BSC IN MEDICINE 2013-2014 Block 1.1: Fundamentals of Medicine · 2013-09-11 · Cells Practical : Blood cells Part of Block 1.1, Fundamentals of Medicine Foreword In these Cell Biology

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


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.

http://www.rug.nl/umcg/onderwijs/nestor/celweb


Cells Practical : Blood cells Part of Block 1.1, Fundamentals of Medicine

Foreword

In these Cell Biology Workshops, you will actively study the material. You will learn to use

the microscope to make close observations, to interpret what you see and to solve problems.

The learning objectives of each practical and workshop are listed in this manual. For you to

achieve these objectives, it is vital that you prepare for each workshop. Microscopy and

discussions with fellow students and supervisors will be your main learning tools. The first

practical is compulsory because it is vital that you learn to use the microscope correctly. The

other workshops are not compulsory. The skills obtained during the practical assignments in

the workshops will be assessed in the Cells practical test. To obtain a pass for this block, you

must pass this practical test. If the pace of a workshop is too fast for you, you can complete

some of the assignments – those for which you do not have to work with the microscope –

after the workshop. Answers to questions and explanations of problems will be published on

Nestor after each practical and workshop. We hope you will enjoy the work you will do for

these workshops.

In addition to this manual, you will have access to other learning sources, which are offered

in various forms.

Digital material: http://www.rug.nl/med/onderwijs/nestor/index:

Go to: Celweb, practicals for students of Medicine

http://www.rug.nl/umcg/onderwijs/nestor/celweb/index

Printed material:

Your own copies of Alberts et al., Essential Cell Biology and Kerr, Functional Histology.

Make sure you bring these textbooks to the practical.

Cells practical: learning objectives

Students will learn to

1. adequately use the light microscope to observe cells at magnifications up to 1000x

2. use the light microscope to recognize types of blood cells in a blood smear

3. develop a systematic approach to counting cell types in a specimen

4. correctly use the concept of ‘normal range’ within the context of diagnostics

5. relate morphological characteristics of the various types of blood cells to their functions

6. explain the principles of tissue preparation for light microscopy

7. derive information about cellular or subcellular components from colours and patterns in

tissue sections or images of such sections (‘interpreting’ images)

8. make a scientific drawing

Assessment

1, 3, 7 and 8: by the supervisor or assistant during the practical

5: self-test on Nestor

2.4, 6: items in the written test, some of which will be based on images

http://www.rug.nl/med/onderwijs/nestor/index

http://www.rug.nl/umcg/onderwijs/nestor/celweb/index


Assignm

ent

Page Content

1 4 Reading the information about the principles of the light microscope

2 6 Adjusting and focusing the microscope

3 8 Recognizing the various types of white blood cells

4 8 Performing a differential count of white blood cells

5 9 Answering the questions about the results and taking the quiz

6 10 Studying the information about staining methods and the accompanying

tables

7 13 Observing and drawing basophil and eosinophil granulocytes stained

with May-Grunwald/Giemsa and answering the questions

You must prepare for assignment 6 and part of assignment 1*. Assignment 5 may be

completed after the practical.

For a proper use of the microscope, you must have some idea of the relevance and function of

its main parts.

* It is assumed that you do not know how to work with a microscope. If you do, continue

with assignment 1 at your own pace.

Assignment 1: Carefully read the following background information about the principles

of light microscopy while referring to the actual microscope. Look at the illustration for

the position of the various parts of the microscope.

Light

The microscopes used in this practical have a light source built into the base, the light of

which shines through the specimen from underneath the slide.

Condenser

The condenser transforms the parallel light from the ‘infinite focus’ light source into a cone-

shaped beam of light. The slide must be situated in the top focus of the condenser. The

condenser is fixed at its highest position.

Objectives and numerical aperture

The beam of light from the condenser illuminates a small area of the slide. This cone-shaped

beam will exit the slide in reverse and is received by the objective. The largest cone of light

that an objective can receive is stated on the objective as the numerical aperture (sin ½ angle

of maximum cone of light). The numerical aperture is the most important parameter of an

objective lens. The larger the aperture, the larger the resolving power (the minimum distance

between distinguishable objects).


Ocular lens (eyepiece)

The upper limit of magnification (objective x ocular lens) is determined by the numerical

aperture of the objective. Usually, this limit is given as 1000 x the numerical aperture. So an

objective with a numerical aperture of 0.65 has an upper limit of magnification of 650x. If

this objective has a magnification of 40, this means that the eyepiece can have a

magnification of around 16x at most (16 x 40 = 640). A higher power eyepiece will result in

deterioration of the image and loss of focus. In practice, a 10x eyepiece is suited to nearly all

purposes.

Diaphragm

Visibility of a normally stained slide is best when the cone of light from the condenser is

almost the same size as the maximum cone of light the objective can receive. This is the case

when 80 to 90 percent of the lens’s numerical aperture is used. If the aperture of the

diaphragm is increased further, the cone of light will be too large and reflected light will enter

the objective, which will make the image appear fuzzy. If the aperture is reduced too much,

the image seems sharper, that is, more details seem to appear, but these are actually artefacts

produced by light-bending phenomena.

There are two methods for setting the proper diaphragm aperture.

1. Take the eyepiece off the eyepiece tube. Near the bottom of the tube, you can see where

the diaphragm opens and closes, letting in more or less light. First close the diaphragm

completely and then open it to such an extent that the resulting opening is almost as large

as the objective opening.

2. Focus on a slide until the image is sharp. Then close the diaphragm to such an extent that

the image clearly deteriorates and open it again slightly until the image becomes sharp

again. This will achieve the optimum compromise between depth of focus, contrast and

imaging.

Oil immersion

In a system of lenses with air/glass boundaries, the aperture of the cone of light and the

objective can never exceed 1 due to the angle of total reflection at the air/glass boundary. By

using immersion oil – which has a similar refractive index as glass – between the slide and

the front lens of the objective, the aperture can be increased to around 1.3. Note that this

cannot be done with normal objectives. Special oil immersion objectives must be used for

this purpose. When working with such objectives, the diaphragm must be fully open.

General guidelines

1. Only put a slide under the microscope when the low-power objective (4x) is in position.

This also applies to slide removal. The higher power objectives (10x, 40x and 100x) have

very short working distances, which means it is easy to damage or soil their front lenses.

Focus on the specimen with the 4x objective before switching to a higher power

objective.

2. Avoid touching the front lenses. If necessary, clean them with a soft cotton cloth (clean

handkerchief) or lens paper, either dry or with a diluted solution of synthetic soap.

3. Keep adjusting the micrometer to bring the entire thickness of the specimen into focus.

4. Relax your eyes; in other words, do not try to accommodate – imagine that you are

looking into the far distance. Adjust the eyepieces of a binocular microscope until you see

a single image when looking with both eyes. (See also assignment 2)


Assignment 2: Adjusting and focusing the microscope

I. Optimum distance between two eyepieces of a binocular microscope

1. Check whether you can move the eyepiece tube. If so, tighten the clamping screw.

2. Ensure maximum illumination.

3. Fully open the diaphragm with the handle under the stage. The condenser will then be

brightly lit.

4. Turn the scale on the left eye piece to zero.

5. Adjust the distance between the eyepieces until you see a single, wide and uniformly lit

area.

WRITE DOWN THE DISTANCE BETWEEN THE TWO EYEPIECES.

INTERPUPILLARY DISTANCE = …………

II. Observation of specimens

1. Rotate the nosepiece to bring the low-power objective (4x) into position.

2. Place a tissue section slide on the stage and move the stage with the stage motion knobs

to position the slide in the light.

3. Locate the specimen with the coarse adjustment knobs and focus with the fine adjustment

knob (=micrometer).

4. Rotate the 10x objective into position and focus. Use this objective to broadly examine

the specimen. Examine interesting sites in more detail with the 40x objective and then go

back to the 10x objective. Use the micrometer constantly to focus on various depth levels

of the specimen. Only remove the slide after rotating the 4x objective into position again.

5. The image may be out of focus when you are using both eyes. Perhaps the acuity of your

right and left eyes is not the same. In that case, focus with your right eye with the left eye

closed. Then close your right eye and focus the image by turning the left eyepiece.

THE POSITION OF THE LEFT EYEPIECE = ……

N.B. Do not accommodate; try to relax both eyes. Use the micrometer to focus, not your

eyes.

6. For proper illumination of the slide, the diaphragm must have the proper aperture (see

‘Diaphragm’ above).

7. Immersion oil must be used with the 100x objective (see ‘Oil immersion’ above and

assignment 7 below).



The following specimens are available for the rest of the assignments.

Slide number/code Type of Cells Staining

99 Whole blood May-Grunwald Giemsa

100 Fractionated blood May-Grunwald Giemsa

Patient A Whole blood May-Grunwald Giemsa

Patient B Whole blood May-Grunwald Giemsa

Patient C Whole blood May-Grunwald Giemsa

Patient D Whole blood May-Grunwald Giemsa

Assignment 3: Identify the various types of white blood cells

A simple centrifugation step suffices to separate most of the red blood cells from the white

blood cells in whole blood. The fraction with predominately white blood cells has been

smeared on slide number 100.

Try to locate at least one example of each type of white blood cell – lymphocytes, monocytes

and granulocytes (neutrophils, eosinophils and basophils) – in slide 100. Use the examples on

the internet (Celweb, Nestor) or in your textbook to help you identify these types.

Assignment 4: Perform a white blood cell differential count

Slides 99, A, B, C and D are blood smears of anticoagulated blood. In these specimens, the

ratio between white and red blood cells is such that you really have to look for the rare white

cells among the abundance of red cells.

Systematically examine slides 99 and patients A, B, C and D in the following manner.

1. First use the low-power objective to locate an area of good quality, that is, an area where

all cells can be seen in isolation.

2. Determine the direction in which you want to examine the specimen further.

3. Rotate the nosepiece to select the 40x objective.

4. Count* (tally) the nucleated cells of the various categories in the visible area and write

the counts in the table below.

5. Now move the slide in the direction you have selected (see 2) and count* the nucleated

cells in this adjacent area.

* Counting method: Determine the type of the first 100 nucleated cells you encounter and

enter the counts in the table below.

Category Slide 99 Patient A Patient B Patient C Patient D

Neutrophil

granulocytes

eosinophil

granulocytes


basophil

granulocytes

lymphocytes

monocytes

Total

N=100

N=100

N=100

N=100

N=100

Assignment 5:

a. What are the normal ranges of the various types of blood cell?

b. Which of the slides (99, Patients A, B, C, D) you have examined has/have a normal

count and which is/are abnormal?

c. Which preliminary diagnosis or diagnoses can be made on the basis of the specimen

or specimens with an abnormal blood count?

d. Compare the counts you have found and your conclusions about these blood counts

by taking the ‘Blood smear test’ quiz on Nestor. Each student must take this quiz

individually, since it also serves to record student attendance. Each student must

therefore log on individually so that their results will be recorded correctly.

Assignment 6: Study the following principles of tissue preparation for light microscopy

To make biological material – usually tissue samples – visible under the light microscope, it

must be prepared. This preparation consists of three main steps.

A. Fixation

B. Embedding and cutting

C. Staining

In the following, the parts in italics plus the two tables are part of the core subject matter.


A. Fixation

The purpose of this step is to fix histological structures in such a way that the image seen

through the microscope is as close to reality as possible. Autolysis must be prevented and

dynamic processes must be stopped. Essentially this means using chemical fixatives to render

insoluble structural components such as proteins, nucleic acid-protein complexes and lipid-

protein complexes. Chemicals that may be used for this purpose include alcohols (ethanol,

methanol), acetone, aldehydes (formaldehyde and glutaraldehyde), acids (acetic acid,

trichloroacetic acid and picric acid = 2,4,6-trinitrophenol) and metallic compounds

(bichloride of mercury = sublimate, chromium(VI)oxide, dichromate ions and osmium

tetroxide).

Each of these compounds has advantages and disadvantages when used as a fixative.

Alcohols and acetone, for example, precipitate proteins through dehydration and denaturation

but do not make nucleic acids insoluble in water. Moreover, they dissolve lipids and shrink

the tissue. Conversely, acetic acid makes the tissue swell and precipitates nucleic acids.

Trichloroacetic acid precipitates both proteins and nucleic acids. Picric acid also has this

effect but tends to hydrolyse (split) nucleic acids. Mercury and chromium create crosslinks in

proteins, while osmium does the same in lipids. Formaldehyde forms methylene bridges

within and between protein molecules. Glutaraldehyde does the same (it has two aldehyde

groups) and is mainly used in electron microscopy. Histologically interesting fixatives usually

consist of a combination of the above compounds. Bouin’s fixative, for example, contains

picric acid (sometimes in alcohol), formaldehyde and acetic acid. Zenker’s fixative consists

of sublimate (bichloride of mercury), dichromate and acetic acid.

Freeze-drying (with liquid nitrogen) is another fixation method. It allows fast processing of

the material and histochemical and cytochemical reactions that are usually not possible in

tissues embedded in paraffin.

B. Embedding and cutting

Light microscopy sections (slices of tissue) are usually less than 10 μm thick; if thicker, the

section will absorb too much light. Moreover, the depth of focus of the microscope is small.

In itself, fixed tissue is too fragile to cut into such thin sections. For this reason, it is first

embedded in a wax such as paraffin. This is only possible, however, after dehydration of the

fixed tissue, which still has a 60 percent water content. This is done with what is known as an

alcohol series. Xylene or another compound with a high refractive index (1.5) is used to

remove the last alcohol and ensures that the tissue is transparent after the molten paraffin has

penetrated into the tissue.

Electron microscopy sections are cut from tissue samples embedded in plastic and are 20 to

100 nm thick.

C. Staining for light microscopy

The molecules of stains consist of two parts – a chromophore group, which provides the

colour, and a binding group, which ‘glues’ the chromophore group to the substrate (proteins,

nucleoproteins, carbohydrates or lipids of the cell or tissue). The chromophore groups consist

of complex organic chemical structures. The binding groups are either

1. amines, in which case the stain is referred to as a basic dye; or

2. carbolyxic acids, sulfonic acids or phenolic hydroxyl groups, in which case the stain is

referred to as an acid dye.


In general, basic dyes are represented by the symbol B+OH-. B+ represents the staining ion –

a cation. Acid dyes are represented by the symbol H+Z-. Here, Z- represents the staining ion,

in this case an anion. The simplest model of the staining reaction is a binding of positive dye

ions to negatively charged substrate molecules or, conversely, a binding of negative dye ions

to positively charged substrate molecules. The charge of both substrate and dye depends on

the acidity of the environment. Sulfonic acid groups are negative at pH>1, phosphoric acid

groups at pH>3, carbolyxic acids at pH>4 and phenolic hydroxyl groups at pH>10. (With

respect to the phenolic-OH, incidentally, this depends strongly on the rest of the molecule;

picric acid, for example, has a negative charge at pH>0.) On the other side of the spectrum

are the amine groups, which are positive at pH<9.

As far as charge is concerned, proteins can be regarded as mosaics of COO- and NH3+

groups, which gain and lose their charge depending on the prevalent pH. There is a pH at

which the sum of all charges is zero. This is referred to as the isoelectric point (IEP). Many

serum and cytoplasm proteins have IEPs between 5 and 7. Eosin, a dye with a negative

charge at pH>3, is able to stain proteins with a nett positive charge, that is, below their IEP.

Nucleic acids have a low IEP (2 for DNA and 3 for RNA). Dyes with a positive charge (e.g.

methylene blue) can stain DNA and RNA if these have a nett negative charge, that is, above

their IEP.

Table I gives an overview of the charges of dyes at neutral pH.

To sum up, material will be stained at a certain pH, namely one at which

proteins are below their IEP (positively charged); and

nucleic acids (and acid polysaccharides) are above their IEP (negatively charged).

A suitable compromise is a pH of around 6.

Staining with hematoxylin is a more complex affair. This ‘nuclear stain’ is the most

frequently used stain in routine histological procedures. It works with a dye solution

consisting of a metal ion such as mordant and hematin (a derivative of hematoxylin). The

staining/binding mechanism cannot be described in terms of interactions between ions.

Instead, coloured complexes are formed with the metal ion (iron or aluminium). The bonds

between metal ion and substrate, but not those between dye molecules and metal ions, can be

broken by acid, which means that superfluous and non-specifically bonded dye can be

washed away. This process is referred to as differentiating. In itself, hematoxylin is useless as

a stain. It must first be oxidized to hematin. This is done in an acid environment with O2 from

the ambient air or by adding Fe3+, which is reduced to Fe2+ in the process. Aluminium-

hematin complexes first stain red. When pH is increased to 7, the stains turn indigo. This

procedure is referred to as blueing. Iron-hematin complexes are coloured blue-black. Hematin

deeply stains the cell nucleus (DNA) but does not stain basophil cytoplasm (RNA) to the

same degree.

If phosphotungstic acid or phosphomolybdic acid is added, some combinations of two acid

dyes will stain both collagen (matrix) and cytoplasmic proteins, with each of the dyes staining

one of these components. The mechanism underlying this effect is not yet understood.

Staining according to Mallory or Masson, for example, has this effect.

Table II provides information about stainings that are often used in histology.

Table I. Commonly used histological stains; classification based on ability to stain either

nucleic acid (primarily nucleus) or protein (primarily cytoplasm) at pH=6


Nuclear stains = basic (+) Cytoplasmic stains = acid (-)

methylene blue eosin (red)

crystal violet acid fuchsin (red)

toluidine blue light green

basic fuchsin (red) aniline blue

methyl green (blue!) orange G (yellow)

pyronin (red) picric acid (yellow)

nuclear fast red

Table II. Commonly used tissue section stains

Nucleus Cytoplasm Medium

(esp. nucleic acid) (esp. protein) (esp. collagen)

H + E hematoxylin eosin eosin

(blue black) (red)

Masson hematoxylin acid fuchsin light green

(red)

Van Gieson hematoxylin picric acid acid fuchsin

(yellow)

Mallory nuclear fast red orange G aniline blue

(yellow)

NB:

Methyl green-pyronin: DNA (blue); RNA (red); protein remains unstained

May-Grunwald/Giemsa (eosin and methylene blue + azure B): particularly suited for

staining blood and bone marrow

Assignment 7: Locate both a basophil and an eosinophil granulocyte in slide 100 (stained

with May-Grunwald/Giemsa) and draw it after examination with the 100x oil immersion

objective.

Instructions for using the oil immersion objective

1. Bring the specimen into focus using the 4x objective.

2. If the diaphragm is not at full aperture already, open it fully.

3. Rotate the nosepiece to the 100x objective and then rotate further to halfway between the

100x and 4x objective.

4. Place a drop of immersion oil on the specimen.

5. Rotate the 100x objective back, so that it touches the oil.

6. Focus with the micrometer.

7. Closely observe several nucleated cells and then draw them (see p. 10 for drawing

instructions).

8. When you remove the slide, ensure that no other objective comes into contact with the oil

by following these instructions:

Rotate the nosepiece to position the 4x objective above the slide.

Remove the slide and clean it immediately with a tissue.

Immediately clean the oil immersion objective also, first with a dry tissue and then

with a tissue soaked in a diluted synthetic soap solution.


Read the instructions on the next page before making your first scientific drawing. You

will do many such drawings during the following workshops and practicals, which will

help you to learn how to interpret micrographs or microscopic drawings. This will be

your first hands-on experience of making such a drawing and interpreting it.


Making a scientific drawing

Why?

The microscope is a tool for searching for and identifying histological and other structures.

Drawings are a good tool for studying a specimen closely. Moreover, they provide a useful

check, also after the actual examination.

How ?

1. Begin by locating a suitable part of the section (good surface area, no artefacts, sensible

diameter).

2. Write down the slide number on the drawing paper.

3. Use B/W and/or coloured pencils.

4. First do a preliminary sketch (on paper or in your mind’s eye); reserve sufficient space for

captions and any detail drawings you may wish to add.

5. Make the drawing.

Be accurate. For example, a nucleus is not a clump of chromatin with a uniform

colour; it has a particular structure that provides clues to the cell’s activity.

Ensure that you only draw what is actually visible. For example, cell membranes

CANNOT be seen under the light microscope.

Adapt the scale of your drawing to the size of the relevant details that must be

included. Perhaps these details will be too small to be rendered faithfully at the scale

you have selected.

It may be necessary, therefore, to make both a general drawing and an enlarged

detail drawing.

If you add a detail view, frame the area in that view on your general drawing.

Make a detailed drawing of just one or only a few cells and sketch in the rest.

Determine what is characteristic of this specimen and bring this out in your drawing.

6. Identify each structure or substructure with a label and connecting line.

7. Indicate the magnification or magnifications used.


Space for your drawing

Basophil granulocyte

Which components of the basophil granulocyte cytoplasm does May-

Grunwald/Giemsa stain and what does this tell us about the function of these blood

cells?


eosinophil granulocyte

Which components of the eosinophil granulocyte cytoplasm does May-

Grunwald/Giemsa stain and what does this tell us about the function of these blood

cells?


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 your lecture notes.


Workshop : Nanotomy and diabetes Part of Block 1.1, Fundamentals of Medicine

INTRODUCTION Nanotomy applied in a rat model for Type 1 diabetes

Nanotomy is a recent development in electron microscopy (EM) that enables us to study

tissues, cells, organelles and macromolecules in a ‘Google Earth’-like fashion.

To be studied in advance:

1. Ravelli et al. SREP01804 (2013), which can be found at:

http://www.nature.com/srep/2013/130508/srep01804/full/srep01804.html

2. As an alternative, study the summary in the Dutch journal of diabetology, which can be

found via Nestor (Ravelli et al. NTD, 2013).

Learning objectives


1. explain the technical principles underlying transmission, scanning and scanning

transmission electron microscopy

2. recognize and interpret various items in images obtained with electron microscopy:

tissue properties

cell types

organelles

macromolecular complexes

3. explain with an example how functional information about cell function (e.g. secretion)

can be obtained with nanotomy

4. describe the general structure of the cell, various cell organelles and macromolecular

complexes and name their functions

5. apply the above knowledge to pathophysiological analyses; in this workshop, this mainly

concerns Type 1 diabetes

Assessment

1 and 5: knowledge items in the written test

2, 3, 4 items in the practical test related to micrographs

http://www.nature.com/srep/2013/130508/srep01804/full/srep01804.html


Assignment

Page

Content

1 19 Naming the cell organelles in an EM drawing

2 20 Studying the technical principles underlying transmission EM

3 21 Recognizing and interpreting a TEM image of a biomembrane

4 22 Studying membrane transport

5 23 Nanotomy: EM of tissues, cells, organelles and macromolecules

6 28 Testing what you have learnt by observing a healthy islet

7 29 Analyzing islets affected by Type 1 diabetes

8 30 Studying the technical principles underlying SEM

9 30 Recognizing and interpreting a SEM image of a cell

10 31 Comparing the main aspects of the various EM techniques

NB: Assignments can be done in advance and may also be completed after the

workshop.


Assignment 1. Name the cell organelles in an EM drawing

A cell contains cell organelles that are essential to the functioning of that cell. Depending on

cellular function, one type of cell will have a higher number of certain organelles than other

types. This workshop and the ones following it pay a lot of attention to cells with various

functions. To check if you know the various cell organelles, examine the following schematic

drawing of a cell (a pancreatic exocrine cell).

1. Identify the various cell organelles by placing the right number at the right line.

2. State the main function(s) of the organelle in the table.

If necessary, consult your textbook.

The answers will be published on Celweb after this workshop.

From: Pappas GS, Laboratory Manual of Histology. Wm. C. Brown, Publ. Dubuque, IA, USA, 1990.

Structure Position Structure Position

1. centriole 9. microtubules

2. cytosol 10. mitochondria

3. Golgi complex 11. microvilli

4. nucleus 12. nucleolus

5. nuclear envelope 13. plasma membrane

6. nuclear pore 14. ribosomes

7. lysosome 15. rough endoplasmic

reticulum

8. microfilaments 16. secretion droplets


Assignment 2: Study the technical principles underlying Transmission Electron

Microscopy (TEM).

In transmission EM (TEM), a high voltage generated between a heated cathode (incandescent

filament) and an anode produces a beam of electrons. One or more condenser lenses focus

this beam onto the plane of focus, in which a very thin section has been placed that can be

penetrated by the electrons. The objective lens produces a magnified image of the object,

which is projected on a screen or recorded with a camera. Modern TEM microscopes provide

magnifications of up to 300,000 times, with a resolution of approximately 2 nm, of sections

which are about 60 nm thick and have a maximum diameter of 3 mm.

The biological material in these very thin sections (mainly consisting of C, H, N and O) does

not scatter electrons sufficiently to provide an image, which is why the specimen is stained

with heavy metals, which do scatter the electrons. The most common contrast medium is

osmium tetroxide (OsO4), which specifically binds to double bonds of lipids and fixes them

by creating crosslinks, thus making membranes visible. Other common contrast media

include uranyl acetate and lead citrate, which are used when the pH is such that all cellular

components (DNA, RNA, proteins, polysaccharides) are negative and, in principle, should

therefore absorb the medium (albeit to varying degrees).


Assignment 3: Recognize and interpret a TEM image of a biomembrane.

Membranes consist of a bilayer of phospholipids, with hydrophobic (apolar) tails pointing

towards each other and hydrophilic (polar) heads forming the outer boundary of the

membrane. The apolar tails may also have double bonds that may react with OsO4, as is

shown in Figure 1 on the left. This will improve the contrast of the two apolar membrane

layers in particular. If this structure is sufficiently magnified with TEM, it manifests itself as a

‘railway track’ (see Figure 1 on the right).

OsO4 binds to apolar tails ‘railway track’

OsO4

OsO4

Fig. 1. Diagram of imaging a membrane with TEM.

Fig. 2. TEM image of part of a plasma membrane.

Questions:

In cross section, the cell membrane appears to have ..... layers at the high magnification

used in Figure 2; at lower magnification, however, it would be seen as having ..... layer(s).

Chemically, the membrane can be regarded as a bilayer consisting of

………………………… .

This bilayer contains islets consisting of ………………………… .

The exterior of the bilayer is lined with ……………………… and ……………………. .

Physiologically, the membrane has ..... layers.


Assignment 4. Study membrane transport

Membranes can form compartments. The plasma membrane is the boundary between the

cytoplasm and the extracellular environment. However, this boundary is dynamic; various

transport processes enable the release and uptake of substances. Examples of such processes

are shown in Fig. 3, which illustrates glucose-induced insulin secretion. Study the figure and

pay specific attention to the various molecules and substances transported across the

membrane.

Which forms of transport can be distinguished?

Possible answers: exocytosis / concentration / voltage-dependent influx / efflux inhibition

The glucose uptake by GLUT2 is mainly dependent on: ...............

The ATP-sensitive K+ pump is a form of ...............

The voltage-gated calcium pump is a form of ...............

The insulin secretion is a form of ...............

Fig 3. Insulin secretion in beta cells caused by rising blood sugar levels. Absorption of

glucose by GLUT2 and glycolytic phosphorylation of glucose cause an increase in ATP/ADP

ratio, which inactivates the potassium channel, which in turn leads to depolarization of the

membrane and the subsequent opening of a voltage-dependent calcium channel. The rise in

calcium concentration leads to the release of insulin. (Source: www.betacell.org)


Fig. 4. The beta cells (Fig. 3) are grouped in islets of Langerhans in the pancreas. The

pancreas further consists of exocrine tissue producing digestive enzymes. In addition to

insulin, the endocrine tissue (islets of Langerhans) also produce other hormones. Source:

http://www.bu.edu/histology/p/10401loa.htm

Assignment 5: Nanotomy: EM of tissues, cells, organelles and macromolecules

As described above, nanotomy was applied in an animal model of diabetes. Study the

ultrastructure of an islet of Langerhans, by visiting www.nanotomy.nl and clicking the large

(grey) islet on the left. This dataset can be studied in a ‘Google Maps’-like fashion. Click the

IIP icon at the top left if you need further instructions. Study the annotations and answer the

following questions. The numbers correspond to the annotations; 1A, for example, refers to 1

Supracellular. If you mouse over this box, you will be presented with a submenu with various

items, e.g. A islet.

1. What is the largest structure that can be recognized? And what is the smallest? What are

their respective dimensions? You can move the annotation menu by right-clicking the grey

bar at the top and dragging the menu to another position to reveal the scale of the image.

Largest structure: Approx. dimensions:

Smallest structure: Approx. dimensions:

1A. What main differences between islets of Langerhans and exocrine pancreas can be used

to distinguish the one from the other?

What are their respective functions?

1B. Which cell can be found in the capillary?

http://www.nanotomy.nl/


1C. Which two cell types can be found in the venule? What are their main differences?

1D. The centroacinar lumen is part of:

a. the endocrine pancreas and contains enzymes

b. the endocrine pancreas and contains hormones

c. the exocrine pancreas and contains enzymes

b. the exocrine pancreas and contains hormones

1E. Shown here is a cross section of a bundle of unmyelinated axons. This bundle shows up

more or less by accident; the axons are electron-lucid (lighter) and contain round tubules and

light-grey filaments. How many axons can be distinguished?

1F. The nuclei of some exocrine cells are not visible. Why?

2A. The exocrine cell contains a lot of ER for protein synthesis. Vesicles secrete it into:

a. the blood

b. the digestive tract

2B. The alpha cell produces glucagon, which is visible in the dark vesicles. What effect does

glucagon have on blood sugar levels?

a. increase

b. decrease

2C. The beta cell in this image is in poor health. The rat has diabetes. We will later study the

differences with a healthy rat. Which organelles can you distinguish? There are only a few

hormone-containing granules visible, particularly on the left below the nucleus. The crystal-

like shape is characteristic and is even more conspicuous in human beta cells. Which

hormone is this?

2D. Although somatostatin-producing delta cells are also a constituent of the islets, they only

account for a few percent of the islets’ cells. We can determine the various cell types because

their granules have different structures. How can somatostatin granules be distinguished from

glucagon or insulin granules?

2E. Is the centroacinar cell important for hormone production or for the development of

secretory ducts? How do you know?

2F. The cell in 2F (a pericyte) separates the hormone-producing ............... pancreas from the

enzyme/proenzyme-producing ............... pancreas.

2G. Inflammatory cells can be seen here because the sick rat has developed an immune

response targeting the islets. What type of leukocyte is shown here? How do you know?


2H. What is the approximate size of this erythrocyte?

2I. What is characteristic of the nucleus of a monocyte?

2J. This phagocyte is (a) passive or (b) active. I know which is correct because ....

2K. The granulocytes studied in the Blood Cell practical had spherical or round shapes. These

clearly look different. What has caused this difference?

2L. The leukocyte contains black specks. What is their approximate size? What could these

be?

2M. It is clear that the small platelets have a more heterogeneous content than the adjacent

erythrocytes. How many platelets can be seen in this venule?

3A. The endoplasmic reticulum (ER) is important for ................. . The black specks are about

.... nm in size; they are ..... at the interior/exterior of the ER.

3B. It is easy to identify a mitochondrion by its:

3C. The cell nucleus contains ......... . It is possible to distinguish several kinds. Which?

Functionally this reflects a process called ........................ .

3D. The Golgi apparatus can be distinguished from the ER at the nanoanatomical scale

because it:

The Golgi apparatus is important for:

4A. Zymogen granules contain enzymes and proenzymes in the ............... cells.

4B. Insulin is produced by the ............ cells.


4C,D,E. Exosomes are secreted vesicles. Is it possible to understand exosome release on the

basis of the various stages? Yes/No

How does exosome release differ from vesicle fusion in, for example, insulin secretion?

4F. Glucagon is produced by the ............ cells.

4G. Somatostatin is produced by the ............ cells.

5. Structure and function of vesicles.

5A. Dense bodies have been given their name because:

5B. Lysozomes play an important role in:

5C. What cell in this micrograph contains numerous caveolae?

5D. Caveolae are just about the smallest vesicles in existence. What is their diameter?

5E. And what is the diameter of the lipid droplets?

5F. On the basis of which two characteristics can an early endosome (as shown here) be

distinguished?

5G. Clathrin-coated pits are typically involved in (a) endocytosis or (b) exocytosis.

5H. If the multivesicular bodies fuse with the plasma membrane, the following may happen:

6A. Crystae are characteristic of:

6B. Which type of atom has accumulated in this ‘tangled’ membrane?

6C What cells can be distinguished? The fenestrae facilitate ............. .

6D. In this image, the basement membrane is situated between two types of cells. These are:

6E. The basement membrane is present as a complex whole. We are still inside the diabetic

rat. Morphologically, the two nucleated cells are obviously leukocytes; they are in different

locations, however. The left leukocyte is situated in ................ but the other is not. Explain

what may be the matter here.

7. Macromolecules can just be distinguished at the magnification used for this image. Various

characteristics ensure that different macromolecules can be recognized.

7A. How many nuclear pores can be distinguished in the ENTIRE cross section of the nuclear

membrane?


7B. This is the top of the nucleus, which again has several nuclear pores. How many?

Draw a 3D reconstruction of one nuclear pore based on 7A/B.

7C. Polysomes consist of:

7D. Draw a model of one polysome with five ribosomes. If you can, indicate the 5'UTR and

3'UTR. If you can, also schematically draw the pro-proteins.

7E. Desmosomes are specialized cell-cell contacts which are especially important for:

(a) tissue firmness or (b) barrier formation.

7F. Tight junctions are specialized cell-cell contacts which are especially important for

barrier formation. Unlike desmosomes, there is no large concentration of intermediate

filaments on the cytoplasmic side. Which barrier can be seen here?

7G/H. Collagen is (a) cytoplasmic or (b) extracellular. Its main function is:

7I/J. Centrioles are often found at perinuclear sites. They mainly consist of:

7K. Each cell has a centriole pair. Give a rough estimate of the number of centrioles that

should be visible in this dataset. Explain your answer.


Assignment 6. Test what you have learnt by observing a healthy islet (dataset 1).

Distinguish ten types of cells. Which characteristics enable you to distinguish one cell type

from the other?

1.

2.

3.

4.

5.

6.

7.

8.

9.

10.

Draw four different organelles.

1.

2.

3.

4.

Draw five different macromolecules or macromolecular complexes and state a function of

each.

1.

2.

3.

4.

5.


Assignment 7. Islets affected by Type 1 diabetes After this introduction to the EM of cells,

organelles and macromolecules, we will now examine the effects of Type 1 diabetes in the rat

model. Go back to the homepage (www.nanotomy.nl) and compare dataset 1 (control) with

dataset 5 (diabetes).

1. What is the blood sugar level of the healthy animal? ...... And what is the level of the

diabetic animal? ......

2. This is caused by a deficiency in:

3. This is caused by the breakdown of beta cells. Insulitis is clearly visible; in dataset 5 we

find many more:

4. Beta cell destruction can be seen from the following (name at least three characteristics):

5. Diabetes patients benefit from the following treatment:

6. Overtreatment leads to ............................... and can be compensated with

.............................. . Comatose patients benefit from: ...............................

You have now examined two stages. If there is still time left after the workshop, test what

you have learnt by studying the other stages. You can also do this at home; the data remains

available.


Assignment 8: Study the technical principles underlying Scanning Electron Microscopy

(SEM).

As with TEM, Scanning EM (SEM) also involves focusing an electron beam through

condenser and objective lenses. The sample is also situated in the plane of focus. In principle,

SEM examines material that is not thin enough for the electrons to pass through. The primary

electron beam is not stationary as in TEM but scans the surface by moving along a grid. The

electron beam scans the sample surface line by line. Either secondary electrons (SE2) are set

free in the sample or the beam electrons are reflected back (backscatter electrons). Both types

can be used to produce an image of the sample surface. If the sample is a very thin section,

the electrons will obviously pass through. If a detector is then placed under the section, a

TEM image can be made with a SEM microscope. This is referred to as STEM: scanning

transmission EM.

Assignment 9: Recognize and interpret a SEM image of a cell.

Figure 5 7 is a micrograph of a liver parenchymal cell. A conspicuous feature of the gall

capillary is a membrane specialization in the form of small membrane protuberances.

Fig. 5. Rat liver parenchymal cell. SEM, 6,100 x (left) and 16,000 x (right).

What is the name given to such protuberances?

What does the cell achieve with this membrane specialization?

In which type of transport is this specialization usually involved?

What does the liver cell transport with the help of this specialization?


Assignment 10: Make a succinct comparison of the EM techniques described above

(TEM, SEM, STEM). Describe the underlying technical principles and give an

application of each.


Workshop : Protein synthesis Part of Block 1.1, Fundamentals of Medicine

Assignment Page Content

1 33 Answering the questions about the nuclear processes involved in

protein synthesis

2 36 Answering the questions about the cytoplasmic processes involved

in protein synthesis

3 39 Converting an EM drawing into the LM image that can be expected

after various stainings

4 41 Studying and drawing plasmablasts and comparing your

expectations with the actual images

5 42 Studying and drawing a pancreatic acinus after HE and MGP

staining. Compare the exocrine pancreatic cell with the plasmablast

Thoroughly prepare for assignments 1 and 2 by studying the relevant material.

Introduction

The biochemistry of protein synthesis will be explained in the theme lectures. This workshop

will deepen your understanding of where and how this protein synthesis occurs in the cell.

Various questions will help you to follow this process as far as it is visible in the cell and to

discuss it with your fellow students. The plasmablast (a cell that produces and secretes

immunoglobulins – antibodies) will serve as the example of a protein-synthesizing cell.

In the first part of the workshop, groups of around eight students must answer the questions

about the micrographs by actively using their textbooks (e.g. Essential Cell Biology and

Functional Histology).

This will be followed by three individual assignments focusing on the interpretation of LM

and EM images of various protein-synthesizing cells.

Learning objectives


1. explain the process of protein synthesis in a eukaryote cell and identify the cell

organelles involved in this process

2. use EM images to explain the involvement of the various cell organelles in various

stages of protein synthesis

3. explain the principle of cellular protein sorting

4. convert an EM image of a cell into an LM image and vice versa

5. where possible, relate morphological characteristics of various types of protein-

synthesizing cells to their functional activity

6. efficiently use various sources of information (textbooks, lecture notes and digital

sources) to carry out the assignments.

Assessment

1, 2, 3 and 5: in the practical and written tests with, for example, items related to EM

micrographs.

4: by the lecturer or assistant during the workshop. 6: in the open-book part of the written

test.


Figure 10. Diagram of a TEM image of a plasmablast.

Assignment 1: Answer the following questions and carry out the following

assignments focusing on the cell nucleus.

The following structures can be recognized in the nucleus:

1. Euchromatin.

2. parietal chromatin

3. nucleolus

4. nuclear membrane

5. nuclear pore

a. Label these structures in Figure 10 with the above numbers.

b. Which chromatin contains the DNA that is in the process of transcripting the DNA into

RNA?


c. Plasmablasts and plasma cells produce immunoglobulins (antibodies). In addition to

mRNA for immunoglobulin synthesis, many other mRNA molecules are produced in the

nucleus. Estimate the percentage of mRNA in a plasma cell that codes for

immunoglobulins. Give arguments for your estimate. For which other types of proteins do

the other mRNA molecules code?

d. What is the function of the nucleolus?

e. What is inside the nucleolus? See also Figure 11.

Figure 11. Bat pancreas, exocrine cell

f. What is the similarity between parietal chromatin and nucleolus-associated chromatin?


g. Explain why the nuclear membrane is sometimes referred to as the nuclear envelope by

referring to Figures 10 and 12.

Figure 12. Rat liver parenchymal cell, RER and nuclear membrane

h. Draw a diagram of the way in which the nuclear envelope encloses the nucleus.

i. During which stage of cellular development is this nuclear envelope structure useful?

j. What can be found on the exterior of the nuclear envelope? See Figure 12.


k. Where does the transport to and from the nucleus take place?

Assignment 2: Answer the following questions and carry out the following

assignments relating to the cytoplasm.

a. Place the numbers below at the correct locations in Figure 10.

6. free ribosomes

7. rough endoplasmic reticulum (RER)

8. Golgi complex

9. mitochondria

10. lysosomes

b. What is the chemical composition of ribosomes?

c. Explain the organization of the ribosomes in Figure 13.

Figure 13. Rat fibroblast, free polysomes

d. Why are there no polysomes visible on the RER in Figure 12?


e. Where is the RER lumen situated in Figure 14? Mark the site with a star.

Figure 14. Rat liver parenchymal cell, RER membranes. R = ribosome.

f. What do the arrows in Figure 14 point to? Take the magnification into account!

g. There are newly formed proteins in the RER lumen. How did they get there?

h. Why is it that some proteins remain in the cytosol?

i. What happens to the primary protein chains in the RER lumen?

j. How are proteins transported from the RER to the Golgi complex?

k. What happens to proteins in the Golgi complex?

l. Figure 15 is a micrograph of a Golgi complex stained with polysaccharide.


Explain the differences in density for the various components.

Figure 15. Golgi complex of human intestinal epithelial cell. Silver staining. The arrow

indicates the direction of the Golgi complex from the cis to the trans side.

m. How do proteins exit the Golgi complex?

n. Indicate the direction of the protein transport in the Golgi complex in Figure 16.

Figure 16. Golgi complex of mouse pancreatic glandular cell

o. Which compartments can the proteins go to after leaving the Golgi complex?


Finally, a task that will help you to summarize the entire process:

p. Describe how and where new ribosomes are made.

Functional morphology of protein synthesis

For a better understanding of the normal or abnormal functioning of certain cells, it is

important that you are able to convert LM colour images to EM B/W images and vice versa.

Sometimes theoretical expectations are not in line with reality. It is often possible to explain

these discrepancies by taking the functional characteristics of cells into account.

Assignment 3: The drawing of a plasmablast in Figure 10 is based on EM micrographs.

Draw a plasmablast as you would expect to see it under the light microscope if stained

with hematoxylin-eosin (HE) and methyl green-pyronin (MGP). (Consult Tables I

and II below, which you will already have encountered during the Blood cells Practical

in Block 1.1.)

Commonly used histological stains; classification based on ability to stain either nucleic

acid (primarily nucleus) or protein (primarily cytoplasm) at pH=6


Nuclear stains = basic (+) Cytoplasmic stains = acid (-)

methylene blue eosin (red)

crystal violet acid fuchsin (red)

toluidine blue light green

basic fuchsin (red) aniline blue

methyl green (blue!) orange G (yellow)

pyronin (red) picric acid (yellow)

nuclear fast red

Table II. Commonly used tissue section stains

Nucleus Cytoplasm Medium

(esp. nucleic acid) (esp. protein) (esp. collagen)

H + E hematoxylin eosin eosin

(blue black) (red)

Masson hematoxylin acid fuchsin light green

(red)

Van Gieson hematoxylin picric acid acid fuchsin

(yellow)

Mallory. nuclear fast red orange G aniline blue

(yellow)

NB: Methyl green pyronin (MGP): DNA = blue; RNA = red.



Theoretically expected LM image of plasma cell after H+E staining


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.


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.



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.


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?


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.


Workshop : Energy metabolism Part of Block 1.1, Fundamentals of Medicine


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


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.


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


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?


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


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.


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.


(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?


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.)


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


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


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.


Draw several cells of the stratified non-keratinizing squamous epithelium.


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.


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.


Draw several dysplastic cells.


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?


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.


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?


Workshop : Cell division and DNA analysis Part of Block 1.1, Fundamentals of Medicine


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.





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


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


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

stimulates their multiplication.

Staining: hematoxylin. Magnification factor: 3000x.

Fig. 22: Interphase

Fig. 23: Prophase 1

Fig. 24: Prophase 2

Fig. 25: Metaphase (frontal view)


Fig. 26: Metaphase (plan view)

Fig. 27: Anaphase 1

Fig. 28: Anaphase 2

Fig. 29: Telophase 1




Cell division characteristics

prophase

Early prophase or Prophase I: Onset of chromosome coiling results in breakdown of

parietal chromatin. Because the parietal chromatin has disappeared, the nuclear envelope

can no longer be seen under the light microscope, but it is still intact.

Late prophase or Prophase II: the nuclear envelope disintegrates; the chromosomes are

released into the cytoplasm. The centriole is duplicated. Both centrioles migrate to the

poles; onset of microtubule formation.

metaphase

All chromosomes are aligned in the equatorial plane.

More microtubules are formed, leading to the formation of the mitotic spindle.

anaphase

The centromere divides, resulting in the compete separation of the two chromatids

(daughter chromosomes) of each metaphase chromosome.

Chromosomes migrate towards the poles.

telophase

Reconstruction of the nuclear envelope.

Uncoiling of the chromosomes.

Note that this division into stages is artificial, in the sense that the transition from one phase

to the next is a gradual process. It is difficult to distinguish cells in the late telophase from

those in the early interphase, but interphase cells are separated from each other, while

telophase cells are joined like ‘Siamese twins’.

Assignment 2: Identify, draw and name the mitotic stages in a cell section with the

help of LM.

Slide 2: Vero cell line, cultivation period 2.5 days. Staining: hematoxylin.

(Cultivated by the Virology department of the Laboratory for Medical Microbiology,

Groningen.) The etymology of the name ‘Vero cells’ is unknown. The lineage was created in

Japan when researchers cultivated renal cells from an African green monkey.

Find at least one cell in interphase, prophase, metaphase, anaphase and telophase

respectively in slide 2. Which phase is the most common? Is there a correlation between

the frequency of incidence and the duration of a particular phase? If so, which?


Draw an anaphase with the help of the 100x oil immersion objective. (If you have

forgotten how, read the instructions on Celweb.)


DNA ANALYSIS: KARYOTYPING

A normal human body cell has 46 chromosomes. These chromosomes come in pairs: 22 pairs

of autosomes and one pair of sex chromosomes (XX in women and XY in men). With the

exception of the X and Y chromosomes in men, the two chromosomes in each pair are very

similar in terms of structure and function.

Chromosomes can be banded with the help of staining techniques that give each chromosome

a characteristic banding pattern. Chromosomes 1 to 22, X and Y each have a different

banding pattern that depends on their structure and function.

It is possible to distinguish and identify each chromosome on the basis of banding pattern,

length and centromere position. Assignment 3: Karyotype the chromosomes in a human cell with the help of a

chromosome map (karyogram or idiogram). Micrographs of metaphase karyograms will be published on Nestor.

Do the micrographs show any chromosomal deviations? If so, what?


Classification of metaphase chromosomes by length (descending order, see Fig. 32).

Group Chromosome number Common characteristic (centromere position)

A 1, 2 and 3 approximately metacentric

B 4 and 5 submetacentric

C 6, 7, 8, 9, 10, 11, 12 and X submetacentric centromere; difficult to distinguish on the basis of banding and length

D 13, 14 and 15 acrocentric

E 16, 17 and 18 approximately metacentric

F 19 and 20 metacentric

G 21, 22 and possibly Y acrocentric

Fig. 32: Example of a chromosome map (karyogram), stained with Giemsa


DNA ANALYSIS: APPLICATIONS OF DNA TECHNOLOGY

In addition to cytogenetics, with which changes in DNA can be identified at chromosomal

level, there are various other molecular biological techniques for identifying changes in

DNA. By using these techniques, the inheritance of certain genetic disorders –

phenylketonuria, Down’s syndrome, Huntington’s disease and others – can be linked to

existing DNA polymorphisms. The techniques are particularly useful for disorders that are

not associated with an abnormal karyotype.

The cases described below concern diagnostic problems that can be solved with the help of

various molecular biological techniques. For a proper application of these techniques to the

following cases, you must understand the principles underlying them. If you have not grasped

these principles yet, you can read up on them in Chapter 10 of Essential Cell Biology.

Assignment 4: Discuss case 1

A family has the following members:

1. Peter (father)

2. Mary (mother)

3. Christine (daughter)

4. William (son)

5. Pauline (daughter)

Christine has PKU, which is inherited recessively. Mary is pregnant again, which is why the

DNA of all family members and the foetus were analysed to probe for 2-allele restriction

fragment length polymorphism (RFLP) close to the PKU locus. The results of a Southern blot

analysis are shown in Figure 33.

Websites with additional karyotyping information and exercises:

www.biology.arizona.edu; go to:

Human Biology>Karyotyping

‘Human biology’ button (bottom) > New Methods in Karyotyping (describes

multicolour spectral karyotyping)

http://www.biology.arizona.edu/


Figure 33. Southern blot for case 1. Lane 6 represents the foetus.

a. Draw up a genealogy of this family. Mark who has the disease and who is a carrier (see

Figure 34 of case 2 for the coding).

b. Does the foetus (no. 6 in the Southern blot) also have PKU, is it a heterozygous carrier or

is it a normal homozygote? Give arguments supporting your answer.

c. What is a restriction enzyme?

d. What is RFLP?


e. What is the procedure for Southern blotting? (See ECB, Fig. 10-14.)

f. Why are the bands in the blot of different sizes?


In a family consisting of father, mother, two daughters and two sons, the father and the two

adult daughters have developed Huntington’s disease, which is inherited autosomal

dominantly. A PCR DNA analysis was performed for a 4-allele variable number of tandem

repeats (VNTR) polymorphism (i.e. a VNTR locus of which there are four variations in this

family). The analysis results and the family genealogy are given in Figure 34.

Figure 34. Genealogy and DNA analysis results for case 2. In the genealogy, the circles

represent the female family members and the squares the males. A filled icon indicates that

the person has the disease.


a. Will the son (no. 6) develop Huntington’s disease? Give arguments supporting your

answer.

b. What does VNTR mean? (See ECB, legend of Fig. 10-30.)

c. What is the procedure for PCR? (See ECB, Fig. 10-27.)


Various members of a family have ornithine transcarbamylase (OTC) deficiency, which is an

X-linked recessive disorder. Male neonates with this enzyme deficiency die very soon after

birth. Enzyme activity in adults can be measured by analysing the blood ornithine level,

which is strongly elevated in heterozygous female carriers.

Figure 35 shows this family’s genealogy and an RFLP haplotyping of DNA cleaved with the

restriction enzyme MspI on which Southern blotting was performed with OTC cDNA as

probe (see ECB, Fig. 10-9, for the principles underlying this technique). The mother (I-2) and

daughters II-1 and II-5 have an elevated blood ornithine level.

Figure 35. Genealogy and DNA analysis results for case 3. The diagonal line across the two

squares means that son III-1 and 3 generation II sons have died. Their DNA was not

analysed.

Generation I

Generation II

Generation III


a. Does the male foetus (III-2) have the deficiency? Give arguments supporting your

answer.

b. How are X-linked disorders inherited?

Assignment 7: Discuss case 4 (ECB, Fig. 10-30)

As part of a forensic investigation, VNTR polymorphisms (3 loci; 2 alleles per locus) were

analysed with PCR. In this analysis, DNA found on the victim (lane F) was compared with

the DNA of three suspects (lanes A, B and C; see Fig. 36).

Figure 36. DNA analysis results for case 4

What is the correlation between the number of VNTR loci being investigated and the

level of certainty with which the suspect can be identified?



Marian is the sister of Charles, who has Down’s syndrome. She wants to know if she is a

carrier of the t(14;21) that has been found in her father, his brother (not tested) and her

paternal grandmother. The test used was Southern blotting. A DNA segment that specifically

binds to chromosome 21 was used as probe. The results of the analysis are given in Figure

37.

Figure 37. DNA analysis results for case 5

a. Does this information indicate that Marian is a carrier?

b. Explain how PCR could have been used for this analysis.

c. Why could Marian’s question also have been answered with cytogenetics (karyotyping)?

For more exercises, with clear explanations, go to the source of case 4:

‘DNA profiling 2001’ in www.biology.arizona.edu

http://www.biology.arizona.edu/

Documents

BSC IN MEDICINE 2013-2014 Block 1.1: Fundamentals of Medicine · 2013-09-11 · Cells Practical : Blood cells Part of Block 1.1, Fundamentals of Medicine Foreword In these Cell Biology