Hey everybody, Professor Karafit here. We're going to talk about the cell membrane lab for Bio1441. You may be writing a lab report over this lab, so make sure to take good notes during lab in terms of what you've done and also from your pre-lab in terms of the meaning of what's happening and some of the background information.
In our previous lab, we made a standard curve based on beta-cyanin light absorption. We're going to use that standard curve in this lab to determine the concentration of beta-cyanin that leaks out of beet. tissue when we damage it with ethanol.
So you might want to bring that standard curve with you. Our basic research question this week is what is the effect of ethanol on the beetroot tonoplast membrane? And some of the skills we'll need include the mass balance equation, which you should have practiced already.
You'll need to be able to interpret your standard curve, so how to use the equation of the line to calculate beta-sion and concentration. You'll need to be able to use a pipette. which you'll learn in class and how to write a predictive hypothesis. Before we get into that I want to point out that if you are in a Bio 1 lab and there is a fire alarm then the procedure is to go down the hall and out the stairs over on this end of the building cross the street and meet in the faculty parking lot. So this is Western Avenue here and you meet in the faculty parking lot.
If you are on this end of the building, maybe in another class or something, you would meet in the yard between the Lewis Science Center and the Ferris Center. And if you are watching this video in the spring semester of 2021, remember that there are designated directions in the hallways in the Conway Corporation Center of Sciences here. So the entrance is officially at the front of the building. and hallways are one-way directions and those are laid out with signs. I'm not going to go over all of that right now.
In case of tornado, what we would do is we would head out from lab and we would we would make our way from the Bio 1 lab here down the hall and go to the basement of Lewis Science Center. And just so you have a heads up, some of the doors in the Bio labs are designated as entrances or exits only. At least so long as we have to be careful about COVID-19.
If this is later, later when you're watching this, then we've all been vaccinated, and perhaps we're lucky enough that that's not an issue anymore. Okay, so a few things we might have to discuss to get something out of this lab is, you know, why are we here? What are we doing? Well, we're going to be looking at beets, but we don't really care about beets as an organism. We're not...
going to go to a beet farming convention or something with this data. We're using beets as a model organism because we know they have beta cyanine inside and that beta cyanine will leak out if beet cell membranes are damaged. So their membranes are a lot like our membranes.
They're a lot like the membranes around the coronavirus or around bacteria. So if we know what can damage their membranes, we'll have a better understanding of how and what damages our membranes. or other cell membranes, these plasma membranes. So what is the function of a membrane?
It's the boundary between the outer world and the inner world of the cell. It's selectively permeable. It allows some things in and keeps other things out. It's composed, if you don't already know, of a phospholipid bilayer where we have these hydrophilic heads, these phosphate-bearing heads that are hydrophilic, they can face water.
And we have these hydrophobic lipid tails that look kind of like a sandwich when you look at a cell membrane in cross-section. What I mean by that is the phospholipid heads are like the bread and the lipid tails, the phosphate heads are the bread and the lipid tails are kind of like the cheese in between. Okay, so this area is hydrophobic.
But then also embedded in these phospholipid bilayers are proteins. So there are proteins that cross to both sides of the membrane. There are proteins that are simply embedded on one side or the other, and those proteins have an number of functions. So what might damage a membrane?
Well bear in mind that these phospholipids are kind of like grease, they're kind of like oil. So soap would damage a membrane because soap is an amphipathic molecule just like a phospholipid is essentially, meaning soap can stick to the polar part and it can stick to the nonpolar parts. And why is that important?
Well soap can stick for example to the nonpolar part of of a phospholipid and it can stick to water. and suddenly The two can interact and the membrane can come apart. Alcohol might damage membranes because alcohol is amphipathic. It has a polar part and a nonpolar part.
It could potentially dissolve the cell membrane or it might actually denature, meaning change the shape, of the proteins in the membrane causing them to leak. Heat might damage membranes causing molecules to move rapidly and come apart. The structure might come apart. Lots of things might damage membranes.
We are going to focus on ethanol. Just bear in mind that we're using plant cells. Plants have a cell wall on the outside made of cellulose. And don't confuse that for the cell membrane, which is made of phospholipids, which is just inside that cell wall typically. And also there's a membrane called the tonoplast that...
surrounds the organelle called the central vacuole inside of a beat cell and that central vacuole is where beta-cyanin is stored. So if we damage the plasma membrane and the central vacuole then that beta-cyanin might leak out into our test tube and then we can measure it using our spectrophotometer. So you're going to need to use the mass balance equation this week because you're going to be making dilutions of alcohol, of ethyl alcohol, ethanol. You'll be given a stock solution of about 95% ethanol, and you're going to make different dilutions ranging from 0% ethanol to 60% ethanol is what you'll be testing. And I'm not going to go over the mass balance equation in this video.
I'll let your lab instructor do that. You're also going to write a hypothesis for this lab, a predictive hypothesis. And so I'm going to give you an example. I like... and your instructor may give you slightly different instructions, but I'm going to give you the way I prefer to see a hypothesis.
If and then. After the word if, you provide your hypothesis. And by definition, a hypothesis is an explanation, not a prediction.
An explanation. Why do you think something's going to happen? After and, you just write a real brief summary of what the experiment is.
And then after then, your prediction. What will happen? So for example. If ethanol denatures membrane proteins and beet segments are treated with varying concentrations of ethanol, then membrane leakage will reflect the amount of damage, the membrane will be resistant to ethanol at low concentrations, and begin to leak when membrane proteins are denatured.
So I would expect one that you come up with to be a little less wordy than that, probably, but if you read the manual you'll know that beets can produce their own ethanol in anaerobic conditions. That's background for your lab report right there. And so if they make their own ethanol, that probably means they're resistant to a little bit of it. Otherwise, they would kill themselves when they make ethanol, right?
Their membranes would come apart. So we have to use logic, have to use background information to make a prediction that takes all of that into consideration. And what you might want to do then is sketch your hypothesis on page 23 of your manual. Unless your manual's changed and it's no longer page 23, but it should be page 23. When you do that, you draw an xy-axis and then you simply label the axes with a title. You don't have to put numbers on it.
Your x-axis, for example, would be ethanol concentration. Your y-axis would be beta-cyanin concentration. You just draw a line showing what your hypothesis would look like graphed out.
What would the major trends of the data look like if your hypothesis were supported? So you want to think about what the dependent and independent variables are for the day. That's something you should always think about ahead of time because that's often a quiz question when you get into lab. You might want to think about what your control would be.
If ethanol mixed with water is what you're testing, you know, you're making dilutions of ethanol, what would a good control be for the week? So what you'll do is you'll set up a data collection table on page 25 and you're going to run your experiment. Now, when you're when you're. Running that experiment, when you're designing it, keep in mind you're probably going to be making five different concentrations of ethanol and a control. So six total concentrations, right?
And each concentration you're going to do three times. That way you can put error bars on your figure, standard error bars, when you make your figure later on. So five concentrations of ethanol plus a control and each one you'll do three different times. Record all your data.
using the spectrophotometer at 535 nanometers. You're going to use that examine tool and you're going to get absorbance at 535 nanometers. Don't forget to make any observations on qualitative data because that's important for your results section of your lab report.
What you will do is record all your data in a data collection table and then you will convert it all to concentration using your standard curve from the last lab. When you are done clean everything up as well as you can, rinse things out, unplug your spectrophotometers, log off the computer. Remember to take really good notes this week as it is important as you will eventually be writing a lab report over this lab. in most lab sections at least. So what you do first, just so you understand what's going on, you're going to make these different concentrations of alcohol, of ethyl alcohol, and you're going to put them into a test tube.
Your lab manual will tell you how much to put into each test tube, or your instructor will, and you'll put ethanol into each test tube. And then what you're going to do is you're going to take a cork borer, which is this thing right here, and you're going to stab it into a beat, and that's going to make a core like this. You're going to carefully cut that into 5mm thick disks like we see here. You're going to do that for all of your test tubes.
You're going to rinse off those disks under cool water to get rid of the beta cyanin that leaked out when you cut them. You're going to plop them into your ethanol solutions and let them incubate, that means sit there, for 5 minutes. There is an example incubation table in your manual that you can use if you want which will help you keep track of when beets went into the alcohol and when they have to come out.
What you do is you let them sit there for five minutes. Your instructor may tell you to agitate them every once in a while. Give it a little swirl to help increase the contact of the beet with ethanol.
Then you'll pipette out that ethanol and beet juice basically and you'll put it into a cuvette which is the little container that goes into the spectrophotometer. You'll record the absorbance at 535, and then you'll go on and test your other samples. So that's basically what you're doing this week. So you'll do that for something like, what is it?
So there's five treatments plus three more. So that's going to be at least 15, about 18 different test tubes you'll be doing and 18 or so beat fragments you'll be using if I did the math right in my head. Okay, so that's what you're doing this week. Make sure you dress appropriately for lab. Have a look at the lab safety stuff in the appendix of the manual.
You want to have closed toe shoes on so we can't see your toes, right? Can't see your feet. You want to cover your midriff with a shirt.
And other than that, just, you know, wash your hands when you're done with lab. Be safe. Wear a mask and you'll have a great lab.
Be ready always for a quiz. Always look over the manual. Watch this video ahead of time.
You should be in good shape for any potential lab quizzes. Lab quizzes often have mass balance equation questions, or questions about what the dependent or independent variable is for the day, or questions about a previous lab. You want to keep on the ball for those because they're easy points if you keep up. Alright, see you soon.