The interaction of electric and magnetic fields with biological materials

THE INTERACTION _{OF ELECTRIC AND}

ý..

MAGNETIC FIELDS _{WITH BIOLOGICAL MATERIALS}

BY

ABDOLHOSSEIN JAFARY-ASL

A THESIS _{SUBMITTED FOR THE DEGREE OF} DOCTOR OF PHILOSOPHY IN THE

DEPARTMENT OF ELECTRONIC AND ELECTRICAL

ENGINEERING _{AT THE UNIVERSITY OF SALFORD}

TO MY PARENTS

ACKNOWLEDGEMENTS

The author would like to express his sincere gratitude and appreciation to Dr. C. W. Smith, for his advice and

encouragement during the supervision of the-work presented in _{this thesis.}

My thanks to Professor G. Carter in whose Department the _{experiments were conducted.}

The continued interest of, and lively discussions with Professor H. Fröhlich _{F. R. S. throughout} the _{course of} these

studies are gratefully acknowledged.

Special _{thanks are also conveyed} to Mr. L. Preston, for his friendly discussions.

Thanks _{also go to Dr.} H. Foster for his supervision with biological methods.

Also _{I would} like _{to thank the workshop staffs and} all of the Electrical Engineering Department.

Finally,

_{I would like}

_{to thank}

Mrs. D. M. Scott

for

ABSTRACT

In the course of the work on the interactions

of

electric and magnetic fields _with biological _materials both living _{and dead, 'it was noticed that published dielectrophoretic} yield curves for biological cells showed unexplained

deviations in _{the region of 2kHz which is the proton magnetic} resonance frequency in a typical laboratory ambient magnetic

field. _{The exact value} is 2.13 _{kHz for a field of 50 UT.} The results of preliminary dielectrophoretic and

dielectric _{measurements show features at} frequencies _which correspond to the NMR condition for that value of steady

magnetic field in which the measurements were made. Very sharp dielectric loss peaks were found corresponding to

1H, 31

p, 23Na, 35C1 _amd 39K _resonances. The electron _spin resonance also showed up in the dielectric loss.

The onset plus NMR conditions

of these

resonances

commences

at the _{value of} the _steady magnetic field strength such that one quantum of magnetic flux (2.07 x 10-15 Wb) would link

the cross-sectional

area of a single

biological

cell

or pair

of cells.

Approximately

1.0 gauss or 0.5 gauss (100iT

or

50pT) _respectively in the _{case of} 5um diameter _yeast cell.

Growing _{cultures of} the _{yeast cells} in fields _which satisfy the _proton NMR conditions as a function of temperature results

in _{a slight reduction} in the _{mean generation} time (MGT). Comparison _of this _with the dielectric _{constant and loss}

and the dielectric increments are greatest.

Steps in _{the voltage-current characteristic of a pearl-} chain of _yeast cells _were found to occur for a few minutes

around the time _{of cytokinesis as they were observed under}

phase _{contrast microscope.} The cells _prepared for _synchronous division _{were collected by dielectrophoresis} into _{a one}

micron gap between two point electrodes mounted on a microscope _slide. Steps _{were observed about} 3 to 4

hours later _{at ambient temperature. The mean generation time} is 4 hours.

An emission of a radio frequency signal from yeast cells in _{the region of 7 MHz and in the range of 50 MHz -}

80 MHz were found to occur about a mean generation time

after starting the incubation of the cells for synchronous growth.

CONTENTS

PAGE Abstract

Chapter

1

" Introduction

1

Chapter 2 _{Yeast Morphology and Growth} 9

2.1 The Yeasts 9

2.1.1 _{The Cell structure} 11

2.2 Growth _{rate of yeast} 14

2.3 Growth _{curve of yeast cells population} 17

2.3.1 The lag _phase 17

2.3.2 _{The exponential phase} 18

2.3.3 _{The negative acceleration phase} 18

2.3.4 _{The stationary phase} 18

2.3.5 The death _phase 20

2.4 Synchronous _growth 22

2.5 Precautions during _experimental 23

measurements

2.5.1 Experimental _culture 23

2.5.2 Sedementation _of the _{yeast cells} 23

2.5.3 Microbiological _techniques 24

for _{electrical and magnetic}

measurements.

Chapter 3 The Biological Cell Membrane 25

3.1 The cell _membrane 25

3.1.1

_{The electrical}

behaviour

27

of the cell _membrane

3.1.2 Active _{transport of solutes across} 35

cell _membranes

CONTENTS CONTINUED _PAGE

3.2 Water in biological _{system 38}

3.3 Movement _{of water across cell 42}

membranes

3.3.1 Diffusion _{and osmosis} 42

3.3.2 Osmosis _{in biological systems} 44

Chapter 4 Dielectric _{Properties of} 45

Biological Materials

4.1 Introduction 45

4.2 Molecular Polarizability _and 46

dielectric dispersion

4.2.1 The Maxwell-Wagner _dispersion 51

4.3 The dielectric _parameters investigated 52

for _{the magnetic resonance measurements}

Chapter 5 Biological Dielectrophoresis 56

5.1 Introduction 56

5.2

Basic

_theory

_{of dielectrophoresis}

57

5.2.1 Comparison _of dielectröphoresi'and 61

electrophoresis

5.2.2 Pearl-chain formation (the _yield) 62

5.2.2.1 Theory _{of pearl-chain growth} from _a 63 cell suspension

5.3 _{The mechanisms involved} in _the 70

response _of the living _cells to _a

non-uniform

_electric

field

_over

the

CONTENTS CONTINUED _PAGE

Chapter 6 Magnetic _{Properties of Biological 72}

Materials

6.1 Introduction ₇₂

6.2

Basic

_magnetism

₇₄

6.2.1 Diamagnetic _{substances 75}

6.2.2 Paramagnetic _substances 79

6.2.3 _{Ferromagnetic substances} 80

6.3 Biological _{effects of the geomagnetic} 82

field (GMF)

6.4 The interaction _{of electromagnetic} fields 84 with biological tissues.

6.4.1 _{The magnetic energy} density (U) 87

associated with a magnetic field.

Chapter

₇

_Aspects

_{of Magnetic}

_{Resonance Theory}

91

Relevant _{to Biological} Materials

7.1 Introduction 91

7.2 Basic _{NMR resonance} theory 91

7.2.1 Nuclear _{magnetic resonance relaxation} 98

times

7.3 Choice _of frequency _{and magnetic} field 100

strength

7.4 Biological _{NMR studies} 100

7.5 Possible dangers _{associated with} NMR 102

studies _{on biological} materials.

7.6

Electron

_{spite resonance}

(ESR)

103

7.6.1 Choice _of frequency _{and magnetic} 104

CONTENTS CONTINUED 7.6.2

Chapter 8

8.1 8.2 8.2.1 8.2.2 8.3 8.3.1 8.3.2

Chapter ₉

9.0 9.1 9.1.1 9.1.2 9.1.3 9.1.4 9.1.5 9.1.6 9.1.7

Biological _{ESR studies}

Biological _{Effects of NMR on The} Sodium _{and Potassium Content of}

Bovine _{Eye Lenses Through Microwave} Modulation

Introduction

The mammalian _{eye lens}

The chemical _{composition of} the lens Sodium _{and potassium content of the} eye lens.

The biological _{effects of} microwaves.

Cataracts

Microwave induced _{cataractogenesis}

Experimental Techniques Design _{and Procedures}

Introduction

Apparatus _used for _measurements of the dielectrophoresis

The cell electrodes The power supply

The conductivity Observations

Electrode _cleaning Source _{of error}

Preparation _{of the samples}

CONTENTS CONTINUED 9.1.8 9.2 9.2.1 9.2.2 9.2.3 9.2.4 9.2.5 9.2.6 9.2.7 9.2.8 9.3

9.3.1

9.5

Experimental _procedure

Apparatus _used for _measurements of the dielectric _properties

Three _{terminal test cell} Capacitance _bridge

Difficulties _{in bridge measurements} The temperature _{control system}

Cell _assembly

Sample _Preparation Sample _concentation

Details _{of the experimental} procedure

Apparatus _used for _{measurements of} the _steps in _{voltage-current}

-characteristics observed using live _{yeast cells}

Sample _preparation

Types _{of electrodes configuration} used

Water

Apparatus _used for _{measurements of} the _{oscillations emitted} from

dividing _{yeast cells}

Apparatus _used for _measurements

of magnetic _resonance effects on

the _{potassium and sodium content of} bovine, _{eye lenses through microwave}

PAGE

Chapter 10 Experimental Results 147

10.1 Dielectrophoresis _measurements 147

10.2 Dielectric _measurements 155

10.3 Steps in _{the voltage-current} 169

characteristics observed using a pearl-chain of live yeast cells

10.4 Oscillations from dividing 178

yeast cells

10.5 _{The effects of NMR on the sodium}

and potassium content of the bovine eye lenses through microwave

modulation.

Chapter 11 Discussion _of Experimental 204

Results

Chapter 12 Conclusion 218

Chapter 13 Appendices 223

13.1 Microbiological Technique 223

13.2 The Procedure Used to Prepare 241

Ag-AgCl Electrode

13.3 Preparation _{of Bovine} Eye Lens 244

References 253

Published Papers

1. Jafary-Asl, A. H., Solanki, S., Aarholt, E.

and Smith,

C. W., J. Biol.

Phys.

11,15-22

(1983)

2. Jafary-Asl, _{and Smith,} C. W., Biological Dielectrics in Electric _{and Magnetic Fields} (in _{print) 1983-}

College, _Cambridge.

LIST _{OF ILLUST: 'ATIONS}

Pt C, E,

Fig. _{1.1 "Ionic conduction through the 3}

biological _membrane".

Fig. 1.2 "Tunneling _of ions _{through a4} membrane".

Fig. 2.1 _{S. Cerevisiae} (normal _{diploid) cells 10} growing in culture.

Ficc. 2.2 _{Budding in Saccliaromyces. 10}

Fig. 2.3 _{The structure of the yeast cell. 12}

Fig. 2.4 _{, rowth curve for a population of 12} yeast _cells.

Fig. 2.5 _{The staining procedure. 21}

Fig. 3.1 _{The Dawson-Danielli model for the 26}

structure _{of cell membranes}

PAGE

Fig. 3.3 _{An electrical model of a typical} 32

biological _cell.

Fig. 3.4 Ion _{movements across} the _cell 37

membrane.

Fig. 3.5 _r Structure _of Water _molecule. 40

Fig. 4.1 Dispersion (f') _{and absorption} (E") 45 curves for a pure polar liquid.

Fig. 4.2 Spherical _{particle of permittiuity} 47

E2 in a continum of permittivity c' and uniform applied field E.

Fig. 4.3 Construction _of the Cole-Cole 54

diagram.

Fig. 5.1 Electrophoresis _{and dielectrophoresis} 61

Fir. 5.2 _{The particles close} to each other in _{a medium with dielectric}

constants £2 > el and their effect on the field

PA'ZE

Fig. 5.3 _{Relation of the volume of suspension} 68 swept out in time t to the volume of a

Pearl-chain _{of particles.}

Fig. 6.1 The _{angular moment vector} P. 76

Precesses _{about B with the} Larmor frequency WL.

Fig. 7.1 The two _{orientations of} the 93

magnetic moments of a spin I=%

nucleus,

Fig. 7.2 _{The splitting of} the _energy levels 94 for _{a spin I=% nucleus when placed}

into _{the magnetic field Bo.}

Fig. 8.1 _{The mammalian eye lens.} 109

Fig. 8.2 Nuclear-cortical _cataract. 119

Fig. 8.3 Temperature effect of depth of 122

various wavelength in the eye

exposed

to microwaves.

Fig. 9.1 The cell _electrodes used in 125

dielectrophoresis _{measurements.}

Fig. 9.2 Experimental _apparatus for 127

PAGE

Fig. 9.3 Three terminal test _{cell. -} 131

Fig. 9.4 Experimental _apparatus for 135

dielectric _{properties measurements.}

Fig. 9.5 _{The cell electrodes used} for 138

voltage-current characteristics.

Fig. 9.6 Experimental _apparatus for 140

measurements _of the _steps in

voltage-current _{characteristics} observed using live _{yeast cells.}

Fig. 9.7 _{Types of electrodes configuration} 142

used for voltage-current

characteristics _measurements of a Pearl-chain _{of yeast cells.}

Fig. 9.8 _{Experimental apparatus for radio 144}

frequency _{measurements of a Pearl-} chain if _{yeast cells.}

Fig. 9.9 Experimental _{apparatus for} 146

irradiation _{with microwaves and} microwaves _{modulated under} NMR

PAGE

Fig. '10.1 Dielectrophoretic _{yield spectrum} 148

of live and dead yeast cells as a function _of frequency _of the _electric

field.

Fig. 10.2 DielectrophoretlC _{yield spectra of} 150 live _{yeast cells as a function of}

the frequency _of the _electric field at different _voltages.

i=i C-

Fig. 10.3 Dielectrophoreeyield _{spectra of 151}

dead _{yeast cells as a funtion of}

the frequency _{of the electric field.} Yeast _cells irradiated _{with W light}

(254nrn)

. Fig. 10.4 Variation of dielectrophoretic 152

yield _with the _{square root of} the length _{of time the field} is _applied V=40Vr _{m. s. frequency = 3kHz.}

Fig. 10.5 Electromagnetic _{yield spectra of} 153

live _{yeast cells as a function of} the _{frequency-of the electric field.}

Fig. 10.6 Measurements _{on lice cells from 154}

o. 5G to 53 (50KT to 500 JT)

PA"E

Fig. 10.7 Dielectric loss _{peaks at ITh} 156

field _{conditions of a Pilo by weight}

bakers' _yeast in deionised _{water at} 24°C.

Fig. 10.8 Variation _of the _{proton NNR peaks} 157 in _{frequency with applied magnetic}

field.

Fig. 10.9, Simultaneous frequency _{and magnetic} 158 field for _{proton N, 3? conditions}

Fig. 10.10 _{The real and imaginary parts of} 159 the _{complex permittivity of} live

yeast _cells as a funtion of frequency.

Fig. 10.11 _{Dead yeast cells.} Sterilized _at 161 121°C.

Fig. 10.12 Chlorine _{and Potassium} NMR 162

Fig. 10.13 Phosphorus _{and Sodium} NMR 163

Fick . 10.14 Proton NNMR 164

Fig. 10.15 Mean generation time _{of yeast} 165

cells.

PAGE

Fig. 10.16 Dielectric increments _ratio to

mean proton NIT, resonance of live yeast cells.

Fig. 10.17 Electron _{spin resonance spectrum} of dielectric loss of live yeast

cells.

166

167

Figs. 10.18 Steps in the Voltage-current 170-176

to 10.24

characteristics _of a Pearl-chain

of live yeast cells collected between two points electrodes.

Fig. 10.25 Control. 177

Figs. 10.26 Radio frequencies emission from a 179-186 to 10.33

Pearl-chain _of live _{yeast cells.}

Fig. 10.34 Similarity in frequencies between 187 yeast _cells and enzyme (ysozyme

Figs.

10.35

Effects

_{of microwave}

irradiation

190-200

to 10.44

and microwave

modulation

by NNMR

frequencies _{on the} Sodium _{and Potassium} content _of the bovine eye lenses.

Fig. 10.45 Dielectric _{measurements on activity} 202 metabolising bovine eye lense (a)

LIST _{OF TABLES}

PAL E

Table 1: The _{activation energies of} 89

molecular effects in biological systems.

Table 2: The _{chemical composition of} 112

the lens.

Table 3: The tissue _{culture medium} 245

1

CHAPTER 1 INTRODUCTION

In recent years living organisms, especially

microorganisms, have become the _subject for study by a large _{and diverse segment of the scientific community.}

They _{are no longer the private domain of the} life _scientists

but _{are now being investigated by chemists, physicists', and} engineers. This influx _of the _{physical sciences} has resulted

in _{the development of many new approaches to biological} problems.

As biological _{systems use the same atoms and molecules} as the _{physical sciences,} it is therefore _reasonable that extent _physical processes should be capable of giving a

quantitative account of the interactions between electromagnetic fields _{and biological dielectrics,} from _zero frequency

upwards.

Fröhlich (1980) _{suggested that} biological _materials

might _{possess a metastable} state with high electric dipole

moment. The experimental evidence. produced by some workers,

for _example Mascarenhas (1975), _{Neumann and Katchalsky} (1972) and Ahmed et al (1976) seems to support these suggestions.

The membranes of the biological

materials

function

through

their

_composites

_structure

of lipid

and protein,

in which

_{the protein}

_could

_exceed

the lipid.

The proteins

in the membrane are lipid

soluble

and not water

soluble

2

especially those seeking to establish the relevance of solid state physical concepts to biological phenomena.

The lipid _{soluble proteins contribute to the most sensitive} part of the biological processes of living systems. On

the _other hand water _{soluble proteins serve as catalyzers}

of chemical reactions and transporters of ions and molecules through biological _compartment fluids.

The diffusion _{of protons and ions across} the _membrane considered _{as the main} factor for the establishment of

electrical _{phenomena associated with} the membranes. The

possibility that _{electron conduction processes} may play a - role in the functioning of some of the membrane proteins

is _{made the more reasonable when it} is _remembered that

biological _membranes in _{their normal physiological state} are subjected to electric field gradients of 107Vm-1

and greater.

Ionic

_conduction

_through

_{the biological}

_{membrane is}

achieved

by "hopping

conduction"

(Pethig,

1979).

If

the

cations _are the only mobile ions within the biological

membrane such as protons that have dissociated from protein molecules for example. These cations migrate through the

membrane by a series of jumps from one position to the next over a coulombic potential energy barrier as shown in

Figure l. la. Before hopping _conduction takes place the cations will be in thermal equilibrium with the surrounding molecular structure and will vibrate in the potential well

3

order of 1012Hz at room temperature. The probability

per second that the cation will pass over the barrier

into _{a neighbouring equilibrium position} in each vibration is _given by

p= fo exp (- j- )

0-4

(0)

r.

1.1

Fig.

1.1

Ionic

conduction

through

the biological

membrane

(a)

The diffusion

_{of cation}

is limited

by a series

of potential

energy

barriers.

(b) _{The rate of migration of cation} increased by modification _of the barrier through external

electric field E.

-do

d-

4

0

If _{an electric} field _{E or chemical} (metatlic) _processes

are supplied, the potential barrier will be modified as

shown in Figure l. lb. A jump in the direction of metabolic activity or the applied field now takes place with an

increased _{probability given} by qEd

-(AW -. 2 _(1.2)

p=f0 exp _kT

The above equations indicating that cations migration increased in the field direction.

If _{on the other} hand _{the potential wells are close} enough (Fig. 1.2) to interact,

B

(o)

(b)

B

Fig. 1.2 Tunneling _of ions _through a membrane

(a) _{The two wells are close enough} to interact

5

but _{they are isolated from any surrounding medium which} can couple them into _{an energy sink or energy source} by means of lattice _vibrations (Phonons) _{or light} interaction

(Photons), _{cation can appear in well B. This is due to} the limitation _{on the energy} loss _{of the cation to get to}

ground state of well B. The cation _will therefor kcontinually oscillating between A and B with _{the average amount of time} spent in _{each well} depending _{on the} relative amplitudes

of the corresponding _{wave functions.} However if the _wells are coupled to an energy _{sink and source,} the _cation that have _appeared in _{B can now fall to the ground state by}

emitting photons or spending its energy as a lattice vibration. The rate at which the electron _{can tunnel} from well A to B

is _{controlled by the width W and height H of the potential}

barrier. If its _{temperature is high enough the cation could} cross the barrier without having to undergo a tunnelling

process.

However, _{a charge carrier} hopping back _{and forth} between two potential wells in _response to an externally applied

alternating electric field will give a dielectric response

indistinguishable from _{that exhibited} by a conventional dipole having _{a permanent} dipolemoment. A set of dipoles _with

distributed _{relaxation times will exist} if the _{charge carriers} move between various potential wells, or if there is a

distribution _{of barrier} heights between _wells.

Since

_all

biological

_structures

_contain

_electric

6

electromagnetic field. More significantly, the orientation of molecules, important in biochemical reactions, may be

altered and biological molecules can be polarized by

permanent dipole aligned or induced dipole moment. These properties are important for the function of a biological

system and so make the system vulnerable to external

perturbation. Because of the extremely small interaction

energy of magnetic dipole moments, as compared with that of electric interaction, it is difficult to explain any

observed response in a biological system as originating from the _{weak magnetic} energy interaction of a system supposedly

in equilibrium.

Sheppard _{and Eisenbud} (1977) _{suggested that small}

amounts of orientational energy may cause steric changes in molecules located _{at the cell surface and that any}

cellular response to a field would imply communication across the _{cell membrane.}

Control _{of chemical activity} is, _{of course} in many cases well _understood in terms _{of relevant enzymes,} which through

their _{catalytic action regulate most} biological _chemical

processes. Enzymes _{are proteins} their electronic configuration can be perturbed by the electromagnetic field energy.

Fröhlich

(1977)

_proposed

_that

in biological

_systems

through which much energy flows, coherent electrical

oscillations would be created at frequencies of the order of 1011 Hz, _{and that} these _oscillations could cause sharp

7

regions could

be created

by biological

water,

specifically the structural water located at the outer

surface of biological molecules _{and cell membrane.} The

resulting

structure

_{would be a sandwich}

_which

could

give

rise to an a. c. Josephson _effect (Bloch, 1968) coupled

to _{the polarization waves thought to occur} in biomolecular structure (Fröhlich 1975).

For several

biological

_systems

involving

_growth

_or

nerve processes, the square of the activation energy is a linear function _{of temperature over a moderate range of}

physiological temperatures. This led Cope (1971) to

suggest that superconductivity was closely associated with the _{growth of cells} having _{superconductive micro-regions}

in _{the DNA and RNA structure, and which electrons tunnelling} along the polypeptide chain, and Shaya et al (1976)

suggested that superconducting Josephson junctions in living systems were sufficiently sensitive to detect variations

in _{the ambient magnetic} field.

On the _assumption that the biological systems possess superconductive properties, Marton (1973) has tried to

associate the local geomagnetic fields with cancer mortality data _{over' a large part of} the _world. His suggestion was

that there might be a (zero voltage) Josephson tunnelling current between the membranes of normal cells in contact.

This _{current might assist} the _{communication} between the cells therefore lead to the normal growth of the cells. This

current

could

be strongly

dependent

on the external

magnetic

8

abnormal growth of the cells with the development of cancerous tissue preferentially in certain geographical

locations.

The experimental

evidence

produced

by some workers,

for _example Ahmed (1975), Shaya (1976), Aarholt (1982)

and Jafary-Rs1 et al (1981-1983) seems to support these suggestions.

The test _{systems chosen} for _{most of} the _{work reported} in _{this thesis was an aqueous suspension of yeast cells.}

The desire _{to use an organism with a simple shape, restricted} the _choice of materials to microorganisms. This in turn

restricted the size and thus led to the decision of using a suspension of cells rather than a single cell. The suspension would also tend to give more reproducible "average"

results when they were all growing at the same rate, that is in the logarithmic phase.

Besides

being

_{of simple}

_shape,

it

_{was also}

desired

the organism should be easy to grow, relatively simple to work with, non-hazardous, non-motile, and as large as

possible. Saccharomyces Cerevisiae (normal diploid strain)

CHAPTER 2 _{YEAST MORPHOLOGY AND GROWTH}

2.1 The Yeasts

Yeasts

_{are classified}

_{as higher}

_protists

_{in the group}

of fungi. They are _{plants which} have _{a well-defined nuclear} membrane and chromosomes. They _{exhibit mitotic cell}

division but lack _{chlorophyll so that they cannot}

synthesize their own food. They obtain _{their energy either} akl

by aerobic oxidative _{processes or by/ erobic} fermentation

depending _{on their circumstances. Yeasts generally occur} as unicellular organisms _{and in a variety of shapes; they} don't _{form colonies.}

In general, yeasts are larger than bacteria.

Saccharomyces

_Cerevisiae

_(normal

_diploid

_strain)

_cells

used in this _{work are spherical and have an average} diameter

of 5 um as observed _{under phase contrast microscope} (Fig. 2.1). They have _{no organs of locomotion and thereby are non-}

motile.

The yeast

_cells

is surrounded

by a rigid

_cell

wall and a cell membrane. The thickness of the cell

membrane is 8 nm and is believed to consist of two protein layers _{separated by a bi-layer of lipid} (Lindegren, _1952).

Yeasts

_{can reproduce}

_vegetatively

_{by budding}

_and

fission, _{or sexually by sporulation. Yeasts such as} the Saccharomyces _used in this _{work multiply} by budding

(Fig. 2.2). According _{to Williams (1976) the nucleus}

does _{not undergo the usual mitotic mechanisms observed} in other fungi. Instead the nuclear _{membrane remains} intact

10

'r

"

r

.r e

' _4n

Fig. 2.1 S. Cerevisiae (normal diploid) cells growing in _culture.

cleavage

of nucleus chromosomes

inside nuclear membrane

mitochondrion

Cý)

nuclei 4-4? v

l

sy.

1

(ii)

bud

vacuole

bud

Mitochondrion

i-- SN

w-

(iv)

Fi'i 2.2

Budding in : 3archaromvices Cerevisiae (, ormal dimension _shown)

extension

of vacuoJc

dividing

t ochondrion

detached bud

birth _scor Iop*

11

then _{undergoes constrictions and the nucleus} is _eventually pinched into two equal _parts. One of the nuclei together with the other organelles _{such as mitochrndria,} then

enters _{a bulge which} develops _{at a point adjacent} to the site of nuclear division. Cytoplasmic _connection between

the bud and parent _cell is _then broken _{and followed} by the laying down of new wall _material.

Although _{yeasts will grow over a wide temperature} range, the _optimum temperature for _most

Their _{nutrient requirements are minimal} usually grow most abundantly _{on complex}

devised for _{the cultivation and isolatii}

be used in liquid form or solidified by of agar.

2.1.1 _{The cell structure}

yeasts is 30°C. but _{they will}

media. Any medium on of yeasts can

the _addition

Lindegren

(1952)

_{and his}

_{collaborators}

_carefully

investigated _{the structure of the yeast cell and the} budding process (Fig. 2.3) and compared their findings with those

of earlier investigators. They reported that each yeast cell contains one or more vacuoles, or transparent

droplets

_{in the cytoplasm,}

_which

_{can be seen with}

_{the light}

microscope

when appropriately

_stained

and which

can be

12

11 Membrane

Cell Wall

I

Vacuole

Nucleus

-Centrosome

Centrochromatin

Mitochondrißtin,

Chromosomes

Nuclear Membrane

yto Plasm

Fig. 2.3 The structure _{of the yeast cell according} to Lindegren (1952).

Mitochondria _{occur as membrane-bound organelles which} on microscopic examination give the appearance of a folded

thread. They consist largely _of lipoprotein-and a small amount of RNA. The mitochondria contain the respiratory

enzymes,

and have beeen termed

the power houses of the

cell.

Although _{studies of the nuclear sub-structures are}

13

the budding _process; (2) the _{nuclear membrane} remains intact during _cell division; _{and (3)} in budding, _the nucleus constricts, one part going to the daughter cell

and one remaining with the parent. Certain structures of more intense electron density have been observed, one

of which is thought to be a nucleus.

A typical _{yeast cell contains what} is termed _cytoplasm this is _{a semifluid containing} finely _{granular materials,}

ribosomes abundent in ribonucleic acid (RNA) and

membrane-bound organelles. The cell membrane with a

thickness _{of approximately} 8 nm, it _consists of two electron dense layers _of lipid _{protein, and ribonucleic acid and}

is believed _{to be a lipoprotein structure,} the inner layer being lipid _{and the outer protein.}

The cell

wall

of yeast

is quite

thin

in young cells

but _{thickens with age. A typical cell wall of yeast} is about 70 nm thick. The major constituents are two

polysaccharides, glucan (30 to 35 percent) and mannan

(30 per cent); there is _{a 6% to} 8% protein _{content some} of which is very likely to be enzymatic, invertase has

been identified in _{cell wall material.} The lipid

concentration range is 8.5% to 13.5% _. The cell _{- wall} protein is linked to both _{the glucan and the mannan and binds} the

cell wall.

The presence of both "bud _scars" and "birth scars"

on the yeast cell had been demonstrated _.A newly detached cell possesses a birth _{scar at} the _{point at which} it _was

joined _to its _{mother cell. When this cell itself produces} bud, _{which gradually become detached, each of the daughter}

14

produced is situated diametrically opposite to the birth scar.

- One. cell was studied by Lindegren which produced 23 successive buds, _{all of which gave rise to macroscopically}

identical _{cultures. The electron microscope was employed} in _{the study, of these bud scars by ', Bar. tholomew and Mittwer}

(1953) _{who found that the reproductive age of a cell is}

limited, but _{that a cell} is _{potentially capable of producing} about 100 daughter cells, since a bud is always formed at a new point on the cell.. They were, however, unable to

find _{any cells with more than} 20 actual bud scars.

2.2 Growth _{rate of yeast}

The prevailing means of _yeast reproduction is by budding

and fission,

one cell

divides,

producing

two cells.

Starting _{with a single yeast cell, the} increase in population is _{by geometric progression} in _{a binary sequence.} The

time interval required for the _cell to divide or for the population to double is known as the generation time.

It has been found _{to be more convenient-to replace} the

generation time bly. the mean generation time (MGT) _. This is because _{not all} the _{cells will produce only one}

daughter _cell, it is _{commonly observed that some cells} produce two or more buds at the _{same time.} The mean

generation time is strongly dependent upon the nutrients in _{the medium and on prevailing physical conditions}

15

Under _{optimum conditions, one can easily determine} the _{mean generation} time by the simple _mathematical

expression of equation (2.5).

The mean generation

time

for

_yeast

_cells

_{are determined}

by inoculating _{the medium with a known number of cells,}

allowing the yeast to grow under _optimum conditions, and then determining _the final _{cell population.}

The experimental data _{required to calculate the MGT} includes (1) _{the number of yeast cells present at the}

beginning, (2) _{the number of yeast cells present at the end} of a given time interval, and (3) the time interval _.

Additional _{measurements would check the validity of the} equation.

The relationship of cell _{numbers and mean generation} time _{can be expressed} by the following _{equations where}

B is _{the number of yeast cells} inoculated into _medium or cell count at zero time. b is the number of cells

at end of the given time period t. G is the mean generation time (MGT) and n number of generations _{each of} time

equal to MGT.

Starting _{with a single cell, the total population}

b at the end of a given time _{period would} be expressed

by

b=1x2n

_2.1

where 2n is the cells population _after the _{nth generation.} However, _{under practical conditions, the number of cells} B introduced into _{the medium at time zero is not 1 but}

16

b=Bx 2n 2.2

Solving Equation (2.2) for _n,

B log b= _log

10 +nl2

10 og 10

bB n=

1og10 _{- 1og10}

2.3 log

12 0

If _{we now substitute the value} for _the log20, _which is 0.301, in _{the above equation, we have}

3.3 log10 b _2.4

B

Thus, by use of equation (2.4) _{we can calculate} the _number

of mean generations

that

have taken place,

providing

we know the initial population B and the population b

after time t and the growth is _only a single exponential.

The mean generation

time

G is equal to t (the

time

which elapsed between b and B) divided by the number of generations n, or

G=t=

n _{3.3 1og10(b/B)}

t

17

2.3 Growth _{curve of yeast cells population}

When a fresh medium is inoculated with a given number of cells, and the yeast cells population determined

intermittently during _{an incubation period of} 48 hours (after _{this period the number of cells that} die _exceeds

the _{number of new cells produced), and the} logarithms _of the _{number of cells versus} time is _plotted, the following

phases of growth can be recognized: lag phase, exponential growth phase, period of negative acceleration, stationary

phase and death phase. Those are shown in figure 2.4.

2.3.1 The lag _phase

The addition of inoculum to a new medium is not

followed by a doubling _{of the population according} to the generation time. Instead, the population remains

temporarily _{unchanged, as illustrated} in the yeast growth curve. But this does not mean that the cells are

quiescent or dormant; on-the contrary, during this stage

the individual _cells increase in _size beyond their normal dimensions. Physiologically _{they are very active and are} synthesizing new protoplasm. The cells in this new

environment may be deficient in enzyme or coenzymes which must first be synthesized in amounts required for

optimal

operation

of the chemical

machinery

of the cell.

Time for _adjustments in _{the physical environment around}

each cell may be required. The organisms _{are metabolizing}

but _there is _{a lag} in _{cell division. At the end of the lag}

18

seen that it takes 4 hours before _{cells start} dividing.

2.3.2 _{The exponential phase}

During _{this period the cells divide steadily at a}

constant rate. As it _{can be seen} in Fig. 2.4, the log of the number of cells plotted against time _results in almost a

straight line. Under _{appropriate conditions} the _{growth rate} is _maximal during _{this phase, and population is then most}

nearly uniform in terms of chemical composition of cells,

metabolic activity etc. Cells in this _{phase are commonly} used for studies of metabolism.

2.3.3 _{The negative acceleration phase}

This is _{due to the decrease in the nutrients of the}

medium due to an enormous increase in cell numbers. Consequently damage _{to the cells may occur as a result of the accumulation}

of metabolic end products.

2.3.4 _{The stationary phase}

-1

In this

_{phase the slowing}

_{of the growth}

_rate

_continues,

after 40 hours it is due more to exhaustion _of the

nutritional factors of the _{medium and the accumulation of}

waste

products.

The population

_{then remains}

_constant

for

a while, perhaps _{as a result of complete cessation of} division _{or the} balancing _{of reproduction rate}

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2.3.5 The death _phase

Following _{the stationary period,} the _{cells may} begin _{to die off} faster _{than new cells are produced,}

if indeed _{any cells are still reproducing.}

The most important conditions contributing to cell death _{are the} depletion _{of essential nutrients and the}

accumulation of inhibitory products. During the death phase, the number of viable cells decreases geometrically,

essentially the inverse of growth during the log phase. An estimate of the proportion of viable yeast cells in _{the cultures can be made bythe methylene} blue _staining method (Baker 1962). 'The basis of the technique is

that _{certain changes occur} in dead cells _{which result} in response to stains different from that of living cells

(Fig. _2.5)

. The staining procedure is carried out in the

following _{way: a cell smear} is _{made and heat} fixed. It is stained with Loeffler's methylene blue solution for ten

minutes _{, rinsed} thoroughly _using tap _{water until} the

smear _appears a pale blue _colour. Carbol fuchsin is _run down the _slide for _{as short a time as possible. The slide}

is _{then washed} immediately _{and blotted dry. Phase contrast} microscope observation shows living _yeast cells _stained

purple and dead cells stained _red.

The number of yeast cells in each phase as a function

22

2.4 Synchronous _growth

The number of cells in a culture usually increases smoothly because at any time at least a few cells are

dividing, _{in other words cell} division is _{not normally}

synchronized. However, a population in which all the cells are undergoing division _{at approximately} the same time

is desirable _particularly for _{some of the work reported} in _{this thesis.} Such a synchronized _{culture can be}

obtained by special methods, but the synchronism generally lasts for _{only a few generations since even the} daughters of a single cell soon get out of step with one another.

Synchronization is _{achieved by ii ulating cells} into a medium at a suboptimal temperature, if they are kept

in _{this condition} for _{some time, they will metabolize slowly} but _{will not} divide. When the _temperature is _gradually

raised to the optimum, the cells undergo a synchronized

division. When these _{cells are observed} by phase _contrast microscope, they are reasonably _well synchronized with

23

2.5 Precautions during _{experimental measurements}

2.5.1 Experimental Culture

It _{must be understood} during _{some phases of growth} the _{cells are young and actively metabolizing} (especially in _{the exponential phase) while during others they are}

dying (especially in _the death _{phase) so that there may} be enormous _{structural and physiological} differences

between _{populations of cells harvested at different times.} The physical and chemical _{environments may be expected} to

affect the organisms differently during different _phases.

2.5.2 Sedementation _{of the yeast cells}

Yeast _{cells are slightly more dense than water, so}

as a result, over _{a period of} time, they _{will settle out of} suspension. This needs to be kept in mind when concentration

measurements are being _{made or when cells of some standard}

concentration prepared for _{a study} have been kept in _{a tube} or syringe for _{several minutes.} To keep the _{concentration}

as uniform as possible from _{one measurement} to the _next,

the _{suspension should be thoroughly} but _{gently mixed and} shaken before each sample is _{removed, although} this _may

24

2.5.3 Microbiological _techniques for _{electrical and} magnetic measurements

To remove variables as far as possible, it is

important _{to use proven} biological _methods for handling

the _{particular organism under study} (Appendix 13.1) _. The original stock of the _{organism should} be kept _refrigerated

so as to be available at any time when it is required to start a new culture having nearly identical characteristics

to preceding one. Otherwise _{over an extended} period

of time, mutations may cause the cells to evolve to give daughter _{cells with very} different _{characteristics.} The stock cultures that are used for producing working cultures must be subcultured periodically, using accepted inoculation

procedures, to keep them alive and free from contamination. Sterilization _{procedures must} be followed _{closely to}

25

CHAPTER 3 _{THE BIOLOGICAL CELL MEMBRANE}

3.1 _{The cell membrane}

Hundreds _{of chemical reactions and transfer processes,} are occuring continuously in _{every active} cell, and

membranes play a vital role in controlling these processes, in _separating incompatible _{substances and in transporting}

materials about the cell. Some of the more complex chemical sequences are accelerated because it appears that the

enzymes which catalyse them are so arranged _{on membranes}

that the _{reactants can move readily} from _{one reaction site} to the _next.

In a packaging role, intracellular _{membranes separate}

components

of the cell

_which

would

be self

destructive

if _{allowed to mix} freely in _{the cytoplasm, they conserve} and maintain regions of local concentrations, regulate

the _{passage of} inorganic ions _{and complexes} between

compartments and provide the principle means for the.

checking ordering and regulation of the metabolic processes which constitute life.

Electron

_microscope

_observations

(Danielli,

1968)

showed that

the cell

_{membrane consists}

of a lipid

bi-layer

which is coated on each _{side-by proteins} (Figure 3.1).

In an early estimate _{of the} dimensions of membranes

Danielli _{predicted an appropriateAof 8 nm on the assumption} that the length _of the lipid _{molecules projecting} into the core _{is about 3 nm and that the thickness of each protein}

coat would be about 1 nm. The advent of the electron

microscope proved this _prediction to be remarkably accurate.

26

Proton _{magnetic resonance studies} indicate _{that a} proportion of the linkages between lipid and protein is

Cell _{v. 1 a(}

I' /\'/

nm

Jcm

/ýý,

...

I.

ý::

.. ý..

.

<'::: :

Globular 6proteiri

Pnýar

end-9rouPs

4,04 1 osa-

IýPtds

FQE acid

ckaitn

3 nm

Poly PepLicie

I/

1ýý

/

º: ý`/ \J; / \:: /

Fig. 3.1 The DaVson-Danielli _model for _{the structure of} cell membranes.

I

by hydrophobic bonds (Wallach, _1968). This _{means that at} least _{some protein} is likely _{to occupy a core position}