Exercise 8

Cell Fractionation Supplement

How to use this supplement:

• Read the first four pages of the exercise in your manual (Introduction & Principles).

• You do not need to complete the prelab exercise that was originally scheduled.

• The following pages replace the Procedures & Worksheet.

• Paper copies of the supplemental materials will be provided in lab. You do not need to print this.

• The Worksheet is due at the end of lab (or a minimal extension if the instructor decides) and a Report of photos is due at the beginning of the next lab (after Spring Break).

Procedures:

Keep fractions on ice whenever possible and work quickly to avoid breakdown of cellular structures and components.

A. Preparation of Yeast Cells

Follow these steps each time it is called for in the procedure:

Pellet Spin the cells for 5 minutes in the appropriate centrifuge for the tube you are using and pour off the supernatant. Be sure to pour the Softening Media into the hazardous waste container.

Stain Stain with Orcein and Methylene Blue

Take an aliquot of your sample for each of the dyes and stain separately

For each dye, on a piece of parafilm, combine and pipet up and down to mix:

7.5µL of sample + 3µL dye + 4.5µL water

Count Using the 1mm square in the center, select 3 of the 25 squares to count, preferably from two corners and the center.

Record each value and take an average. Calculate the concentration of cells per mL appropriately taking into account the volume. Hint: the volume under a 1mm square is 0.1 µL. (Refer to the Hemoglobin/RBC lab for more info)

Be sure to record your observations throughout the procedure. In addition to drawings of what you see under the microscope, also take photos that will be submitted as a report.

1. Each group should obtain a portion of the yeast culture per the instructions of your lab instructor. Remove aliquots and stain your cells.

2. Count the cells using a hemocytometer. If the concentration is too high, dilute an aliquot of your sample with water so you can obtain the best estimate of cell number.

Note:

Remember: a cell count between 25 and 250 is the best. Record your data and observations on the data sheet.

The cells still have their cell walls, so they are resistant to osmotic stress and can be resuspended in water until we digest their cell walls.

3. Based on your counts, transfer 5 x 107 cells to an appropriate size tube (1.5mL microfuge tube or 15mL tube). If you have fewer cells than called for, transfer the entire amount. If you have cells leftover, save the remainder of your cells until the end of lab in case you need them.

4. Pellet the cells using a tube appropriate for the amount of culture you need to use: use the microfuge for ≤1.5mL; use the clinical centrifuge for >1.5 mL.

5. Resuspend the cells in 500 µL of water by gently pipetting up and down. This step removes any residual culture media that may interfere with subsequent steps. If your cells are not already in a 1.5 mL microfuge tube, transfer them to one. Stain your cells and Count.

6. Pellet the cells in the microfuge. Resuspend the cells in 250 µL of Softening Media. Add the Softening Media to your tube under the snorkel. Incubate for 15 min at room temperature before continuing to the next step.

7. While the cells are in softening media, pre-cool the Lysis Buffer and Dounce homogenizer on ice (see Step 10 and Section B).

8. Pellet the cells in the microfuge and pour off the supernatant into the hazardous waste container. Resuspend the cells in 250µL Spheroplasting Media. Reserve the tip to mix your sample in the next step.

9. Add 2.5 µL Lyticase, mix by pipetting up and down with the 1mL tip you used to add the spheroplasting media. Incubate for 15 minutes at room temperature.

10. At the end of the incubation, place the tube in a microfuge at 4°C and pellet the cells by spinning for 5 minutes. Removal of the lyticase solution is important so that continued digestion does not damage the cells. Pour off the lyticase solution and resuspend the cells in 250 µL cold Lysis Buffer and leave them on ice.

11. Gently mix by flicking and remove aliquots for staining. Stain your samples and Count.

B. Homogenization & Centrifugation:

1. Transfer your cells to the Dounce homogenizer containing 1mL of Lysis Buffer that has been prechilled on ice. Use 5 strokes with Pestle A to lyse the cells. Check an aliquot of the cells by Staining and observing with the hemocytometer. The hemocytometer will allow you to better judge what you are seeing based on the expected size. If you have cells/nuclei to count, Count them and record your results. Continue to lyse the cells using 5 strokes and then checking them under the microscope. Evaluate your observations to determine the number of strokes that yields the best results.

2. When lysis is sufficient, add 3 mL of cold Lysis Buffer to the homogenizer, pipet up and down gently to mix and aliquot 1 mL into each of four microfuge tubes labeled A through D. Centrifuge as indicated in part C.

Since we are still developing the procedure, we may choose to omit sections of the lab, but you should understand all of the principles and be able to answer questions related to this. Your instructor will tell you which, if any, sections to omit.

C. Centrifugation:

1. Centrifuge each tube using the Spectrafuge microcentrifuge:

Table 1. Centrifugation Parameters1

Tube Speed (in rpm) RCF Time (in min) RCF-min

A --- --- --- 0

B 3,500 1,000 xg 10 1 x 104

C 8,000 5,000 xg 10 5 x 104

D 14,000 16,000 xg 30 5 x 105

1 Abbreviations: rpm = revolutions per minutes, RCF (see 2), min = minutes, g = gravitational force 2 RCF, relative centrifugal force (sometimes known as g force), is dependent on the speed and radius of the rotor. 3 RCF-min is a measure of centrifugal force during centrifugation and is the product of the g force and minutes.

2. Label three 1.5 mL microfuge tubes B-s, C-s, D-s. As each centrifugation is completed, immediately remove the supernatant from the tube carefully and transfer the contents to the appropriate tube.

3. Analyze each supernatant for the presence of cells (using Methylene blue), nuclei (using Orcein)

and for alkaline phosphatase activity. The analysis will be divided among students in a group of 4. Two students (one for each dye) will be responsible for staining and taking photos. The remaining students will be responsible for analyzing enzymatic activity. Data will be collected and shared within a group.

D. Analysis of Fractions:

Assay for Acid Phosphatase:

1. Label six glass tubes: A, B, C, D, Blank, Enzyme. Pipet the following into each of these tubes:

0.75 mL (i.e. 750 µL) Citrate Buffer

0.75 mL Substrate (i.e. pNPP)

2. Start the reactions by adding one of the following as appropriate:

0.75 mL Fraction (A, B, C, or D as appropriate)

0.75 mL Citrate Buffer (for the blank)

0.75 mL Enzyme (for a positive control)

Note the starting time of each reaction and incubate all tubes at 37C for 15 minutes.

4. At the appropriate time, stop each reaction by adding 4 mL of NaOH.

5. Set the spectrophotometer to 405 nm, zero the spectrophotometer using the blank and measure the absorbance of the remaining 5 tubes. Observe the color in the tubes and record your observations in the table

Analysis for presence of nuclei:

Take an aliquot of each fraction and stain as indicated at the beginning of the procedure. Tabulate your data in the tables provided.

Report

See instructions on the next page

Report

In addition to the Worksheets below, your group will also compile the photos you collected from observations in the microscope. The worksheet will receive an individual grade and the report will receive a group grade, so each student’s grade will be compiled from these two components. For the report, use the Sample Descriptions from Table 4 as headers and insert your photos. Give the report the title indicated below. Be sure to list all of your group members (first and last names). Submit this to your instructor in whatever form requested (e.g. electronically, in paper, or both). Your report should look something like the example shown below.

Exercise 8 Cell Fractionation Report

Group Members: _____________________________________________

Sample: Yeast Culture before Centrifugation (Methylene blue)

Sample: Yeast before Softening (Methylene blue)

Sample: After Lyticase (Orcein)

WORKSHEET: NAME ____________________________________

Cell Fractionation Lab period/Instructor ______________________

A. Observations Before Cell Lysis:

1. Draw what you see in one medium square (i.e. 1 of the 25 in the large central 1mm2 square) containing the yeast cells before lysis.

Methylene Blue Orcein

2. Based on what you see in the hemocytometer, estimate the size of the cells. Show your

calculations. You may want to refer to the earlier red blood cell lab.

B. Observations After Cell Lysis:

1. For one sample, draw what you see in one medium square. Indicate which sample you observed to make the drawing.

C. Analysis of Acid Phosphatase Enzyme Activity:

1. In the table below, record absorbance readings and observations of the color of the samples.

Table 2. Acid Phosphatase Activity in Fractions

Fraction Absorbance, A405 Color (scale 0 to 5)§

A

B

C

D

Enzyme

Blank NA‡

§ Estimate the color observed in each tube

‡ Non-applicable for the absorbance reading since used to blank the samples

D. Observations Throughout the procedure: 1. Record your general observations and counts in Table 4 at the end of this document. 2. Based on your data, answer the questions below to explain your conclusions? Depending on the

progress your section makes, you may be able to answer more or fewer of the questions. Bullet

your answers to represent each question.

a. Some technical questions you may answer: Was it easy to see the cells? Did the orcein

stain the nuclei well? Did the nuclei only stain when cells were lysed or were the nuclei

evident when cells were intact? Was it easy to tell when the yeast cells were intact? Was

there a lot of debris obscuring the view?

b. Some general observations: What size were the yeast cells (see your earlier answer)?

Using this procedure, did the spheroplasts appear smaller, larger or about the same size as

the yeast cells? What size did the nuclei appear to be?

c. Some questions related to the purpose of the procedure: Were the cells intact (noting if

they should have been intact or lysed at the step you are observing)? What proportion of

cells were lysed based on the number of intact cells and nuclei? Does it appear that some

cells were lysed and their organelles destroyed (based on the number of cells plus nuclei

retrieved)?

E. Overall Conclusions

Summarize your overall conclusion: How did the procedure and experiment work? What should be the next thing to test? Suggest parameters if possible (e.g. incubate with lyticase longer).

F. Fractionation by Centrifugation: Applying what you know

If you were separating mitochondria, cytosolic components (e.g. enzymes of glycolysis), nuclei and intact cells using differential centrifugation, in what fraction would you predict to find them based on their relative size? Assume that you use conditions that are able to separate all of the components listed. Be specific in your answers.

Table 3. Prediction of Cellular Components During Cell Fractionation

Fraction Primary Cellular Component(s) in the Fraction

H

P1

P2

P3

S

Abbreviations: H = homogenate (or lysate), P1 to P3 = 1st, 2nd, 3rd pellets, S = final supernatant

Table 4. Cell Fractionation Exercise Supplemental Data Table Sample Description In the space provided also provide observations

Counts in each of 3 squares

Avg Count Diluted Conc Original conc Total

Yeast culture before centrifugation

(Methylene blue only)

1

2

3

Yeast before softening

(Methylene blue only)

1

2

3

After lyticase

(Orcein)

1

2

3

After lyticase

(Methylene Blue)

1

2

3

After Homogenization

(Orcein)

Strokes =

1

2

3

After Homogenization

(Methylene Blue)

Strokes =

1

2

3

Sample Description In the space provided also provide observations

Counts in each of 3 squares

Avg Count Diluted Conc Original conc Total

After Homogenization

(Orcein)

Strokes =

1

2

3

After Homogenization

(Methylene Blue)

Strokes =

1

2

3

After Homogenization

(Orcein)

Strokes =

1

2

3

After Homogenization

(Methylene Blue)

Strokes =

1

2

3

Fraction A

(Orcein & Methylene Blue)

1

2

3

Fraction B

(Orcein & Methylene Blue)

1

2

3

Sample Description In the space provided also provide observations

Counts in each of 3 squares

Avg Count Diluted Conc Original conc Total

Fraction C

(Orcein & Methylene Blue)

1

2

3

Fraction D

(Orcein & Methylene Blue)

1

2

3

SAMPLE CALCULATIONS:

Show a set of sample calculations that represent how you calculated the values in the table.