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Postby mikki » Wed Nov 09, 2005 9:57 pm

Anaerobic respiration in yeast:
In this exercise, we will examine the effect of inhibitors and enayme specificity on the fermentation pathway employed by yeast cells. The product of fermentation, CO2, can be collected and measured as an indicator of the respiration rate. The enzyme enolase catalyzes the conversion of phosphoglycerate into phosphoenol pyruvate. This reaction is dependent on Mg ions and is inhibited by fluroide ions (sodium flouroide, NaF, percipitates Mg ions and thus inhibits the pathway). Fluoride is toxic to humans so HANDLE WITH CARE!

Construct FIVE respirometers (indentical to the enzymometer used in previous lab) and label the tubes well with a group name and the tube number. Fill a small test tube with the liquides indicated below. Slide a large test tube over the small one as far as possible, then quickly invert the assembly so that no liquid is lost. Immediately measure the length of the airspace aboce the liquid in millimeters. Place the tubes in a 37 degress Celsius water bath. The airspace should be carefully measured at 15 minute intervals for one hour.

Tube 1. 5 Ml yeast suspension, 10 mL distilled H2O
Tube 2. 5 Ml yeast suspension, 5 mL 5% glucose, 5 mL h2O
Tube 3. 5 Ml yeast suspension, 5 mL 5% glucose, 5 mL 0.01M NaF
tube 4. 5 Ml yeast suspension, 5 mL 5% glucose, 5 mL 0.05M NaF
Tube 5. 5 Ml yeast suspension, 5 mL 5% glucose, 5 mL 0.10M NaF

What was the effect of NaF on carbon dioxide production? Did the different concertrations of NaF inhibitor affect respiration?

Second part of lab

The effect of enzyme specificity on yeast respiration will be evaluated using lactose. Baker's yeast is unable to metabolize lactose, which is a dissacharide. The enzyme that will be used in this exercise will demonstrate how a nonutilizable sugar can be enzymatically converted into a sugar that can be metabolized. Lactaid is a lactase (galactosidase) that breaks apart lactose into the monosaccharides glactose and glucose.

Construct Four Respirometers and label the tubes well with a group name and the tube number. Fill a small test tube (same directions go here as up top. Place in a 37 degree's Celsius bath water for an hour and measure every 15mins of that hour.


Tube 1. 10 mL 5% + 5 mL yeast suspension
Tube2. 10 mL 5% glucose + 2 drops of lactaid solution (one tablet crushed into powder and added to 5 mL of water) + 5 mL yeast suspension
Tube 3. 10 mL whole milk (5% lactose) + 5 mL yeast suspension
Tube 4 10 mL whole milk (5% lactose) + 2 drops of lactaid solution (one tablet crushed inot powder and added to 5 mL of water) + 5 mL yeast suspension.

Questions on this experiment on the following post. I want post this before something happens and I accidentally delete it or something.
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Re: Questions

Postby biostudent84 » Wed Nov 09, 2005 10:00 pm

mikki wrote:Questions on this experiment on the following post. I want post this before something happens and I accidentally delete it or something.


A good plan ;)
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Postby mikki » Wed Nov 09, 2005 10:00 pm

According to your results, were the yeast cells able to metabolize lactose?

How did the addition of lactase affect the production of carbon dioxide (respiration rate)? Why?

Lactose intolerance is a condition in which people are unable to digest lactose. How does the product Lactaid assist these people?

Why were the respirometers incubated at 37 degrees Celsius?
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Postby mikki » Wed Nov 09, 2005 10:04 pm

Oh and I have to have a hypothesis for each of these tubes in each experiment. God I will be so glad when this thing is over with Thursday.
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Postby mikki » Wed Nov 09, 2005 10:06 pm

This is the experiment that I kept posting questions on and nobody could help me because they didn't understand my questions. They were a bit unclear, but that is because I wasn't there for the lab. I hope this will help you guys to help me finish writing my lab report. I do appreciate the time taken to help us bio challenged students.
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Postby biostudent84 » Wed Nov 09, 2005 10:10 pm

A quick question before I write out the answers...I've done this lab before.

Have you done this lab yet in class? Or are you just writing your hypotheses?
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Postby mikki » Wed Nov 09, 2005 10:13 pm

Last post I promise on this topic. :-) This is what she wants in my discussion part of the report.
Discuss or explain (in detail) the results.
Make conclusions based ont he results.
Do the results show anything surpirising? If so, what could have caused such results?
Are the results what were expected? Explain the use of any controls. Discuss sources of error that may have occurred.
Make reference to the hypothesis. Was the hypothesis accepted or rejected? Explain why

MUST INCLUDE:
Address each hypothesis for each tube
Address experimental error and make suggestions to imporve the procedure in the future.
Make Conclusions based on reults

I wonder if she will want a urine sample, blood test and my first born along with my report?
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Postby mikki » Wed Nov 09, 2005 10:14 pm

No they did the lab on Halloween and I had to miss class to take my 4 year old trick or treating.
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Postby biostudent84 » Wed Nov 09, 2005 10:28 pm

Lactose, the disaccharide, does react, but it only releases a large amount of carbon dioxide gas. This is because yeast cells do not possess the ability (enzymys) to break the disaccharides down to the monoccharide: glucose. Thus, bloating and gas is often experienced by individuals that are unable to make lactase...lactose intolerant folk.

Adding Lactaid (lactase synthesized in a lab) to the solution broke down the lactose to glucose so that the yeast could then ferment THAT molecule.

Incubating the solutions increased the rates of reactions by raising the temperature to the optimum...in the case of glucase (enzymes that break down glucose), 37*C is the optimum temperature, and the reaction will proceed at the highest rate.

I know I did not specifically answer your questions. However, I did give you the background information that your professor would have given you in your lab. *grins* I gave you just enough rope to hang yourself with.
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Postby mikki » Wed Nov 09, 2005 10:34 pm

lol, and hang myself I know I can do. Thank you, honestly, I'd be pulling my hair out right now if I had no help at all.
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Postby mikki » Thu Nov 10, 2005 2:38 am

Can someone take a look at this website and tell me if I can incorporate all of these items with in my discussion?
Code: Select all
http://www.harford.edu/faculty/fgrimm/newhome/bio/lab11.htm


I don't know that all of them apply to the lab I posted. Is Mg ions the same thing as Magnesium?
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Can I include these?

Postby mikki » Thu Nov 10, 2005 2:39 am

sorry I'm having problems on the site for some reason. Everything is crawling and sometimes the browswer gives me a MySql server errors. I don't have sql so I am assuming it is referring to this websites server.
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