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 Contents i

MICROBIOLOGICAL TECHNIQUES
 MICROBIOLOGICAL TECHNIQUES

N. Murugalatha
Assistant Professor and Head
Lali Growther J. Vimalin Hena
Assistant Professor Assistant Professor
N. Hema Shenpagam R. Anitha
Assistant Professor Assistant Professor
D. Kanchana Devi G. Rajalakshmi
Assistant Professor Assistant Professor

Department of Microbiology and Biotechnology

Hindusthan College of Arts and Science
Coimbatore, Tamil Nadu

MJP PUBLISHERS
Chennai New Delhi Tirunelveli
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Cataloguing-in-Publication Data
Microbiological techniques / by N. Muruga-
Latha ... [et al.].- chennai: MJP
Publishers, 2012.
xiv, 442 p.; 24 cm.
Includes Appendix, References.
ISBN 978-81-8094-107-8 (hb.)
1. Microbiology 2. Techniques-Microbiology.
I. Murugalatha, N ... [et al.].
579 dx22 MIC MJP 142

ISBN 978-81-8094-107-8 MJP PUBLISHERS

© Publishers, 2012 New No. 5, Muthu Kalathy Street
All rights reserved Triplicane
Printed and bound in India Chennai 600 005
Publisher : J.C. Pillai
CIP Data : Prof. K. Hariharan, Librarian
RKM Vivekananda College, Chennai.

This book has been published in good faith that the work of the author is original. All efforts have been taken to make the material error-free. However,
the author and publisher disclaim responsibility for any inadvertent errors.
 PREFACE
Microbiological Techniques is designed for the students, to explore the world of microorganisms and
how the process of scientific discovery is carried out, with an ease. The study of microbiology is dynamic
because of the ubiquitous nature of the microbes and the variability inherent in every living organism. The
broad nature of the subject and diversity of topics from the fundamentals to its unique fields can make
the way of presentation a little difficult; but it is also a part of what makes microbiology an interesting
and challenging subject.
The book primarily focuses on the basic microbiological techniques with applications for undergraduate
and postgraduate students in diverse area of biological techniques. This book is the outcome of nearly a
decade of teaching and research experience. The manual comprises twelve parts in which exercises in first
three parts provide sequential developments of fundamental techniques. The remaining exercises are as
independent as possible to allow the instructor to select the desirable sequence. Exercises are pursued in a
normal scale providing maximum details so that one can perform the experiment independently and safely.
The style and simplicity of expression have been our twin objectives. All exercises have been thoroughly
tested in our laboratory by our students with wide variety of real talents and enthusiasm.
We wish to express our immense gratitude to the Management of Hindusthan College of Arts and
Science for allowing us to pursue this work, during our service in their Institution.
We have immense pleasure in expressing our deepest gratitude to our principal Dr. N. Baluswamy,
Hindusthan College of Arts & Science, for his valuable support which has been a great help in this endeavour.
We extend our gratefulness to all our colleagues in the Department of Biotechnology and Microbiology,
for encouraging us right through our work. We are most grateful to every soul involved in making up of
this manual spending their time, talent and interest in the work.
We look forward for the support, constructive ideas and valuable suggestions from the users of the book.

N. Murugalatha
Lali Growther
J. Vimalin Hena
N. Hema Shenpagam
R. Anitha
D. Kanchana Devi
G. Rajalakshmi
 CONTENTS

1. Introduction to Microbiology 1
Introduction 1
Bacteria 1
Stages of bacterial growth 2
Factors affecting bacterial growth 3
Cyanobacteria 7
2. Tools of Microbiology 9
Microscopy 9
General principles of microscopy 9
Light microscopy 11
Electron Microscopy 18
Scanned-Probe Microscopy 20
Use and Care of Microscopes 22
Autoclave 26
Hot Air Oven 28
Incubator 29
Water Bath 31
BOD Incubator 32
Colony Counter 34
Haemocytometer 35
Oxygen Electrode 38
Lyophilizer 40
Laminar Air Flow (Vertical) 43
3. Fundamentals of Microbiology 45
Laboratory Precautions 45
Principles of Aseptic Techniques 46
Cleaning of Glassware 48
viii Contents

Safety 51
Control of Microorganisms 52
Sterilization 52
Disinfection 61
Culture Media Preparation 64
Different types of Media 65
Sterilization 68
Media Inoculation and Incubation 69
Pure Culture Techniques 71
Aerobic Culture Techniques 71
Anaerobic Culture Techniques 78
Robertson’s Cooked Meat (Rcm) Medium (Clostridium Sp.) 78
Anaerobic Jar (Total Anaerobes) 79
Wright’s Tube Method 82
Staining Techniques 84
Smear preparation 84
Types of staining techniques 85
Simple Staining 85
Gram Staining 86
Capsule Staining 89
Spore Staining (Schaeffer and Fulton Method) 90
Acid-Fast Staining (Ziehl–Neelsen’s Method) 92
Flagellar Staining 94
Negative Staining 96
Granule Staining 97
Giemsa stain for thin films (Blood) 97
Fontana’s stain for Leptospires 98
Periodic Acid—Schiff (PAS) Stain 98
Fungal wet mount—Lactophenol Cotton Blue Staining 99
Slide Culture Method 100
Micrometry 102
Micrometry and Measurement of Microorganisms 102
Motility Determination 105
Enumeration of bacteria, fungi and actinomycetes from soil 110
Phenol Coefficient Test 113
Maintenance and Preservation of Cultures 114
 Contents ix

4. Microbial Physiology 117

Growth Curve 117
Direct Count 117
Viable Count 122
Turbidity Method 125
Biochemical Tests 128
IMViC Reactions 128
Indole Production Test 128
Methyl Red–Voges Proskauer Test 130
Citrate Utilization Test 131
Oxidase Test 133
Catalase Test 134
Urease Test 135
Hydrogen Sulphide Test 136
Triple Sugar Iron Agar Test 137
Nitrate Reduction Test 138
Polymer (Starch, Casein, Gelatin, Lipid) Degradation 140
Carbohydrate Fermentation Test 142
Amino Acid Decarboxylase Test 143
Phenylalanine Deaminase Test 145
Coagulase Test 146
Esculin Hydrolysis 147
ONPG ( O-Nitrophenyl β-d-Galactopyranoside) Test 149
OF Test for Carbohydrate Utilization 150
Malonate Utilization Test 152
Lecithinase Test 153
Pigment Extraction From Algae 154
Effect of Temperature on the Growth of Bacteria and Fungi 158
Effect of Osmotic Pressure on the Growth of Bacteria and Yeast 162
Effect of pH on the Growth of Bacteria and Fungi 164
5. Industrial Microbiology 167
Wine Production 167
Citric Acid Production 169
Production of Glutamic Acid 172
Protease Estimation 175
Production of Extracellular Cellulase by Solid-state Fermentation 180
x Contents

Mushroom Cultivation 183

Extracellular Enzyme Production—Amylase 185
Immobilization of Cells 186
6. Environmental Microbiology 189
Isolation of Nitrogen Fixers 189
Isolation of Symbiotic Nitrogen Fixers (Rhizobium Sp.) 189
Isolation of Free-living Nitrogen Fixers (Azotobacter Sp.) 190
Isolation of Phosphate Solubilizers 191
Standard Qualitative Analysis of Water by MPN Method 192
Quantitative Analysis of Water by Membrane Filter Technique 196
Dissolved Oxygen 200
Biological Oxygen Demand (BOD) 202
Chemical Oxygen Demand (COD) 204
Total Suspended Solids 206
Decolorization of Dye and Dye-containing Effluents 209
Nitrogen Cycle 210
Ammonification 211
Nitrification 212
Denitrification 213
Enumeration of Microorganisms from Wood and Paint 214
Assay of Microorganisms from Biomedical Waste 215
Isolation and Culture of Algae 216
Spraying 216
Single Cell/Colony/Filament Isolations 217
7. Food Microbiology 219
Methylene Blue Reduction Test 219
Phosphatase Test for Pasteurisation in Liquid Milk 221
Turbidity, Colony and Coliform Tests for Pasteurized Milk 223
Isolation of Food Spoilers 224
Analysis of Food Sample for Mycotoxin (Aflatoxin) 226
8. Genetics 229
Isolation of Chromosomal DNA from Bacteria 229
Isolation of Genomic DNA from Cauliflower by Supaquick Method 232
Isolation of Plasmid DNA 234
Agarose Gel Electrophoresis 235
 Contents xi

Isolation of RNA 239

Isolation of Mutants 241
Induced Mutation—Isolation of Antibiotic-resistant Mutants 241
Isolation of Auxotrophic Mutants (Replica-plating Technique) 242
Restriction Digestion of Lambda DNA 244
Bacterial Conjugation 245
Bacterial Transformation 247
Southern Blotting 249
Western Blotting 253
Random Amplified Polymorphic DNA (RAPD) 255
Restriction Fragment Length Polymorphism (RFLP) 259
9. Immunology 265
Blood Grouping 265
Antistreptolysin O 267
Widal Test 269
Slide Agglutination Method 270
Tube Agglutination Method 271
Serological Tests for Diagnosis of Syphilis 272
VDRL (Venereal Disease Research Laboratory) test 273
RPR (Rapid Plasma Reagin) test 274
Enzyme-linked Immunosorbent assay (Elisa) 276
C-reactive Protein (CRP) 277
Rheumatoid Arthritis (RA) Factor 279
Countercurrent Immunoelectrophoresis 281
Immunodiffusion 282
Ouchterlony’s Double Diffusion (ODD) Technique 282
Radial Immunodiffusion (RID) Technique 285
Isolation and Characterization of Antigens 287
Purification of Immunoglobulins by Precipitation and Dialysis 288
10. Medical Microbiology 291
Isolation and Characterization of Pathogens from Clinical Samples 291
Sample Collection 291
Identification of Microorganisms 295
Sample Processing 295
Identification and Enumeration of Lymphocytes 310
xii Contents

Identification of Fungal Pathogens 313

Yeasts and Yeast-like Fungi 318
Morphology of Cryptococci 320
Germ Tube test 320
Growth on Cornmeal agar for Chlamydospore or True Hypae Production 321
Carbohydrate Assimilation Test 323
Carbohydrate Fermentation Test 323
Urease Test 324
Examination of Blood Smear for Malarial Parasite 325
Examination of Parasites From Faeces 326
Anti Microbial Susceptibility Testing (Kirby–Bauer Disc Diffusion Method) 329
Determination of Minimum Inhibitory Concentration 330
Antibiotic Susceptibility Testing 330
E-test 331
Broth Dilution Method 333
11. Biochemical Methodology 335
Centrifugation 335
Components 335
Types 336
Applications 338
Separation of Amino Acids 339
Paper Chromatography 339
Thin-layer Chromatography 343
Column Chromatography 345
Colorimeter and Spectrophotometer 347
Laws of Absorption 348
Visible Spectrophotometer 348
UV Spectrophotometer 351
Fluorscence Spectroscopy 352
Atomic Absorption Spectrophotometer (AAS) 356
Turbidometry 359
Bomb Calorimeter 362
Total Protein Estimation 364
Lowry’s Method 364
Bradford’s Method 367
Creatinine Estimation 368
 Contents xiii

Fractionation and Size (Determination of Proteins Using SDS–PAGE 369

Buffer Solutions 374
Concentration Units (Molarity, Normality, Molality) 380
12. Virology 385
Isolation of Coliphages 385
Phage Titration 386
Egg Inoculation 389
Cultivation of Animal and Plant Viruses 395
Cultivation of Animal Viruses 395
Cultivation of Plant Viruses 401

Appendix 407
References 439
 1
INTRODUCTION TO MICROBIOLOGY

INTRODUCTION
Microbiology is the study of microorganisms, that is, the organisms which are of microscopic
dimensions. These organisms are too small to be clearly perceived by the unaided human eye.
If an object has a diameter of less than 0.1 mm, the eye cannot perceive (or more correctly resolve)
it,  and very little detail can be perceived in an object with a diameter of 1 mm. Roughly speaking,
organisms with a diameter of 1 mm or less are microorganisms and fall into the broad domain
of microbiology. Since most microorganisms are only a few thousandths of a millimetre in size,
they can be seen only with the aid of microscope.
These microorganisms are classified into protozoa, algae, fungi, bacteria and viruses. Viruses
are ultramicroscopic and have an obligate parasitic relationship, but for practical purposes these
still come under the domain of microbiology.
At present, there is a general agreement to include five major groups as microorganisms:
viruses, bacteria, fungi, algae and protozoa (the studies of which are called as virology, bacteriology,
mycology, phycology and protozoology respectively).

BACTERIA
Bacteria are unicellular organisms that lack chlorophyll and are among the smallest living things
on earth. Multiplying rapidly under favourable conditions, bacteria can aggregate into colonies
of millions or even billions of organisms within a space as small as a drop of water. The Dutch
merchant and amateur scientist, Antony Van Leeuwenhoek, was the first to observe bacteria and
other microorganisms. Using single-lens microscopes of his own design, he described bacteria and
other microorganisms (called animalcules) in a series of letters to the Royal Society of London
between 1674 and 1723.
Bacteria are classified under Prokaryotes. Broadly, this taxonomic ranking reflects the fact
that the genetic material of bacteria is contained in a single, circular chain of deoxyribonucleic
acid (DNA) that is not enclosed within a nuclear membrane. The word “prokaryote” is derived
2 Microbiological Techniques

from a Greek word meaning “prenucleus”. Moreover, the DNA of prokaryotes is not associated
with the special chromosome proteins called histones, which are found in higher organisms.
In addition, prokaryotic cells lack other membrane-bound organelles, such as mitochondria.
Prokaryotes belong to the kingdom, Monera. Some scientists have proposed splitting this
designation into the kingdoms, Eubacteria and Archaebacteria. Eubacteria, or true bacteria,
consist of more common species, while Archaebacteria (with the prefix “archae”, meaning
“ancient”) represent strange bacteria that inhabit very hostile environments. Scientists believe
that these bacteria are most closely related to the bacteria, which lived when the earth was formed.
Examples of archaebacteria are those bacteria which currently live in extremely salty environments
or extremely hot environments, like geothermal vents of the ocean floor. Some examples of
archaebacteria are Methanococcus jannaschiii, Haloferax, Thermus aquaticus and Thermococcus litoralis.

Outer membrane Storage granule

Cytoplasm Capsule
Periplasmic space (sometimes)

Cytoplasmic (inner)
membrane
Peptidoglycan

Flagellum
Basal body

Ribosome
Mesosome
Chromosome
Pilus

Figure 1.1 Structure of a bacterial cell

STAGES OF BACTERIAL GROWTH

Under ideal conditions, the growth of a population of bacteria occurs in several stages, termed
as lag, log, stationary and death phase. During the lag phase, active metabolic activity occurs
involving synthesis of DNA and enzymes, but no growth. Geometric population growth occurs
during the log or exponential phase, when metabolic activity is most intense and cell reproduction
exceeds cell death. Following the log phase, the growth rate slows and the production of new
cells equals the rate of cell death. This period, known as the stationary phase, involves the
establishment of an equilibrium in population numbers and a slowing of the metabolic activities
of individual cells. The stationary phase reflects a change in growing condition, for example, a
 Introduction to Microbiology 3

lack of nutrients and/or the accumulation of waste products. When the rate of cell death exceeds
the number of new cells formed, the population equilibrium shifts to a net reduction in numbers
and the population enters the death phase, or logarithmic decline phase. The population may
diminish until only a few cells remain, or the population may die out entirely.

Stationary phase

Death
Log phase
Log phase
phase

Figure 1.2 Growth Curve

FACTORS AFFECTING BACTERIAL GROWTH

1. Temperature
The lowest temperature at which a particular species will grow is the minimum growth temperature,
while the maximum growth temperature is the highest temperature at which they will grow. The
temperature at which their growth is optimal is called the optimum growth temperature. In
general, the maximum and minimum growth temperatures of any particular type of bacteria are
about 30°F (–1°C) apart. Most bacteria thrive at temperatures at or around that of the human body,
i.e., 98.6°F (37°C), and some, such as Escherichia coli, are normal parts of the human intestinal
flora. These organisms are mesophiles (moderate temperature-loving), with an optimum growth
temperature between 77°F (25°C) and 104°F (40°C). Mesophiles adapt themselves to thrive in
temperatures close to that of their host. Psychrophiles, which prefer cold temperatures, are
divided into two groups. One group has an optimal growth temperature of about 59°F (15°C),
but can grow at temperatures as low as 32°F (0°C). These organisms live in ocean depths or Arctic
regions. Other psychrophiles that can also grow at 32°F (0°C) have an optimal growth temperature
between 68°F (20°C) and 86°F (30°C). These organisms, sometimes called psychrotrophs, are
often those associated with spoilage of food under refrigeration. Examples of pschychrotrophs are
Arthrobacter sp., Psychrobacter sp. and members of the genera Halomonas, Pseudomonas, Hyphomonas
and Sphingomonas. Thermophiles thrive in very hot environments, many having an optimum
growth temperature between 122°F (50°C) and 140°F (60°C), similar to that of hot springs in
4 Microbiological Techniques

Yellowstone National Park. Such organisms thrive in compost piles, where temperatures can rise
as high as 140°F (60°C). Extreme thermophiles grow at temperatures above 195°F (91°C), along
the sides of hydrothermal vents on the ocean bottom 217 mi (350 km) north of the Galapagos
Islands. Some bacteria grow in temperatures that can reach as high as 662°F (350°C). Some
examples of thermophiles are Thermus aquaticus and Thermococcus litoralis.

2. pH
Like temperature, pH also plays a role in determining the ability of bacteria to grow or thrive in
particular environments. Most commonly, bacteria grow optimally within a narrow range of pH
between 6.7 and 7.5. Acidophiles, however, prefer acidic conditions. For example, Thiobacillus
ferrooxidans, which occurs in drainage water from coal mines, can survive at pH 1. Other bacteria,
such as Vibrio cholerae, which causes cholera, can thrive at a pH as high as 9.0.

3. Osmotic Pressure
Osmotic pressure is another limiting factor for the growth of bacteria. Bacteria contain about
80–90% water; they require moisture to grow because they obtain most of their nutrients from
aqueous environment. Cell walls protect prokaryotes against changes in osmotic pressure over a
wide range. However, sufficiently hypertonic media at concentrations greater than those inside
the cell (such as 20% sucrose) cause water loss from the cell by osmosis. Fluid leaves the bacteria
causing the cell to contract, which, in turn, causes the cell membrane to separate from the
overlying cell wall. This process of cell shrinkage is called plasmolysis. Since plasmolysis inhibits
bacterial cell growth, the addition of salts or other solutes to a solution inhibits food spoilage by
bacteria, for example, in the salting of meat and fish. Some types of bacteria, called extreme or
obligate halophiles, are adapted to—and require—high salt concentrations, such as those found
in the Dead Sea, where salt concentrations can reach 30%. Facultative halophiles do not require
high salt environments to survive, but are capable of tolerating these conditions. Halophiles can
grow in salt concentrations up to 2%, a level that would inhibit the growth of other bacteria.
However, some facultative halophiles, such as Halobacterium halobium grow in salt lakes, salt flats,
and other environments where the concentration of salts is up to seven times greater than that
of the oceans. When bacteria are placed in hypotonic media with concentrations weaker than
the inside of the cell, water tends to enter by osmosis. The accumulation of this water causes the
cell to swell and then to burst, a process called osmotic lysis.

4. Carbon, Nitrogen and other Growth Factors

In addition to water and correct salt balance, bacteria also require a wide variety of elements,
especially carbon, hydrogen, nitrogen, sulphur, phosphorus, potassium, iron, magnesium and
calcium. Growth factors, such as vitamins, pyrimidines and purines (the building blocks of DNA),
 Introduction to Microbiology 5

are also necessary. Carbon is the fundamental building block of all the organic compounds needed
by living things, including nucleic acids, carbohydrates, proteins and fats. Chemoheterotrophs
are bacteria that use organic compounds such as proteins, carbohydrates and lipids as their carbon
source, and electrons from organic compounds as their energy source. Most bacteria (as well as
all fungi, protozoans and animals) are chemoheterotrophs. Chemoautotrophs (for example,
hydrogen, sulphur, iron and nitrifying bacteria) use carbon dioxide as their carbon source and
electrons from inorganic compounds as their energy source. Saprophytes are heterotrophs that
obtain their carbon from dead and decayed organic matter. Many different soil bacteria release
plant nitrogen as ammonia (ammonification). Bacteria, such as the Nitrosomonas, convert ammonia
to nitrite, while Nitrobacter convert nitrite to nitrate. Other bacteria, especially Pseudomonas, convert
nitrate to nitrogen gas. These bacteria complement the activity of nitrogen-fixing bacteria (for
example, Rhizobium, which fix nitrogen from the atmosphere and make it available to leguminous
plants, and Azotobacter, which are also found in fresh and marine waters). Together, the activities
of these bacteria are responsible for the nitrogen cycle by which the gas is taken up by living
organisms, used to make proteins and other organic compounds, returned to the soil during
decay, then released into the atmosphere to be reused by living things. Phototrophs use light as
their primary source of energy, but may differ in their carbon sources. Photoheterotrophs (purple
non-sulphur and green non-sulphur bacteria) use organic compounds as their carbon source, while
photoautotrophs (for example, photosynthetic green sulphur and purple sulphur bacteria) use
carbon dioxide as a source of carbon. Oxygen may or may not be a requirement for a particular
species of bacteria, depending on the type of metabolism used to extract energy from food (aerobic
or anaerobic). In all cases, the initial breakdown of glucose to pyruvic acid occurs during glycolysis,
which produces a net gain of two molecules of the energy-rich adenosine triphosphate (ATP).

5. Gaseous Requirements
Most bacteria may be placed into one of three groups based on their response to gaseous oxygen.
Aerobic bacteria thrive in the presence of oxygen and require it for their continued growth and
existence (Staphylococcus sp. Streptcoccus sp. Enterobacteriacae sp. Myobacterium tuberculosis). Other
bacteria are anaerobic (C. perfringens, C. botulinum), and cannot tolerate gaseous oxygen, such
as those bacteria which live in deep underwater sediments, or those which cause bacterial food
poisoning. The third group are the facultative anaerobes (Escherichia coli), which prefer growing
in the presence of oxygen, but can continue to grow without it. Let us discuss about each of
them in detail.
Aerobic bacteria Aerobic bacteria use oxygen to break down pyruvic acid, releasing much
more ATP than is produced during glycolysis, by the process known as aerobic respiration.
In addition, aerobic bacteria have enzymes such as superoxide dismutase, capable of breaking
down toxic forms of oxygen, such as superoxide free radicals, which are also formed by aerobic
respiration. During aerobic respiration, enzymes remove electrons from the organic substrate
and transfer them to the electron transport chain, which is located in the membrane of the
6 Microbiological Techniques

mitochondrion. The electrons are transferred along a chain of electron carrier molecules. At the
final transfer position, the electrons combine with atoms of oxygen—the final electron accept
or—which in turn combines with protons (H+) to produce water molecules. Energy, in the form
of ATP, is also produced. Along the chain of electron carriers, protons that are pumped across
the mitochondrial membrane re-enter the mitochondrion. This flow of electrons across the
membrane fuels oxidative phosphorylation, the chemical reaction that adds a phosphate group
to adenosine diphosphate (ADP) to produce ATP. Obligate aerobes must have oxygen in order
to live. Facultative aerobes can exist in the absence of oxygen also by using fermentative or
anaerobic respiration. Anaerobic respiration and fermentation occur in the absence of oxygen,
and produce substantially less ATP than aerobic respiration.
Anaerobic bacteria Anaerobic bacteria use inorganic substances other than oxygen as the
final electron acceptor. For example, Pseudomonas and Bacillus reduce nitrate ion (NO3-) to nitrite
ion (NO2-), nitrous oxide (N2O) or nitrogen gas (N2). Clostridium sp. which include those that
cause tetanus and botulism, are obligate anaerobes, that is, they are not only unable to use
molecular oxygen to produce ATP, but are also harmed by toxic forms of oxygen formed during
aerobic respiration. Unlike aerobic bacteria, obligate anaerobes lack the ability to synthesize
enzymes that neutralize these toxic forms of oxygen.
Microaerophilic bacteria Microaerophilic bacteria are a specific type of microorganism
(especially bacteria) that require oxygen to survive, but require environments containing
lower levels of oxygen than that are present in the atmosphere (~20% concentration). Many
microaerophiles are also capnophiles, as they require an elevated concentration of carbon dioxide.
In the laboratory they can be easily cultivated in a candle jar, a container into which a lit candle
is introduced before sealing the airtight lid. The flame burns until extinguished by oxygen
deprivation, creating a carbon dioxide-rich, oxygen-poor atmosphere.
Some of the examples are as follows.
 Borrelia burgdorferi, a species of spirochaete bacteria that causes Lyme disease in humans.
 Helicobacter pylori, a species of proteobacteria that has been linked to peptic ulcers and
some types of gastritis. Some do not consider it a true obligate microaerophile.
 Campylobacter has been described as microaerophilic.
 Streptococcus intermedius has also been described as microaerophilic.

CYANOBACTERIA
Cyanobacteria are photosynthetic, that is, they can manufacture their own food. Because they
are bacteria, they are quite small and usually unicellular, though they often grow in colonies
 Introduction to Microbiology 7

large enough to be seen. They have the distinction of being the oldest known fossils, more than
3.5 billion years old. They are one of the largest and most important groups of bacteria on earth.
The other great contribution of the cyanobacteria is the origin of plants. The chloroplast with
which plants make food for themselves is actually a cyanobacterium living within the plant’s cells.
Sometime in the late Proterozoic, or in the early Cambrian, cyanobacteria began to take up residence
within certain eukaryotic cells, making food for the eukaryote host in return for a home. This event
is known as endosymbiosis, and is also the origin of the eukaryotic mitochondrion.

Figure 1.3 Nostoc

Trichomes

Single trichome

dead cell

Figure 1.4 Oscillatoria

Cyanobacteria are often called blue-green algae because of their photosynthetic nature.
This name is convenient for talking about organisms that make their own food, but does not
reflect any relationship between the cyanobacteria and other organisms called algae. Cyanobacteria
8 Microbiological Techniques

are relatives of the bacteria, not eukaryotes, and it is only the chloroplast in eukaryotic algae to
which the cyanobacteria are related. Cyanobacteria are found throughout the world in terrestrial,
freshwater and marine habitats, but blooms typically occur in fresh water.

CHARACTERISTICS OF CYANOBACTERIA
1. Cyanobacteria or blue-green algae are gram-negative bacteria with a number of unusual
traits.
2. They are the largest and most diverse group of photosynthetic bacteria, which was previously
known as blue-green algae.
3. Cyanobacteria are true prokaryotes.
4. They vary greatly in shape and appearance.
5. They range in diameter from about 1 to 10 microns.
6. They may be unicellular and exist as colonies of many shapes, or form filaments called
trichomes.
7. They have normal gram-negative type cell wall.
8. They often use gas vesicles to move in the water, and many filamentous species have gliding
motility.
9. Their photosynthetic system closely resemble that of eukaryotes because they have
chlorophyll a and photosystem II. They carry out oxygenic photosynthesis, i.e., they use
water as an electron donor and generate oxygen during photosynthesis.
10. Cyanobacteria use pigments and electron transport chain components that are located
in thylakoid membranes linked with particles called phycobilisomes. They contain
phycocyanin pigment, and the CO2 in them is assimilated through the Calvin cycle.
11. Reserve food material in cyanobacteria is glycogen.
12. In cyanobacteria, hormogonia, akinetes and heterocysts are also produced.
 2
TOOLS OF MICROBIOLOGY

MICROSCOPY
INTRODUCTION
Microscopy refers to the use of light or electrons to magnify objects. Microorganisms are
too small to be seen with the unaided eye, and hence they are observed with a microscope.
The word “Microscope” is derived from the latin word Micro which means “small”, and the
Greek word skopos, which means “to look at”. Modern microbiologists use microscopes that
produce with great clarity, magnification that ranges from 10–1000 times greater than those of
Leeuwenhoek’s single lens. This chapter describes how different types of microscopes function
and why one type is used in preference to the other.
Microorganisms and their structural components are measured in even smaller units, such as
micrometre and nanometre. A micrometre is equal to 0.000001 m (10–6 m).

GENERAL PRINCIPLES OF MICROSCOPY

Wavelength of Radiation
Visible light is one part of the spectrum of electromagnetic radiation that includes X-rays,
microwaves and radio waves. The beams of radiation may be referred to as either rays or waves.
These various forms of radiation differ in wavelength—the distance between two corresponding
parts of a wave. The human eye discriminates among different wavelengths of visible light and
sends patterns of nerve impulses to the brain, which interprets the impulses as different colours.
For example, we see wavelengths of 400 nm as violet and wavelengths of 650 nm as red.
White light, composed of many colours (wavelengths), has an average wavelength of 550 nm.
Using radiations of smaller wavelengths results in enhanced microscopy.
10 Microbiological Techniques

Magnification
A light microscope usually has at least three objective lenses: the low power, high power and
oil-immersion lenses. In general, these lenses magnify an object 10, 40 and 100 times, respectively
(Magnification is represented by the multiplication (×) sign).This intermediate image is remagnified
by the ocular lens with a standard 10X ocular lens, the total magnification achieved is
100×, 400× and 1000×, respectively. The total magnification is determined by the following formula:

Total magnification = Magnification of objective lens × Magnification of ocular lens

Resolution
Resolution, which is also called resolving power, is the ability to distinguish between objects that
are close together as distinct and separate. Leeuwenhoek’s microscopes had a resolving power of
about 1mm i.e., it could distinguish between objects if they were more than about 1mm apart,
and objects close together (1mm) appear as a single object.
Limit of resolution is the smallest distance by which two objects can be separated and still
be distinguishable as two separate objects. The better the resolution, the better the ability to
distinguish two objects that are close to one another. Modern microscopes can distinguish between
objects as close together as 0.2mm
The resolving power (RP) of a lens system is important in microscopy because it denotes the
size of the smallest object that can be seen clearly. The resolving power varies for each objective
lens and is calculated using the following formula:

λ
RP =
2 × NA
In this formula, the Greek letter l (lambda) represents the wavelength of light and is usually
set at 500 nm, the halfway point between the limits of visible light. The symbol NA stands for the
numerical aperture of the lens and refers to the size of the cone of light that enters the objective
lens after passing through the specimen. The numerical aperture of an objective lens is defined
by NA=n sin q where n is the index of refraction of the medium in which the lens is working
(1.0 for air, 1.33 for pure water, and up to 1.56 for oils), and q is the half-angle of the maximum cone
of light that can enter or exit the lens. This number generally is printed on the side of the objective
lens. For a low-power objective with a NA of 0.25, the resolving power is calculated as follows:

500 nm 550
RP = = = 1,000 nm or 1.0 µm
2 × 0.25 0.50

Since the resolving power for this lens system is 1.0mm, any object smaller than 1.0mm could
not be seen as a clear distinct object. An object larger than 1.0mm would be resolved. Resolving
 Tools of Microbiology 11

power is calculated with the lens suspended in oil rather than air, a factor that increases the
numerical aperture to 1.25. Both low-power and high-power objectives are wide enough to capture
sufficient light for viewing. The oil-immersion objective, on the other hand, is so narrow that
most of the light would bend away and would miss the objective lens if oil were not used. The
index of refraction (or refractive index) is a measure of the light-bending ability of a medium.
Immersion oil has a refractive index of 1.5, which is almost identical to the refractive index of
glass. Because the refractive index is same for oil and glass, the light does not bend as it passes
from the glass slide and the specimen into the oil. By comparison, air has a refractive index of
1.0, which accounts for the abrupt bending of light as it enters. The oil, thus, provides a
homogeneous pathway for light from the slide to the objective, and the resolution of the object
increases. With the oil-immersion lens having a numerical aperture of 1.25, the highest resolution
possible with light microscope is 0.2mm (200 nm).

Contrast
Contrast refers to differences in intensity between two objects or between an object and its
background. Contrast is important in determining the resolution. For example, although you
can easily distinguish between two golf balls lying side by side on a green surface 15 m away, at
that distance it is much more difficult to distinguish between them if they lie on a white towel.
Most microorganisms are colourless and have very little contrast whether one uses light or
electrons. One way to increase the contrast between organisms and their background is to stain them.

LIGHT MICROSCOPY
1. Bright-field Microscopes
i. Simple microscope Leeuwenhoek first reported his observation of microorganisms
using a simple microscope in 1673. A simple microscope contains a single magnifying lens
(Figure 2.1).
ii. Compound microscope Simple microscopes have been replaced in modern laboratories
by compound microscopes. A compound microscope uses a series of lenses for magnification.
The microscope consists of a sturdy metal body or stand composed of a base and an arm to which
the remaining parts are attached. A light source, either a mirror or an electric illuminator, is
located at the base. Two focusing knobs, the fine and coarse adjustment knobs are located on
the arm, which can move the stage or the nosepiece to focus the image.
The stage is positioned halfway up the arm and holds the microscope slides by either simple
slide clips or a mechanical stage clip. A mechanical stage allows the operator to move a slide
around smoothly during viewing by use of stage control knobs. The substage condenser is mounted
within or beneath the stage and focuses a cone of light on the slide. Its position often is fixed in
simpler microscopes but can be adjusted vertically in more advanced models.
12 Microbiological Techniques

Lens
Folded Arm

Vertical limb

Stage

Stand

mirror

Foot

Figure 2.1 Simple microscope

The curved upper part of the arm holds the body assembly to which a nosepiece and one or
more eyepieces or oculars are attached. More advanced microscopes have eyepieces for both eyes
and are called binocular microscopes. The body assembly itself contains a series of mirrors and
prisms so that the barrel holding the eyepiece may be tilted for ease in viewing. The nosepiece
holds three to five objectives with lenses of differing magnifying power and can be rotated to
position any objective beneath the body assembly. (Figure 2.2)
The objective lenses on a typical microscope are: scanning objective lens (4×), low-power
objective lens (10×), high-power lens or high dry objective lens (40×) and oil-immersion
objective lens (100×). Ideally, a microscope should be parfocal, i.e., the image should remain
in focus when objectives are changed. Under usual operating conditions, the field of vision in a
compound light microscope is brightly illuminated. By focusing the light, the condenser produces a
bright-field illumination.
 Tools of Microbiology 13

Eyepiece lenses

Revolving nosepiece

Objective lenses Arm

Stage with clips

Condenser Coarse and fine focus

Illuminator Stage Controls

Base

Figure 2.2 Compound microscope

2. Dark-field Microscope
Pale objects are best observed with dark-field microscopes. These microscopes utilize a dark-
field stop in the condenser that prevents light from directly entering the objective. Instead,
light rays are reflected inside the condenser so that they pass into the slide at such an oblique
angle that they miss the objective lens. Only those light rays that are scattered by the specimen
enter the objective lens and are seen, so the specimen appears light against a dark background.
This increases contrast and enables observation of more details than that are visible in
bright-field microscopy.
Dark-field microscopy helps in the diagnosis of diseases caused by spiral bacteria because
these organisms are near the limit of resolution of the light microscope and do not stain well.
For example, syphilis caused by the spiral bacterium Treponema pallidum has a diameter of about 0.15mm
This bacterium may be observed in scrapings taken from a lesion of a person who has the disease.
Dark-field microscopy also is the preferred way to study motility of live prokaryotic and eukaryotic
cells.
14 Microbiological Techniques

Objective lens
Light that strikes
specimen

Microbes on
slide
Condenser lens
Dark field ring

Light rays

Figure 2.3 Dark-field microscope

3. Phase Microscopes
There are two types of phase microscopes: phase-contrast microscopes and differential
interference contrast microscopes.
i. Phase-contrast microscope Another way to observe microorganisms is by using a
phase-contrast microscope. Phase-contrast microscope is the simplest phase microscope that
produces sharply defined images in which fine structures can be seen in living cells. These
microscopes are particularly useful for observing cilia and flagella. Phase-contrast microscopy
is especially useful because it permits detailed examination of internal structures in living
microorganisms. In addition, it is not necessary to fix (attach the microbes to the microscope
slide) or stain the specimen, a procedure that could distort or kill the microorganisms.
The principle of phase-contrast microscopy is based on the wave nature of light rays, and
the fact that light rays can be in phase (their peaks and valleys match) or out of phase. If the
wave peak of light rays from one source coincides with the wave peak of light rays from another
source, the rays interact to produce reinforcement (relatively brighter image). However, if the
wave peak from one light source coincides with the wave trough from another light source, the
rays interact to produce interference (relatively darker image).
Light rays passing through a specimen naturally slow down and are shifted about
1/4 wavelength out of phase. A special filter called a phase plate, which is mounted in a phase
objective lens, retards these rays another 1/4 wavelength, so that they are 1/2 wavelength out of
phase with their neighbours. When the phase microscope lens brings the two sets of rays together,
troughs of one wave interfere with the crests of the other because they are out of phase, and thus,
contrast is created (Figure 2.4).
 Tools of Microbiology 15

Elevated (or excavated) ring on phase plate is coated to

reduce the intensity of direct light. Differences in length of light
path for direct and indirect light creates a 41 λ phase shift.

Diffracted light
1
λ
4

Undeviated
light

Objective Phase plate

lens

Specimen

Condenser

Phase annulus

Phase annulus creates a ring of oblique

illumination

Dim, indirect light deviated by passage through specimen

1
has been delayed 4 λ due to diffraction.

Direct light
(undeviated)

Now the direct light has been dimmed

1
and delayed 4 λ by passage through
phase plate elevations. Interference
with deviated light is now accentuated.

Figure 2.4 Phase-contrast microscope

ii. Differential interference contrast microscope In the mid-1950s, a French optics

theoretician named Georges Nomarski modified the Wollaston prism used for detecting optical
gradients in specimens and converting them into intensity differences. Today there are several
implementations of this design, which are collectively called differential interference contrast
(DIC). Living or stained specimens, which often yield poor images when viewed in bright-field
illumination, are made clearly visible by optical rather than chemical means.
16 Microbiological Techniques

Light detector
Polarizing filter

Wollaston prism

Objective lens

Sample

Condenser lens

Wollaston filter
Polarizing filter

Light source

Figure 2.5 Differential interference contrast microscope

In transmitted light DIC, light from the lamp is passed through a polarizer located beneath
the substage condenser, in a manner similar to polarized light microscopy. Next in the light path
(but still beneath the condenser) is a modified Wollaston prism that splits the entering beam of
polarized light into two beams travelling in slightly different directions. The Wollaston prism is
composed of two quartz wedges cemented together, from which emerging light rays vibrate at
90 degrees relative to each other with a slight path difference. A different Wollaston prism is
needed for each objective of different magnification. Wollaston prisms are usually loaded into a
revolving turret on the condenser, which allows the microscopist to rotate the appropriate prism
into the light path when changing magnifications.
The plane-polarized light vibrating only in one direction (East-West) perpendicular to the
propagation direction of the light beam, enters the beam-splitting modified Wollaston prism and
is split into two rays vibrating perpendicular to each other. These two rays travel close together
but in slightly different directions. The rays intersect at the front focal plane of the condenser,
where they pass travelling parallel and extremely close together with a slight path difference,
but they are vibrating perpendicular to each other and are therefore unable to cause interference.
The distance between the rays, called the shear, is so minute that it is less than the resolving
ability of the objective.
The split beams enter and pass through the specimen where their wave paths are altered in
accordance with the specimen’s varying thickness, slopes, and refractive indices. These variations
 Tools of Microbiology 17

cause alterations in the wave path of both beams passing through areas of any specimen details
lying close together. When the parallel beams enter the objective, they are focused above the rear
focal plane where they enter a second modified Wollaston prism that combines the two beams at a
defined distance outside of the prism itself. This removes the shear and the original path difference
between the beam pairs. As a result of having traversed the specimen, the paths of the parallel beams
are not of the same length (optical path difference) for differing areas of the specimen.
In order for the beams to interfere, the vibrations of the beams of different path length must be
brought into the same plane and axis. This is accomplished by placing a second polarizer (analyser)
above the upper Wollaston beam-combining prism. The light then proceeds toward the eyepiece
where it can be observed as differences in intensity and colour. The design results in one side of
the image appearing bright (or possibly in colour) while the other side appears darker (or another
colour). This shadow effect bestows a pseudo three-dimensional appearance to the specimen.
There are numerous advantages in DIC microscopy as compared to phase-contrast microscopy.
With DIC, it is possible to make fuller use of the numerical aperture of the system and to provide
optical staining (colour). DIC also allows the microscope to achieve excellent resolution. Use of
full objective aperture enables the microscopist to focus on a thin plane section of a thick specimen
without confusing images from above or below the plane.

4. Fluorescence Microscope
Fluorescence microscopy takes advantage of fluorescence, the ability of substances to absorb
short wavelengths of light (ultraviolet) and give off light at a longer wavelength (visible).
Some organisms fluoresce naturally under ultraviolet light. If the specimen to be viewed does
not naturally fluoresce, it is stained with one group of fluorescent dyes called fluorochromes.
The fluorescence microscope exposes a specimen to ultraviolet, violet or blue light and
forms an image of the object with the resulting fluorescent light. A mercury vapour arc lamp or
other source produces an intense beam, and heat transfer is limited by a special infrared filter.
The light passes through an exciter filter that transmits only the desired wavelength. A dark-
field condenser provides a black background against which the fluorescent objects glow.
The microscope forms the image of the fluorochrome-labelled microorganisms from the light
emitted when they fluoresce. A barrier filter positioned after the objective lenses removes any
remaining ultraviolet light, which would damage the viewer’s eyes, or blue and violet light, which
would reduce the contrast of the image (Figure 2.6).
An important application of fluorescence microscopy is the fluorescent antibody technique
used to identify an unknown organism. In one variation of this procedure, fluoresein is
chemically attached to antibodies, the protein molecules produced by the body’s immune system.
These “tagged” antibodies are mixed with a sample of unknown organism. If the antibodies are
specific for that organism, they will bind to it and coat the cells with the dye. When subjected to
UV light, the organism will fluoresce. The organism fails to fluoresce, if the antibodies are not
specific to that organism and a different tagged antibody is tried.
18 Microbiological Techniques

Eyepiece

Barrier filter

HBO
lamp
house Dichroic beam splitter
Excitation
filter

Exciting Objective
light
Emitted
fluorescence
light Specimen

Figure 2.6 Fluorescent microscope

Fluorescent microscope is also used to stain certain pathogens. For example, the dye fluorescein
isothiocyanate attaches to cells of Bacillus anthracis, the causative agent of anthrax, and appears
apple green. Another dye, auramine O, which fluoresces yellow, stains Mycobacterium tuberculosis.

ELECTRON MICROSCOPY
Objects smaller than about 0.2mm such as viruses or the internal structures of cells, must be
examined with an electron microscope. In electron microscopy, a beam of electrons is used,
instead of light. Free electrons travel in waves. The resolving power of the electron microscope
is far greater than that of the other light microscopes.
The better resolution of electron microscope is due to the shorter wavelengths of electrons;
the wavelength of the electrons are about 100,000 times smaller than the wavelength of visible
light. Thus, electron microscopes are used to examine structures too small to be resolved with
light microscopes. Images produced by electron microscope are always black and white, but they
may be coloured artificially to accentuate certain details.
Instead of using glass lenses, an electron microscope uses electromagnetic lenses to focus a
beam of electrons onto the specimen. There are two types of electron microscopes: the transmission
electron microscope and the scanning electron microscope.
 Tools of Microbiology 19

1. Transmission Electron Microscope

In the transmission electron microscope (TEM), (Figure 2.7(a)) a finely focused beam of electrons
from an electron gun passes through a specially prepared ultrathin section of the specimen.
The beam is focused on a small area of the specimen by an electromagnetic condenser lens that
performs roughly the function of the condenser of the light microscope, directing the beam of
electrons in a straight line to illuminate the specimen.
Electron microscope uses electromagnetic lenses to control illumination focus and
magnification. Instead of being placed on a glass slide, as in light microscopes, the specimen
is usually placed on a copper mesh grid. The beam of electron passes through the specimen
and then through an electromagnetic objective lens, which magnifies the image. Finally, the
electrons are focused by an electromagnetic projector lens, rather than by an ocular lens as in
a light microscope, onto a fluorescent screen or photographic plate. The final image, called a
transmission electron micrograph, appears as many light and dark areas, depending on the
number of electrons absorbed by different areas of the specimen.
In practice, the transmission electron microscope can resolve objects as close together as
2.5 nm and objects are generally magnified 10,000–100,000×. TEM is used to view and record
detailed structures, such as organelles within cells. Ultrathin sections of the object must be prepared
because the electron beam can penetrate matter only for a very short distance. After embedding
the specimen in a suitable plastic mounting medium or freezing it, scientists cut the specimen into
sections with a diamond knife. In this manner, a single bacterium can be sliced, like a loaf of bread,
into hundreds of thin sections. Several of the sections are placed on a small grid and stained with
heavy metals such as lead and osmium, to provide contrast (positive staining) or used to increase
the electron opacity of the surrounding field (negative staining). Negative staining is useful for
the study of the smallest specimens, such as virus particles, bacterial flagella and protein molecules.
In addition to positive and negative staining, a microbe can be viewed by a technique called
shadow casting. In this procedure, a heavy metal, such as platinum or gold, is sprayed at an
angle of about 45 degrees so that it strikes the microbe from only one side. The metal piles up on
one side of the specimen and the uncoated area on the opposite side of the specimen leaves a clear
area behind it as a shadow. This gives a three-dimensional effect to the specimen and provides
a general idea of the size and shape of the specimen. Transmission electron microscopy has high
resolution and is extremely valuable for examining different layers of specimens. However, it
does have certain disadvantages. Because electrons have limited penetrating power, only a very
thin section of specimen (about 100 nm) can be studied effectively. Thus, the specimen has no
three-dimensional aspect. In addition, specimens must be fixed, dehydrated and viewed under
a high vacuum to prevent electron scattering. These treatments not only kill the specimen but
also cause some shrinkage and distortion, sometimes to the extent that there may appear to be
additional structures in a prepared cell. Such additional structures that appear as a result of the
method of preparation are called artifacts.
20 Microbiological Techniques

2. Scanning Electron Microscope

The scanning electron microscope (SEM) was developed in the late 1960s to enable researchers
to see the surfaces of objects in the natural state and without sectioning. In scanning electron
microscopy, an electron gun produces a finely focused beam of electrons called the primary electron
beam (Figure 2.7 (b)). These electrons pass through electromagnetic lenses and are directed over
the surface of the specimen. The primary electron beam knocks electrons out of the surface of the
specimens and the secondary electrons thus produced are transmitted to an electron collector,
amplified and used to produce an image on a viewing screen or photographic plate. The image
is called a scanning electron micrograph.
This microscope is especially useful in studying the surface structures of intact cells and
viruses. In practice, it can resolve objects as close together as 20 nm, and magnication produced
is generally of 1,000–10,000×.

Electron gun
Primary electron beam
Electron beam
Electromagnetic
condenser lens Electromagnetic lenses
Viewing screen
Electromagnetic Viewing
objective lens eyepiece
Electron
collector
Electromagnetic Secondary
projector lens electrons

Fluorescent Specimen
screen or photo-
graphic plate Amplifier

(a) Transmission electron microscope (b) Scanning electron microscope

Figure 2.7 Electron microscope

SCANNED-PROBE MICROSCOPY
Since the early 1980s, several new types of microscopes, called scanned-probe microscopes,
have been developed. They use various kinds of probes to examine the surface of a specimen at
a very close range, and they do so without modifying the specimen or exposing it to damaging
high-energy radiation. Such microscopes can be used to map atomic and molecular shapes, to
characterize magnetic and chemical properties and to determine temperature variations inside
cells. Among the new scanned-probe microscopes are the scanning tunnelling microscope and
the atomic force microscope.
 Tools of Microbiology 21

1. Scanning Tunnelling Microscope

Scanning tunnelling microscope (STM) uses a thin metal (tungsten) probe that scans a specimen
and produces an image revealing the bumps and depressions of the atoms on the surface of the
specimen (Figure 2.8). The resolving power of a STM is much greater than that of an electron
microscope, it can resolve features that are only about 1/100th the size of an atom. Moreover,
special preparation of the specimen for observation is not needed. STMs are used to provide
detailed views of molecules, such as DNA.

Control voltages for piezotube

Piezoelectric tube
with electrodes

Tunneling Distance control

Current amplifier and scanning unit

D Tip

Sample

Tunnelling
Voltage
Data processing
and display

Figure 2.8 Schematic diagram of scanning tunneling microscope

2. Atomic Force Microscope

In atomic force microscopy (AFM), a metal and diamond probe is gently forced down onto a
specimen. As the probe moves along the surface of the specimen, its movements are recorded
and a three-dimensional image is produced. The deflection of a cantilever holding the tip is
monitored by a photodiode. As with STM, AFM does not require special specimen preparation.
AFM is used to image both biological substances (upto atomic level) and molecular processes
(such as the assembly of fibrin, a component of a blood clot).
22 Microbiological Techniques

Recent studies using atomic force microscopes have provided insight into the molecular folding
of lipopolysaccharides on the surfaces of gram-negative pathogenic bacteria.

Laser

Photo diode

Cantilever

Tip

t
un
Sample

mo
le
mp
Sa
Z

X
Piezoelectric
scanner
Piezo movement

Figure 2.9 Atomic force microscope

The various types of microscopes described above are summarized in Table 2.1.

USE AND CARE OF MICROSCOPES

 Always carry the microscope with two hands, one hand on the arm and one hand placed
under the base for support.
 Remove cover, plug into outlet, turn on light.
 Place slide in mechanical slide holder.
 Set the stage in the “all the way up” position.
 Using scanning objective, locate the object; using the coarse adjustment knob, focus to obtain
the clearest image possible.
 Using the fine adjustment knob, bring the object into the clearest image possible; centre the
object in the field of view.
 Bring the low-power objective into place.
 Focus the object using minor adjustments with the fine adjustment knob.
 Centre object in field of view; adjust light source, if needed.
 Tools of Microbiology 23

 Bring the high-power objective into place.

 Using only the fine adjustment knob, bring the object into the clearest focus possible. Adjust
light, if needed and centre the object in the field of view.
 When the work is finished with your microscope:
 Remove the slide.

 Centre the mechanical stage holder.

 Place the scanning objective in place.

 Return the stage to the “all the way up” position.

 Turn off the light.

 Fold up and tie the cord.

 Replace the cover.

 Return the microscope to the cabinet.

Cleaning the Microscope

 Do not let the microscope get too dirty—always use the dust cover when not in use.
 To clean the eyepiece, use a high-quality lens paper. First, brush any visible dust from the
lens, and then wipe the lens.
 Do not use facial tissues; they are made from ground-up wood fibres and could damage the
lenses.
 To clean the objective lenses, use a fresh piece of the lens paper each time so that you do not
transfer dust from one lens to another.
 Use lens paper on all glass parts of the microscope. (A cotton swab (Q-tipTM) can be used in
place of lens paper).
Things to Remember
 Never use the coarse adjustment knob when on high power.
 Never swing the oil-immersion objective into place.
 If you lose your object (microscope gets bumped, slide is displaced) always start from the
beginning (scanning objective, stage “all the way up”) to relocate your object.
 Total magnification = Magnification of the eyepiece × Magnification of the objective
10× 4 (scanning) = 40×

10× 10 (low) = 100×

10× 40 (high) = 400×

 Table 2.1 A summary of various types of microscopes 24

Microscope type Distinguishing features Principal use

I. Light microscopes
1. Bright-field microscope Uses visible light as a source of illumination. To observe various stained specimens and to
Cannot resolve structures smaller than about count microbes. Does not resolve very small
0.2 µm; specimen appears against a bright specimen, such as viruses.
Microbiological Techniques

background; inexpensive and very easy to use.

2. Dark-Field microscope Uses a special condenser with an opaque disc To examine living microorganisms that are
that blocks light from entering the objective invisible in bright-field microscopy; do not
lens directly; light reflected by specimen enters stain easily or are distorted by staining.
the objective lens and the specimen appear Frequently used to detect Treponema
light against dark background. pallidum in the diagnosis of syphilis.
3. Phase contrast Uses a special condenser containing an annular To facilitate detailed examination of the
microscope (ring-shaped) diaphragm. The diaphragm internal structures of the living specimens.
allows direct light to pass through the
condenser, focusing light on the specimen and
a diffraction plate in the objective lens. Direct
and reflected or diffracted light rays are
brought together to produce the image; no
staining is required.
4. Differential interference Like phase-contrast, this uses differences in To provide three-dimensional images.
contrast (DIC) refractive indices, to produce image; uses two
microscope beams of light separated by prisms. The
specimen appears coloured as a result of the
prism effect; no staining required.
5. Fluorescent microscope Uses an ultraviolet or near ultraviolet source of To rapidly detect and identify microbes in
illumination that causes fluorescent microbes tissues or clinical specimens, for
(green-coloured) in a specimen to emit light. fluorescent–antibody technique (immuno-
fluorescence).
(Contd.)
 Table 2.1 (Continued)

Microscope type Distinguishing features Principal use

II. Electron microscopes
1. Transmission electron Uses a beam of electrons instead of light; To examine viruses or the internal ultra
microscope electron passes through the specimen structures structure in thin sections of cells (usually
smaller than 0.2µm can be resolved. The magnified 10,000–100,000×).
image produced is two-dimensional.
2. Scanning electron Uses a beam of electrons instead of light; To study the surface features of cells and
microscope electrons are reflected from the specimen viruses (usually magnified
structures smaller than 0.2µm can be resolved. 1,000–10,000×).
The image produced appears
three-dimensional.
III. Scanned-probe microscopes
1. Scanning tunneling Uses a thin metal probe that scans a specimen To provide detailed view of molecules
microscope and produces an image revealing the bumps inside cells.
and depressions of the atoms on the surface of
the specimen. Resolving power is much greater
than that of the electron microscope. No
special preparation required.
2. Atomic force Uses a metal and diamond probe gently forced To provide images of biological molecules
microscope down along the surface of the specimen. and molecular processes.
Produces a three-dimensional image. No special
preparation required.
Tools of Microbiology 25
26 Microbiological Techniques

AUTOCLAVE
Definition
Autoclave is a device used for sterilization. It uses moist heat for killing all the microbes, including
heat-resistant spores. It was discovered by Chamberland in 1884.

Principle
One of the important agents used in sterilization is heat. Autoclave uses moist heat
(121°C at 15 lb/in2 pressure for 15 minutes). The moist heat is highly penetrative. Heat coagulates
organisms. Autoclave has the mechanism for regulating steam pressure in ensuring complete
evacuation of air from the chamber. A suitable safety valve is included to avoid explosion.
The pressure of air allows increase in pressure within the chamber without increase in temperature.
Hence, all the air is expelled out of the chamber.

Structure
It has a double-walled, high gauze steel jacket in a hollow cylindrical manner (Figure 2.10).
Three sides are closed with double-walled steel. Upper side is closed with an air-tight thick door.
The door is fixed with bakelite handle. There is a safety lock within the door. Inside the cylinder,
the working space is found. There are many types of autoclaves—vertical and horizontal—of
varying size.
A water tank with an electric heater is found to be fixed at the bottom of the chamber. In it,
the water is heated and steam is produced, which is sent into the chamber. The steam of correct
pressure is sent into the chamber. The pressure regulator is fixed with the steam line pipe to
cut off the steam. The steam intake pipe from the tank is split into 2 or 3 small pipes so that
steam is supplied to all parts of the chamber in a uniform manner with the same pressure. Two
pipelines from the chamber are fitted with the chamber pressure gauze to measure the pressure
and another to the thermometer, to measure the temperature. The pressure is measured in lb/in2
and temperature in °C. On the bottom side of the chamber, a drain pipeline is seen that removes
excess water. Another vent is seen that removes excess steam faster after sterilization. The vent
lines are connected to a waste line, which is let out of the autoclaving room. The vent pipe lines
are fixed with the operating valves which should be in a closed condition to build the pressure
inside the chamber while autoclaving. At the top of the autoclave, safety air valves are fixed.
If the pressure goes above 15 lb/in2, it automatically bursts off to release the steam pressure
inside the autoclave. The materials should not touch the walls, as it might lead to breakage of
the glasswares.
In autoclaves, vertical and horizontal types with built-in water tanks and electric heater are
also available.
 Tools of Microbiology 27

Steam exhaust pipe

Steam from jacket into chamber

Inner chamber Door

Steam in
Culture
medium

Jacket

To drain Steam supply

Figure 2.10 Autoclave

Method of Operation
The temperature is set by turning the temperature setting knob at the bottom of the water bath.
It raises the temperature of the thermostat. The pilot indicator lamp starts to glow and indicates
that the temperature has risen. The temperature in the thermometer is watched till it reaches
the desired temperature.

Checking the Efficiency of Autoclave

To check the efficiency, strips containing Bacillus stearothermophilus are placed along with the
material to be sterilized. After sterilization, the strips are placed in the nutrient agar medium to
check for the survivors. Chemical sterilization check tapes also can be used.

Uses
1. Used to sterilize media, nutrient solution.
2. Used to sterilize clothes, cotton and other easily charable substances.
28 Microbiological Techniques

HOT AIR OVEN

Definition
Hot air oven is a device that is used to sterilize objects with the help of dry heat as the sterilizing agent.

Principle
This is an air-dry type of sterilizer. It is used for the sterilization of the articles made of glass or
metals. It is used to sterilize pipettes, Petri dishes and flasks. Direct contact of glassware with
the walls of hot air oven should be prevented. Otherwise, it will lead to breakage of glasswares.
Hence, glasswares are kept in Petri cans and pipette cans. Dry heat is less penetrative than moist
heat. Their ability to attack the enzymes in microbes is very slow. Hence, a high temperature
treatment for a long time is required for killing all organisms. All the materials are treated at
160°C for one hour. Laboratory coats, rubber, culture media would burn or boil to dryness, and
hence, cannot be sterilized by hot air oven.

Figure 2.11 Hot air oven

 Tools of Microbiology 29

Structure
It is a cube-like structure, approximately of equal height × length × breadth (Figure 2.11).
It is made of three walls with two air spaces. The outer wall is covered with asbestos pad to reduce
the radiation by heat. A burner manifold runs along both sides. The coil is connected to an electric
circuit and conventional current travels in a complete circuit through the wall spaces and interior
of the oven. Two openings are present on the two sides for the products of combustion to escape
through. At the point where the electric circuit enters into the coil, a thermostat is fixed. It regulates
the current flow. The thermostat is operated on both directions by adjusting a temperature setting
knob. A rise in temperature is indicated by the thermostat by the glow of pilot lamp. Adjustable
shelves made up of perforated steel plates are present for keeping the materials to be sterilized. The
whole instrument is kept in use or out of use by an “On/Off” switch.

Method of Operation
The temperature is set by turning the temperature setting knob at the bottom of the oven.
It raises the temperature of the thermostat. The pilot indicator lamp starts to glow and indicates
that the temperature has risen. The temperature in the thermometer is watched till it reaches
the desired temperature of 160ºC. Once the temperature is set, the setting knob should not be
disturbed so that the temperature is maintained.

Checking the Efficiency of Hot Air Oven

To check the efficiency, strips containing Bacillus stearothermophilus are placed along with the
material to be sterilized. After sterilization, the strips are placed in the nutrient agar medium to
check for survivors. Chemical sterilization check tapes also can be used.

Use
It is used to sterilize all glassware and equipment.

INCUBATOR
Definition
Incubator is an instrument that is used to maintain optimum temperature for organisms to grow
and perform maximum metabolic activity.

Principle
Temperature is considered to be an important factor for the growth of microorganisms. In the
energy-producing process in an organism, the metabolic pathway consists of many complex
enzymes by which the complex substrates are converted into simpler ones. The enzymes catalyse
chemical reactions in which the energy-rich compounds, called the ATP molecules, are produced.
30 Microbiological Techniques

These enzymes have their own speed of catalysing the reactions. Many external factors are found
to be playing a major role in the determination of speed of an enzyme. When the temperature
is found to be optimum, the enzyme activity is found to be at peak level, which results in faster
growth and multiplication of the organism. Each organism produces different extracellular enzymes
and each enzyme has its own temperature for maximum activity. This temperature is known as
optimum temperature of the organisms. By maintaining the optimum temperature, enzymes
are activated at high speed, and growth can be seen in large amounts.

Structure
It is an instrument similar to that of hot air oven in structure (Figure 2.12). The usual temperature
of a bacteriological incubator is 37°C since most of the bacteria are mesophilic in nature.
For fungal cultures, the optimum temperature is around 25ºC. It is constructed with two walls.
The outer wall is covered with asbestos pad to reduce the loss of heat. The incubator is fitted with
an insulator door. In the door, plexi glass seal is fixed, which helps in inspecting the specimen
without disturbing the temperature. The inner chamber is made of stainless steel with trays to
keep the materials to be incubated. The shelf positions of the trays are adjustable. All the controls
are mounted at the bottom of the incubator, which comprises of self-illuminated “On/Off ” switch,
heat energy regulator, solid-state fully automated temperature controller cum indicator, indicator
lamps and protective fuses.

Figure 2.12 Incubator

Method of Operation
The heat energy is radiated manifold through a coil burner running along the rear side.
The electric current is passed through a thermostat of low range. The thermostat is connected
 Tools of Microbiology 31

with capacitors for the continuous usage of the instrument. A thermometer is seen at the top
to know the temperature inside the incubator. The power supply can be checked through pilot
lamps. Many incubators of different ranges are used for the growth of organisms.

Uses
Incubator is used to incubate inoculated Petri plates. Various types of microbes can be incubated
by setting various temperatures.

WATER BATH
Definition
Water bath is a covered glass or metal tank containing water at a thermostatically controlled
temperature, which is used in the incubation of microbes.

Principle
The contents placed in a water bath are raised to the required temperature much more rapidly
than in an incubator. In an incubator, the heat is radiated through air molecules, where a
loss of energy is seen. The penetration of heat energy is slow. Hence, heat waves are radiated
through water molecules in a water bath. These water baths are used for short-term incubation.
The material to be incubated is placed in a vessel suspended in the water. The level of water has
to be maintained at one-half to two-thirds of liquid in containers to be incubated. This results
in convention current, which heats the contents of the vessel, and hastens reactions, such as
agglutination. A motor-driven propeller ensures rapid mixing of the water and the maintenance
of the given temperature in all parts of the tank. Greater temperature stability can be achieved
in a water bath than in a hot air incubator. By maintaining the incubation temperature in water
bath, the enzyme action is found to be maximum than in an incubator.

Structure
Water bath is made up of thick steel with heat resistant material (Figure 2.13). Bakelite present
in between the two walls prevents heat loss through walls. The water bath is constructed with
electrical heating coils at the bottom which is connected with a thermostat. The heating coils are
covered with perforated steel stands to prevent the direct contact of vessels with the coils. The
thermostat is controlled with a temperature control knob. The temperature range is marked in
°C in the knob. By turning the knob left or right, the temperature can be lowered or increased.
The turning of the knob alters the current supply to the thermostat, which in turn heats the
electric coil to increase the temperature of water or cuts the electric supply to the electric heating
coil. This reduces the temperature of the water. A pilot lamp indicates the increase in temperature
and is seen near the temperature setting knob. The thermostat is connected with a capacitor
32 Microbiological Techniques

for continuous use of water bath. The water bath is fitted with lids to prevent the heat loss and
evaporation. Distilled deionized water should be used in water bath to avoid chalky deposits.
A thermometer is attached in such a way that its temperature-sensitive region is in touch with
the water. The current temperature inside the water bath can be determined.

Figure 2.13 Water bath

Refrigerated Water Bath

It is used for maintaining temperature below the ambient temperature. It includes a cooling coil,
which operates continuously and maintains a steady temperature by means of the thermostat
and heater of the water bath.

Method of Operation
The temperature is set by turning the temperature setting knob at the bottom of the water bath.
It raises the temperature of the thermostat. The pilot indicator lamp starts to glow and indicates
that the temperature has risen. The temperature in the thermometer is watched till it reaches
the desired temperature.

Uses
1. It is used for short-time incubation.
2. It is used to sterilize temperature-sensitive compounds at high temperature.
3. It is used to incubate organisms at correct temperature, so that its enzyme activity is at
maximum level and hence, can be used for enzyme extraction.

BOD INCUBATOR
Definition
BOD incubator is an instrument used for measuring the biological oxygen demand, i.e., the
amount of oxygen used in the respiratory process of microorganisms in oxidizing the organic
matter in sewage.
 Tools of Microbiology 33

Principle
The amount of oxygen used is measured by calculating the dissolved oxygen. These environmental
test chambers are devised so that it can satisfy a variety of environmental conditions. They are
especially suitable for the stimulation of fixed temperature and relative humidity values above
freezing point.

Structure
They have double walls, the exterior wall is made of sheet steel and the interior is made of
stainless steel, to avoid corrosion (Figure 2.14). A flush fitting insulated door has leak gasket.
The hand-operated wiper allows a clear view of the interior. To the inside of the door, a glass door
with tight closure is seen. The rear chamber of the interior is fitted with a refrigeration evaporator
to decrease the temperature; heater is seen to increase the temperature of powerful air-circulating
fan to create a positive air flow in the chamber. A thermostatically sealed compressor below the
chamber provides temperature below ambient. The same compartment houses a boiler with an
immersion heater and water level controller for creating humidity. The control panel comprises
of an “On/Off ” switch, heat energy regulators, fully automatic solid-state digital electronic
controller cum indicators, indicator lamps and protective fuses.

Figure 2.14 BOD Incubator

34 Microbiological Techniques

Method of Operation
The temperature is set by turning the temperature setting knob at the bottom of the BOD
incubator. It raises the temperature of the thermostat. The thermostat is connected with capacitors
for the continuous usage of the instrument.

Use
It is used to estimate the amount of oxygen consumed. If all the oxygen is utilized in the given
sample, it indicates a high level of sewage pollution.

COLONY COUNTER
Definition
A colony counter is an instrument that is used to count colonies of bacteria or other microorganisms
growing on an agar plate. Early counters were merely lighted surfaces on which the plate was
placed, with the colonies marked off with a felt-tipped pen on the outer surface of the plate
while the operator kept the count manually. More recent counters attempt to count the colonies
electronically, by identifying individual areas of dark and light according to automatic or user-set
thresholds, and counting the resulting contrasting spots.

Principle and Method of Operation

An appropriate dilution or several dilutions within the estimated appropriate range, is spread
using sterile technique on the agar plate, which is then incubated under the appropriate conditions
for growth until individual colonies appear. Each colony marks the spot where a single organism
was originally placed, thus the number of colonies on the plate equals the number of organisms
within the volume of liquid spread on the plate. That concentration is then extrapolated by the
known dilution from the original culture, to estimate the concentration of organisms within that
original culture.
The maximum number of colonies which may be effectively counted on a single plate is
somewhere between 100 and 1,000, depending on the size of the colony and the type of organism.

Structure
The machine is operated by “On/Off ” switch. It also has a light switch to switch on the light
source. The colony counter has a centring device at the centre which has an illumination screen
holder to place the plate to be counted. There is a reset button to reset the colony counter. It has
digital counter which shows the number of colonies counted. A felt-tipped pen marks off the
colony. A magnifying glass is attached to the centring device by a rod (Figure 2.15).

Use
Colony counter is used to estimate the density of microorganisms within a culture.
 Tools of Microbiology 35

2
3

4 Front part
5 1. Threaded hole for holder rod
2. Reset push-button
3. Digital counter
4. IIIuminated screen holder for reticule
5. Centring device
Side part
6 6. Magnifying glass holder
7. Feeder for top light
7 8. Bracket
Rear part
9. Socket for contact pointer
10. Socket for fiber marker
11. Light switch
. 8 12. On/off switch
13. Fuse
14. Mains cable
9 13
10 14
12
11

Figure 2.15 Colony counter

HAEMOCYTOMETER
Definition
The simplest, most convenient and cheapest means for accurately determining the numbers of cells
in a sample is to use a haemocytometer and a microscope. A haemocytometer is a specialized
slide that has a counting chamber with a known volume of liquid.

Principle and Method of Operation

 The haemocytometer consists of a heavy glass slide with two counting chambers, each of
which is divided into nine large 1 mm squares, on an etched and silvered surface separated
by a trough.
 A coverslip sits on top of the raised supports of the ‘H’-shaped troughs enclosing both chambers.
There is a ‘V’ or notch at either end where the cell suspension is loaded into the haemocytometer.
When loaded with the cell suspension, it contains a defined volume of liquid.
36 Microbiological Techniques

 The engraved grid on the surface of the counting chamber ensures that the number of particles
in a defined volume of liquid is counted.
 The haemocytometer is placed on the microscope stage and the cell suspension is counted.
Figure 2.16 shows the haemocytometer system.

Figure 2.16 Haemocytometer system

1 mm
corner square

Middle square

Figure 2.17 Standard haemocytometer chamber

Exploring the Variety of Random
Documents with Different Content
distinctly recognized that, in the material world, there is neither
break nor pause; so that what we call the divisions of nature have
no existence, except in our minds.[776] He was even almost
prepared to do away with that imaginary difference between the
organic and inorganic world, which still troubles many of our
physicists, and prevents them from comprehending the unity and
uninterrupted march of affairs. They, with their old notions of
inanimate matter, are unable to see that all matter is living, and that
what we term death is a mere expression by which we signify a
fresh form of life. Towards this conclusion, all our knowledge is now
converging; and it is certainly no small merit in Leslie, that he, sixty
years ago, when really comprehensive views, embracing the whole
creation, were scarcely known among scientific men, should have
strongly insisted that all forces are of the same kind, and that we
have no right to distinguish between them, as if some were living,
and others were dead.[777]
We owe much to him, by whom such views were advocated. But
they were then, and in a certain, though far smaller degree, they are
now, so out of the domain of physical experience, that Leslie never
could have obtained them by generalizing in the way which the
inductive philosophy enjoins. His great work on heat was executed,
as well as conceived, on the opposite plan;[778] and his prejudices
on this point were so strong, that we are assured by his biographer,
that he would allow no merit to Bacon, who organized the inductive
method into a system, and to whose authority we in England pay a
willing, and I had almost said a servile, homage.[779]
Another curious illustration of the skill with which the Scotch mind,
when once possessed of a principle, worked from it deductively,
appears in the geological speculations of Hutton, late in the
eighteenth century. It is well known, that the two great powers
which have altered the condition of our planet, and made it what it
is, are fire and water. Each has played so considerable a part, that
we can hardly measure their relative importance. Judging, however,
from the present appearance of the crust of the earth, there is
reason to believe, that the older rocks are chiefly the result of
fusion, and that the younger are aqueous deposits. It is, therefore,
not unlikely, that, in the order in which the energies of nature have
unfolded themselves, fire preceded water, and was its necessary
precursor.[780] But, all that we are as yet justified in asserting is,
that these two causes, the igneous and the aqueous, were in full
operation long before man existed, and are still busily working.
Perhaps they are preparing another change in our habitation,
suitable to new forms of life, as superior to man, as man is superior
to the beings who occupied the earth before his time. Be this as it
may, fire and water are the two most important and most general
principles with which geologists are concerned; and though, on a
superficial view, each is extremely destructive, it is certain that they
can really destroy nothing, but can only decompose and recompose;
shifting the arrangements of nature, but leaving nature herself
intact. Whether one of these elements will ever again get the upper
hand of its opponent, is a speculation of extreme interest. For, there
is reason to suspect, that, at one period, fire was more active than
water, and that, at another period, water was more active than fire.
That they are engaged in incessant warfare, is a fact with which
geologists are perfectly familiar, though, in this, as in many other
cases, the poets were the first to discern the truth. To the eye of the
geologist, water is constantly labouring to reduce all the inequalities
of the earth to a single level; while fire, with its volcanic action, is
equally busy in restoring those inequalities, by throwing up matter to
the surface, and in various ways disturbing the crust of the globe.
[781] And as the beauty of the material world mainly depends on
that irregularity of aspect, without which scenery would have
presented no variety of form, and but little variety of colour, we
shall, I think, not be guilty of too refined a subtlety, if we say that
fire, by saving us from the monotony to which water would have
condemned us, has been the remote cause of that development of
the imagination which has given us our poetry, our painting, and our
sculpture, and has thereby not only wonderfully increased the
pleasures of life, but has imparted to the human mind a
completeness of function, to which, in the absence of such a
stimulus, it could not have attained.
When geologists began to study the laws according to which fire
and water had altered the structure of the earth, two different
courses were open to them, namely, the inductive and the
deductive. The deductive plan was to compute the probable
consequences of fire and water, by reasoning from the sciences of
thermotics and hydrodynamics; tracking each element by an
independent line of argument, and afterwards coördinating into a
single scheme the results which had been separately obtained. It
would then only remain to inquire, how far this imaginary scheme
harmonized with the actual state of things; and if the discrepancy
between the ideal and the actual were not greater than might fairly
be expected from the perturbations produced by other causes, the
ratiocination would be complete, and geology would, in its inorganic
department, become a deductive science. That our knowledge is ripe
for such a process, I am far, indeed, from supposing; but this is the
path which a deductive mind would take, so far as it was able. On
the other hand, an inductive mind, instead of beginning with fire and
water, would begin with the effects which fire and water had
produced, and would first study these two agents, not in their own
separate sciences, but in their united action as exhibited on the crust
of the earth. An inquirer of this sort would assume, that the best
way of arriving at truth would be to proceed from effects to causes,
observing what had actually happened, and rising from the complex
results up to a knowledge of the simple agents, by whose power the
results have been brought about.
If the reader has followed the train of thought which I have
endeavoured to establish in this chapter, and in the first volume, he
will be prepared to expect that when, in the latter half of the
eighteenth century, geology was first seriously studied, the inductive
plan of proceeding from effects to causes became the favourite one
in England; while the deductive plan of proceeding from causes to
effects, was adopted in Scotland and in Germany. And such was
really the case. It is generally admitted, that, in England, scientific
geology owes its origin to William Smith, whose mind was singularly
averse to system, and who, believing that the best way of
understanding former causes was to study present effects, occupied
himself, between the years 1790 and 1815, in a laborious
examination of different strata.[782] In 1815, he, after traversing the
whole of England on foot, published the first complete geological
map which ever appeared, and thus took the first great step towards
accumulating the materials for an inductive generalization.[783] In
1807, and, therefore, before he had brought his arduous task to an
end, there was formed in London the Geological Society, the express
object of which, we are assured, was, to observe the condition of
the earth, but by no means to generalize the causes which had
produced that condition.[784] The resolution was, perhaps, a wise
one. At all events, it was highly characteristic of the sober and
patient spirit of the English intellect. With what energy and
unsparing toil it has been executed, and how the most eminent
members of the Geological Society have, in the pursuit of truth, not
only explored every part of Europe, but examined the shell of the
earth in America and in Northern Asia, is well known to all who are
interested in these matters; nor can it be denied, that the great
works of Lyell and Murchison prove that the men who are capable of
such laborious enterprises, are also capable of the still more difficult
achievement of generalizing their facts and refining them into ideas.
They did not go as mere observers, but they went with the noble
object of making their observations subservient to a discovery of the
laws of nature. That was their aim; and all honour be to them for it.
Still, it is evident, that their process is essentially inductive; it is a
procedure from the observation of complex phenomena, up to the
elements to which those phenomena are owing; it is, in other words,
a study of natural effects, in order to learn the operation of natural
causes.
Very different was the process in Germany and Scotland. In 1787,
that is, only three years before William Smith began his labours,
Werner, by his work on the classification of mountains, laid the
foundation of the German school of geology.[785] His influence was
immense; and among his pupils we find the names of Mohs, Raumer,
and Von Buch, and even that of Alexander Humboldt.[786] But the
geological theory which he propounded, depended entirely on a
chain of argument from cause to effect. He assumed, that all the
great changes through which the earth had passed, were due to the
action of water. Taking this for granted, he reasoned deductively
from premisses with which his knowledge of water supplied him.
Without entering into details respecting his system, it is enough to
say, that, according to it, there was originally one vast and primeval
sea, which, in the course of time, deposited the primitive rocks. The
base of all was granite; then gneiss; and others followed in their
order. In the bosom of the water, which at first was tranquil,
agitations gradually arose, which, destroying part of the earliest
deposits, gave birth to new rocks, formed out of their ruins. The
stratified thus succeeded to the unstratified, and something like
variety was established. Then came another period, in which the
face of the waters, instead of being merely agitated, was convulsed
by tempests, and, amid their play and collision, life was generated,
and plants and animals sprung into existence. The vast solitude was
slowly peopled, the sea gradually retired; and a foundation was laid
for that epoch, during which man entered the scene, bringing with
him the rudiments of order and of social improvement.[787]
These were the leading views of a system which, we must
remember, exercised great sway in the scientific world, and won
over to its side minds of considerable power. Erroneous and far-
fetched though it was, it had the merit of calling attention to one of
the two chief principles which have determined the present condition
of our planet. It had the further merit of provoking a controversy,
which was eminently serviceable to the interests of truth. For, the
great enemy of knowledge is not error, but inertness. All that we
want is discussion, and then we are sure to do well, no matter what
our blunders may be. One error conflicts with another; each destroys
its opponent, and truth is evolved. This is the course of the human
mind, and it is from this point of view that the authors of new ideas,
the proposers of new contrivances, and the originators of new
heresies, are benefactors of their species. Whether they are right or
wrong, is the least part of the question. They tend to excite the
mind; they open up the faculties; they stimulate us to fresh inquiry;
they place old subjects under new aspects; they disturb the public
sloth; and they interrupt, rudely, but with most salutary effect, that
love of routine, which, by inducing men to go grovelling on in the
ways of their ancestors, stands in the path of every improvement, as
a constant, an outlying, and, too often, a fatal obstacle.
The method adopted by Werner was evidently deductive, since he
argued from a supposed cause, and reasoned from it to the effects.
In that cause, he found his major premiss, and thence he worked
downwards to his conclusion, until he reached the world of sense
and of reality. He trusted in his one great idea, and he handled that
idea with consummate skill. On that very account, did he pay less
attention to existing facts. Had he chosen, he, like other men, could
have collected them, and subjected them to an inductive
generalization. But he preferred the opposite path. To reproach him
with this is irrational; for, in his journey after truth, he chose one of
the only two roads which are open to the human mind. In England,
indeed, we are apt to take for granted that one road is infinitely
preferable to the other. It may be so; but on this, as on many other
subjects, assertions are current which have never been proved. At all
events, Werner was so satisfied with his method, that he would not
be at the pains of examining the position of rocks and their strata,
as they are variously exhibited in different countries; he did not even
explore his own country, but, confining himself to a corner of
Germany, he began and completed his celebrated system, without
investigating the facts on which, according to the inductive method,
that system should have been built.[788]
Exactly the same process, on the same subject, and at the same
time, was going on in Scotland. Hutton, who was the founder of
Scotch geology, and who, in 1788, published his Theory of the Earth,
conducted the inquiry just as Werner did; though, when he began
his speculations, he had no knowledge of what Werner was doing.
[789] The only difference between them was, that while Werner
reasoned from the agency of water, Hutton reasoned from the
agency of fire. The cause of this may, I think, be explained. Hutton
lived in a country where some of the most important laws of heat
had, for the first time, been generalized, and where consequently,
that department of inorganic physics had acquired great reputation.
It was natural for a Scotchman to take more than ordinary interest in
a subject in which Scotland had been so successful, and had
obtained so much fame. We need not, therefore, wonder that
Hutton, who, like all men, felt the intellectual bent of the time in
which he lived, should have yielded to an influence of which he was,
perhaps, unconscious. In obedience to the general mental habits of
his country he adopted the deductive method. In further obedience
to the more special circumstances connected with his own
immediate pursuits, he gathered the principles from which he
reasoned from a study of fire, instead of gathering them, as Werner
did, from a study of water.
Hence it is, that, in the history of geology, the followers of Werner
are known as Neptunists, and those of Hutton as Plutonists.[790] And
these terms represent the only difference between the two great
masters. In the most important points, namely their method, they
were entirely agreed. Both were essentially one-sided; both paid a
too exclusive attention to one of the two principal agents which have
altered, and are still altering, the crust of the earth; both reasoned
from those agents, instead of reasoning to them; and both
constructed their system without sufficiently studying the actual and
existing facts; committing, in this respect, an error which the English
geologists were the first to rectify.
As I am writing a history, not of science, but of scientific method, I
can only briefly glance at the nature of those services which Hutton
rendered to geology, and which are so considerable, that his system
has been called its present basis.[791] This, however, is too strongly
expressed; for, though Hutton was far from denying the influence of
water,[792] he did not concede enough to it, and there is a tendency
among several geologists to admit that the system of Werner
considered as an aqueous theory, contains a larger amount of truth
than the advocates of the igneous theory are willing to allow. Still,
what Hutton did was most remarkable, especially in reference to
what are now termed metamorphic rocks, the theory of whose
formation he was the first to conceive.[793] Into this, and into their
connexion, on the one hand, with the sedimentary rocks, and, on
the other hand, with those rocks whose origin is perhaps purely
igneous, I could not enter without treading on debatable ground.
But, putting aside what is yet uncertain, I will mention two
circumstances respecting Hutton which are undisputed, and which
will give some idea of his method, and of the turn of his mind. The
first circumstance is, that, although he ascribed to subterranean
heat, as exhibited in volcanic action, a greater and more constant
energy than any previous inquirers had ventured to do,[794] he
preferred speculating on the probable consequences of that action,
rather than drawing inferences from the facts which the action
presented; he being on this point so indifferent, that he arrived at
his conclusions without inspecting even a single region of active
volcanoes, where he might have watched the workings of nature,
and seen what she was really about.[795] The other circumstance is
equally characteristic. Hutton, in his speculations concerning the
geological effects of heat, naturally availed himself of the laws which
Black had unfolded. One of those laws was, that certain earths owe
their fusibility to the presence of fixed air in them before heat has
expelled it; so that if it were possible to force them to retain their
fixed air, or carbonic acid gas, as we now call it, no amount of heat
could deprive them of the capability of being fused. The fertile mind
of Hutton saw, in this discovery, a principle from which he could
construct a geological argument. It occurred to him, that great
pressure would prevent the escape of fixed air from heated rocks,
and would thus enable them to be fused, notwithstanding their
elevated temperature. He then supposed that, at a period anterior to
the existence of man, such a process had taken place under the
surface of the sea, and that the weight of so great a column of
water had prevented the rocks from being decomposed while they
were subjected to the action of fire. In this way, their volatile parts
were held together, and they themselves might be melted, which
could not have happened except for this enormous pressure. By
following this line of argument, he accounted for the consolidation of
strata by heat; since, according to the premisses from which he
started, the oily, or bituminous parts, would remain, in spite of the
efforts of heat to disperse them.[796] This striking speculation led to
the inference, that the volatile components of a substance, and its
fixed components, may be made to cohere, in the very teeth of that
apparently irresistible agent whose business it is to effect their
separation. Such an inference was contrary to all experience; or, to
say the least, no man had ever seen an instance of it.[797] Indeed,
the event was only supposed to happen in consequence of
circumstances which were never met with on the surface of the
globe, and which, therefore, were out of the range of all human
observation.[798] The utmost that could be expected was, that, by
means of our instruments, we might, perhaps, on a small scale,
imitate the process which Hutton had imagined. It was possible, that
a direct experiment might artificially combine great pressure with
great heat, and that the result might be, that the senses would
realize what the intellect had conceived.[799] But the experiment had
never been tried, and Hutton, who delighted in reasoning from ideas
rather than from facts, was not likely to undertake it.[800] He cast his
speculation on the world, and left it to its fate.[801] Fortunately,
however, for the reception of his system, a very ingenious and skilful
experimenter of that day, Sir James Hall, determined to test the
speculation by an appeal to facts; and as nature did not supply the
facts which he wanted, he created them for himself. He applied heat
to powdered chalk, while, at the same time, with great delicacy of
manipulation, he subjected the chalk to a pressure about equal to
the weight of a column of water half a mile high. The result was,
that, under that pressure, the volatile parts of the chalk were held
together; the carbonic acid gas was unable to escape; the
generation of quicklime was stopped; the ordinary operations of
nature were baffled, and the whole composition, being preserved in
its integrity, was fused, and, on subsequently cooling, actually
crystallized into solid marble.[802] Never was triumph more
complete. Never did a fact more fully confirm an idea.[803] But, in
the mind of Hutton, the idea preceded the fact by a long interval;
since, before the fact was known, the theory had been raised, and
the system which was built upon it had, indeed, been published
several years. It, therefore, appears that one of the chief parts of
the Huttonian Theory, and certainly its most successful part, was
conceived in opposition to all preceding experience; that it pre-
supposed a combination of events which no one had ever observed,
and the mere possibility of which nothing but artificial experiment
could prove; and, finally, that Hutton was so confident of the validity
of his own method of inquiry, that he disdained to make the
experiment himself, but left to another mind that empirical branch of
the investigation which he deemed of little moment, but which we,
in England, are taught to believe is the only safe foundation of
physical research.[804]
I have now given an account of all the most important discoveries
made by Scotland, in the eighteenth century, respecting the laws of
the inorganic world. I have said nothing of Watt, because, although
the steam-engine, which we owe to him, is of incalculable
importance, it is not a discovery, but an invention. An invention it
may justly be termed, rather than an improvement.[805]
Notwithstanding what had been effected in the seventeenth century,
by De Caus, Worcester, Papin, and Savery, and notwithstanding the
later additions of Newcomen and others, the real originality of Watt
is unimpeachable. His engine was, essentially, a new invention; but,
under its scientific aspect, it was merely a skilful adaptation of laws
previously known; and one of its most important points, namely, the
economy of heat, was a practical application of ideas promulgated
by Black.[806] The only discovery made by Watt, was that of the
composition of water. Though his claims are disputed by the friends
of Cavendish, it would appear that he was the first who ascertained
that water, instead of being an element, is a compound of two
gases.[807] This discovery was a considerable step in the history of
chemical analysis, but it neither involved nor suggested any new law
of nature, and has, therefore, no claim to mark an epoch in the
history of the human mind.[808] There is, however, one circumstance
connected with it which is too characteristic to be passed over in
silence. The discovery was made in 1783, by Watt, the Scotchman,
and by Cavendish, the Englishman, neither of whom seems to have
been aware of what the other was doing.[809] But between the two
there was this difference. Watt, for several years previously, had
been speculating on the subject of water in connexion with air, and
having, by Black's law of latent heat, associated them together, he
was prepared to believe that one is convertible into the other.[810]
The idea of an intimate analogy between the two bodies having
once entered his mind, gradually ripened; and when he, at last,
completed the discovery, it was merely by reasoning from data
which others possessed besides himself. Instead of bringing to light
new facts, he drew new conclusions from former ideas.[811]
Cavendish, on the other hand, obtained his result by the method
natural to an Englishman. He did not venture to draw a fresh
inference, until he had first ascertained some fresh facts. Indeed, his
discovery was so completely an induction from his own experiments,
that he omitted to take into consideration the theory of latent heat,
from which Watt had reasoned, and where that eminent Scotchman
had found the premisses of his argument.[812] Both of these great
inquirers arrived at truth, but each accomplished his journey by a
different path. And this antithesis is accurately expressed by one of
the most celebrated of living chemists, who, in his remarks on the
composition of water, truly says, that while Cavendish established
the facts, Watt established the idea.[813]
 Thus much, as to what was effected by the Scotch in the
department of inorganic science. If we now turn to organic science,
we shall find that, there also, their labours were very remarkable. To
those who are capable of a certain elevation and compass of
thought, it will appear, in the highest degree, probable, that,
between the organic and inorganic world, there is no real difference.
That they are separated, as is commonly asserted, by a sharp line of
demarcation, which indicates where one abruptly ends, and the
other abruptly begins, seems to be a supposition altogether
untenable. Nature does not pause, and break off in this fitful and
irregular manner. In her works there is neither gap nor chasm. To a
really scientific mind, the material world presents one vast and
uninterrupted series, gradually rising from the lowest to the highest
forms, but never stopping. In one part of that series, we find a
particular structure, which, so far as our observations have yet
extended, we, in another part, cannot find. We also observe
particular functions, which correspond to the structure, and, as we
believe, result from it. This is all we know. Yet, from these scanty
facts, we, who, at present, are still in the infancy of knowledge, and
have but skimmed the surface of things, are expected to infer, that
there must be a point, in the chain of existence, where both
structure and function suddenly cease, and, after which, we may
vainly search for signs of life. It would be difficult to conceive a
conclusion more repugnant to the whole march and analogy of
modern thought. In every department, the speculations of the
greatest thinkers are constantly tending to coördinate all
phenomena, and to regard them as different, indeed, in degree, but
by no means as different in kind. Formerly, men were content to
ground their conviction of this difference in kind, on the evidence of
the eye, which, on a cursory inspection, saw an organization in some
bodies, and not in others. From the organization, they inferred the
life, and supposed that plants, for instance, had life, but that
minerals had none. This sort of argument was long deemed
satisfactory; but, in the course of time, it broke down; more
evidence was required, and, since the middle of the seventeenth
century, it has been universally admitted, that the eye, by itself, is an
untrustworthy witness, and that we must employ the microscope,
instead of relying on the unaided testimony of our own puny and
precarious senses. But the microscope is steadily improving, and we
cannot tell what limits there are to its capacity for improvement.
Consequently, we cannot tell what fresh secrets it may disclose.
Neither can we say, that it may not be altogether superseded by
some new artificial resource, which shall furnish us with evidence, as
superior to any yet supplied, as our present evidence is superior to
that of the naked eye. Even already, and notwithstanding the
shortness of time during which the microscope has been a really
effective instrument, it has revealed to us organizations, the
existence of which no one had previously suspected. It has proved,
that what, for thousands of years, had been deemed mere specks of
inert matter, are, in truth, animals possessing most of the functions
which we possess, reproducing their species in regular and orderly
succession, and endowed with a nervous system, which shows that
they must be susceptible of pain and enjoyment. It has detected life
hidden in the glaciers of Switzerland; it has found it embedded in the
polar ice, and, if it can flourish there, it is hard to say from what
quarter it can be shut out. So unwilling, however, are most men to
relinquish old notions, that the resources of chemistry have been
called in, to ascertain the supposed difference between organic and
inorganic matter: it being asserted, that, in the organic world, there
is a greater complexity of molecular combination, than in the
inorganic.[814] Chemists further assert, that, in organic nature, there
is a predominance of carbon, and, in inorganic, a predominance of
silicon.[815] But chemical analysis, like microscopic observation, is
making such rapid strides, that each generation, I had almost said
each year, is unsettling some of the conclusions previously
established; so that, now, and for a long time hence, we must
regard those conclusions as empirical, and, indeed, as merely
tentative. Surely a permanent and universal inference cannot be
drawn from shifting and precarious facts, which are admitted to-day,
and may be overthrown to-morrow. It would, therefore, appear that,
in favour of the opinion, that some bodies are living, and that others
are dead, we have nothing, except the circumstance, that our
researches, so far as they have yet gone, have shown that cellular
structure, growth, and reproduction, are not the invariable properties
of matter, but are excluded from a large part of the visible world,
which, on that account, we call inanimate. This is the whole of the
argument on that side of the question. On the other side, we have
the fact, that our sight, and the artificial instruments, by whose aid
we have arrived at this conclusion, are confessedly imperfect; and
we have the further fact, that, imperfect as they are, they have
proved, that the organic kingdom is infinitely more extensive than
the boldest dreamer had ever imagined, while they have not been
able to enlarge the boundaries of the inorganic kingdom to any thing
like the same amount. This shows, that, so far as our opinions are
concerned, the balance is steadily inclining in one given direction; in
other words, as our knowledge advances, a belief in the organic is
encroaching upon a belief in the inorganic.[816] When we, moreover,
add, that all science is manifestly converging towards one simple
and general theory, which shall cover the whole range of material
phenomena, and that, at each successive step, some irregularities
are explained away, and some inequalities are reduced, it can hardly
be doubted, that such a movement tends to weaken those old
distinctions, the reality of which has been too hastily assumed; and
that, in their place, we must, sooner or later, substitute the more
comprehensive view, that life is a property of all matter, and that the
classification of bodies into animate and inanimate, or into organic
and inorganic, is merely a provisional arrangement, convenient,
perhaps, for our present purposes, but which, like all similar
divisions, will eventually be merged in a higher and wider scheme.
Until, however, that step is taken, we must be content to reason
according to the evidence supplied by our imperfect instruments, or
by our still more imperfect senses. We, therefore, recognize the
difference between organic and inorganic nature, not as a scientific
truth, but as a scientific artifice, by which we separate in idea, what
is inseparable in fact; hoping, in this way, to pursue our course with
the greater ease, and ultimately to obtain results, which will make
the artifice needless. Assuming, then, this division, we may refer all
investigations of organic bodies to one of two objects. The first
object is, to ascertain the law of those bodies, in their usual, healthy,
or, as we somewhat erroneously phrase it, normal course. The other
object is, to ascertain their law, in their unusual, unhealthy, or
abnormal course. When we attempt to do the first of these things,
we are physiologists. When we attempt to do the second, we are
pathologists.[817]
Physiology and pathology are thus the two fundamental divisions
of all organic science.[818] Each is intimately connected with the
other; and eventually, no doubt, both will be fused into a single
study, by discovering laws which will prove that here, as elsewhere,
nothing is really abnormal, or irregular. Hitherto, however, the
physiologists have immeasurably outstripped the pathologists in the
comprehensiveness of their views, and, therefore, in the value of
their results. For, the best physiologists distinctly recognize that the
basis of their science must include, not only the animals below man,
but also the entire vegetable kingdom, and that, without this
commanding survey of the whole realm of organic nature, we cannot
possibly understand even human physiology, still less general
physiology. The pathologists, on the other hand, are so much in
arrear, that the diseases of the lower animals rarely form part of
their plan; while the diseases of plants are almost entirely neglected,
although it is certain that, until all these have been studied, and
some steps taken to generalize them, every pathological conclusion
will be eminently empirical, on account of the narrowness of the field
from which it is collected.
The science of pathology being still so backward in the conception
as well as in the execution, that even men of real ability believe that
it can be raised from a mere study of the human frame, it will hardly
be expected that the Scotch, notwithstanding the marvellous
boldness of their speculations, should have been able, in the
eighteenth century, to anticipate a method which the nineteenth
century has yet to employ. But they produced two pathologists of
great ability, and to whom we owe considerable obligations. These
were, Cullen and John Hunter.[819] Cullen was eminent only as a
pathologist; but Hunter, whose fine and discursive genius took a
much wider range, was great both in physiology and in pathology. A
short account of their generalizations respecting organic science, will
be a fitting sequel to the notices I have already given of what was
done by their countrymen for inorganic science, during the same
period. It will complete our survey of the Scotch intellect, and will
enable the reader to form some idea of the brilliant achievements of
that most remarkable people, who, contrary to the course of affairs
in all other modern nations, have shown that scientific discoveries do
not necessarily weaken superstition, and that it is possible for two
hostile principles to flourish side by side, without ever coming into
actual collision, or without sensibly impairing each other's vigour.
 In 1751, Cullen was appointed professor of medicine in the
University of Glasgow;[820] from which, however, in 1756, he was
removed to the University of Edinburgh,[821] where he delivered
those celebrated lectures, on which his fame now depends. During
the early part of his career, he paid great attention to inorganic
physics, and propounded some remarkable speculations, which are
supposed to have suggested the theory of latent heat to Black, who
was his pupil.[822] But, to follow out those views, would have
required a number of minute experiments, which it did not suit the
habit of his mind to make. Having, therefore, put forth his ideas, he
left them to germinate, and passed on to his arduous attempt to
generalize the laws of disease as they are exhibited in the human
frame. In the study of disease, the phenomena being more obscure
and less amenable to experiment, there was greater latitude for
speculation; hence, he could more easily indulge in that love of
theory, which was his ruling passion, and with an extreme devotion
to which he has been reproached.[823] That the reproach is not
altogether unjust, must, I think, be admitted, since we find him
laying down the doctrine, that, inasmuch as, in the treatment of
disease, theory could not be separated from practice, it was
unimportant which came first.[824] This was tantamount to saying,
that a medical practitioner might allow his theories to control his
observations; for it is certain that, in an immense majority of cases,
men are so tenacious of the opinions they imbibe, that whatever, in
any pursuit, first occupies their understanding, is likely to mould all
that comes afterwards. In ordinary minds, associations of ideas, if
firmly established, become indissoluble; and the power of separating
them, and of arranging them in new combinations, is one of the
rarest of our endowments. An average intellect, when once
possessed by a theory, can hardly ever escape from it. Hence, in
practical matters, theory should be feared, just as, in scientific
matters, it should be cherished; because practical pursuits are
chiefly engrossed by the lower class of minds, where associations
and the force of prejudice are extremely strong, while scientific
pursuits concern the higher class, where such prepossessions are
comparatively weak, and where close associations are more easily
severed. The most powerful intellects are most accustomed to new
arrangements of thought, and are, therefore, most able to break up
old ones. On them, belief sits lightly, because they well know how
little evidence we have for many of even our oldest beliefs. But the
average, or, as we must say, without meaning offence, the inferior,
minds, are not disturbed by these refinements. Theories, which they
have once heartily embraced, they can hardly ever get rid of, and
they often dignify them with the name of essential truths, and resent
every attack upon them as a personal injury. Having inherited such
theories from their fathers, they regard them with a sort of filial
piety, and cling to them as if they were some rich acquisition, which
no one has a right to touch.
To this latter class, nearly all men belong, who are more engaged
in practical pursuits than in speculative ones. Among them, are the
ordinary practitioners, whether in medicine or in any other
department, extremely few of whom are willing to break up trains of
thought to which they are inured.[825] Though they profess to
despise theory, they are, in reality, enslaved by it. All that they can
do, is to conceal their subjection, by terming their theory a
necessary belief. It must, therefore, be deemed a remarkable proof
of Cullen's love of deductive reasoning, that he, sagacious and clear-
sighted as he was, should have supposed that, in so practical an art
as medicine, theory could, with impunity, precede practice. For, it is
most assuredly true, that, taking men in the average, their minds are
so constructed, that it cannot precede it without controlling it. It is
equally true, that such control must be hurtful. Even now, and
notwithstanding the great steps which have been taken in morbid
anatomy, in animal chemistry, and in the microscopic investigation
both of the fluids and solids of the human frame, the treatment of
disease is a question of art, far more than a question of science.
What chiefly characterizes the most eminent physicians, and gives
them their real superiority, is not so much the extent of their
theoretical knowledge,—though that, too, is often considerable,—but
it is that fine and delicate perception which they owe, partly to
experience, and partly to a natural quickness in detecting analogies
and differences which escape ordinary observers. The process which
they follow, is one of rapid, and, in some degree, unconscious,
induction. And this is the reason why the greatest physiologists and
chemists, which the medical profession possesses, are not, as a
matter of course, the best curers of disease. If medicine were a
science, they would always be the best. But medicine, being still
essentially an art, depends mainly upon qualities which each
practitioner has to acquire for himself, and which no scientific theory
can teach. The time for a general theory has not yet come, and
probably many generations will have to elapse before it does come.
To suppose, therefore, that a theory of disease should, as a matter
of education, precede the treatment of disease, is not only
practically dangerous, but logically false. With its practical danger I
am not now concerned; but its logical aspect is a curious illustration
of that passion for systematic and dialectic reasoning which
characterized Scotland. It shows that Cullen, in his eagerness to
argue from principles to facts, instead of from facts to principles,
could, in the most important of all arts, recommend a method of
procedure, for which even our knowledge is not ripe, but which, in
his time, was so singularly rash and immature, that nothing can
explain its adoption by a man of such vigorous understanding,
except the circumstance of his living in a country in which that
peculiar method reigned supreme.
It must, however, be admitted that Cullen wielded the method
with great ability, especially in his application of it to the science of
pathology, to which it was far better suited than to the art of
therapeutics. For, we must always remember, that the science which
investigates the laws of disease, is quite a different thing from the
art which cures it. The science has a speculative interest, which is
irrespective of all practical considerations, and which depends simply
on the fact, that, when it is completed, it will explain the aberrations
of the whole organic world. Pathology aims at ascertaining the
causes which determine every departure from the natural type,
whether of form or of function. Hence it is, that no one can take a
comprehensive view of the actual state of knowledge, without
studying the theoretic relations between pathology and other
departments of inquiry. To do this, is the business, not of practical
men, but of philosophers, properly so called. The philosophic
pathologist is as different from the physician, as a jurist is different
from an advocate, or as an agricultural chemist is different from a
farmer, or as a political economist is different from a statesman, or
as an astronomer, who generalizes the laws of the heavenly bodies,
is different from a captain, who navigates his ship by a practical
application of those laws. The two sets of functions may be united,
and occasionally, though very rarely, they are, but there is no
necessity for their being so. While, therefore, it would be absurdly
presumptuous for an unprofessional person to pass judgment on the
therapeutical system of Cullen, it is perfectly legitimate for any one,
who has studied the theory of these matters, to examine his
pathological system; because that, like all scientific systems, must
be amenable to general considerations, which are to be taken, partly
from the adjoining sciences, and partly from the universal logic of
philosophic method.
It is from this latter, or logical, point of view, that Cullen's
pathology is interesting for the purposes of the present chapter. The
character of his investigations may be illustrated by saying, that his
method in pathology is analogous to that which Adam Smith
adopted at the same time, though in a very different field. Both were
deductive; and both, before arguing deductively, suppressed some of
the premisses from which they reasoned. That this suppression is
the key to Adam Smith's method, and was an intentional part of his
plan, I have already shown; as also that, in each of his two works,
he supplied the premisses in which the other work was deficient. In
this respect, he was far superior to Cullen. For, though Cullen, like
Smith, began by mutilating his problem in order to solve it more
readily, he, unlike Smith, did not see the necessity of instituting
another and parallel inquiry, which should complete the scheme, by
starting from the premisses that had been previously omitted.
 What I have termed the mutilation of the problem, was effected
by Cullen in the following manner. His object was, to generalize the
phenomena of disease, as they are exhibited in the human frame;
and it was obvious to him, as to every one else, that the human
frame consists partly of solids and partly of fluids. The peculiarity of
his pathology is, that he reasons almost entirely from the laws of the
solids, and makes so little account of the fluids, that he will only
allow them to be the indirect causes of disease, which, in a scientific
view, are to be deemed strictly subordinate to the direct causes, as
represented by the solid constituents of our body.[826] This
assumption, though false, was perfectly justifiable, since, by
curtailing the problem, he simplified its study; just as Adam Smith, in
his Wealth of Nations, simplified the study of human nature, by
curtailing it of all its sympathy. But this most comprehensive thinker
was careful, in his Theory of Moral Sentiments, to restore to human
nature the quality of which the Wealth of Nations had deprived it;
and, by thus establishing two different lines of argument, he
embraced the whole subject. In the same way, it was incumbent on
Cullen, after having constructed a theory of disease by reasoning
from the solids, to have constructed another theory by reasoning
from the fluids; so that a coördination of the two theories might
have raised a science of pathology, as complete as the then state of
knowledge allowed.[827] But to this, his mind was unequal. Able
though he was, he lacked the grasp of intellect which characterized
Adam Smith, and which made that great man perceive, that every
deductive argument which is founded on a suppression of premisses,
must be compensated by a parallel argument, which takes those
premisses into account.[828] So little was Cullen aware of this, that,
having built up that system of pathology which is known to medical
writers as Solidism, he never took the pains to accompany it by
another system, which gave the first rank to the fluids. On the
contrary, he believed that his plan was complete and exhaustive, and
that what is termed Humoral Pathology was a fiction, which had too
long usurped the place of truth.[829]
 Several of the views advocated by Cullen were taken from
Hoffmann, and several of the facts from Gaubius; but that his
pathology, considered as a whole, is essentially original, is evident
from a certain unity of design which is inconsistent with extensive
plagiarism, and which proves that he had thoroughly thought out his
subject for himself. Without, however, stopping to inquire how much
he borrowed from others, I will briefly indicate a few of the salient
points of his system, in order to enable the reader to understand its
general character.
According to Cullen, all the solids in the human body are either
simple or vital. The simple solids retain, after death, the properties
which they possessed during life. But the vital solids, which form the
fundamental part of the nervous system, are marked by properties,
which disappear directly death occurs.[830] Hence, the simple solids,
having fewer functions than the vital, have also fewer diseases; and
the maladies to which they are liable admit of easy classification.
[831] The real difficulty lies in the vital solids, because on their
peculiarities the whole nervous system depends, and nearly all
disorders are immediately due to changes in them. Cullen, therefore,
made the nervous system the basis of his pathology; and, in
speculating on its functions, he assigned the chief place to an occult
principle, which he termed the Animal Power, or Energy, of the brain.
[832] This principle acted on the vital solids. When the principle
worked well, the body was healthy; when it worked ill, the body was
unhealthy. Since, then, the state of the vital solids was the main
cause of disorder, and since the Energy of the brain was the main
cause of the state of the vital solids, it became important to know
what the influences were which acted on the Energy, because in
them we should find the beginning of the series. Those influences
were divided by Cullen into physical and mental. The physical were,
heat, cold, and effluvia, the three most potent of the material
disturbers of the human frame.[833] The mental influences, which
excited the brain to act on the solids, were comprised under six
different heads, namely, the will, the emotions, the appetites, the
propensities, and, finally, the two great principles of habit and of
imitation, on which he, with good reason, laid considerable stress.
[834] In arguing from these mental causes, and in generalizing the
relations between them and the sensations of the body, he, faithful
to his favourite method, proceeded deductively from the
metaphysical principles then in vogue, without inquiring inductively
into their validity, such an induction being, he thought, no part of his
duty.[835] He was too anxious to get on with his dialectic, to be
interrupted by so trifling a matter as the truth or falsehood of the
premisses on which the reason rested. What he did in the
metaphysical part of his pathology, he also did in its physical part.
Although the blood and the nerves are the two leading features of
the human economy, he did not search into them by a separate
induction; he subjected them neither to chemical experiments in
order to learn their composition, nor to microscopic observations in
order to learn their structure.[836] This is the more observable,
because though we must admit that animal chemistry was then
generally neglected, and that its real meaning was scarcely
understood until the wonderful labours of Berzelius revealed its
importance, still the microscope was ready to Cullen's hands; it
having been invented a hundred and fifty years before he completed
his pathology, and having been in common scientific use for about a
hundred years. But his love of synthesis overcame him. His system is
constructed by reasoning from general principles; and of that
process, he certainly was a consummate master. Between the
premisses and the conclusion, he hardly ever lets error creep in.
And, in reference to the results of his speculations, he had one
immense merit, which will always secure to him a conspicuous place
in the history of pathology. By insisting on the importance of the
solids, he, one-sided though he was, corrected the equal one-
sidedness of his predecessors; for, with extremely few exceptions, all
the best pathologists, from Galen downwards, had erred in ascribing
too much to the fluids, and had upheld a purely humoral pathology.
Cullen turned the minds of men in the other direction; and though,
in teaching them that the nervous system is the sole primary seat of
disease, he committed a great mistake, it was a mistake of the most
salutary kind. By leaning on that side, he restored the balance.
Hence, I have no doubt, he indirectly encouraged those minute
researches into the nerves, which he would not himself stop to
make, but which, in the next generation, gave rise to the capital
discoveries of Bell, Shaw, Mayo, and Marshall Hall. At the same time,
the old humoral pathology, which had prevailed for many centuries,
was practically pernicious, because, assuming that all diseases are in
the blood, it produced that constant and indiscriminate venesection,
which destroyed innumerable lives, besides the irreparable injury it
often inflicted both on body and mind; weakening those whom it
was unable to slay. Against this merciless onslaught, which made
medicine the curse of mankind, the Solid Pathology was the first
effective barrier.[837] Practically, therefore, as well as speculatively,
we must hail Cullen as a great benefactor of his species; and we
must regard his appearance as an epoch in the history of human
comfort, as well as in the history of human thought.
It may, perhaps, facilitate the conceptions of unprofessional
readers, if I give, in as few words as possible, a specimen of the way
in which Cullen employed his method, in investigating the theory of
some one class of diseases. For this purpose, I will select his
doctrine of fever, which, though now generally abandoned, once
exercised more influence than any other part of his pathology. Here,
as elsewhere, he reasons from the solids.[838] Disregarding the state
of the blood, he says, that the cause of all fever is a diminished
energy of the brain.[839] Such diminution may be produced by
various sedatives, the most common of which are effluvia, whether
marsh or human, intemperance, fear, and cold.[840] Directly the
energy of the brain is impaired, the disease begins. Rapidly passing
through the nervous system, its first palpable effect is a chill, or cold
fit, which is accompanied by a spasm on the extremities of the
arteries, particularly where they touch the surface of the body.[841]
This spasm on the extreme vessels, irritates the heart and arteries,
and the irritation continues till the spasm is relaxed.[842] At the same
time, the increased action of the heart restores the energy of the
brain; the system rallies; the extreme vessels are relieved; while, as
a consequence of the whole movement, sweat is excreted, and the
fever abates.[843] Shutting out, therefore, all consideration of the
fluids of the body, the successive stages of languor, cold fit, and hot
fit, might, in Cullen's opinion, be generalized by reasoning merely
from the solids, which, furthermore, produced his well-known
distinction between fevers, the continuance of which is owing to an
excess of spasm, and those, the continuance of which is owing to an
excess of debility.[844]
A similar process of thought gave birth to his Nosology, or general
classification of diseases, which some have regarded as the most
valuable part of his labours;[845] though, for reasons already
mentioned, we must, I think, reject all such attempts as premature,
and as likely to work more harm than good, unless they are simply
used as a contrivance to aid the memory. At all events, the Nosology
of Cullen, though it exhibits clear traces of his powerful and
organizing mind, is fast falling into disrepute, and we may be sure,
that, for a long time yet, a similar fate will await its successors. Our
pathological knowledge is still too young for so great an enterprise.
[846] We have every reason to expect, that, with the aid of
chemistry, and of the microscope, it will continue to grow more
rapidly than it has hitherto done. Without venturing to predict the
rate of its increase, we may form some idea of it, by considering
what has been effected with resources very inferior to those we now
possess. In a work of great authority, published in the year 1848, it
is stated, that since the appearance of Cullen's Nosology, our mere
enumeration of diseases has almost doubled, while our knowledge of
the facts relating to disease has more than doubled.[847]
I have now only one more name to add to this splendid catalogue
of the great Scotchmen of the eighteenth century.[848] But it is the
name of a man, who, for comprehensive and original genius, comes
immediately after Adam Smith, and must be placed far above any
other philosopher whom Scotland has produced. I mean, of course,
John Hunter, whose only fault was, an occasional obscurity, not
merely of language, but also of thought. In this respect, and,
perhaps, in this alone, Adam Smith had the advantage; for his mind
was so flexible, and moved so freely, that even the vastest designs
were unable to oppress it. With Hunter, on the contrary, it
sometimes seemed as if the understanding was troubled by the
grandeur of his own conceptions, and doubted what path it ought to
take. He hesitated; the utterance of his intellect was indistinct.[849]
Still, his powers were so extraordinary, that, among the great
masters of organic science, he belongs, I apprehend, to the same
rank as Aristotle, Harvey, and Bichat, and is somewhat superior
either to Haller or Cuvier. As to this classification, men will differ,
according to their different ideas of the nature of science, and,
above all, according to the extent to which they appreciate the
importance of philosophic method. It is from this latter point of view
that I have, at present, to consider the character of John Hunter;
and, in tracing the movements of his most remarkable mind, we
shall find, that, in it, deduction and induction were more intimately
united than in any other Scotch intellect, either of the seventeenth
or eighteenth century. The causes of this unusual combination, I will
now endeavour to ascertain. When they are understood, they will
not only explain many peculiarities in his works, but will afford
materials for speculation, to those who love to examine the
development of ideas, and who are able to discern the way in which
different schemes of national thought have given different shapes to
national character, and have thereby modified the whole course of
human affairs, to an extent of which the ordinary compilers of
history have not the slightest suspicion.
Hunter remained in Scotland till the age of twenty, when he
settled in London; and, though he was abroad for about three years,
he abandoned his own country, and became, socially and
intellectually, a native of England.[850] Hence, the early associations
of his mind were formed in the midst of a deductive nation; the later
associations, in the midst of an inductive one. For twenty years he
lived among a people, who are, perhaps, the acutest reasoners in
Europe, if you concede to them the principles from which they
reason; but who, on the other hand, owing to their proneness to this
method, are so greedy after general principles, that they will accept
them on almost any evidence, and are, therefore, at once very
credulous and very logical. In that school, and surrounded by those
habits, the intellect of John Hunter was nurtured during the most
impressible period of his life. Then the scene suddenly shifted.
Coming to England, he passed forty years in the heart of the most
empirical nation in Europe; a nation utterly abhorring all general
principles, priding itself on its common sense, boasting, and with
good reason too, of its practical sagacity, proclaiming aloud the
superiority of facts over ideas, and despising every theory, unless
some direct and immediate benefit could be expected to accrue from
it. The young and ardent Scotchman found himself transplanted into
a country totally different from that which he had just quitted; and
such a difference could not fail to influence his mind. He saw, on
every side, marks of prosperity, and of long and uninterrupted
success, not only in practical, but also in speculative, life; and he
was told that these things were effected by a system which made
facts the first consideration. He was ambitious of fame, but he
perceived that the road to fame was not the same in England as in
Scotland. In Scotland, a great logician would be deemed a great
man; in England, little account would be made of the beauty of his
logic, unless he was careful that the premisses from which he
argued, were trustworthy, and verified by experience. A new
machine, a new experiment, the discovery of a salt, or of a bone,
would, in England, receive a wider homage, than the most profound
speculation from which no obvious results were apprehended. That
this way of contemplating affairs has produced great good, is
certain. But it is also certain, that it is a one-sided way, and satisfies
only part of the human mind. Many of the noblest intellects crave for
something which it cannot supply. In England, however, during the
greater part of the eighteenth century, it was even more supreme
than it is now, and was, indeed, so universal, that, from the year
1727 until nearly the close of the century, our country did not
possess, in any branch of science, a speculator who had sufficient
force to raise himself above those narrow views which were then
deemed the perfection of wisdom.[851] Much was added to our
knowledge, but its distant boundaries were not enlarged. Though
there was an increase of curious and valuable details, and though
several of the small and proximate laws of nature were generalized,
it must be admitted, that those lofty generalizations, which we owe
to the seventeenth century, remained stationary, and that no
attempt was made to push beyond them. When John Hunter arrived
in London, in 1748, Newton had been dead more than twenty years,
and the English people, absorbed in practical pursuits, and now
beginning, for the first time, to enter into political life, had become
more averse than ever to inquiries which aimed at truth without
regard to utility, and had accustomed themselves to value science
chiefly for the sake of the direct and tangible benefit which they
might hope to derive from it.
That Hunter must have been influenced by these circumstances,
will be obvious to whoever considers how impossible it is for any
single mind to escape from the pressure of contemporary opinion.
But, inasmuch as all his early associations had inclined him in
another direction, we perceive that, during his long residence in
England, he was acted on by two conflicting forces. The country of
his birth made him deductive; the country of his adoption made him
inductive. As a Scotchman, he preferred reasoning from general
principles to particular facts; as an inhabitant of England, he became
inured to the opposite plan of reasoning from particular facts to
general principles. In every country, men naturally give the first
place to what is most valued. The English respect facts more than
principles, and therefore begin with the facts. The Scotch consider
principles as most important, and therefore begin with the principles.
And, I make no doubt that one of the reasons why Hunter, in
investigating a subject, is often obscure, is that, on such occasions,
his mind was divided between these two hostile methods, and that,
leaning sometimes to one and sometimes to the other, he was
unable to determine which he should choose. The conflict darkened
his understanding. Adam Smith, on the other hand, in common with
all the great Scotchmen who remained in Scotland, was remarkably
clear. He, like Hume, Black, and Cullen, never wavered in his
method. These eminent men were not acted on by English influence.
Of all the most illustrious Scotchmen of the eighteenth century,
Hunter alone underwent that influence, and he alone displayed a
certain hesitation and perplexity of thought, which seems unnatural
to so great a mind, and which, as it appears to me, is best explained
by the peculiar circumstances in which he was placed.
One of the ablest of his commentators has justly observed, that
his natural inclination was, to conjecture what the laws of nature
were, and then reason from them, instead of reasoning to them by
slow and gradual induction.[852] This process of deduction was, as I
have shown, the favourite method of all Scotchmen, and, therefore,
was precisely the course which we should have expected him to
adopt. But, inasmuch as he was surrounded by the followers of
Bacon,[853] this natural bias was warped, and a large part of his
marvellous activity was employed in observations and experiments,
such as no Scotch thinker, living in Scotland, would ever have
engaged in. He himself declared, that thinking was his delight;[854]
and there can be no doubt that, had he been differently situated,
thinking would have been his principal pursuit. As it was, the
industry with which he collected facts, is one of the most
conspicuous features in his career. His researches covered the whole
range of the animal kingdom, and were conducted with such untiring
zeal, that he dissected upwards of five hundred different species,
exclusive of dissections of different individuals, and exclusive, too, of
dissections of a large number of plants.[855] The results were
carefully arranged and stored up in that noble collection which he
formed, and of the magnitude of which we may gain some idea from
the statement, that, at his death, it contained upwards of ten
thousand preparations illustrative of the phenomena of nature.[856]
By this means, he became so intimately acquainted with the animal
kingdom, that he made a vast number of discoveries, which,
considered singly, are curious, but which, when put together,
constitute an invaluable body of new truths. Of these, the most
important are, the true nature of the circulation in crustacea and
insects;[857] the organ of hearing in cephalopods;[858] the power
possessed by mollusks of absorbing their shells;[859] the fact that
bees do not collect wax, but secrete it;[860] the semicircular canals
of the cetacea;[861] the lymphatics of birds;[862] and the air-cells in
the bones of birds.[863] We are also assured, that he anticipated the
recent discoveries respecting the embryo of the kangaroo;[864] and
his published works prove, that, in the human subject, he discovered
the muscularity of the arteries,[865] the muscularity of the iris,[866]
and the digestion of the stomach after death by its own juice.[867]
Although, in his time, animal chemistry was not yet raised to a
system, and was consequently little heeded by physiologists, Hunter
endeavoured, by its aid, to search out the qualities of the blood, so
as to ascertain the properties of its constituents.[868] He also
examined it in different stages of embryonic life, and by minutely
tracking it through its periods of development, he made the capital
discovery, that the red globules of the blood are formed later than its
other components. His contemporaries, however, were so little alive
to the importance of this great physiological truth, that it fell dead
upon them, and, being forgotten, it was, about fifty years
afterwards, rediscovered, and was announced, in 1832, as a law of
nature which had just been brought to light.[869] This is one of many
instances in the history of our knowledge, which proves how useless
it is for a man to advance too far beyond the age in which he lives.
[870] But Hunter, besides making the discovery, also saw its
meaning. From it, he inferred that the function of the red globules is
to minister to the strength of the system, rather than to its repair.
[871] This is now universally admitted; but it was not admitted till
long after his death. Its recognition is chiefly owing to the rapid
advance of animal chemistry, and to improvements in the
microscope. For, by the employment of these resources, it has
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