'll work with yeast
when we do the computation experiments it will be the yeast. So eukaryote cells so now
we're into the eukaryote, so for lab you'll be looking at some demo slides for this.
[Instructor writing on board] And for this, you'll be making your own slides
so these are various wet mounts. Now, these do have true membrane bound organelles
you'll be pretty easy to see the nucleus, especially when you do the cheek stain. So
with eukaryote cells this is a plant cell here we'll be looking at the [Indiscernible]
we'll see the green chloroplast, you might possibly be able to see the nucleus, but if
we don't stain for it it's hard to see. But there are membrane organelles, Dr.ﾊPauly
has time to go over this. Some of the protists you'll be looking at is the amoeba. It has
the pseudopods that they use for movement, they look like what white blood cells, they
move this way, amoeba motion. These are difficult to find.
So, whenever you make a wet mount, whenever you put this light on always use the lowest
power objective first. Lower the stage, all the way, put your slide in place.
Then what I do, is I usually crank the light from the side I can see the lights going through
the wet liquid that covers stuff, whatever and then I know that right there's my focal
point so what I have to do is move the stage to get it in focus, and I just use the course
focus it's quick, it's there. Okay. Now, why do you want to use the low
power? First off it gives you the biggest depth of focus so when you're looking for
that specimen, realize and it's not easy to realize this, but here's the slide, the cover
slip is here, there's actually a column of liquid here, where you're specimens can be.
So by using a big lower power magnification and a [Indiscernible] the entire column of
liquid is in focus if you started a high power, only a small portion of that will be in focus.
So it will be very difficult to find the specimens. Further more, the build of view is much bigger
with a low power than with a high power. So when you're searching for this, the area you're
scanning is much greater here this cylinder, it's a cylinder, because round field of view,
depth of focus is bigger with a low power. I probably helped 200 students this first
lab find things and the very first thing is I do the first thing I do is drop the magnification
to low power. I always use it because it makes it much faster. I no he you will go to the
higher power but once you set it at the low power, the next higher power is going to be
a slight changes. So the amoeba, if you use low power you will find them, the other thing
that sometimes people have a problem with they have too much light and there's no contrast.
So I'll show you shortly what that looks like, paramecium, nigh tell la, these are quite
big. These cells are huge, I mean huge, these cells are 2 to 3ﾊmillimeters. Even bigger.
That's huge for a cell. I mean, most cells are maybe 10 micrometers.
Because these cells are so huge, the time it would make for a molecule to diffuse from
there to the other end of the cell, tens of minutes that cell would be dead if it were
to rely strictly on that. The way to get not diffusing, but bulk mixing and you will see
that it's called cytoplasmic streaming. So it's like when you stir the container that's
what's going on. You will see it's a eye spot there it does sense light and the left verses
the right, so there's a series of steps called: Clumination(?), this is the with the [Indiscernible]
which increases contrast, and that is with it open.
Now, I can barely see that amoeba there but that's the exact same image, so what happens
is you have too much light and it's hard to find these things so make sure you're dealing
with a [Indiscernible] that's fairly close it's fairly subjective I can can't tell you
it varies with the specimen, you will learn it.
Question? >>STUDENT: [Indiscernible]
>>INSTRUCTOR: Absolutely you can stain eukaryotes. With the cheek cells you'll be using you will
take them, you'll have your slide premade with a drop of dye, methylene blue and it's
in saline that saline is isotonic to your cheek cells so when you take and scrape you
have of the cells you will put the toothpick in the saline transfer it from the toothpick
to the saline so you can stain eukaryote cells, people typically don't do the gram stain on
eukaryote cells because it's not reflective of cell walls. There are eukaryotes that do
have cell walls, plants do have cell walls but they use a different staining procedure.
Any other questions? >>STUDENT: [Indiscernible]
>>INSTRUCTOR: So, yes, the field of view is greater for low power, so if the low power
is like this field is view is the circle image you see looking down.
The depth of focus is the vertical distance so it is really a cylinder, so at low power
the cylinder is big, as you increase the magnification, what happens is, you take that field of view
and you slink it down so you go like this to this in the center. So when you change
the magnification it is extremely helpful and I'll show you a top view, it is extremely
helpful before you change magnification, if this is your field of view, to center your
specimen because when you change the magnification, the field of view will be there. Smaller diameter,
if you haven't done that and your specimen is off to the side and you change your field
of view, it disappears from your field of view. So really is important to ruse use that
smallest field of view and realize of course that this is a cylinder, and the depth here
is much greater than that. Okay. I probably can't stress that enough.
Low power first. Okay. All right.
Here it shows you those cheek cells you'll be doing. Your own cheek cells, there's the
nucleus there. And a lot of people say these look like a fried egg because these are epithelial
cells they're fun and the epithelial represents the egg yolk. It's not from the side, this
is the top, so it's hard to see that. This is tongue scraping so you see long chains
there. This is a low power view, this is a higher
power view, here are the chloroplasts. So if you think about this, I guess the best
analogy I can think of imagine you bought a basketball signed by Michael Jordan worth
lots of money so I that ship it to you, they put packing peanuts down in the bottom, they
put the ball in there. They ship it to you. That's like a plant cell. So what happens
is the box represents the cell wall. The packing peanuts are the chloroplasts that basketball
is a huge central vacuole. So if you were to section that cell, because you can do that,
the sections are thin enough, so let's just think about that. There's the box.
There's that large central vacuole, in this case we have chloroplasts, scattered through
out. So think about if we were to offer the exceptions and we're looking down from above,
if we're at the top of that, we would see that outline of the box, looking down, and
we would see the chloroplasts here, scattered through out.
So at the very top view, [Instructor writing on board], we would see the chloroplasts through
out, does that make sense? What we've done is optically sections. With the top of the
cell, but we can move the stage so we move the point of focus so we can then be say in
the very middle we still have the outside cell wall. So that would still look the same,
but this case, we can't have those chloroplasts in the center because they're excluded by
that central vacuole, so they would only be out here in the perimeter. Does that make
sense? And like wise, at the very bottom we would
see something that resembles if this is A B and C, A B and C, C resembles A.
>>STUDENT: [Indiscernible] >>INSTRUCTOR: So, in this case, the objective
we would be using would probably be the 40 acts objective, the highest power to give
us the thinnest section. If I use the low power too much of this would be in focus,
my sections would be too thick. Okay. >>STUDENT: [Indiscernible]
>>INSTRUCTOR: So, okay. So theﾊ what perspective is this? This was the side view of the cell,
but when we use the microscope we look down from the top.
Okay. So this is the top view, section like this,
A, this is the middle section B, and this is the bottom section C.
Okay. Did that help?
Yeah, so if you need clarification please ask.
So if you look here, what we see isﾊ you see the chloroplasts through out, there's
the nucleus, tell me if you don't see this, there's the nucleolus. Here's one cell wall,
adjacent cell wall, in fact when you use this, they're large for the cells, when you're at
the top of the cell, you'll see the chloroplast move in this direction, if this is a compartment,
the chloroplast have to go around like this, just like a bathtub, so if you're at the top
here looking you see the chloroplasts are moving in the direction. When you're at the
bottom you see the chloroplast here at the bottom. When you're at the middle but the
chloroplasts are going down but because they're going out of view it's hard to see that.
Optical section is the key to the microscope. Question on that? So you'll be looking at
various thing it is difficult part is to take the images and reconstruct them into one.
For example like when they do a CAT Scan you've seen this on House, which isn't on TV anywhere.
So you see, but the CAT Scan they're taking a section, a section, a section, and they're
collecting that data, and then they basically take each section and they use computer to
compile the various sections together to replication that 3 D image, that's the same thing you'll
be doing. Thin sections, light sections and reconstruct
them. Anybody have a 3 D printer? No not yet. Soon. For the [Indiscernible] work I'm going
to go over this briefly. So we have FWC plates. See water, these do not have antibiotic in
them. Now, I put the dye down here, you're not putting dye you're putting seawater. So
the volumes 100, so you'll use a tip, you'll rotate that, you'll rotate the box open, to
grab your tip, you'll plate 100 microliters in the middle of this and then you'll use
thorough technique to plate it. The only thing I want to illustrate is there are two ways
to hold this. You can hold it like this, which I find awkward, which is like thisﾊ so if
you hold it. You will see this to demonstrate this. Hold it like this, rotate, remove this.
Rotate. And rotate.
Then what we'll do isﾊ you want to sterilize it again, we're going to incubate these plates
and then next lab we should see some isolated colonies, hopefully. But when we see colony
on the plate, we spread that seawater out when we see the colonies one the plate next
week, we're going to not be sure if that was one initial back trim that founded that colony,
right? Maybe there were two that were close together. So we're going to need to do, take
that one colony, select it and streak it out. So we're going to use tech knee teaks, sterile
techniques to streak it out and our aim here is to end up with a single colony that we're
confident is a single colony from one single bacterium. So we'll streak out in one lab
and then in another lab. We keep streaking it out to make sure we have isolated one colony.
The reason is because we want to take and obtain gene sequences from the bacterium;
if there were two species of bacteria there when we go to the PCR we get two PCR products
and when we go to sequence it we'll have a difficult time. We need to have with our technique
one there. We have to make sure we have one of these. We'll do PCR, go over this again
and again, so just to plant the seed right now of PCR, here is the bacterial chromosome
it's 1 double stranded molecule, usually circular in bacteria, not usually, there's a gene here
that corresponds to the 16 S ribosome gene, there's another gene that corresponds to [Indiscernible]
and I'm illustrated to this for the 16 S we know ahead of time the sequence that flanks
this gene. So we know the sequence to the right and to
the left, so we can generate what's called universal primers it doesn't matter what the
bacterial species it the universal primers should work. What they will do is allow us
to make tens of thousands, millions of copies of this DNA.
So our PCR product will be double stranded. Okay.
So we'll start with one single double strand like this. We'll separate them, do you guys
know how these are held together? Hydrogen bonds so it's really separate to
separate them, you heat them up. You separate the two strands, one is primary specific for
one strand it's primary specific for the other. People always ask which is forward and which
is reverse, doesn't matter all you need to know is they come in pairs. One's a forward
one is a reverse, one person would called that forward and the other person would call
this reverse. The only thing that matters is they come in pairs. That allows us to make
copies of this. When we go to sequence the DNA we need to have copies of this that are
identical. So once we get that the we get the PCR product we have to make sure it works
so we'll take your PCR action, jell, had some dyes that bind the DNA so we can see how much
DNA is there and we'll separate. The DNA will migrate to the jell, based on
the size and charge, so basically we'll partition this and we'll go over this more later. We'll
get different sizes separately and we're looking a on the jell to see does that PCR reaction
have a product, does it match the expected size and if so what's the quantity of it so
we can set it up for sequencing section so we send that DNA out we'll give it primer
and then we'll send out for sequencing and get data back and from this DNA data this
is what you're going to get. You're going to profile like this: Ewe you're going to
take that DNA sequence enter it into a database, using tools, make a consensus because you're
going have multiple sequencing of that PCR product, you will blast it and look for what
organisms corresponds to. So this example of the various organisms that corresponded
to in this lab, this is one, another one, then we can use a cladistic program these
are closely related and these went to this clave. Notice the terminology here, everything
can be a clave, so that is a clave everyone. These two are claves, this one with that is
a clave, so clave's just refer to interrelationships this is closely and this. So we'll go backwards
here, we'll do it in lab, not the easiest thing to do but we'll work with that: I have
to spend a little bit more time on one other presentation. If I can get the computer to
work here. And that is since we're working with animals.
We need to go a little bit over the ethics of animal use here.
Okay. So, the bottom line is that at UC Berkeley
there are many organisms that look after the health and well being of the various animals
used on this campus, whether it's research or teaching since you'll be working with some
of the animals you have to know the ethics evolved around this and contract information
if you think they should be informed of things so we'll do animal dissection in here, in
particular a rat dissection that's basically vertebrates are the ones we care about the
most, but we'll dissect crayfish, animal care use committee and also animal care. All three
of these we're responsible for following various guidelines in terms of animal use. Now, why
we use them, they're used certainly for models of human diseases so a lot of times the animals
are used for teaching purposes but sometimes they're used to model human disease. So we
might infect the animal with the disease, see how it progresses, what treatments we
can use, how would it work on a human or maybe on that organism. So we may be studying how
you can treat a particular infection, in chickens or things like that, itﾕs important to deal
with that. There has be been some real abuse done by
labs in the past, and unfortunately I'm sure there are still current labs that are doing
abuse, okay. So, when we work with the animals in lab,
treat them with respect, I would prefer you not give your rats a nickname when you dissect
it. Okay.
Treat it with some respect, now. What we're doing here is the reason why we still do dissections
is you learn valuable dissection skills, and there's a lot of variability in the physiology
and the anatomy of these animals and some disease states.
So in lab, if you come across a disease state please point it out to your fellow class mates
and stuff like that we'll find examples of various abnormals, infections, there are no
computer programs now that suddenly introduce these diseases into these sections the other
thing is for the rats, all of the rats that we use you'll use pens to pin them out. You'll
count the number of pins when you start and when you end because all of the rats we donate
to an organization that uses them for a food source for basically various raptors and stuff
so if we didn't rotate the rats for them they would go out and purchase them. So they're
not treated with formaldehyde, or anything like, they're fresh rats.
We'll collect the rats we give them to the organization they use them to the feed the
birds if we didn't do it they would still be buying them so all we've done is including
a teaching step in the process so we've done what we can to minimize this. So there should
be proper treatment of these, there are various laws and policy in place, there are various
regulations, national and state regarding this.
We have very strict regulations in terms of housing these animals, in fact, I have been
very tempted to [Indiscernible] rat, I have not had myﾊ you guys will see this when
you come to my office hours do I not have any ventilation control in my room, it will
be a varies from 55 to 85ﾊdegrees in my office in a time frame from 3 to 4 hours,
sometimes students will come in and say, it's hot in here.
If I was a rat that temp can vary by 6 degrees. So there are strict regulations, I haven't
had that success here on campus, specific training procedures how many of you do research
in labs with various animals? So there's a fair number of you who are aware of the specific
guidelines that are required. When we work with the rats, we're going have
you wear resistance pair raptors. If you have any known allergies. Tell me ahead of time.
So we need documentation so all of a sudden ewe all you can't be allergic, but we'll take
precautions to help you. We have had students very allergic to cockroaches
we dissect a cockroach. Crayfish things like that. If you have a known allergy, crayfish.
So we can have you stake precautions if you feel faint or itchy or anything like that.
Leave the lab. Get fresh air. We've had to call 911 on several occasions.
Usually it's around finals when you haven't eaten for 5 days and only had 45 cans of red
bull. So and you're like this. We get that way too. Okay.
Anyway, as you go through this, remember to treat the animals with respect, there are
3 Rs here, replacement. Unfortunately as I mentioned we've looked into these various
models to see if we could replace the dissections with the various online models they're not
very good. If you look at the literature, people promote these things and say they're
great, they're actually pretty low quality I'm not saying they can can't get there eventually
none of them introduce the disease state but we try to take the extra step to donate them
to the facility that rehabilitates the wildlife. We use as full rats as possible. However to
encourage active learning you almost always will work as a pair of students in lab.
Almost always. Except for with the microscopy where each of you will have your own microscope,
don't share them. Each of you has your own so you learn the skills better. And refinement
we've had various protocols that we take care of. For the rats when we kill them it's an
overdose of CO2 we have to have basically a muffler so the noise of the CO2 doesn't
frighten them so there are precautions we take to minimize them. And respect, which
I said, don't name them. Okay.
We have veterinarians on duty at all times. We will not be able to do it this semester
because of the construction, but we have a diversity lab, which is pretty cool where
we have a live crocodile and various birds and things like that, tortoise, things like
that. But upper division uses our animals they do surgery here. Anyway, if there's any
ever issues about this, please take advantage of the resources to contact, contact myself,
and enjoy your lab. Please take sure you do that pre lab, no lab this week, do the pre
lab before you come and good luck. Enjoy it.