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UNIT 1 INTRODUCTION

BASICS OF MICROBIAL EXISTENCE

Microbiology defined - The study of microbiology is the study of microorganisms, that


are invisible to the naked eye. Because objects less than about one millimeter in diameter cannot
be seen clearly and must be examined with a microscope, microbiology is concerned primarily
with organisms. It includes viruses, bacteria, many algae fungi, and protozoa. For example, bread
molds and filamentous algae are studied by microbiologists, yet are visible to the naked eye.
Two bacteria that are visible without a microscope, Thiomargarita and Epulopiscium,
also have been discovered. The difficulty in setting the boundaries of microbiology led Roger
Stanier to suggest that the field be defined not only in terms of the size of its subjects but also in
terms of its techniques. A microbiologist usually first isolates a specific microorganism from a
population and then cultures it. Thus microbiology employs techniques such as sterilization and
the use of culture media that are necessary for successful isolation and growth of
microorganisms.

IMPORTANCE OF MICROBIOLOGY
1. Impact on Human Health
2. Balance of Nature - food source, play a role in decomposition, help other animals
digest grass (cattle, sheep, termites).
3. Environmental provide safe drinking water; development of biodegradable
products; use bacteria to clean up oil spills, etc. calledbioremediation.
4. Industrial foodstuffs (beer, wine, cheese, bread), antibiotics, insulin, genetic
engineering
5. Agricultural - research has led to healthier livestock and disease-free crops.

Even before microorganisms were seen, some investigators suspected their existence and
responsibility for disease. Among others, the Roman philosopher Lucretius (about 9855 B.C.)
and the physician Girolamo Fracastoro (14781553) suggested that disease was caused by
invisible living creatures. The earliest microscopic observations appear to have been made
between 1625 and 1630 on bees and weevils by the Italian Francesco Stelluti, using a
Microscope probably supplied by Galileo. However, the first person to observe and describe
microorganisms accurately was the amateur microscopist Antony van Leeuwenhoek (1632
1723) of Delft, Holland.

Leeuwenhoek earned his living as a draper and haberdasher (a dealer in mens clothing
and accessories), but spent much of his spare time constructing simple microscopes composed of
double convex glass lenses held between two silver plates. His microscopes could magnify
around 50 to 300 times, and he may have illuminated his liquid specimens by placing them
between two pieces of glass and shining light on them at a 45 angle to the specimen plane. This
would have provided a form of dark-field illumination and made bacteria clearly visible.
Beginning in 1673 Leeuwenhoek sent detailed letters describing his discoveries to the Royal
Society of London. It is clear from his descriptions that he saw both bacteria and protozoa.

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HISTORY OF MICROBIOLOGY
Some Important Events in the Development of Microbiology

Date Microbiological History


1546 Fracastoro suggests that invisible organisms cause disease
15901608 Jansen develops first useful compound microscope
1676 Leeuwenhoek discovers animalcules
1688 Redi publishes work on spontaneous generation of maggots
17651776 Spallanzani attacks spontaneous generation
1786 Mller produces first classification of bacteria
1798 Jenner introduces cowpox vaccination for smallpox
18381839 Schwann and Schleiden, the Cell Theory
18351844 Bassi discovers that silkworm disease is caused by a fungus
and proposes that many diseases are microbial in origin
18471850 Semmelweis shows that childbed fever is transmitted by Velocity of light
1849 Graham distinguishes colloids and crystalloids
1857 Pasteur shows that lactic acid fermentation is due to a microorganism
1858 Virchow states that all cells come from cells Darwins On the Origin of sp
1861 Pasteur shows that microorganisms do not arise by spontaneous generation
1867 Lister publishes his work on antiseptic surgery
1869 Miescher discovers nucleic acids
18761877 Koch demonstrates that anthrax is caused by Bacillus anthracis
1880 Laveran discovers Plasmodium, the cause of malaria
1881 Koch cultures bacteria on gelatin Iives produces first color
1882 Koch discovers tubercle bacillus, Mycobacterium tuberculosis First
1884 Koch postulates phagocytosis
1885 Pasteur develops rabies vaccine
1886 Fraenkel discovers Streptococcus pneumoniae, a cause of pneumonia
1887 Petri dish (plate) developed by Richard Petri
18871890 Winogradsky studies sulfur and nitrifying bacteria
1889 Beijerinck isolates root nodule bacteria Eastman makes box camera (1888)
1890 Von Behring prepares antitoxins for diphtheria and tetanus
1892 Ivanowsky provides evidence for virus causation of tobacco mosaic
1894 Kitasato and Yersin discover Yersinia pestis, the cause of plague
1895 Bordet discovers complement Rntgen discovers X rays (1895)
1896 Van Ermengem discovers Clostridium botulinum, the cause of botulism
1897 Buchner prepares extract of yeast that ferments
1899 Beijerinck proves that a virus particle causes the tobacco mosaic disease
1900 Reed proves that yellow fever is transmitted by the mosquito
1902 Landsteiner discovers blood groups
1903 Wright and others discover antibodies in the blood of immunized
1905 Schaudinn and Hoffmann show Treponema pallidum causes syphilis
1906 Wassermann develops complement fixation test for syphilis
1909 Ricketts shows that Rocky Mountain spotted fever is transmitted by ticks
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1910 Ehrlich develops chemotherapeutic agent
1911 Rous discovers a virus that causes cancer in chickens
19151917 DHerelle and Twort discover bacterial viruses
1921 Fleming discovers lysozyme
1923 First edition of Bergeys Manual
1928 Griffith discovers bacterial transformation
1929 Fleming discovers penicillin Stock market crash (1929)
1931 Van Niel shows that photosynthetic bacteria use reduced
1933 Ruska develops first transmission electron microscope
1935 Stanley crystallizes the tobacco mosaic virus
1937 Chatton divides living organisms into procaryotes
1941 Beadle and Tatum, one-gene-one-enzyme hypothesis
1944 Avery shows that DNA carries information during transformation
1946 Lederberg and Tatum describe bacterial
1949 Enders,Weller, and Robbins grow poliovirus in human tissue cultures
1950 Lwoff induces lysogenic bacteriophages
1952 Hershey and Chase show that bacteriophages inject DNA into host
1953 Phase-contrast microscope developed
Medawar discovers immune tolerance
Watson and Crick propose the double helix structure for DNA
1955 Jacob and Wollman discover the F factor is a plasmid Montgomery
Jerne and Burnet propose the clonal selection theory
1959 Yalow develops the radioimmunoassay technique
1961 Jacob and Monod propose the operon model of gene regulation
1961 1966 Nirenberg, Khorana, and others elucidate the genetic code
1962 Porter proposes the basic structure for immunoglobulin G
First quinolone antimicrobial (nalidixic acid) synthesized
1970 Discovery of restriction endonucleases by Arber and Smith
Discovery of reverse transcriptase in retroviruses by Temin and Baltimore
1973 Ames develops a bacterial assay for the detection of mutagens
1975 Kohler and Milstein develop technique for the production monoclonal antibodies
1977 Recognition of archaea as a distinct microbial group by Woese and Fox
Gilbert and Sanger develop techniques for DNA sequencing
1979 Insulin synthesized using recombinant DNA techniques
Smallpox declared officially eliminated
1980 Development of the scanning tunneling microscope
1982 Recombinant hepatitis B vaccine developed AIDS first recognized (1981)
19821983 Discovery of catalytic RNA by Cech and Altman, First artificial heart implanted
19831984 The human immunodeficiency virus isolated and identified by by Gallo
and Montagnier
1986 First vaccine (hepatitis B vaccine) produced by genetic engineering
1990 First human gene-therapy testing
1992 First human trials of antisense therapy
1995 Chickenpox vaccine approved for U.S. use Haemophilus influenzae genome
1996 Methanococcus jannaschii genome sequenced Water found on the Yeast genome
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1997 Discovery of Thiomargarita namibiensis, the largest known bacterium E.coli
2000 Discovery that Vibrio cholerae has two separate chromosomes

CLASSIFICATION AND NOMENCLATURE OF MICROORGANISM

Nomenclature is the set of rules and conventions that govern the names of taxa. Binomial
nomenclature (also called binominal nomenclature or binary nomenclature) is a formal system of
naming species of living things by giving each a name composed of two parts, both of which use
Latin grammatical forms, although they can be based on words from other languages. Such a
name is called a binomial name (which may be shortened to just "binomial"), a binomen or a
scientific name.
For example, the bacteria used in yogurt production would be classified as follows
Kingdom: Bacteria
Phylum: Firmicutes
Class: Bacilli
Order: Lactobacillales
Family: Lactobacillaceae
Genus: Lactobacillus
Species: L. delbrueckii
Subspecies: L. d. bulgaricus
Rules of Nomenclature
1. Use Binary Names - Binary names consisting of a generic name and a species epithet
(e.g., Escherichia coli), must be used for all microorganisms. Names of categories at or above
the genus level may be used alone, but species and subspecies names (species names) may not.
In other words, never use a species name alone.
2. When to Capitalize The genus name is always capitalized, the species name is never
capitalized, e.g. Bacillus anthracis.
3. When to Italicize - Names of all taxa (kingdoms, phyla, classes, orders, families,
genera,
species, and subspecies) are printed in italics and should be underlined if handwritten; strain
designations and numbers are not. If all the surrounding text is italic, then the binary name would
be non-italic (Roman typeface) or underlined (e.g. A common cause of diarrhea is E. coli).
4. When to use Initials - A specific epithet must be preceded by a generic name, written
out in full the first time it is used in a paper. Thereafter, the generic name should be abbreviated
to the initial capital letter (e.g., E. coli), provided there can be no confusion with other genera
used in the paper. Be careful with the S words; Salmonella, Shigella, Serratia, Staphylococcus,
Streptococcus, etc.
5. Common Names - Vernacular (common) names should be in lower case roman type,
non italic (e.g., streptococcus, brucella). However when referring to the actual genus name (or
above) always capitalize and italicize.
6. Subspecies and Serovars - For Salmonella, genus, species, and subspecies names
should be rendered in standard form: Salmonella enterica at first use, S. enterica thereafter;
Salmonella enterica subsp. arizonae at first use, S. enterica subsp. arizonae thereafter. Names of
serovars should be in roman type with the first letter capitalized: Salmonella enterica Serovar
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typhimurium. After the first use, the serovar may also be given without a species name:
Salmonella serovar Typhimurium.
7. Abbreviations for Species use sp. for a particular species, spp. for several species
(spp stands for species plural). These abbreviations are not italicized; e.g. Clostridium sp. or
Clostridium spp.
8. Plural Forms. Plural of genus is genera Plural of species (sp.) is species (spp.)
9. Listing References
Always use the Journal of Clinical Microbiology as a guideline. List the authors (in
bold), publication date, name of article, name of journal, volume (in bold), then pages.

EUKARYOTIC CLASSIFICATION
Four kingdoms:
1. Kingdom Protista (unicellular eukaryotes)
algae and protozoa
simple eukaryotes, dont t elsewhere
nutritionally diverse: autotrophs, heterotrophs, intracellular parasite, etc.
2. Kingdom Fungi
yeasts, molds, mushrooms
absorb organic material through plasma membrane
3. Kingdom Animalia
multicellular animals
ingest organic food through a mouth
have cells organized into tissues
PROKARYOTIC CLASSIFICATION
Prokaryotes = two domains:
1. Bacteria
all pathogenic prokaryotes
many non pathogenic prokaryotes
all photoautotrophic prokaryotes
2. Archaea
all prokaryotes with walls that are not peptidoglycan
often carryout unusual metabolism and live in extreme environments
no kingdoms, but all other taxonomic groups
groupings based entirely on gene sequencing since most look similar
VIRAL CLASSICATION
viruses do not t domain system as they are acellular
usually only classied by Family and Genus
usually only referred to by common name
e.g. HIV (human immuno-deciency virus)
Genus = Lentivirus
Family = Retroviridae

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Viral species = dened as a population of viruses with similar characteristics (including
morphology, genes and enzymes) that occupy a particular ecological niche viruses are obligate
intracellular parasites:
they evolved to infect cells
they usually only infect one type of cell: the one that best supports the viral
replication
thus viruses tend to be very specic about their niche
e.g. HIV: infects only human T helper cells

MICROSCOPIC EXAMINATION OF MICROORGANISMS

The Light Microscope


Microbiologists currently employ a variety of light microscopes in their work; bright-
field, dark-field, phase-contrast, and fluorescence microscopes are most commonly used. Modern
microscopes are all compound microscopes. That is, the magnified image formed by the
objective lens is further enlarged by one or more additional lenses.

The Bright-Field Microscope


The ordinary microscope is called a bright-field microscope because it forms a dark
image against a brighter background. 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 in the base. Two focusing knobs, the fine and coarse
adjustment knobs, are located on the arm and can move either the stage or the nosepiece to focus
the image.

The stage is positioned about halfway up the arm and holds 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. 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.

Mount the specimen on stage

The cover slip must be up if there is one. High magnification objective lenses can't focus
through a thick glass slide; they must be brought close to the specimen, which is why coverslips
are so thin. The stage may be equipped with simple clips (less expensive microscopes), or with
some type of slide holder. The slide may require manual positioning, or there may be a
mechanical stage (preferred) that allows precise positioning without touching the slide.

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Optimise the lighting

A light source should have a wide dynamic range, to provide high intensity illumination
at high magnifications, and lower intensities so that the user can view comfortably at low
magnifications. Better microscopes have a built-in illuminator, and the best microscopes have
controls over light intensity and shape of the light beam. If your microscope requires an external
light source, make sure that the light is aimed toward the middle of the condenser. Adjust
illumination so that the field is bright without hurting the eyes.

Adjust the condenser

To adjust and align the microscope, start by reading the manual. If no manual is available,
try using these guidelines. If the condenser is focusable, position it with the lens as close to the
opening in the stage as you can get it. If the condenser has selectable options, set it to bright
field. Start with the aperture diaphragm stopped down (high contrast). You should see the light
that comes up through the specimen change brightness as you move the aperture diaphragm
lever.

ELECTRON MICROSCOPE
An electron microscope (EM) is a type of microscope that uses an electron beam to
illuminate a specimen and produce a magnified image.An EM has greater resolving power than
a light microscope and can reveal the structure of smaller objects because electrons have
wavelengths about 100,000 times shorter than visible light photons. They can achieve better than
50 pm resolution and magnifications of up to about 10,000,000x whereas ordinary, non-confocal
light microscopes are limited by diffraction to about 200 nm resolution and useful magnifications
below 2000x.
The electron microscope uses electrostatic and electromagnetic lenses to control the
electron beam and focus it to form an image. These electron optical lenses are analogous to the
glass lenses of a light optical microscope.
Electron microscopes are used to investigate the ultrastructure of a wide range of
biological and inorganic specimens including microorganisms, cells, biopsy samples, metals,
and crystals. Industrially, the electron microscope is often used for quality control and failure
analysis. Modern electron microscopes produce electron micrographs, using specialized digital
cameras or frame grabbers to capture the image.
TYPES:
1. SEM Scanning Electron Microscope
2. TEM Transmission Electron Microscope

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STAINING TECHNIQUES

GRAM STAINING
Gram staining is one of the most useful staining procedures in the traditional
bacteriological laboratory.The technique is used as a tool for the differentiation of Gram-positive
and Gram-negative bacteria, as a first step to determine the identity of a particular bacterial
sample.

Staining mechanism

Gram-positive bacteria have a thick mesh-like cell wall made of peptidoglycan (90% of
cell wall), which stain purple and Gram-negative bacteria have a thinner layer (10% of cell wall),
which stain pink. Gram-negative bacteria also have an additional outer membrane which
contains lipids, and is separated from the cell wall by the periplasmic space. There are four basic
steps of the Gram stain, which include applying a primary stain (crystal violet) to a heat-fixed
smear of a bacterial culture, followed by the addition of a mordant (Gram's iodine), rapid
decolorization with alcohol or acetone, and counterstaining with safranin.

Crystal violet (CV) dissociates in aqueous solutions into CV + and chloride (Cl) ions.
These ions penetrate through the cell wall and cell membrane of both Gram-positive and Gram-
negative cells. The CV+ ion interacts with negatively charged components of bacterial cells and
stains the cells purple. Iodine (I or I3) interacts with CV+ and forms large complexes of crystal
violet and iodine (CVI) within the inner and outer layers of the cell. When a decolorizer such as
alcohol or acetone is added, it interacts with the lipids of the cell membrane. A Gram-negative
cell will lose its outer membrane and the peptidoglycan layer is left exposed. The CVI
complexes are washed from the Gram-negative cell along with the outer membrane. In contrast,
a Gram-positive cell becomes dehydrated from an ethanol treatment. The large CVI complexes
become trapped within the Gram-positive cell due to the multilayered nature of its
peptidoglycan. The decolorization step is critical and must be timed correctly; the crystal violet
stain will be removed from both Gram-positive and negative cells if the decolorizing agent is left
on too long (a matter of seconds).

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After decolorization, the Gram-positive cell remains purple and the Gram-negative cell
loses its purple color. Counterstain, which is usually positively-charged safranin or basic fuchsin,
is applied last to give decolorized Gram-negative bacteria a pink or red color.

Some bacteria, after staining with the Gram stain, yield a Gram-variable pattern: a mix of
pink and purple cells are seen. The genera Actinomyces, Arthobacter, Corynebacterium,
Mycobacterium, and Propionibacterium have cell walls particularly sensitive to breakage during
cell division, resulting in Gram-negative staining of these Gram-positive cells. In cultures of
Bacillus, Butyrivibrio, and Clostridium a decrease in peptidoglycan thickness during growth
coincides with an increase in the number of cells that stain Gram-negative In addition, in all
bacteria stained using the Gram stain, the age of the culture may influence the results of the stain.

Gram-staining is a four part procedure which uses certain dyes to make a bacterial cell stand
out against against its background. The specimen should be mounted and fixed on a slide before
you proceed to stain it. The reagents used are:

Crystal Violet (the Primary Stain)


Iodine Solution (the Mordant)

Decolorizer (ethanol is a good choice)

Safranin (the Counterstain)

Water (preferably in a squirt bottle)

ACID FAST STAINING

Acid-fastness is a physical property of some bacteria referring to their resistance to


decolorization by acids during staining procedures.

Acid-fast organisms are difficult to characterize using standard microbiological


techniques (e.g. Gram staining), though they can be stained using concentrated dyes, particularly
when the staining process is combined with heat. Once stained, these organisms resist the dilute
acid and/or ethanol-based de-colorization procedures common in many staining protocols, hence
the name acid-fast.

The high mycolic acid content of certain bacterial cell walls, like those of
Mycobacterium, is responsible for the staining pattern of poor absorption followed by high
retention. The most common staining technique used to identify acid-fast bacteria is the Ziehl-
Neelsen stain, in which the bacteria are stained bright red and stand out clearly against a blue
background. Acid-fast bacteria can also be visualized by fluorescence microscopy using specific
fluorescent dyes (auramine-rhodamine stain, for example). Some bacteria may also be partially
acid-fast.

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Only a few types of bacteria are acid-fast, notably members of the Mycobacterium. Most
are rod-shaped, termed acid-fast bacilli (AFB), but other forms also occur. Medically the most
important AFB is Mycobacterium tuberculosis. Genera like Nocardia and Corynebacterium are
also notable examples.

FLAGELLAR STAINING

The flagella stain allows observation of bacterial flagella under the light microscope.
Bacterial flagella are normally too thin to be seen under such conditions. The flagella stains
employ a mordant to coat the flagella with stain until they are thick enough to be seen. These
staining techniques are typically very difficult.

Many bacteria are motile; some accomplish this motility by means of flagella. Flagella
can vary by number and location. Some bacteria only have one flagella; this is called
monotrichous. In most monotrichous bacteria, the flagella are at the end of the cell; this
placement is called polar. Some bacteria have flagella at either end of the cell; this arrangement
is called amphitrichous. Many bacteria have multiple flagella; these may all be located in a tuft at
one end of the cell, in which case the arrangement is lophotrichous, or they may be all over the
cell, in which case the arrangement is peritrichous.

Procedure:

A. Bacterial Suspension
1. From an agar slant culture:
a. Suspend a loop full of bacteria in 2 ml distilled water to obtain an opalescent suspension.
b. Allow the suspension to stand undisturbed for 15 to 20 minutes while flagella are
regenerated and extended.

B. Slides
1. Select new or unscratched slides.
2. Clean slides with cleaning powder as directed.
3. Flame one of the slides and allow it to cool.
4. Make a heavy wax line on the flamed side along the margin of one end, along both sides to
within about one inch of the other end, and across the slide to complete the rectangle. Handle the
slide by the unmarked end only. The wax line creates a retaining wall to allow a pool of stain to
surround the cells during the 15 to 15 minute staining period.

C. Preparation of smear
1. Handling the suspension carefully remove a large loopful of the suspension and place it at one
end of the rectangular area.
2. Tilt the slide to permit the suspension to run down to the other end of the slide. If the drop
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fails to run, add another drop.
3. Air dries the film. Do not heat.
4. Place the slide on a horizontal staining rack.

D. Staining procedure
1. Add about 1 ml. of the Flagella Stain solution (one dropper full) to the smear and allow
staining for 10-15 minutes. The solution should not be allowed to flow outside the waxed area.
2. Flood off the stain by adding tap water to the slide while it remains on the rack. Do not tip
the slide before this is done.
3. Drain and flood the slide with carbol fuchsin for one minute. Rinse by flooding. Drain and
air dry. Do not blot.

E. Examination of slide
Examine using oil immersion lens and identify cells as distinguished from dye and debris which
will adhere to the slide because of the mordant. Cells will be small rods and have regular
outlines.

CAPSULAR STAINING

The cell walls of many bacterial species are surrounded by a polymeric substance
referred to as a capsule (if discrete) or slime later (if amorphous). These capsules perform
numerous physiological functions, including conferring resistance to phagocytosis and allowing
adhesion to sites with favorable environments. The size of such capsules varies with species and
among strains of a species, and may also be affected by the composition of the growth medium.

Because most capsule materials are water soluble, simple stains will not adhere to them,
thus making capsule staining a difficult process. As a result of this limitation, capsule staining
works by staining the bacteria and background more intensely than the capsule itself. Capsule
staining is diagnostically useful since the presence of large capsules generally indicates the
presence of a virulent form of some bacterial species (e.g. pneumococci); by forcing capsular
swelling (e.g. through the exposure the certain anti-bodies), a condition known as the Quellung
phenomenon, the strain of bacteria can be easily characterized.

Uses of Capsule Staining

Detection of dental plaque


Detection of Anthrax
Detection of Cataracts

Capsules are formed by organisms such as Klebsiella pneumoniae. Most capsules are
composed of polysaccharides, but some are composed of polypeptides. The capsule differs from
the slime layer that most bacterial cells produce in that it is a thick, detectable, discrete layer
outside the cell wall. Some capsules have well-defined boundaries, and some have fuzzy, trailing
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edges. Capsules protect bacteria from the phagocytic action of leukocytes and allow pathogens to
invade the body. If a pathogen loses its ability to form capsules, it can become avirulent.

Bacterial capsules are non-ionic, so neither acidic nor basic stains will adhere to their
surfaces. Therefore, the best way to visualize them is to stain the background using an acidic
stain and to stain the cell itself using a basic stain. The medium in which the culture is grown as
well as the temperature at which it is grown and the age of the culture will affect capsule
formation. Older cultures are more likely to exhibit capsule production.

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UNIT II MICROBES-STRUCTURE AND MULTIPLICATION

Bacteria Cell Structure

They are as unrelated to human beings as living things can be, but bacteria are essential
to human life and life on planet Earth. Although they are notorious for their role in causing
human diseases, from tooth decay to the Black Plague, there are beneficial species that are
essential to good health.

For example, one species that lives symbiotically in the large intestine manufactures
vitamin K, an essential blood clotting factor. Other species are beneficial indirectly. Bacteria give
yogurt its tangy flavor and sourdough breads its sour taste. They make it possible for ruminant
animals (cows, sheep, and goats) to digest plant cellulose and for some plants, (soybean, peas,
and alfalfa) to convert nitrogen to a more usable form. Bacteria are prokaryotes, lacking well-
defined nuclei and membrane-bound organelles, and with chromosomes composed of a single
closed DNA circle. They come in many shapes and sizes, from minute spheres, cylinders and
spiral threads, to flagellated rods, and filamentous chains. They are found practically everywhere
on Earth and live in some of the most unusual and seemingly inhospitable places.

There are two different ways of grouping bacteria. They can be divided into three types
based on their response to gaseous oxygen. Aerobic bacteria require oxygen for their health and
existence and will die without it. Anaerobic bacteria can't tolerate gaseous oxygen at all and die
when exposed to it. Facultative anaerobes prefer oxygen, but can live without it.

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The second way of grouping them is by how they obtain their energy. Bacteria that have to
consume and break down complex organic compounds are heterotrophs. This includes species
that are found in decaying material as well as those that utilize fermentation or respiration.
Bacteria that create their own energy, fueled by light or through chemical reactions, are
autotrophs.

Capsule - Some species of bacteria have a third protective covering, a capsule made up
of polysaccharides (complex carbohydrates). Capsules play a number of roles, but the
most important are to keep the bacterium from drying out and to protect it from
phagocytosis (engulfing) by larger microorganisms. The capsule is a major virulence
factor in the major disease-causing bacteria, such as Escherichia coli and Streptococcus
pneumoniae. Nonencapsulated mutants of these organisms are avirulent, i.e. they don't
cause disease.
Cell Envelope - The cell envelope is made up of two to three layers: the interior
cytoplasmic membrane, the cell wall, and -- in some species of bacteria -- an outer
capsule.
Cell Wall - Each bacterium is enclosed by a rigid cell wall composed of peptidoglycan, a
protein-sugar (polysaccharide) molecule. The wall gives the cell its shape and surrounds
the cytoplasmic membrane, protecting it from the environment. It also helps to anchor
appendages like the pili and flagella, which originate in the cytoplasm membrane and
protrude through the wall to the outside. The strength of the wall is responsible for
keeping the cell from bursting when there are large differences in osmotic pressure
between the cytoplasm and the environment.

Cell wall composition varies widely amongst bacteria and is one of the most important
factors in bacterial species analysis and differentiation. For example, a relatively thick,
mesh like structure that makes it possible to distinguish two basic types of bacteria. A
technique devised by Danish physician Hans Christian Gram in 1884, uses a staining and
washing technique to differentiate between the two forms. When exposed to a gram stain,
gram-positive bacteria retain the purple color of the stain because the structure of their
cell walls traps the dye. In gram-negative bacteria, the cell wall is thin and releases the
dye readily when washed with an alcohol or acetone solution.

Cytoplasm - The cytoplasm, or protoplasm, of bacterial cells is where the functions for
cell growth, metabolism, and replication are carried out. It is a gel-like matrix composed
of water, enzymes, nutrients, wastes, and gases and contains cell structures such as
ribosomes, a chromosome, and plasmids. The cell envelope encases the cytoplasm and all
its components. Unlike the eukaryotic (true) cells, bacteria do not have a membrane
enclosed nucleus. The chromosome, a single, continuous strand of DNA, is localized, but
not contained, in a region of the cell called the nucleoid. All the other cellular
components are scattered throughout the cytoplasm.

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Cytoplasmic Membrane - A layer of phospholipids and proteins, called the cytoplasmic
membrane, encloses the interior of the bacterium, regulating the flow of materials in and
out of the cell. This is a structural trait bacteria share with all other living cells; a barrier
that allows them to selectively interact with their environment. Membranes are highly
organized and asymmetric having two sides, each side with a different surface and
different functions. Membranes are also dynamic, constantly adapting to different
conditions.

One of those components, plasmids, is small, extra chromosomal genetic structures


carried by many strains of bacteria. Like the chromosome, plasmids are made of a
circular piece of DNA. Unlike the chromosome, they are not involved in reproduction.
Only the chromosome has the genetic instructions for initiating and carrying out cell
division, or binary fission, the primary means of reproduction in bacteria. Plasmids
replicate independently of the chromosome and, while not essential for survival, appear
to give bacteria a selective advantage.

Plasmids are passed on to other bacteria through two means. For most plasmid types,
copies in the cytoplasm are passed on to daughter cells during binary fission. Other types
of plasmids, however, form a tube like structure at the surface called a pilus that passes
copies of the plasmid to other bacteria during conjugation, a process by which bacteria
exchange genetic information. Plasmids have been shown to be instrumental in the
transmission of special properties, such as antibiotic drug resistance, resistance to heavy
metals, and virulence factors necessary for infection of animal or plant hosts. The ability
to insert specific genes into plasmids has made them extremely useful tools in the fields
of molecular biology and genetics, specifically in the area of genetic engineering.

Flagella - Flagella (singular, flagellum) are hair like structures that provide a means of
locomotion for those bacteria that have them. They can be found at either or both ends of
a bacterium or all over its surface. The flagella beat in a propeller-like motion to help the
bacterium move toward nutrients; away from toxic chemicals; or, in the case of the
photosynthetic cyanobacteria; toward the light.
Nucleoid - The nucleoid is a region of cytoplasm where the chromosomal DNA is
located. It is not a membrane bound nucleus, but simply an area of the cytoplasm where
the strands of DNA are found. Most bacteria have a single, circular chromosome that is
responsible for replication, although a few species do have two or more. Smaller circular
auxiliary DNA strands, called plasmids, are also found in the cytoplasm.
Pili - Many species of bacteria have pili (singular, pilus), small hair like projections
emerging from the outside cell surface. These outgrowths assist the bacteria in attaching
to other cells and surfaces, such as teeth, intestines, and rocks. Without pili, many
disease-causing bacteria lose their ability to infect because they're unable to attach to host
tissue. Specialized pili are used for conjugation, during which two bacteria exchange
fragments of plasmid DNA.
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Ribosomes - Ribosomes are microscopic "factories" found in all cells, including bacteria.
They translate the genetic code from the molecular language of nucleic acid to that of
amino acidsthe building blocks of proteins. Proteins are the molecules that perform all
the functions of cells and living organisms. Bacterial ribosomes are similar to those of
eukaryotes, but are smaller and have a slightly different composition and molecular
structure. Bacterial ribosomes are never bound to other organelles as they sometimes are
(bound to the endoplasmic reticulum) in eukaryotes, but are free-standing structures
distributed throughout the cytoplasm. There are sufficient differences between bacterial
ribosomes and eukaryotic ribosomes that some antibiotics will inhibit the functioning of
bacterial ribosomes, but not a eukaryote's, thus killing bacteria but not the eukaryotic
organisms they are infecting.

VIRUSES

A virus (from the Latin noun virus, meaning toxin or poison) is a sub-microscopic
particle (ranging in size from 20300 nm) that can infect the cells of a biological
organism. Viruses can replicate themselves only by infecting a host cell. They therefore
cannot reproduce on their own. At the most basic level, viruses consist of genetic material
contained within a protective protein coat called a capsid. They infect a wide variety of
organisms: both eukaryotes (animals, plants, protists, and fungi) and prokaryotes
(bacteria and archaea). A virus that infects bacteria is known as a bacteriophage, often
shortened to phage. The study of viruses is known as virology and people who study
viruses are known as virologists. Viruses cause several serious human diseases, such as
AIDS, influenza and rabies. Therapy is difficult for viral diseases as antibiotics have no
effect on viruses and few antiviral drugs are known. The best way to prevent viral
diseases is with a vaccine, which produces immunity.

Viral classification
Group I: double-stranded DNA viruses
Group II: single-stranded DNA viruses

Group III: double-stranded RNA viruses

Group IV: positive-sense single-stranded RNA viruses

Group V: negative-sense single-stranded RNA viruses

Group VI: reverse transcribing Diploid single-stranded RNA viruses

Group VII: reverse transcribing Circular double-stranded DNA viruses

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Replication

Viral populations do not grow through cell division, because they are acellular; instead,
they use the machinery and metabolism of a host cell to produce multiple copies of themselves.
A virus can still cause degenerative effects within a cell without causing its death; collectively
these are termed cytopathic effects. Released virions can be passed between hosts through either
direct contact, often via body fluids, or through a vector. In aqueous environments, viruses float
free in the water.

Lytic or lysogenic

Viruses may have a lytic or a lysogenic cycle, with some viruses capable of carrying out
both.

Applications

Life sciences
Material science and nanotechnology

ALGAE

Algae are a very large and diverse group of simple, typically autotrophic organisms,
ranging from unicellular to multicellular forms, such as the giant kelp (large brown alga), that
may grow up to 50 meters in length. Most are photosynthetic and "simple" because they lack
many of the distinct cell organelles and cell types found in land plants. The largest and most
complex marine forms are called seaweeds.

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Though the prokaryotic cyanobacteria are informally referred to as blue-green algae, this
usage is incorrect since they are regarded as bacteria. The term algae is now restricted
eukaryotic organisms. All true algal cells therefore have a nucleus enclosed within
a membrane and plastids bound in one or more membranes. Algae constitute
a polyphyletic group since they do not include a common ancestor, and although their plastids
seem to have a single origin, from cyanobacteria, they were acquired in different ways. Green
algae are examples of algae that have primary chloroplasts derived
from endosymbiotic cyanobacteria. Diatoms are examples of algae with secondary chloroplasts
derived from an endosymbiotic red alga.

While cyanobacteria were traditionally considered algae, they are now classified
as bacteria and therefore prokaryotes due to large differences such as the lack of a cell nucleus or
membrane-bound organelles, the presence of a single circular chromosome, the presence
of peptidoglycan in the cell walls, and ribosomes different in size and content from those of
the eukaryotes.[15][16] Rather than in chloroplasts, they conduct photosynthesis on specialized
infolded cytoplasmic membranes called thylakoid membranes. Therefore, they differ
significantly from algae despite occupying similar ecological niches.

By modern definitions, algae are eukaryotes that conduct photosynthesis within


membrane-bound organelles called chloroplasts. Chloroplasts contain circular DNA and are
similar in structure to cyanobacteria, presumably representing reduced
cyanobacterial endosymbionts. The exact nature of the chloroplasts is different among separate
lineages of algae, reflecting different endosymbiotic events. The table below describes the
composition of the three major groups of algae. Their lineage relationships are shown in the
figure in the upper right. Many of these groups contain some members that are no longer
photosynthetic. Some retain plastids, but not chloroplasts, while others have lost plastids entirely.

FUNGI

Fungi (singular fungus) are a kingdom of eukaryotic organisms. The fungi are
heterotrophic organisms characterized by a chitinous cell wall, and in the majority of species,
filamentous growth as multicellular hyphae forming a mycelium; some fungal species also grow
as single cells. Sexual and asexual reproduction is via spores, often produced on specialized
structures or in fruiting bodies. Yeasts, molds, and mushrooms are examples of fungi. The
discipline of biology devoted to the study of fungi is known as mycology.

Importance for human use

Human use of fungi for food preparation or preservation and other purposes is extensive
and has a long history: yeasts are required for fermentation of beer and bread, some other fungal
species are used in the production of soy sauce and tempeh, and mushroom farming and
gathering is a large industry in many countries. Many fungi are producers of antibiotics,
including -lactam antibiotics such as penicillin and cephalosporin.Widespread use of these

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antibiotics for the treatment of bacterial diseases, such as tuberculosis, syphilis, leprosy, and
many others began in the early 20th Century and continues to play a major part in anti-bacterial
chemotherapy. The study of the historical uses and sociological impact of fungi is known as
ethnomycology.

Edible and poisonous fungi


Some of the most well-known types of fungi are the edible and poisonous mushrooms.
Many species are commercially raised, but others must be harvested from the wild. Agaricus
bisporus, sold as button mushrooms when small or Portobello musrooms when larger, are the
most commonly eaten species, used in salads, soups, and many other dishes. Other commercially
grown mushrooms that have gained in popularity in the West and are often available fresh in
grocery stores include straw mushrooms (Volvariella volvacea), oyster mushrooms (Pleurotus
ostreatus), shiitakes (Lentinula edodes), and enokitake (Flammulina spp.).

There are many more mushroom species that are harvested from the wild for personal
consumption or commercial sale. Milk mushrooms, morels, chanterelles, truffles, black trumpets,
and porcini mushrooms (also known as king boletes) all demand a high price on the market.
They are often used in gourmet dishes.

For certain types of cheeses, it is also a common practice to inoculate milk curds with
fungal spores to foment the growth of specific species of mold that impart a unique flavor and
texture to the cheese. This accounts for the blue colour in cheeses such as Stilton or Roquefort
which is created using Penicillium roqueforti spores.Molds used in cheese production are usually
non-toxic and are thus safe for human consumption; however, toxic fungal metabolites (e.g.,
aflatoxins, roquefortine C, patulin, or others) may accumulate due to fungal spoilage during
cheese ripening or storage.

Many mushroom species are toxic to humans, with toxicities ranging from slight
digestive problems or allergic reactions as well as hallucination to severe organ failures and
death. Some of the most deadly mushrooms belong to the genera Inocybe, Cortinarius, and most
infamously, Amanita, which includes the destroying angel (A. virosa) and the death cap (A.
phalloides), the most common cause of deadly mushroom poisoning. Fly agaric mushrooms (A.
muscaria) also cause occasional poisonings, mostly as a result of ingestion for use as a
recreational drug for its hallucinogenic properties.

Morphology

Mold covering a decaying peach over a period of six days. The frames were taken
approximately 12 hours apart. Though fungi are part of the opisthokont clade, all phyla except
for the chytrids have lost their posterior flagella.Fungi are unusual among the eukaryotes in
having a cell wall that, besides glucans (e.g., -1,3-glucan) and other typical components,
contains the biopolymer chitin. Many fungi grow as thread-like filamentous macroscopic
structures called hyphae, and an assemblage of intertwined and interconnected hyphae is called a
mycelium. Fungal mycelia can become visible macroscopically, for example, as concentric rings
on various surfaces, such as damp walls, and on other substrates, such as spoilt food (see figure),
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and are commonly and generically called mould; fungal mycelia grown on solid agar media in
laboratory petri dishes are usually referred to as colonies, with many species exhibiting
characteristic macroscopic growth morphologies and colours, due to spores or pigmentation.
Hyphae can be septate, i.e., divided into hyphal compartments separated by a septum,
each compartment containing one or more nuclei or can be coenocytic, i.e., lacking hyphal
compartmentalization. However, septa have pores, such as the doliporus in the basidiomycetes
that allow cytoplasm, organelles, and sometimes nuclei to pass through. Coenocytic hyphae are
essentially multinucleate supercells. In some cases, fungi have developed specialized structures
for nutrient uptake from living hosts; examples include haustoria in plant-parasitic fungi of
nearly all divisions, and arbuscules of several mycorrhizal fungi, which penetrate into the host
cells for nutrient uptake by the fungus. Specialized fungal structures important in sexual
reproduction are the apothecia, perithecia, and cleistothecia in the ascomycetes, and the fruiting
bodies of the basidiomycetes, and a few ascomycetes, which can sometimes grow very large and
are well known as mushrooms.

ACTINOMYCETES

Actinobacteria are a group of Gram-positive bacteria. They were all believed to have
high guanine and cytosine content in their DNA. However, recently it has been shown that
several freshwater Actinobacteria actually have low G+C content. The G+C content of
freshwater Actinobacteria can be as low as 42%. In view of this, use of the epithet high G+C
Gram positive organisms to refer to Actinobacteria needs to be discontinued. They can be
terrestrial or aquatic. Although understood primarily as soil bacteria, they might be more
abundant in freshwaters.] Actinobacteria is one of the dominant bacterial phyla and contains one
of the largest of bacterial genera, Streptomyces. Analysis of glutamine synthetase sequence has
been suggested for phylogenetic analysis of Actinobacteria.

Characteristics

Actinobacteria include some of the most common soil life, freshwater life, and marine
life, playing an important role in the decomposition of organic materials, such
as cellulose and chitin, and thereby playing a vital part in organic matter turnover and the carbon
cycle. In the soil, this replenishes the supply of nutrients and is an important part
of humus formation. Other Actinobacteria inhabit plants and animals, and include a
few pathogens, such as Corynebacterium, Nocardia and Rhodococcus. An example of the latter
is Rhodococcus equi, an equine pathogen.

Actinobacteria are well known as secondary metabolite producers and hence of high
pharmacological and commercial interest. In 1942 Selman Waksman discovered that the
soil bacteria he was studying made actinomycin, a discovery for which he received a Nobel
Prize. Since then, hundreds of other naturally occurring antibiotics have been discovered in these
terrestrial microorganisms, especially from the genus Streptomyces.

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Some Actinobacteria form branching filaments, which somewhat resemble
the mycelia of fungi, among which they were originally classified under the older
name Actinomycetes. Most members are aerobic, but a few, such as Actinomyces israelii, can
grow under anaerobic conditions. Unlike the Firmicutes, the other main group of Gram-positive
bacteria, they have DNA with a high GC-content, and some Actinomycetes species produce
external spores.

Some types of Actinobacteria, the Actinomycetes are responsible for the peculiar odor
emanating from the soil after rain (petrichor), mainly in warmer climates. The chemical that
produces this odor is known as geosmin. The Actinomycetes are also known to form intracellular
inclusions of polyhydroxyalkanoates under certain environmental conditions (e.g. lack of
elements such as phosphorus, nitrogen, or oxygen combined with an excessive supply of carbon
sources).

YEAST AND MYCOPLASMA

Yeasts are eukaryotic microorganisms classified in the kingdom Fungi, with ,500 species
(estimated to be 1% of all fungal species). Yeasts are unicellular, although some species with
yeast forms may become multicellular through the formation of strings of connected budding
cells known as pseudohyphae, or false hyphae, as seen in most molds. Yeast size can vary
Greatly depending on the species, typically measuring 34 m in diameter, although some yeasts
can reach over 40 m. Most yeasts reproduce asexually by mitosis, and many do so by an
asymmetric division process called budding.

By fermentation, the yeast species S. cerevisiae converts carbohydrates to carbon


dioxide and alcohols for thousands of years the carbon dioxide has been used in baking and the
alcohol in alcoholic beverages. It is also a centrally important model organism in modern cell
biology research, and is one of the most thoroughly researched eukaryotic microorganisms.
Researchers have used it to gather information about the biology of the eukaryotic cell and
ultimately human biology. Other species of yeasts, such as Candida albicans, are opportunistic
pathogens and can cause infections in humans. Yeasts have recently been used to generate
electricity in microbial fuel cells, and produce ethanol for the biofuel industry.

Yeasts do not form a single taxonomic or phylogenetic grouping. The term "yeast" is
often taken as a synonym for Saccharomyces cerevisiae, but the phylogenetic diversity of yeasts
is shown by their placement in two separate phyla: the Ascomycota and the Basidiomycota. The
budding yeasts ("true yeasts") are classified in the order Saccharomycetales.

Nutrition and growth

Yeasts are chemoorganotrophs, as they use organic compounds as a source of energy and
do not require sunlight to grow. Carbon is obtained mostly from hexose sugars, such
as glucose and fructose, or disaccharides such as sucrose and maltose. Some species can
metabolize pentose sugars such as ribose, alcohols, and organic acids. Yeast species either
require oxygen for aerobic cellular respiration (obligate aerobes) or are anaerobic, but also have
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aerobic methods of energy production (facultative anaerobes). Unlike bacteria, no known yeast
species grow only anaerobically (obligate anaerobes). Yeasts grow best in a neutral or slightly
acidic pH environment.

Yeasts vary in what temperature range they grow best. For example, Leucosporidium
frigidum grows at 2 to 20 C (28 to 68 F), Saccharomyces telluris at 5 to 35 C (41 to 95 F),
and Candida slooffi at 28 to 45 C (82 to 113 F).[18] The cells can survive freezing under certain
conditions, with viability decreasing over time.

Uses

The useful physiological properties of yeast have led to their use in the field
of biotechnology. Fermentation of sugars by yeast is the oldest and largest application of this
technology. Many types of yeasts are used for making many foods: baker's yeast in bread
production; brewer's yeast in beer fermentation; yeast in wine fermentation, and
for xylitol production. So-called red rice yeast is actually a mold, Monascus purpureus. Yeasts
include some of the most widely used model organisms for genetics and cell biology.

Bioremediation

Some yeasts can find potential application in the field of bioremediation. One such
yeast, Yarrowia lipolytica, is known to degrade palm oil mill effluent, TNT (an explosive
material), and other hydrocarbons, such asalkanes, fatty acids, fats and oils. It can also tolerate
high concentrations of salt and heavy metals, and is being investigated for its potential as a heavy
metal biosorbent. Baker Yeast Saccharomyces cerevisiae has potential to bioremediate the toxic
pollutants from ground water like Arsenic. Bronze statues are known to be degraded by certain
species of yeast.

MYCOPLASMA

Mycoplasma refers to a genus of bacteria that lack a cell wall. Without a cell wall, they
are unaffected by many common antibiotics such as penicillin or other beta-lactam antibiotics
that target cell wall synthesis. They can be parasitic or saprotrophic. Several species
are pathogenic in humans, including M. pneumoniae, which is an important cause of atypical
pneumonia and other respiratory disorders, and M. genitalium, which is believed to be involved
in pelvic inflammatory diseases. Mycoplasma are the smallest living cells yet discovered, can
survive without oxygen and are typically about 0.1 m in diameter.

History of Mycoplasma research

The discovery of mycoplasmas is on 1898. Initially, due to their unknown nature and
relationships with other organisms, while being minute in size and not being qualified as bacteria
they were considered viruses for years. However, many years later with further discovery
mycoplasmas were confused with the L-forms, which are bacteria that have lost their cell walls
either completely or partially. Nevertheless, in 1950s and 1960s this confusion came to end when
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first genomic analysis data through DNA hybridization were obtained. This analysis ruled out
any relationship of mycoplasmas to the L-forms.

Currently, mycoplasmas are considered to have evolved from gram-positive, walled


eubacteria by degenerative evolution, meaning their evolutionary history appears to include the
loss of cell wall. Further, in 1960s and 1970s knowledge about ultrastructure, cell membrane,
genome and metabolic pathways of mycoplasmas resulted in conclusion that they are the
smallest and simplest self-replicating organisms.

The lack of cell wall convey some unique properties of mycoplasmas such as sensitivity
to osmotic shock and detergents, resistance to penicillin, and formation of odd fried-egg shaped
colonies. Sections of mycoplasmas reveal that their cells are essentially built of three organelles,
such as: the cell membrane, ribosomes, and circular double stranded DNA tightly packed
molecule. Their mode of replication is no different from that of prokaryotes dividing by binary
fission. However for binary fission to happen, cytoplasmic division must fully synchronize with
genome replication and in mycoplasmas cytoplasmic replication lags behind genome replication,
which ultimately results in the formation of multinucleated filaments.

From in vitro cultivation of mycoplasmas it has been discovered that they are
"fastidious", i.e. difficult to cultivate. The reasons for these difficulties for species such
as Mycoplasma genitalium and Mycoplasma pneumoniae is the lack of all the genes involved in
amino acid synthesis, making them dependent on exogenous supply of amino acids.

Dependence on exogenous supplies of fatty acids and cholesterol serves as an advantage


to conduct further studies on these organisms. In order to compensate for these deficiencies
mycoplasmas are grown on complex media, usually consisting of beef heart infusion, peptone,
yeast extract, and serum with various supplements.

Mycoplasmas are known to consist of just plasma membrane which makes them good
models for membrane studies. Due to this reason the availability of these membranes in pure
state have enabled their chemical, enzymatic and antigenic characterization. The membrane
mostly consists of 60% to 70% of proteins and the rest of it being 20% to 30% of lipid.

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UNIT III MICROBIAL NUTRITION GROWTH AND METABOLISM

The Common Nutrient Requirements

Analysis of microbial cell composition shows that over 95% of cell dry weight is made
up of a few major elements: carbon, oxygen, hydrogen, nitrogen, sulfur, phosphorus, potassium,
calcium, magnesium, and iron. These are called macroelements or macronutrients because they
are required by microorganisms in relatively large amounts. The first six (C, O, H, N, S, and P)
are components of carbohydrates, lipids, proteins, and nucleic acids. The remaining four
macroelements exist in the cell as cations and play a variety of roles.

For example, potassium is required for activity by a number of enzymes, including some
of those involved in protein synthesis. Calcium, among other functions, contributes to the heat
resistance of bacterial endospores. Magnesium serves as a cofactor for many enzymes,
complexes with ATP, and stabilizes ribosomes and cell membranes. Iron is a part of cytochromes
and a cofactor for enzymes and electron-carrying proteins.

All organisms, including microorganisms, require several micronutrients or trace


elements besides macroelements. The micronutrients manganese, zinc, cobalt, molybdenum,
nickel, and copper are needed by most cells. However, cells require such small amounts that
contaminants in water, glassware, and regular media components often are adequate for growth.
Therefore it is very difficult to demonstrate a micronutrient requirement. In nature,
micronutrients are ubiquitous and probably do not usually limit growth. Micronutrients are
normally a part of enzymes and cofactors, and they aid in the catalysis of reactions and
maintenance of protein structure.

Sources of Carbon, Energy, and Electrons

Carbon Sources
Autotrophs CO2 sole or principal biosynthetic carbon source
Heterotrophs Reduced, preformed, organic molecules from other organisms
Energy Sources
Phototrophs Light
Chemotrophs Oxidation of organic or inorganic compounds
Electron Sources
Lithotrophs Reduced inorganic molecules
Organotrophs Organic molecules

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CULTURE MEDIA
A culture medium is a solid or liquid preparation used to grow, transport, and store
microorganisms. To be effective, the medium must contain all the nutrients the microorganism
requires for growth. Specialized media are essential in the isolation and identification of
microorganisms, the testing of antibiotic sensitivities, water and food analysis, industrial
microbiology, and other activities.

Although all microorganisms need sources of energy, carbon, nitrogen, phosphorus,


sulfur, and various minerals, the precise composition of a satisfactory medium will depend on the
species one is trying to cultivate because nutritional requirements vary so greatly. Knowledge of
a microorganisms normal habitat often is useful in selecting an appropriate culture medium
because its nutrient requirements reflect its natural surroundings. Frequently a medium is used to
select and grow specific microorganisms or to help identify a particular species. In such cases the
function of the medium also will determine its composition.

Synthetic or Defined Media

Some microorganisms, particularly photolithotrophic autotrophs such as cyanobacteria


and eucaryotic algae, can be grown on relatively simple media containing CO2 as a carbon,nitrate
or ammonia as a nitrogen source, sulfate, phosphate, and a variety of minerals. Such a medium in
which all components are known is a defined medium or synthetic medium.

Complex Media

Media that contain some ingredients of unknown chemical composition are complex
media. Such media are very useful, as a single complex medium may be sufficiently rich and
complete to meet the nutritional requirements of many different microorganisms. In addition,
complex media often are needed because the nutritional requirements of a particular
microorganism are unknown, and thus a defined medium cannot be constructed. This is the
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situation with many fastidious bacteria, some of which may even require a medium containing
blood or serum.

TYPES OF MEDIA

Media such as tryptic soy broth and tryptic soy agar are called general purpose media
because they support the growth of many microorganisms. Blood and other special nutrients may
be added to general purpose media to encourage the growth of fastidious heterotrophs. These
specially fortified media (e.g., blood agar) are called enriched media.

Selective media favor the growth of particular microorganisms. Bile salts or dyes like
crystal violet favor the growth of gram-negative bacteria by inhibiting the growth of gram-
positive bacteria without affecting gram-negative organisms. Endo agar, eosin methylene blue
agar and MacConkey agar three media widely used for the detection of E. coli and related
bacteria in water supplies and elsewhere, contain dyes that suppress gram-positive bacterial
growth. MacConkey agar also contains bile salts. Bacteria also may be selected by incubation
with nutrients that they specifically can use. A medium containing only cellulose as a carbon and
energy source is quite effective in the isolation of cellulose-digesting bacteria. The possibilities
for selection are endless, and there are dozens of special selective media in use.

Differential media are media that distinguish between different groups of bacteria and
even permit tentative identification of microorganisms based on their biological characteristics.
Blood agar is both a differential medium and an enriched one. It distinguishes between hemolytic
and nonhemolytic bacteria. Hemolytic bacteria (e.g., many streptococci and staphylococci
isolated from throats) produce clear zones around their colonies because
of red blood cell destruction. MacConkey agar is both differential and selective. Since it contains
lactose and neutral red dye, lactose-fermenting colonies appear pink to red in color and are easily
distinguished from colonies of non-fermenters.

THE GROWTH CURVE

Population growth is studied by analyzing the growth curve of a microbial culture. When
microorganisms are cultivated in liquid medium, they usually are grown in a batch culture or
closed system that is, they are incubated in a closed culture vessel with a single batch of medium.
Because no fresh medium is provided during incubation, nutrient concentrations decline and
concentrations of wastes increase. The growth of microorganisms reproducing by binary fission
can be plotted as the logarithm of the number of viable cells versus the incubation time. The
resulting curve has four distinct phases

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Lag Phase

When microorganisms are introduced into fresh culture medium, usually no immediate
increase in cell number occurs, and therefore this period is called the lag phase. Although cell
division does not take place right away and there is no net increase in mass, the cell is
synthesizing new components. A lag phase prior to the start of cell division can be necessary for
a variety of reasons. The cells may be old and depleted of ATP, essential cofactors, and
ribosomes; these must be synthesized before growth can begin. The medium may be different
from the one the microorganism was growing in previously. Here new enzymes would be needed
to use different nutrients. Possibly the microorganisms have been injured and require time to
recover. Whatever the causes, eventually the cells retool, replicate their DNA, begin to increase
in mass, and finally divide. The lag phase varies considerably in length with the condition of the
microorganisms and the nature of the medium. This phase may be quite long if the inoculum is
from an old culture or one that has been refrigerated. Inoculation of a culture into a chemically
different medium also results in a longer lag phase.

Exponential Phase

During the exponential or log phase, microorganisms are growing and dividing at the
maximal rate given their genetic potential, the nature of the medium, and the conditions under
which they are growing. Their rate of growth is constant during the exponential phase; that is, the
microorganisms are dividing and doubling in number at regular intervals. Because each
individual divides at a slightly different moment, the growth curve rises smoothly rather than in
discrete jumps. The population is most uniform in terms of chemical and physiological properties
during this phase; therefore exponential phase cultures are usually used in biochemical and
physiological studies.

Exponential growth is balanced growth. That is, all cellular constituents are
manufactured at constant rates relative to each other. If nutrient levels or other environmental
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conditions change, unbalanced growth results. This is growth during which the rates of
synthesis of cell components vary relative to one another until a new balanced state is reached.
This response is readily observed in a shift-up experiment in which bacteria are transferred from
a nutritionally poor medium to a richer one. The cells first construct new ribosomes to enhance
their capacity for protein synthesis. This is followed by increases in protein and DNA synthesis.
Finally, the expected rise in reproductive rate takes place.

Stationary Phase

Eventually population growth ceases and the growth curve becomes horizontal. This
stationary phase usually is attained by bacteria at a population level of around 10 9 cells per ml.
Other microorganisms normally do not reach such high population densities, protozoan and algal
cultures often having maximum concentrations of about 106 cells per ml. Of course final
population size depends on nutrient availability and other factors, as well as the type of
microorganism being cultured. In the stationary phase the total number of viable microorganisms
remains constant. This may result from a balance between cell division and cell death, or the
population may simply cease to divide though remaining metabolically active.

Death Phase

Detrimental environmental changes like nutrient deprivation and the buildup of toxic
wastes lead to the decline in the number of viable cells characteristic of the death phase. The
death of a microbial population, like its growth during the exponential phase, is usually
logarithmic (that is, a constant proportion of cells dies every hour). This pattern in viable cell
count holds even when the total cell number remains constant because the cells simply fail to
lyse after dying. Often the only way of deciding whether a bacterial cell is viable is by incubating
it in fresh medium; if it does not grow and reproduce, it is assumed to be dead. That is, death is
defined to be the irreversible loss of the ability to reproduce.

MICROBIAL GROWTH

Growth of Bacterial Cultures


Bacterial Division:
Occurs mainly by binary fission.
A few bacterial species reproduce by budding.
Generation Time:
Time required for a cell to divide, and its population to double.
Generation time varies considerably:
E. coli divides every 20 minutes.
Most bacteria divide every 1 to 3 hours.
Some bacteria require over 24 hours to divide.
Measuring Microbial Growth
Direct Methods of Measurement
1. Plate count:

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Most frequently used method of measuring bacterial populations.
Inoculate plate with a sample and count number of colonies.
Assumptions:
Each colony originates from a single bacterial cell.
Original inoculum is homogeneous.
No cell aggregates are present.
Advantages:
Measures viable cells
Disadvantages:
Takes 24 hours or more for visible colonies to appear.
Only counts between 25 and 250 colonies are accurate.
Must perform serial dilutions to get appropriate numbers/plate.
Pour Plate:
Introduce a 1.0 or 0.1 ml inoculuminto an emptyPetri dish.
Add liquid nutrient medium kept at 50oC.
Gently mix, allow to solidify, and incubate.
Disadvantages:
Not useful for heat sensitive organisms.
Colonies appear under agar surface.
Spread Plate:
Introduce a 0.1 ml inoculum onto the surface of Petri dish and Spread with a sterile glass rod.
Advantages: Colonies will be on surface and not exposed to melted agar.

Serial Dilutions are Used with the Plate Count Pour Plate versus Spread plate
Method to Measure Numbers of Bacteria

2. Filtration:

Used to measure small quantities of bacteria.


Example: Fecal bacteria in a lake or in ocean water.
A large sample (100 ml or more) is filtered to retain bacteria.
Filter is transferred onto a Petri dish.
Incubate and count colonies.
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3. Most Probable Number (MPN):

Used mainly to measure bacteria that will not grow on solid medium.
Dilute a sample repeatedly and inoculate several broth tubes for each dilution point.
Count the number of positive tubes in each set.
Statistical method: Determines 95% probability that a bacterial population falls within a certain
range.

4. Direct Microscopic Count:

A specific volume of a bacterial suspension (0.01 ml) is placed on a microscope slide with a
special grid.
Stain is added to visualize bacteria. Cells are counted and multiplied by a factor to obtain
concentration.

Advantages:
No incubation time required.

Disadvantages:
Cannot always distinguish between live and dead bacteria.
Motile bacteria are difficult to count.
Requires a high concentration of bacteria (10 million/ml).

Indirect Methods of Measurement

1. Turbidity:
As bacteria multiply in media, it becomes turbid.
Use a spectrophotometer to determine % transmission or absorbance.
Multiply by a factor to determine concentration.
Advantages:
No incubation time required.
Disadvantages:
Cannot distinguish between live and dead bacteria.
Requires a high concentration of bacteria (10 to 100 million cells/ml).

2. Metabolic Activity:
As bacteria multiply in media, they produce certain products: Carbon dioxide & Acids.
Measure metabolic products and Expensive.

3. Dry Weight: Bacteria or fungi in liquid media are centrifuged. Resulting cell pellet is
weighed.

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UNIT IV
CONTROL OF MICROOGANISMS

Physical Controls
A. Heat

1. Advantages - simple, inexpensive, effective penetrates to kill microbes throughout


the object; best method if material being treated is not damaged by heat.

2. Mode of Action - denatures proteins.

3. Treatments

a. Dry Heat Sterilization - ex. flaming loops, tubes in lab & hot air ovens
(171oC, 1hr., 160oC for 2 hr., 121oC for 16 hrs.); used to sterilize materials that
can withstand high temps. & any materials damaged by moisture.

b. Moist Heat Sterilization - ex. boiling or in autoclaves; effective at a lower


temperature than dry heat & it penetrates more quickly; disadvantages of
boiling - does not kill thermophiles, endospores; autoclave is more effective
than boiling- it uses pressure to raise the temperature above that of boiling
(121oC, 15psi, for 20 min.); used to sterilize liquids and material easily
charred; used in food canning & the lab to sterilize glassware & media.

c. Pasteurization - limits growth, but does not sterilize; used to slow spoilage
of milk & dairy products, wine, beer; advantage: causes minimal damage to
the product; developed by Louis Pasteur; standard treatment: heat to 63 oC for
30 min. or 72oC for 15 sec.

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B. Cold

1. Effect - microbiostatic; does not sterilize; slows down enzymes.

a. Refrigeration - preserves food because it stops the growth of most species


of microbes (slows chemical reactions); most disease-causing microbes are
mesophiles, not psychrophiles; an exception is Listeria spp., which causes
listeriosis (food poisoning).

b. Freezing - kills most bacteria, but survivors can remain alive for long
periods in the frozen state; bacteria cultures can be preserved by rapid
freezing, sometimes with the addition of a compound called DMSO, milk, or
glycerol to protect proteins.

C. Radiation

1. Electromagnetic Spectrum - Radiation is classified by wavelength with ionizing


and UV light radiation at the short-wavelength end, visible light in the middle, & radio
waves at the long-wavelength end. The shorter the wavelength, the greater its energy,
& the more lethal it is. Mode of Action: denatures DNA.

2. Two types of radiation that kill bacteria directly are UV (ultraviolet) Light &
Ionizing Radiation. The effect of both is sterilization.

a. UV Light - bacteria actually have special enzymes that can correct some damage
done by UV light!; in the lab mercury vapor lamps (germicidal lamps) are used;
disadvantages: kills only on surfaces & these wavelengths can also be harmful to humans.

b. Ionizing Radiation - 2 forms; both cause a chain of ionizations by stripping


electrons from atoms, resulting in cell death; disadvantages: technically complex; is being used
to sterilize some produce, much to the public's dismay.

1.) X rays

2.) Gamma rays

D. Membrane Filtration

1. Effect - physically removes cellular organisms (not viruses - they are too small).

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2. Uses - in lab, used with media, antibiotics, & other heat sensitive materials;
filtration is replacing pasteurization in some causes, because filtration causes even
less damage; you may have heard of the new "cold filtered" beers.

E. Drying

1. Defined - the removal of water.

2. Two processes:

a. evaporation involving heat - effect - kills many microbes; rarely used in


lab because the high heat causes chemical changes (denaturation); is used in
food industry.

b. lyophilization [freeze drying] - removes water directly by converting water


from a solid state (ice) to a gaseous state; materials are frozen & placed in a
chamber to which a partial vacuum is applied; avoids the chemical changes
caused by heat drying; effect - stops microbial growth by stopping most
chemical reactions (just like regular freezing) frequently used in the
microbiology lab to preserve perishable materials such as proteins, blood
products, & reference cultures of microbes; used in food industry to make
instant coffee, etc.; disadvantage - expensive.

F. Osmotic Strength

1. Method - high concentrations of salt or sugar.

2. Mode of action - microbes cannot grow if they are deprived of water; also,
crenation or shrinkage can occur (you're placing the microbes in a hypertonic
environment).

3. Disadvantage - once added, solutes (such as salt or sugar) cannot be easily


removed; not used in lab.

II. Chemical Control The effectiveness of a chemical anitmicrobial agent is affected


by time, temperature, pH, and concentration.

A. Testing Germicides 3 ways:

1. Phenol coefficients: Germicides can be tested by comparing their effectiveness


to phenol, a traditional germicide. It was phenol that Lister first used - he called it
carbolic acid. The procedure involves preparing several dilutions of a chemical agent,

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inoculating them with the bacteria Salmonella typhi (a digestive tract pathogen)
or Staphylococcus aureus (a wound pathogen), incubating the tubes, and then checking
for cloudiness in the tubes, indicating growth. The ratio of the effective dilution of the
chemical agent to the dilution of phenol that has the same effect is the phenol
coefficient. A disinfectant with a phenol coefficient of 1.0 has the same effectiveness as
phenol. Less than 1.0 means its less effective. Greater than 1.0 means its more
effective.

2. Paper disc method paper discs are saturated with the chemical agent and placed
on the surface of an agar plate inoculated with a test organism. Clear zones of
inhibition appear around the discs if the chemical agent is effective.

3. Use-dilution test The test microbe is added to dilutions of the chemical


agent. The highest dilution that remains clear after incubation indicates a germicides
effectiveness.

B. Mechanisms of Action

1. Affect Proteins The alteration of protein structure is called denaturation. Denaturation


can be permanent (bacteriocidal) or temporary (normal structure can be restored
bacteriostatic). Mechanisms of denaturation include:

a. Hydrolysis breakdown of a molecule by addition of water

b. Oxidation addition of oxygen or removal of hydrogen

c. Attachment of atoms or chemical groups ex. heavy metals (mercury),


alkylating agents (ex. CH )

2. Affect Membranes

a. membrane proteins denaturation

b. membrane lipids can be dissolved.

3. Affect Cell Wall Formation

4. Affect Nucleic Acid Structure

5. Affect Metabolism

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C. Types of Germicides

1. Surfactants

a. Structure - compounds with hydrophilic & hydrophobic parts.

b. Mode of action - Penetrate oily substances in water & break them apart into
small droplets that become coated with surfactant molecules. The
hydrophobic end of the surfactant stick into the droplets & the hydrophilic end
is attracted to the water. The result is an emulsion, a fine suspension of oily
droplets in water, which can now be rinsed away.

c. Effect of soaps & detergents - wash away microbes, but do not kill them.

d. Wetting agents are surfactants that are often used with other chemical
agents to help the agent penetrate fatty substances. Surfactants are not
germicidal by themselves!

e. Quaternary ammonium salts four organic groups attached to a nitrogen


atom. Effect: kill all classes of cellular microbes & enveloped viruses by
disrupting membranes.

Uses: nontoxic & widely used in the home, industry, labs, &
hospitals. Their effectiveness is decreased in the presence of soap.

2. Phenol & Phenolics

a. Structure - compounds with hydroxyl groups (-OH) attached to a benzene


ring.

Mode of Action - denature cell proteins, disrupt cell membranes.

b. Effect - kill most organisms; action is not impaired by organic materials


(remain active even in the presence of blood, feces, etc.)

c. Examples:

1.) Lysol

2.) Cresol found in creosote; plant derivative used to prevent the


rotting of wooden posts, fences, railroad ties.

3.) Hexachlorophene - chlorinated phenolic; effective as an antiseptic;


once widely used as an ingredient in soaps & lotions; in 1970's was
found to increase risk of brain damage in babies; has now been
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replaced with chlorhexidine in hospitals good agent for surgical
scrubs.

3. Alcohols

a. Structure - compounds with a hydroxyl group (-OH).

b. Mode of Action when mixed with water disrupt lipids in cell membranes
& denature proteins.

c. Ethanol & Isopropanol - widely used as skin antiseptics; a 50 to 70%


solution in water is the most effective concentration (one of the few
exceptions to the rule: increase effectiveness by increasing concentration);
does not sterilize skin because it evaporates quickly and does not penetrate
deeply enough into skin pores.

d. Main disadvantage - do not kill endospores.

4. Halogens

a. Mode of Action - inactivates enzymes by oxidation.

b. Examples

1.) Iodine antiseptic

a.) Tincture iodine in a dilute alcohol solution; one of first skin


antiseptics.

b.) Iodophor mixture of iodine and surfactants;


ex. Betadine and Isodine (used for surgical scrubs and to
prepare skin for surgery)

2.) Chlorine disinfectant; ingredient in household bleach; added to


drinking water and swimming pools; inactivated by the presence of
organic materials.

5. Hydrogen peroxide

a. Mode of Action oxidizing agent (denatures proteins)

b. Uses of H2O2: antiseptic for cleaning wounds, disinfect medical instruments


& soft contact lenses. When H2O2 comes into contact with tissue, it bubbles
producing oxygen gas. This is because all aerobes (incl. eukaryotes) produce
the enzymes catalase & peroxidase which decompose H2O2 into oxygen &
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H2O. H2O2 generally kills microbes before it is destroyed by catalase or
peroxidase. Used to clean deep puncture wounds, because the oxygen
produced kills obligate anaerobes present in the wound (ex. Clostridium).

6. Heavy Metals

a. Mode of Action - heavy metals (mercury, copper, silver) react with the
sulfhydryl groups of proteins (denaturation)

b. Effect - kills many microbes.

c. Examples:

1.) Mercuric chloride - once widely used as an antiseptic; highly toxic;


merthiolate & mercurochrome are used (less toxic); merthiolate is
prepared as a tincture; use - basic first aid kit supplies for disinfecting
skin & mucous membranes.

2.) Silver Nitrate - once applied to eyes of newborns to prevent


gonorrhea; the trend for a while was toward using antibiotics instead,
but the development of antibiotic-resistant strains has necessitated the
use of silver nitrate again.

3.) Selenium sulfide kills fungi, including spores; commonly used to


treat fungal skin infections; included in dandruff shampoos (dandruff
is often caused by a fungus).

7. Alkylating Agents

1. Mode of action - they alkylate (attach short chains of carbon atoms) to


proteins and nucleic acids. It should not be used where they may effect
human cells (these agents are carcinogenic).

2. Formalin - 37% solution of formaldehyde used to preserve tissues & kill all
microbes, including spores; lower concentrations are used to inactivate
microbes for killed vaccines.

3. Glutaraldehyde - used to sterilize surgical instruments if equipment for


heat sterilization is not readily available.

4. Ethylene oxide - gas; advantages: disappears from the object after


treatment; disadvantage: extremely toxic to humans so must be used in a
sealed chamber; kills all bacteria, including endospores; used to sterilize

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materials destroyed by heat (plastic, rubber gloves, animal feed, mattresses,
telephones).

8. Dyes Ex. Crystal violet blocks cell wall synthesis. It effectively inhibits growth of
bacteria in cultures and in skin infections. It can be used to treat yeast infections.

CONDITIONS INFLUENCING THE EFFECTIVENESS OF ANTIMICROBIAL AGENT


ACTIVITY

Destruction of microorganisms and inhibition of microbial growth are not simple matters
because the efficiency of an antimicrobial agent (an agent that kills microorganisms or inhibits
their growth) is affected by at least six factors.

1. Population size. Because an equal fraction of a microbial population is killed during


each interval, a larger population requires a longer time to die than a smaller one.
2. Population composition. The effectiveness of an agent varies greatly with the nature of
the organisms being treated because microorganisms differ markedly in susceptibility. Bacterial
endospores are much more resistant to most antimicrobial agents than are vegetative
forms, and younger cells are usually more readily destroyed than mature organisms. Some
species are able to withstand adverse conditions better than others. Mycobacterium tuberculosis,
which causes tuberculosis, is much more resistant to antimicrobial agents than most other
bacteria.
3. Concentration or intensity of an antimicrobial agent. Often, but not always, the more
concentrated a chemical agent or intense a physical agent, the more rapidly microorganisms are
destroyed. However, agent effectiveness usually is not directly related to concentration or
intensity. Over a short range a small increase in concentration leads to an exponential rise in
effectiveness; beyond a certain point, increases may not raise the killing rate much at all.
Sometimes an agent is more effective at lower concentrations. For example, 70% ethanol is more
effective than 95% ethanol because its activity is enhanced by the presence of water.
4. Duration of exposure. The longer a population is exposed to a microbicidal agent, the
more organisms are killed. To achieve sterilization, an exposure duration sufficient to reduce the
probability of survival to 106 or less should be used.
5. Temperature. An increase in the temperature at which a chemical acts often enhances
its activity. Frequently a lower concentration of disinfectant or sterilizing agent can be used at a
higher temperature.
6. Local environment. The population to be controlled is not isolated but surrounded by
environmental factors that may either offer protection or aid in its destruction. For example,
because heat kills more readily at an acid pH, acid foods and beverages such as fruits and
tomatoes are easier to pasteurize than foods with higher pH like milk. A second important
environmental factor is organic matter that can protect microorganisms against heating and
chemical disinfectants. Biofilms are a good example. The organic matter in a surface biofilm will
protect the biofilms microorganisms; furthermore, the biofilm and its microbes often will be
hard to remove. It may be necessary to clean an object before it is disinfected or sterilized.

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Syringes and medical or dental equipment should be cleaned before sterilization because the
presence of too much organic matter could protect pathogens and increase the risk of infection.
The same care must be taken when pathogens are destroyed during the preparation of drinking
water. When a citys water supply has a high content of organic material, more chlorine must be
added to disinfect it.

The Use of Chemical Agents in Control

Phenolics
Phenol was the first widely used antiseptic and disinfectant.
In 1867 Joseph Lister employed it to reduce the risk of infection during operations.
Today phenol and phenolics (phenolderivatives) such as cresols, xylenols, and
orthophenylphenol are used as disinfectants in laboratories and hospitals. The commercial
disinfectant Lysol is made of a mixture of phenolics.
Phenolics act by denaturing proteins and disrupting cell membranes. They have some real
advantages as disinfectants:
phenolics are tuberculocidal, effective in the presence of organic material, and remain
active on surfaces long after application.
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However, they do have a disagreeable odor and can cause skin irritation.
Hexachlorophene has been one of the most popular antiseptics because it persists
on the skin once applied and reduces skin bacteria for long periods. However, it
can cause brain damage and is now used in hospital nurseries only in response to
a staphylococcal outbreak.

Alcohols

Alcohols are among the most widely used disinfectants and antiseptics.
They are bactericidal and fungicidal but not sporicidal.
Some lipid-containing viruses are also destroyed. The two most popular alcohol
germicides are ethanol and isopropanol, usually used in about 70 to 80% concentration.
They act by denaturing proteins and possibly by dissolving membrane lipids. A 10 to 15
minute soaking is sufficient to disinfect thermometers and small instruments.

Halogens

A halogen is any of the five elements (fluorine, chlorine, bromine, iodine, and astatine) in
group VIIA of the periodic table. They exist as diatomic molecules in the free state and form
saltlike compoundswith sodium and most other metals. The halogens iodine and chlorine are
important antimicrobial agents. Iodine is used as a skin antiseptic and kills by oxidizing cell
constituents and iodinating cell proteins. At higher concentrations, it may even kill some spores.
Iodine often has been applied as tincture of iodine, 2% or more iodine in a water-ethanol solution
of potassium iodide. Although it is an effective antiseptic, the skin may be damaged, a stain is
left, and iodine allergies can result.

More recently iodine has been complexed with an organic carrier to form an iodophor.
Iodophors are water soluble, stable, and nonstaining, and release iodine slowly to minimize
skin burns and irritation. They are used in hospitals for preoperative skin degerming and in
hospitals and laboratories for disinfecting. Some popular brands are Wescodyne for skin and
laboratory disinfection and Betadine for wounds. Chlorine is the usual disinfectant for municipal
water supplies and swimming pools and is also employed in the dairy and food industries.
It may be applied as chlorine gas, sodium hypochlorite, or calcium hypochlorite, all of which
yield hypochlorous acid (HClO) and then atomic oxygen. The result is oxidation of cellular
materials and destruction of vegetative bacteria and fungi, although not spores.

Death of almost all microorganisms usually occurs within 30 minutes. Since organic
material interferes with chlorine action by reacting with chlorine and its products, an excess of
chlorine is added to ensure microbial destruction. One potential problem is that chlorine reacts
with organic compounds to form carcinogenic trihalomethanes, which must be monitored in
drinking water. Ozone sometimes has been used successfully as an alternative to chlorination in
Europe and Canada. Chlorine is also an excellent disinfectant for individual use because it is
effective, inexpensive, and easy to employ. Small quantities of drinking water can be disinfected
with halazone tablets.

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Halazone (parasulfone dichloramidobenzoic acid) slowly releases chloride when added to
water and disinfects it in about a half hour. It is frequently used by campers lacking access to
uncontaminated drinking water. Chlorine solutions make very effective laboratory and household
disinfectants. An excellent disinfectant-detergent combination can be prepared if a 1/100 dilution
of household bleach (e.g., 1.3 fl oz of Clorox or Purex bleach in 1 gal or 10 ml/liter) is combined
with sufficient nonionic detergent (about 1 oz/gal or 7.8 ml/liter) to give a 0.8% detergent
concentration. This mixture will remove both dirt and bacteria.

Heavy Metals

The ions of heavy metals such as mercury, silver, arsenic, zinc, and copper are used as
germicides. More recently these have been superseded by other less toxic and more effective
germicides (many heavy metals are more bacteriostatic than bactericidal). There are a few
exceptions. A 1% solution of silver nitrate is often added to the eyes of infants to prevent
ophthalmic gonorrhea (in many hospitals, erythromycin is used instead of silver nitrate because it
is effective against Chlamydia as well as Neisseria). Silver sulfadiazine is used on burns. Copper
sulfate is an effective algicide in lakes and swimming pools. Heavy metals combine with
proteins, often with their sulfhydryl groups, and inactivate them. They may also precipitate cell
proteins.

Quaternary Ammonium Compounds

Detergents are organic molecules that serve as wetting agents and emulsifiers because
they have both polar hydrophilic and nonpolar hydrophobic ends. Due to their amphipathic
nature detergents solubilize otherwise insoluble residues and are very effective cleansing agents.
They are different than soaps, which are derived from fats. Although anionic detergents have
some antimicrobial properties, only cationic detergents are effective disinfectants.

The most popular of these disinfectants are quaternary ammonium compounds


characterized by a positively charged quaternary nitrogen and a long hydrophobic aliphatic
chain. They disrupt microbial membranes and may also denature proteins.

Cationic detergents like benzalkonium chloride and cetylpyridinium chloride kill most
bacteria but not M. tuberculosis or endospores. They do have the advantages of being stable,
nontoxic, and bland but they are inactivated by hard water and soap. Cationic detergents are
often used as disinfectants for food utensils and small instruments and as skin antiseptics.
Several brands are on the market. Zephiran contains benzalkonium chloride and Ceepryn,
cetylpyridinium chloride.

Aldehydes

Both of the commonly used aldehydes, formaldehyde and glutaraldehyde, are highly
reactive molecules that combine with nucleic acids and proteins and inactivate them, probably by
crosslinking and alkylating molecules. They are sporicidal and can be used as chemical sterilants.
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Formaldehyde is usually dissolved in water or alcohol before use. A 2% buffered solution of
glutaraldehyde is an effective disinfectant. It is less irritating than formaldehyde and is used to
disinfect hospital and laboratory equipment. Glutaraldehyde usually disinfects objects within
about 10 minutes but may require as long as 12 hours to destroy all spores.

Sterilizing Gases

Many heat-sensitive items such as disposable plastic petri dishes and syringes, heart-lung
machine components, sutures, and catheters are now sterilized with ethylene oxide gas. Ethylene
oxide (EtO) is both microbicidal and sporicidal and kills by combining with cell proteins. It is a
particularly effective sterilizing agent because it rapidly penetrates packing materials, even
plastic wraps.

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