Manual for the Cell Biology practicals and workshops Page 1 BSC IN MEDICINE 2013-2014 Block 1.1: Fundamentals of Medicine Authors: Prof. N.A. Bos and Dr D. Opstelten Cell Biology Discipline Group Faculty of Medical Sciences University of Groningen MANUAL for the CELL BIOLOGY PRACTICALS AND WORKSHOPS
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Manual for the Cell Biology practicals and workshops Page 1
BSC IN MEDICINE
2013-2014
Block 1.1: Fundamentals of Medicine
Authors:
Prof. N.A. Bos and Dr D. Opstelten
Cell Biology Discipline Group
Faculty of Medical Sciences
University of Groningen
MANUAL
for the
CELL BIOLOGY PRACTICALS AND
WORKSHOPS
Manual for the Cell Biology practicals and workshops Page 2
Table of Contents
Activity Subject Page
Practical
Microscopic examination of blood cells 3
Workshop
Nanotomy and diabetes 17
Workshop
Protein synthesis 32
Workshop
Energy metabolism 45
Workshop
Pathology 53
Workshop
Cell division and DNA analysis 57
The following sources of information can be used during the workshops to supplement
this manual:
Digital material: Celweb
http://www.rug.nl/umcg/onderwijs/nestor/celweb
or:
via Nestor, Block 1.1,
Fundamentals of Medicine/Workshops
All assignments can be found on the web, which also contains colour versions of the
illustrations in this manual. Celweb also contains the answers to the questions in this manual.
These will be made available after a workshop has ended.
Printed material:
Your own copies of Alberts et al., Essential Cell Biology, Kerr, Functional Histology and
Jorde et al., Medical Genetics. Make sure you bring these textbooks to every workshop.
NB: Methyl green pyronin (MGP): DNA = blue; RNA = red.
Manual for the Cell Biology practicals and workshops Page 41
Space for your drawing
Theoretically expected LM image of plasma cell after H+E staining
Space for your drawing
Theoretically expected LM image of plasma cell after MGP staining
Assignment 4. Observe plasmablasts in tissue sections and compare the results with
the theoretical expectations.
a. Take slide 34 (HE) and read the description.
Slides 33 and 34. Rabbit spleen, 2.5 days after intravenous injection of paratyphoid
fever, stained with methyl green pyronin (33) or hematoxylin-eosin (34). Among other
details, these specimens show the white pulpa with periarteriolar lymphoid sheath and
the follicle with follicle centre, lymphocyte ring and peripheral zone with weakly
basophilic peripheral zone cells, which are often distinguished by a light nucleus also.
A relevant feature is the presence of plasmablasts with strongly basophilic cytoplasm in
the periarteriolar lymphoid sheath.
b. Locate the plasmablasts with objectives 4x and 10x and study them at 40x. Properly
adjust the aperture diaphragm. Only then will you be able to see all the plasmablast
details, for example the parietal chromatin. You can also use the 100x lens in combination
with immersion oil for a more detailed view (see Chapter 1 Practical, Block 1.1 or
Celweb).
c. Does your observation match the drawing of your expectation made in Assignment 3? If
necessary, change your drawing.
Manual for the Cell Biology practicals and workshops Page 42
d. Also answer question (c) for slide 33 (MGP) and change your drawing if necessary.
e. How can the differences between expectation and observation be explained?
Assignment 5: Study and draw exocrine pancreatic cells in sections in which they
have been stained with various stainings. On the basis of your observations, explain
the functional difference between protein synthesis in the exocrine pancreatic cell
and in the plasmablast.
a. Study the descriptions of slides 90 and 92, Guinea pig pancreas, stained with
hematoxylin-eosin (90) or methyl green-pyronin (92).
Slide 92: Guinea pig pancreas, stained with hematoxylin-eosin.
Slide 90: Guinea pig pancreas, stained with methyl green-pyronin.
The pancreas is a protein-synthesizing alveolar gland that consists of lobules
interspersed with fatty tissue containing the larger secretion ducts, arteries and veins.
The lobules consist of acini, secretion ducts and isthmi (strips of tissue connecting the
acini and the secretion ducts).
The cytoplasm of the acinar cells exhibit strong basal basophilia (rough endoplasmic
reticulum) and strong apical acidophilia (secretion droplets).
In addition to this exocrine glandular tissue, the pancreas also contains endocrine
tissue: the pancreatic islets, where insulin and other hormones are produced. These will
be dealt with in Workshop 9, Endocrine organs.
b. Try to identify acini, isthmi, blood vessels and pancreatic islets in slide 92 under low
magnification.
c. Find a detailed section of an acinus.
d. Draw several acinar cells and label the structures that you have drawn.
Space for your drawing
Manual for the Cell Biology practicals and workshops Page 43
e. In which part of the cell (apex or base) is most of the stained protein located?
f. What does hematoxylin stain?
g. Take slide 90 (Guinea pig pancreas, stained with methyl green-pyronin) and draw some
of the acinar cells as they appear with this staining.
Space for your drawing
h. What are the differences with the HE specimen?
i. At which side of the cell is most of the RNA located?
j. Which EM-observable structures are the cause of the strong basal basophilia and strong
apical acidophilia? (See Figure 11, p. 18.)
k. How does the processing of the proteins produced in the plasma cell differ from the
processing of those produced in the pancreatic cell?
Manual for the Cell Biology practicals and workshops Page 44
The next workshop cannot be
completed successfully without
extensive preparation (study of the
relevant material) in advance.
Do not forget to bring your textbook
(Alberts) and lecture notes.
Manual for the Cell Biology practicals and workshops Page 45
Workshop : Energy metabolism Part of Block 1.1, Fundamentals of Medicine
Assignment Page Content
1 46 Studying the available energy sources.
2 47 Comparing carbohydrates and fats as energy sources.
3 47 Comparing aerobic and anaerobic metabolism.
4 48 Studying how and where glycolysis occurs
5 48 Studying how and where the citric acid cycle occurs
6 50 Studying how and where oxidative phosphorylation occurs
7 51 Answering questions about mitochondrial genetics
8 52 Calculating the energy yield of cellular respiration.
Prepare yourself for this workshop by studying the relevant material.
Introduction
Cells use ATP as an energy source. The two main sources of ATP are glycolysis and
oxidative phosphorylation. This workshop contains various assignments that will help you
understand this cellular process.
Learning objectives
Students will learn to
1. recognize and explain the structure of the mitochondrion
2. explain how the cell produces useful energy through cellular respiration
3. explain the differences between the breakdown of sugars and fats
4. explain the difference between aerobic and anaerobic metabolism
5. explain the relevance of glycolysis, citric acid cycle and oxidative phosphorylation to
energy metabolism
6. apply the principles of mitochondrial inheritance to patient diagnostics.
Assessment 1 and 5: practical test and written test.
1-8 (incl.) items in the written test testing the students’ knowledge and understanding.
Manual for the Cell Biology practicals and workshops Page 46
Assignment 1: Study the available energy sources.
The most common sugar in our body is glucose. This substance is stored in the form of
glycogen. Because the synthesis of glucose can only be achieved by green plants, we
must obtain this important fuel from our food. Our main source of glucose is starch.
a. Examine the nature of starch from a chemical perspective.
b. Where in the body is starch broken down into glucose?
c. Chemically speaking, how does this metabolic process take place?
d. Where in our body is glucose that we do not immediately need as fuel stored? Locate it
on the micrograph below (Figure 19).
Figure 19, rat liver, TEM 50,000x
Manual for the Cell Biology practicals and workshops Page 47
Assignment 2: Compare carbohydrates and fats as energy sources.
Figure 20. Chemical structure of glycogen (left) and triacylglycerol (right)
Discuss these structures to explain why fat is a much more practical storage medium than
carbohydrates.
Assignment 3: Compare aerobic and anaerobic metabolism.
When we exercise intensely for a long time, our muscles will incur an oxygen debt at some
point, when the fuels required for additional muscular effort can no longer be completely
metabolized.
a. Explain why we can still use extra carbohydrates (glucose) in such situations but not extra
fat or fatty acids to meet the increased muscular energy needs.
b. To double the muscular effort, how many times more glucose do we need under these
anaerobic circumstances than in the aerobic situation?
During the anaerobic breakdown of glucose, the muscle produces a lot of lactic acid.
c. From which substance is this lactic acid formed?
d. Give the complete chemical equation for this conversion.
e. What is the name of this type of reaction?
Manual for the Cell Biology practicals and workshops Page 48
f. Why is it highly important for the muscle that this conversion takes place?
Assignment 4: Study how and where glycolysis occurs.
Glycolysis only has one oxidation-reduction step.
a. Which step is that?
b. What precisely is oxidized and what is reduced?
c. Where does glycolysis take place in the cell and what are the three main products of
glycolysis in terms of cellular energy production?
Assignment 5: Study the citric acid cycle.
Cellular respiration takes place in the mitochondria. Figure 21 is an EM micrograph of a
mitochondrion.
a. Identify the following mitochondrial structures by placing the corresponding number at
the correct site:
1. outer membrane
2. inner membrane
3. cristae
4. matrix
5. intermembrane space
Manual for the Cell Biology practicals and workshops Page 49
Figure 21, EM of mitochondrion, liver cell, 50,000x
A cell can obtain much more energy from the same fuels by means of the citric acid cycle and
oxidative phosphorylation than through glycolysis alone.
b. Where are the enzymes of the citric acid cycle located?
Carbohydrate and fat metabolism have a common intermediate substance.
c. Which intermediate is this and how is it formed during carbohydrate metabolism and fat
metabolism respectively?
d. What is the main product of the citric acid cycle from the perspective of cellular ATP
production? How many molecules of this product are formed for each glucose molecule?
e. The citric acid cycle itself does not consume oxygen. Why then does the cell still need
oxygen to keep the citric acid cycle going?
f. Write down the net reaction of the citric acid cycle in such a way that it is clear that there
are an equal number of C, H and O atoms on either side of the reaction arrow.
Manual for the Cell Biology practicals and workshops Page 50
g. Suppose that it is possible to have the citric acid cycle run only once in a cell, with acetyl
CoA – with both C atoms radioactively marked – as the substrate. How many molecules
of radioactive CO2 would be formed during this one cycle? Explain your answer.
Assignment 6: Study oxidative phosphorylation.
a. What is the essential function of the mitochondrial inner membrane in ATP production?
b. Which general characteristics of the biological membrane allow the inner membrane to
perform that function?
Before you start on the next assignment, we advise you to study pages 93-100 of
ECB, which provide a brief summary of Chapter 3 reviewing free energy (G), free
energy change (ΔG) and other concepts. Pay special attention to the ‘chemical
equilibria’ section of Panel 3-1 and the explanation given of the relationship
between ΔG and K, the reaction equilibrium constant. Then study the following
sections in Chapter 14: ‘The redox potential is a measure of electron affinities’ (pp.
469-470) and ‘Electron transfers release large amounts of energy’ (pp. 470-472).
c. From an energy viewpoint, how many mol ATP can be produced by the oxidation of 1
mol NADH by O2? Follow this procedure to arrive at the answer:
1. Look up the values of the standard redox potentials of the NADH/NAD+ and
O2/H2O redox pairs (Panel 14-1).
2. Use these values to calculate the ΔEo of the NADH + H
+ + ½ O2 NAD
+ + H2O
reaction.
3. Then use the outcome of step 2 in the formula on page 471 to calculate the ΔGo of
this reaction.
4. Look up the value of the ΔGo for the ATP + H2O ADP + Pi reaction (Figure
13.7, p. 470).
5. Use the values found in steps 3 and 4 to calculate the maximum number of mol
ATP that can be formed by the oxidation of 1 mol NADH.
Manual for the Cell Biology practicals and workshops Page 51
(Pay attention to the + and - signs!)
d. How does the calculated value differ from the number of mol ATP that is actually
formed?
e. Can this be regarded as an efficient process? Compare it to other energy-producing
processes, for example a combustion engine.
f. What function does the proton gradient formed during the electron transport across the
inner membrane have in ATP synthesis?
The ATP formed by oxidative phosphorylation is released into the matrix.
g. How does this ATP end up in the cytosol?
Assignment 7: Answer questions about mitochondrial genetics.
Mitochondria have their own protein-synthesizing apparatus that produces several of the
enzymes involved in cellular respiration. In MELAS patients, the mechanism for producing a
particular mitochondrial tRNA is defective.
a. What consequences could this have for the energy metabolism of these patients?
b. Do transcription and translation occur in separate sites in mitochondria?
c. Where in nature do we find a protein-synthesizing mechanism similar to the one found in
the mitochondrial system?
Manual for the Cell Biology practicals and workshops Page 52
d. How do mitochondria multiply?
e. How is mitochondrial DNA inherited?
f. What effects does this have on genetic disorders that are caused by a mutation in
mitochondrial DNA?
Assignment 8: Calculate the energy yield of cellular respiration.
This final assignment may be completed at home: calculate how much ATP the cell can
obtain from carbohydrates and fats.
a. Calculate the number of ATP molecules produced per mol NADH to O2, when
1. 5 protons are pumped across the inner membrane for each electron passed on by the
respiration-enzyme complex
2. 3 protons have to undergo ATP synthase for the production of 1 ATP molecule from
ADP and Pi
3. 1 proton is used for the transport of ATP from the mitochondrion to the cytosol.
b. How much ATP is then produced by the oxidation of 1 glucose molecule through
glycolysis, pyruvate dehydrogenase and the citric acid cycle?
c. How much ATP is then produced by the oxidation of 1 palmitate molecule through fatty
acid oxidation and the citric acid cycle?
(Assume that NADH in the cytosol can yield as much ATP as NADH in the
mitochondrion and that the electrons of FADH2 can yield as much ATP as those of
NADH.)
Manual for the Cell Biology practicals and workshops Page 53
Workshop : Pathology
Part of Block 1.1, Fundamentals of Medicine
Assignme
nt
Page
Content
1 54 Normal cervix
2 54 Metaplasia
3 55 Dysplasia
4 56 Hyperplasia and atrophy
Introduction
Cells are highly adaptable to changing circumstances. Such changes are often evident from
changes in cell characteristics. The study of changes in individual cells is the subject of
Cytology. Often such changes can best be seen by investigating the cell complexes that make
up tissues. Similar changes in cells and tissues will also occur during illness. In Pathology,
the following terms are used to describe this type of adaptation or change.
Atrophy = cell size reduction
Hypertrophy = cell size increase
Hyperplasia = increase in number of cells
Metaplasia = a reversible change from one cell type to another cell type
Dysplasia = abnormal change in certain cell types
Cell death is another option for tissues to adapt to changing circumstances. Two types of cell
death are distinguished.
Apoptosis = programmed cell death (does not result in damage to surrounding tissue)
Necrosis = cell death resulting in damage to surrounding tissue
In this workshop, various physiological and pathological changes in cells and tissues will be
demonstrated with the help of several sections of epithelial tissue from the cervix and
endometrium. The function and form of epithelial cells have been explained in detail in Block
1.1.
Learning objectives
Students will learn to
1. use the concepts of atrophy, hypertrophy, hyperplasia, metaplasia and dysplasia to
describe changes in cells and tissues
2. explain the difference between apoptosis and necrosis
3. distinguish between normal and abnormal epithelium.
Assessment:
1 and 2 with knowledge items in the written test
3 with items in the practical and written tests involving the study of micrographs
Manual for the Cell Biology practicals and workshops Page 54
Assignment 1: normal cervix
To identify changes in a tissue, it is first necessary to study the normal structure of that tissue.
Micrograph 1 (see Celweb) is a histological section of the cervicovaginal junction. Study the
description. The endocervix consists of mucous epithelium, while the ectocervix consists of
stratified non-keratinizing squamous epithelium.
Draw several cells of the mucous epithelium.
Space for your drawing
Draw several cells of the stratified non-keratinizing squamous epithelium.
Space for your drawing
What is the fate of differentiated epithelial cells and how does this work?
Assignment 2: metaplasia
Epithelial cells may change from one cell type to another in response to injury. When the
damaged tissue has a normal structure but is located at the wrong site, this process is called
metaplasia. Metaplasia can be observed in the cervix, for example, when non-keratinizing
squamous epithelium is found among the cylindrical epithelium of the endocervix.
Study micrographs of the cervix taken with various magnifications (2a, 2b and 2c).
Does this epithelium look the same as the non-keratinizing squamous epithelium in image
1?
What type of injury could have caused this type of change in this tissue?
Assignment 3: dysplasia
Dysplasia occurs when the structure of the tissue becomes abnormal. Dysplasia is often
regarded as a precursor of neoplasms and can thus serve as an early indication of malignant
changes.
Manual for the Cell Biology practicals and workshops Page 55
Such changes are often first seen in cervical smears taken to detect possible tissue changes by
screening various cytological characteristics.
A database of micrographs of cytological sections has been created for this classification.
Two of these micrographs are available as examples. The first contains cells that can be
identified as normal squamous epithelium (image 3). The second contains dysplastic cells
(image 4) of which the original cell type is difficult to identify.
Draw several cells of normal squamous epithelium.
Space for your drawing
Draw several dysplastic cells.
Space for your drawing
Describe the differences between the cells in these two cytological sections.
The entire database can be accessed for further study.
When a cervical smear contains suspect cells, taking a biopsy is the next step. The next
micrograph is an example of a biopsy section.
Micrograph 5 shows poorly differentiated squamous epithelium among the normal
epithelium of the cervical crypts.
What is the ratio between nucleus and cytoplasm in normal tissue and in dysplastic
tissue?
How does the structure of the nucleus and cytoplasm differ between normal and
dysplastic tissue?
Manual for the Cell Biology practicals and workshops Page 56
Assignment 4: hyperplasia and atrophy
An example of physiological hyperplasia is the thickening of the endometrium (wall of the
uterus) during the menstrual cycle.
The number of endometrium cells increases to prepare for the implantation of a fertilized
ovum.
A large number of cell divisions can be seen in this tissue, even at higher magnifications.
Study micrograph 6 of an adult endometrium.
Draw several of the dividing endometrial cells.
Space for your drawing
Compare image 6 with image 7, a micrograph of the endometrium of a 70-year-old woman. It
clearly shows the atrophy of this tissue.
How many cell layers does the endometrium of image 6 contain and how many layers can
you count in image 7?
Manual for the Cell Biology practicals and workshops Page 57
Workshop : Cell division and DNA analysis Part of Block 1.1, Fundamentals of Medicine
Assignment Page Content
1 57 Studying cells photographed during various mitotic stages
2 60 Identifying, drawing and naming the mitotic stages in a cell section
with the help of LM.
3 61 Karyotyping the chromosomes in a human cell with the help of a
chromosome map
4 63 Discussing case 1.
5 65 Discussing case 2.
6 66 Discussing case 3.
7 67 Discussing case 4.
8 68 Discussing case 5.
Assignment 1 can be completed before the workshop begins. You can prepare for
assignments 4-8 by studying Essential Cell Biology, and finish them after the workshop,
if necessary, with the help of what you have learned.
Background information for assignments 1-3 can be found in Kerr and in this manual.
Learning objectives
Students will learn to
1. identify mitotic stages and explain the process involved
(a) in LM images of cells and tissues; and
(b) with the help of LM in sections of cultivated cells
2. identify numerical and structural chromosomal deviations in a karyogram
3. explain how a polymerase chain reaction (PCR) works
4. explain the principle underlying Southern blotting
5. explain Restriction Fragment Length Polymorphism (RFLP) and how it can be detected
6. interpret the results of an RFLP analysis for DNA diagnostic purposes
7. explain the concept of variable number of tandem repeat (VNTR)
8. interpret the results of a VNTR analysis for DNA diagnostic purposes. Assessment 1: Items in the practical and written tests involving the examination of micrographs 2: Items in the practical and written tests involving the examination of a karyogram 3-5, 7: Items in the written test, testing the student’s knowledge and understanding 6, 8: Items in the practical and written tests concerning analysis results
Manual for the Cell Biology practicals and workshops Page 58
Assignment 1: Study cells photographed during various mitotic stages.
(Use the description of cell division characteristics in the box on p. 55.)
Figures 22 to 31 are images of cells in interphase, prophase, metaphase, anaphase and
telophase in a fibroblast (connective tissue) culture. Fibroblasts are elongated cells that
sometimes have cytoplasmic processes. The cells were cultivated in a suitable medium that