HISTORY OF MICROBIOLOGY

CONTRIBUTORS IN MICROBIOLOGY:
 Antonie van Leeuwenhoek
 Louis Pasteur
 Robert Koch
 Joseph Lister
 Edward Jenner

ANTONIE VAN LEEUWENHOEK(1676):

-He earned his living as a Draper and haberdasher ( a dealer in men's clothes and accessories).

-Used magnifying glasses to check the quality of clothes.

-He built simple microscope with single biconvex lens with magnification power of 200 times to examine blood,
yeasts, insects and other tiny objects

-Made observation on the plaque between his own teeth, his wife, daughter and two old man who had never
cleaned their teeth in life. Looking at these samples with his microscope, Leeuwenhoek reported the small
organisms as “animalcules”.

-He described many animalcules including three major morphological forms of bacteria (rod, sphere, and spiral),
various free living and parasitic protozoa from human and animal feces except for viruses.

LOUIS PASTEUR(1822-1895):

-Known as ‘Father of Microbiology’

-Professor of Chemistry in France

-Proposed the principle of fermentation for preservation of food

-Introduced the sterilization techniques and developed steam sterilizer, hot air oven and autoclave.

-Described the method of pasteurization of milk.

-Contributed for vaccine development against several diseases such as anthrax, fowl cholera and rabies

-Postulated ‘Germ Theory of Disease’

-Liquid Media concept: used nutrient broth to grow microorganisms.

-Founder of the Pasteur Institute, Paris.

ROBERT KOCH(1843-1910):

-German general practitioner

-Introduced solid media (Using agar as solidifying agents)

-Introduced method for isolation of bacteria in pure culture

-Described hanging drop method for testing motility

-Discovered bacteria such as the anthrax bacilli, tubercle bacilli and cholera bacilli.

-Introduced staining techniques by using aniline dye

-Koch’s phenomenon: Guinea pigs already infected with tubercle bacillus developed a hypersensitivity reaction
when injected with tubercle bacilli or its protein.

Koch’s postulates:

1)The microorganism should be constantly associated with the lesions of the disease.

2) It should be possible to isolate the organism in pure culture from the lesions of the disease.

3) The same disease must result when the isolated microorganism is inoculated into a suitable laboratory animal.

4)It should be possible to re-isolate the organism in pure culture from the lesions produced in the experimental
animal.

5) An additional fifth criterion was introduced stating that antibody to the causative organism should be
demonstrable in the patient’s serum.
Exceptions of Koch’s Postulates:

-Mycobacterium leprae and Treponema pallidum: They cannot be grown in vitro; however, they can be maintained
in experimental animals

-Neisseria gonorrhoeae: There is no animal model; however, it can be grown in vitro.

JOSEPH LISTER(1867):

-Father of Antiseptic surgery.

-Observed that postoperative infections were greatly reduced by using disinfectants such as diluted carbolic acid
during surgery to sterilize the instruments and to clean the wounds.

EDWARD JENNER(1796):

-Developed first vaccine of the world, the small pox vaccine

-Used cowpox virus (Variolae vaccinae) to immunize children against smallpox from which the term “vaccine” has
been derived.

-During his experiment he inoculated 8 years old boy with cowpox and after 8 weeks later the same boy was
inoculated with the pus from small pox lesion. The boy showed no effect.

OTHER IMPORTANT CONTRIBUTORS:

-Hans Christian Gram (in 1884): developed “Gram staining” method

-Ernst Ruska: founder of electron microscope (1931)

-Alexander Fleming (in 1929): Discovered penicillin

-Goodpasture: He described the viral culture technique in chick embryo

-Walter Gillbert and Frederick Sanger: Developed the method of DNA sequencing (in 1977)

-Karry B Mullis: Discovered polymerase chain reaction (PCR) and was awarded Noble prize in 1993

-PAUL EHRLICH:

*Father of chemotherapy

*First to report acid-fast nature of tubercle bacillus

*Developed techniques to stain tissues and blood cells

*Proposed a toxin antitoxin interaction called Ehrlich phenomenon and also introduced methods of standardizing
toxin and anti toxin.
*Proposed ‘Side chain theory’ for antibody production

*Discovered Salvarsan, an arsenical compound (also called “magic bullet”) as first effective treatment for Syphilis,
initiating and also naming concept of chemotherapy

*Founder and first director of the Paul Ehrlich Institute, Germany.

SPONTANEOUS GENERATION VS GERM THEORY OF DISEASE

John Needham, an Irish priest in 1745, published the experiments purposing

the spontaneous generation..He boiled a “broth” or “soup” which should kill
any microbes. Left it sitting out. It spoiled. He concluded that microbes arose
spontaneously.

-Lazzaro Spallanzani, an Italian naturalist in 1775 attempted to refuse Needham’s work by performing extensive
experiment.

-He boiled “broth” in glass containers and melted the glass to closed.

-Nothing grew and disproved the spontaneous generation.

-Pasteur rejected the theory of Spontaneous Generation of microorganisms. He gave germ theory of disease.

-Hypothesis: Microbes come from cells of organisms on dust particles in the air, not the air itself.

-Pasteur put broth into several special S-shaped flasks.

-Each flask was boiled and placed at various locations.

HANGING DROP TEST:

-Hanging drop preparation is one of the commonest and easiest method to demonstrate bacterial motility.

-After the drop is prepared on a cover slip and kept over the cavity slide, the edge of the drop is focused. The edge
is focused because:

-Better contrast at the edge: Due to difference in the refractive index of the drop and the cover slip.

-As the drop hangs, it thins towards the edge, containing less number of bacteria and less overcrowding; hence
motility can be clearly appreciated.

-Live aerobic bacteria come towards the edge to get more oxygen for respiration.

Types of Bacterial Motility: 3 types of movements: Active, Passive and

Brownian movement; where active movement is the true motility
exhibited by motile bacteria which needs to be differentiated from
passive and Brownian movement.

*Active movement: Organism move in different directions and change

their positions in the flied.

*Passive movement: Drifting of the organisms in the same direction

along the current of the fluid.

*Brownian movement: It is an oscillatory movement about a nearly fixed

point by all small bodies suspended in fluid.
DEVELOPMENT OF IMMUNOLOGY

-Edward Jenner(1749-1823),an English physician introduced and invented vaccine against smallpox.

- During his experiment he inoculated 8 years old boy with cowpox and after 8 weeks later the same boy was
inoculated with the pus from small pox lesion. The boy showed no effect.

-Louis Pasteur in 1880, concluded a theory that bacterial virulence could be attenuated by culture in vitro & can be
used as vaccine. On that basis he Prepared live vaccine against chicken cholera , Anthrax & Rabies.

-Development of vaccine against hydrophobia by Pasteur. He took an extract of spinal cord of rabid rabbit,
suspended in sterile dry air for several days and gave 13 inoculation to 9 year old boy Joseph Meister , who was
bitten by rabid dog on hands, legs and thighs.

-Jonas Salk in 1953, developed Salk killed polio vacci

-Sabin in 1961, used live oral attenuated polio vaccine for active immunization all infants. ne for Poliomyelitis.

-Concept of cloning of cell arise where George Kohler & Cesar Milstein in 1975 1st generate “Monoclonal
Antibodies” which is used for commercial production of different antibodies.

- Hepatitis B Vaccine developed in 1986 by Genetic Recombinant.

HISTORICAL DEVELOPMENT IN VIROLOGY

-Viral diseases such as smallpox, rabies, polio and others had been known from centuries. The exact nature of
infectious agent for infections did not become apparent until 1890s.

-Charles Chamberland in 1884, made a porcelain filter designed for sterilizing drinking water that gave concept
that the invisible infective material less than the known organisms could cause disease in plants and animals.

- Ivanowski in 1892, a Russian scientist was probably the 1st to explained mosaic disease in tobacco plant, is by
such filterable microbes that can’t be visible by light microscope or smaller than bacteria.

-Beijerinck in 1898, confirmed the Tobacco Mosaic Disease. Viruses are particulate smaller than bacteria and
unable to multiply outside the living cells. He also recognized viral dependence on living cells for reproduction in
1899.

-Loeffler and Frosch in 1898, demonstrated a filterable agent responsible for foot & mouth disease in cattle.

-Buist in 1887, visualized the largest vaccinia virus after staining it with aniline methyl voilet (100-500 nm Size)

-Walter Read in 1902, explained the first human disease proved to have a viral etiology was yellow fever in Cuba.

-Ellerman &Bang in 1908 gave the concept of viral infection could lead to malignancy

-Landsteinner& Popper in 1909, demonstrated causative agent of Poliomyelitis.

-Rous in 1911, discovered a virus that produce malignant tumors in chickens.

-Twort & d’Herelle, independently discovered the viruses that multiply in bacteria, i.e. Bacteriophage in 1915.

-Goodpasture in 1930, discovered the cultivation of viruses on chick embryo.

-Ruska in 1934: By the end of 19th century virus had been identified in terms of infectivity & filterability which
became more clear only after the invention of Electron Microscope. Ruska in 1940, obtained first picture of a virus
by using electron microscope.

-George Hirst in 1941, described Phenomenon of hemagglutination, that has provided a rapid accurate &
quantitative methods for counting virus.

HISTORY OF PARASITES

-The term protozoa was 1st used by a German Goldfuss in 1817.

-Alphonse Laveran, discovered malaria parasites in the blood of patient suffering from malaria.

-Ronald Ross, born in Almora, India first demonstrate the different morphological forms of malaria parasite in his
own lab in Calcutta. For his brilliant work he got Nobel prize in 1902.

-William Leishman & Charles Donovan, independently discovered Leishmania donovani in 1903.

-Linnaeus in 1758, is credited with the discovery of many helminthes.

HISTORY OF FUNGI:

-Term mycoses derived from mykes,a Greek word mushroom.

-Raymond Jacques Sabouraud (1864-1936), published his work on dermatophytes.

- He introduced Sabouraud’s Dextrose agar.

-Father of Mycology.

-In 1842 John Gill, described the disease mycetoma for the 1st time in Madurai, India.

-Samuel Taylor Darling on 7th Dec 1905, reported Histoplasma capsulatum while performing an autopsy on patient
in Panama.

CHARACTERISTICS OF MICROORGANISMS
-Microorganisms are grouped under both prokaryotes and eukaryotes.

-Bacteria and blue green algae are placed under prokaryotes. They have a primitive nucleus, and other properties
of a prokaryotic cell.

-Whereas other algae, fungi and parasites (protozoa and helminthes) belong to eukaryotes; having a well-defined
nucleus and various eukaryotic cellular organelles.

-Viruses are considered neither prokaryotes nor eukaryotes because they lack the characteristics of living things,
except the ability to replicate.

-Prions consist of abnormal infectious protein molecules without nucleic acid.

-They are highly resistant to physical and chemical agents. Prions produce slow infections in humans having long
incubation period (in years) called prion disease (a neurodegenerative condition of brain).
* Svedberg unit (S) is used as a measuring unit for sedimentation speed in ultracentrifuge which is related to size,
shape and density of the particle.

Size of the microorganisms:

-Microorganisms are extremely small. The size of the bacteria is expressed in micrometers (1µm = 10^-3 mm)
whereas viruses are measured in nanometers (1nm = 10^-3µm).

-Most of the bacteria of medical importance generally measure 0.2-1.5 µm in diameter and about 3-5 µm in length,
while the majority of the human pathogenic viruses range 20-300 nm in diameter.
- Because of the small size, microorganisms cannot be seen distinctly with the unaided eye but need a microscope
for their visualization. Most bacteria can be observed by light microscope whereas viruses need an electron
microscope.

CLASSIFICATION OF BACTERIA
Phenotypic classification:

 Morphological
 Anatomical
 Staining
 Cultural characteristics
 Nutrition
 Environmental factors
 Biochemical reactions
 Antigenic structure

Genotypic classification:

 DNA-DNA hybridization
 G+C content

A)Bacteria can be classified into six major groups on MORPHOLOGICAL BASIS:

1. TRUE BACTERIA

2.ACTINOMYCETES (actin- ray, mykes-fungus)

3. Spirochaetes

4. Mycoplasmas

5. Rickettsiae

6. Chlamydiae

1. TRUE BACTERIA

I) Cocci – These are spherical or oval cells. On the basis of arrangement of individual organisms they can be
described as:

 Monococci (Cocci in singles) – Monococcus spp.

 Diplococci (Cocci in pairs) – Streptococcus
pneumoniae
 Staphylococci (Cocci in grape-like clusters) –
Staphylococcus aureus
 Streptococci (Cocci in chains) – Streptococcus
pyogenes
 Tetrad (Cocci in group of four) - Micrococcus spp.
 Sarcina (Cocci in group of eight)
II)Bacilli – These are rod-shaped
bacteria.

On the basis of arrangement of

organisms, they can be described as

 Diplobacilli
 Streptobacilli
 Pallisades
 Chinese-letterform
 Coccobacilli
 Comma-shaped

2.Actinomycetes (actin- ray, mykes-fungus) :These are rigid organisms like true
bacteria but they resemble fungi in that they exhibit branching and tend to form
filaments. They are termed such because of their resemblance to sun rays when seen
in tissue sections.

3. Spirochaetes : These are relatively longer, slender, non-branched microorganisms of spiral shape
having several coils.

4. Mycoplasmas : These bacteria lack in rigid cell wall (cell wall lacking) and are highly pleomorphic and of
indefinite shape. They occur in round or oval bodies and in interlacing filaments.

5. Rickettsiae and Chlamydiae: These are very small, obligate parasites, and at one time were considered closely
related to the viruses. Now, these are regarded as bacteria.

B) BASED ON ANATOMICAL FEATURES:

1)Capsule:

o Some bacteria possess a layer of amorphous viscid material lying outside the
cell wall called glycocalyx. When the glycocalyx layer is well organized and not
easily washed off, it is called capsule.
o Capsulate– Streptococcus pneumoniae
o Non-capsulate – Viridans streptococci

Demonstration of Capsule:

o Negative staining by India ink and nigrosin stain: Capsule appears as a clear refractile halo around the
bacteria; where as both the bacteria and the background appear black.
o M'Faydean capsule stain: It is used for demonstration of capsule of Bacillus anthracis by using polychrome
methylene blue stain.
o Serological test: Capsular material is antigenic and can be demonstrated by mixing it with a specific
anticapsular serum.
o Quellung reaction: Capsular serotypes of Streptococcus pneumoniae can be detected by adding antisera
mixed with methylene blue. Capsule becomes swollen, refractile and delineated.
o Capsular antigen: It can be detected in the sample (e.g. CSF) by latex agglutination test by using specific
anricapsular antibodies coated on latex particles. This is available for pneumococcus, Cryptococcus,
Haemophilus influenzae and meningococcus.
2) Flagella (organs of locomotion):

Flagella are thread-like appendages, protruding from the cell wall, that confer motility to the bacteria.

o Monotrichous: Single polar flagellum

o Lophotrichous: Multiple polar flagella
o Amphitrichous: Single flagellum at both the ends
o Peritrichous: flagella distributed over the entire cell surface

Detection for flagella:

o Tannic acid staining (Leifson’s method and Ryu’s method): uses tannic
acid as a mordant, to coat and thicken the flagellum so it can be
observed by light microscopy.
o Electron microscopy
o Hanging drop method
o Semisolid medium
o Dark ground microscopy and phase contrast microscopy.

3) Spore:

Spores are highly resistant resting (or dormam) stage of die bacteria formed in unfavorable environmemal
conditions as a result of the depletion of exogenous nutrients. Bacterial spores formed within the parent cell, are
called endospores.

o Spore-forming – Bacillus spp.

o Non-sporing – Escherichia coli

Demonstration of spores:

o Gram Staining: Spores appear as unstained refractile

bodies within the cells.
o Modified Ziehl-Neelsen staining: Spores are weakly
acid-fast and appear red color when ZN staining is
performed using 0.25% sulfuric acid as decolorizer.
o Special techniques for endospore staining include the
Schaeffer-Fulton stain and the Moeller stain.

C) BASED ON STAINING REACTION:

1)GRAM’S STAIN

o Gram-positive cocci – Staphylococcus aureus

o Gram-negative cocci – Neisseria gonorrhoeae
o Gram-positive rods – Clostridium spp.
o Gram-negative rods – E. coli
Principle of gram staining:

1)pH theory:

Cytoplasm of gram-positive bacteria is more acidic, hence, can retain the basic dye (e.g. crystal violet) for longer
time. Iodine serves as mordant, i.e. it combines with die primary stain to form a dye-iodine complex which gets
retained inside the cell.

2) Cell wall theory:

Gram-positive cell wall has a thick peptidoglycan layer (50-100 layers thick), which are tightly cross linked to each
other.

The peptidoglycan itself is not stained; instead, it seems to act as a permeability barrier preventing loss of crystal
violet.

Gram-negative cell wall is more permeable thus allowing the outflow of crystal violet easily. This is due to:

 The thin peptidoglycan layer in gram-negative cell wall which is not tightly cross linked.
 Presence of lipopolysaccharide layer in the cell wall of gram-negative bacteria, which gets disrupted easily
by the action of acetone or alcohol; thus allowing the primary stain to come out of the cytoplasm.

After mordanting with Gram's iodine, bigger dye-iodine complexes are formed in the cytoplasm. Following
decolorization, as more lipid content in gram-negative bacterial cell wall gets dissolved leading to formation of
larger pores through which the dye-iodine complexes escape. Due to less lipid in gram-positive bacterial cell wall
smaller pores are formed which do not allow the dye-iodine complexes to escape.

3) ACID FAST STAIN

 Acid-fast bacteria–Mycobacterium tuberculosis

 Non-acid-fast bacteria– Staphylococcus aureus
 Principle: Acidfastness is due to presence of mycolic acid in the cell wall.

D) BASED ON CULTURAL CHARACTERISTICS:

1)Extra growth factors requirements:

 Fastidious – Hemophilus influenzae

 Non-fastidious – Escherichia coli

2) Hemolysis on Sheep Blood Agar

 Alpha-hemolysis – Streptococcus pneumoniae

 Beta-hemolysis – Streptococcus pyogenes

3) Utilization of carbohydrates

 Oxidative - Micrococcus
 Fermentative – Escherichia coli

4) Growth rate

 Rapid growers– Vibrio cholerae

 Slow growers – Mycobacterium tuberculosis
5) Pigment production

 Pigment producer – Staphylococcus aureus

 Pigment non-producer – Escherichia coli

E) BASED ON NUTRITION:

1)Autotrophs: Autotrophic bacteria synthesize their own food. They derive energy from light or chemical reactions.
They utilize simple inorganic compounds like carbon dioxide, water, hydrogen sulfide, etc. and convert them into
organic compounds like carbohydrates, proteins, etc. to supplement their energy requirements.

2)Heterotrophs: Heterotrophs cannot synthesize their own food and rely on other organisms for nutrition.

F) BASED ON ENVIRONMENTAL FACTORS:

1) Temperature :

• Psychrophiles (15-200C) – Pseudomonas fluorescens

• Mesophiles (20-400C) – Escherichia coli, Salmonella enterica, Staphylococcus aureus
• Thermophiles (50-600C)- Bacillus stearothermophilus
• Extremely thermophiles (as high as 2500C)

2)Oxygen dependence:

• Aerobe (grow in ambient temperature, which contains 21% O2 and a small amount of CO2, 0.03%)
• Obligate aerobes – Strictly require O2 for their growth (Pseudomonas aeruginosa)
• Microaerophilic (grow under reduced O2, 5-10% and increased CO2, 8-10%)- Campylobacter jejuni,
Helicobacter pylori
• Facultative anaerobe (capable of growing either in presence or absence of O2)- E. coli
• Obligate anaerobe – grow in the absence of O2 e.g.,Clostridium spp.
• Capnophilic (require increased concentration of CO2, i.e., 5-10%) – H. influenzae, N. gonorrhoeae
• Aerotolerant: does not require oxygen for growth but can tolerate its presence.

3) pH :

• Acidophiles (Lactobacillus acidophilus)

• Alkaliphiles (Vibrio)
• Neutralophiles (pH 6-8)
• Majority of the medically important bacteria grow best at neutral or slightly alkaline reaction (pH 7.2-7.6)

4)Salt concentration :

• Halophiles: The halophiles, named after the Greek word for "salt-loving", that thrive in high salt
concentrations. Non-halophiles

5)Atmospheric pressure:

G)OTHER WAYS OF CLASSIFICATION:

• Motile/Non-motile
• Pathogenic/Non-pathogenic
• Sensitive/Resistant (to particular antibiotic/ chemicals)
• Lactose fermenter/Lactose non-fermenter
 BACTERIAL TAXONOMY
-Taxonomy is the science dealing with four major components: characterization, classification, identification and
nomenclature of organisms.

-Taxonomy provides basic understanding about the components of biodiversity which is necessary for effective
decision-making about conservation and sustainable use.

-Taxonomy's first father was the philosopher Aristotle(384-322 BC), also known as the “father of science.“

-He was the first to attempt to classify all the animals according to their similarities: animals with blood and
animals without blood (Invertebrates ). He further divided the animals with blood into live-bearing and egg
bearing.

-Aristotle's view of life was hierarchical. He assumed that creatures could be grouped in order from lowest to
highest, with the human species being the highest.

HISTORY: FATHER OF TAXONOMY

-A Swedish naturalist named Carolus Linnaeus is considered the 'Father of Taxonomy‘ since 1700s. His two most
important contributions to taxonomy were:

•A hierarchical classification system

•The system of binomial nomenclature

-He proposed that there were three broad groups, called kingdoms, into which the whole of nature could fit. These
kingdoms were animals, plants, and minerals.

COMPONENTS OF TAXONOMY:

A. Characterization:

- In order to identify and classify organisms, their characteristics in groups should be learnt. In bacteriology, we
study the characteristics of a culture, strictly pure (axenic) culture.

-The majority characteristics (of bacteria) fall in following categories:

i. Morphological characteristics: Cell shape, size, structure, cell arrangement, occurrence of special
structure, developmental forms, staining reactions, motility, flagellar arrangement etc.
ii. Chemical composition: When cellular components are subject to chemical analysis, a characteristic
chemical composition may be detected in a particular micro-organism. Eg detection of LPS in cell wall of
Gram Negative bacteria but not in Gram Positive bacteria, Teichoic acid in cell wall of Gram Positive
bacteria but absent in cell wall of Gram Negative bacteria. This is of greater importance in performing
viral classification.
iii. Cultural characteristics: Based on nutritional requirements, physical conditions, pattern of growth, colony
morphology study.
iv. Metabolic characters: The way in which cells obtain and use their energy, carry out chemical reactions,
and regulate these reactions. Eg some bacteria obtain energy from sunlight while others by oxidizing/
fermentating organic or inorganic compounds.
 v. Antigenic characteristics: Some antigens which are distinctive for particular bacteria is detected by their
antibodies.
vi. Pathogenicity: It is also another quality in characterizing bacteria.
vii. Ecological characteristics: The habitat is important in characterizing bacteria. And similarly the distribution
and interactions among species in an environment.

B. Classification:

-The characters are then weighed, greater emphasis being given to some than others, or they may be given equal
importance depending on the method of classification

-The first attempt on bacterial classification was made by Mueller (1786) and then by Ehrenberg (1838) when very
little was known about bacteria.

TWO KINGDOM CLASSIFICATION

The two kingdom classification system was given by Carlous Linaaeus in 1758. He then divided each kingdom into
classes and later grouped the classes into phyla for animals and divisions for plants.

THREE KINGDOM CLASSIFICATION

-Then in the 1860s, the German investigator Ernst Haeckel proposed a three-kingdom system of classification.
Haeckel's three kingdoms were Animalia, Plantae, and Protista. Members of the kingdom Protista were believed to
have evolved from common ancestors of plants and animals with relatively little change. The kingdom Protista
includes the protozoa, fungi, bacteria, and other microorganisms.

-Haeckel in 1866 classified all unicellular organisms into a third kingdom Protista.
 FOUR KINGDOM CLASSIFICATION

The development of optic and electronic microscopy showed important differences in cells (Protista) , mainly
according to the presence or absence of distinct nucleus, leading Édouard Chatton to distinguish organisms in
prokaryotes (without a distinct nucleus) and eukaryotes (with a distinct nucleus) in a paper from 1925.

Based on it, Copeland proposed a four-kingdom system, moving prokaryotic organisms, bacteria and “blue-green
algae”, into the kingdom Monera.

FIVE SYSTEM CLASSIFICATION

The position of fungi was not well established, oscillating between

kingdoms Protista and Plantae. So, in 1969, Robert Whittaker proposed a
fifth kingdom to include them, the called Kingdom Fungi.

BACTERIAL CLASSIFICATION:

-Bacterial classification is difficult unlike the classification of other higher organisms. This is because of the
following reasons:

 Infinite variety
 Natural capacity for variation and adaptation
 Absence of sexual reproduction, the use of inbreeding as a test for differentiation is prevented
 Absence of fossil remains
 Insufficient morphological differences

-Therefore, no standard and rigid classification of bacteria is universally accepted, although Bergey’s Manual of
Systematic Bacteriology is widely used as an authoritative source

-In bacterial taxonomy, the taxonomical units (taxa) are initially constructed from strains. A strain is made up of all
the descendants of a pure culture; it is usually a succession of culture derived from an initial colony.

-The basic taxonomic group (taxon) is the species which is a collection of strains having similar characteristics as
follows:

a. Structural traits of shape, size, mode of movement, resting stage, gram stain, macroscopic growth.
b. Biochemical and nutritional traits, end products
c. Physiologic traits relative to oxygen, temperature, pH, and response to antibacterial growth.
d. Ecological traits
e. DNA based composition, homology, and genetic traits.
-Just as bacterial species is composed of a collection of similar strains, bacterial genus is composed of similar
species.

-The taxonomic groups of higher ranks than genus are listed:

 Family: A group of similar genera

 Order: A group of similar families
 Class: A group of similar orders
 Division: A group of similar classes
 Kingdom: A group of similar divisions
A full taxonomical
classification of S.aureus

METHODS FOR BACTERIAL CLASSIFICATION:

There are three methods of bacterial classification:

1)Phylogenetic classification: It implies an evolutionary arrangement of species. Here, some characteristics are
given special weightage. Depending upon characteristics so chosen, the classification may give different patterns.
For example, the intestinal Gram Negative Bacilli have been traditionally classified depending on whether they
ferment lactose or not. This method is convenient, however, the characters selected may not always be valid.

2)Numerical/ Adansonian classification (taxometrics): Phylogenetic classification for bacteria based on only a few
arbitrarily weighed characteristics is difficult and may be less valid. Since 1950’s, classification of many species
have been revised in the form of computerized numerical comparisons of large number of phenotypic traits.

This system was devised by Michel Adanson in 18th century. It gives equal weight to all the traits for comparison.
If enough (50 to several hundred) phenotypic traits are examined, occurrence of a few variable traits (those due to
extrachromosomal elements) are minimized.

Numerical taxonomy reveals similarities, resemblances and differences on the phenotypic characters without
reference to the genetic origin.

The numeric al taxonomical comparisons may be expressed as a Jaccard Similarity Coefficient (Sj) which includes
only positive features or as a Simple matching Coefficient (Ssm) which includes both positive and negative
features.

Other coefficients may be used for other special purposes. These coefficients express the percentage of traits held
in common between organisms. Designations of taxa are then made on the basis of the ranges of degree of
similarity.

 Similarity coefficient Sj
= a/(a+b+c)
 Simple matching coefficient Ssm
= (a+d)/(a+b+c+d)

where, a= no. of features present in both strains

 b= no. of features present in first strain only

c= no. of features present in second strain only

d= no. of features absent in both strains

3)Genetic classification:

a. DNA composition: there is a very wide range in the G+C component of DNA of bacteria, varying from 25 –
80% in the different genera. However, for any one species, the G+C content is relatively fixed or falls
within a very narrow range, this provides a basis for classification.
b. DNA homology: this is another approach in which organisms are arranged on the basis of homology of
their DNA base sequence. The degree of similarity can used at species level of classification.
c. rRNA homology and rRNA oligonucleotide cataloging: the nucleotide sequence of DNA changes more
rapidly than rRNA as rRNA cistrons are highly conserved. The degree of similarity can therefore be used as
a measure of relatedness between organism but at a level beyond that of species (at the level of genus ,
family order etc.

4) Intraspecies classification:

- Used in the classification of bacterial species for the purposes of diagnosis and epidemiological studies.

-This classification may be based on biotyping, serotyping, phagetyping, bacteriocin typing etc.

C. IDENTIFICATION:

-A proper classification is required for identification and nomenclature. The characteristics chosen should be such
that they are easy to determine, such as shape, staining reactions, sugar fermentations etc.

-DNA homology experiments, though useful for classification, would be unsatisfactory for identification of an
organism because of complexity of the procedure.

D. NOMENCLATURE:

-Each species of well identified microorganism has only one officially accepted name, by international agreement.
This system provides precise communication.

-Example: if an organism is called Escherichia coli in one country and Coprobacterium intestinale in another, it
creates chaos

The name of species is merely a convenient label, not necessarily even descriptive, although some names are –
e.g., Micrococcus luteus means yellow berry in Latin. Proteus vulgaris is Latin word for common organism of many
shapes.

Some organisms are named after persons, e.g., Escherichia coli- the organism of colon, named after Theodor
Escherich, a German bacteriologist. Names may be non-sensible as well.

Bacterial naming also follows binomial system.

Bacteria are sometimes referred by colloquial names e.g., Typhoid bacillus, colon bacillus etc.

Any new name should be published in the Bergey’s Manual of Systematic Bacteriology to achieve official
recognition.
 BACTERIAL GROWTH CURVE:

BACTERIAL CELL DIVISION:

o Divide by binary fission

o Cell division commences when bacterial cell reaches a
critical mass in its cellular constituents.
o The nuclear division precedes cytoplasmic division.

GENERATION TIME:

Generation time is the time required for a bacterium to give rise to two daughter cells under optimum condition.

o Escherichia coli: 20 mins

o Mycobacterium tuberculosis: 10-15 hrs
o Mycobacterium leprae: 12-13 days

BACTERIAL COUNT:

o Total count: total no. of bacteria (live or dead) in the specimen. This is done by counting bacteria under
microscope using counting chamber
o Viable count: no. of living (viable) cells in the specimen. This is done by pour plate method or by spreading
method.

BACTERIAL GROWTH CURVE:

o When a bacterium is inoculated into a suitable liquid culture medium and incubated, its growth follows a
definite course.
o When bacterial count of such culture is determined at different intervals and plotted in relation to time, a
bacterial growth curve is obtained.
Bacterial Growth Curve consists of four phases:

a. Lag phase
b. Log phase
c. Stationary phase
d. Decline phase

a.Lag phase:

• Period between inoculation and beginning of multiplication of bacteria

• Enzymes and metabolites build up
• Bacterial cell increase in size
• Bacteria reaches their maximum size at the end of lag phase

b. Log phase:

• Bacteria divides exponentially

• Bacteria become smaller in size
• Biochemically active
• Uniformly stained

c. Stationary phase:

• After log phase, bacterial growth ceases almost completely due to exhaustion of nutrients, accumulation
of toxic products and autolytic enzymes.
• The no. of progeny cells formed is just enough to replace the no. of cells that die.
• Sporulation occurs in this phase
• More storage granules are formed
• Bacteria produce exotoxins, antibiotics and bacteriocins

d. Decline phase:

• Gradually, the bacteria stop diving completely; while the cell death continues due to exhaustion of
nutrients, and accumulation of toxic products.
• There is decline in viable count and not in total count
ENVIRONMENTAL FACTORS AFFECTING BACTERIAL
GROWTH:
 Environmental factors can significantly effect microbial cells
 Microbes are affected by changes in physical and chemical nature of their environment and often
adapting to severe environmental conditions (extremophiles)
 The main environmental influences are:
a. Temperature :
Microbes have distinct cardinal temperatures:
• Minimum
• Optimum
• Maximum

Based on temperature range the microbes can grow in:

o Psychrophiles: 15 – 20 C
o Mesophiles: 20 – 40 C
o Thermophiles: 50 – 60 C
o Hyperthermophiles (or extremely thermophiles): usually around 95 C but can grow upto 250 C

b. pH:
• Acidophiles: pH 0 – 5.5
• Neutrophiles: pH 5.5 – 8.0
• Alkaliphiles: pH 8.0 – 11.5

pH dramatically affects microbial growth by:

• Disrupting the plasma membrane

• Altering the activity of enzymes
• Altering the activity of membrane transport proteins.

c. Osmolarity and water activity:

 • Isotonic: No net movement of water occurs
• Hypotonic (low osmolarity): water moves into the cell. If cell wall is strong it contains the
swelling. If the cell wall is weak or damaged, the cell bursts which is known as osmotic lysis.
• Hypertonic (high osmolarity): water moves out of the cell, causing its cytoplasm to shrink which
is known as plasmolysis.

Classification based on osmolarity:

1. Non-halophile: Can not tolerate high salt

2. Halotolerant: can grow over a wide range of water activity
3. Halophiles: optimum growth in the presence of high salt > 0.4 M salt
4. Extreme halophiles: optimum growth in presence of very high salt > 2 M salt

d. Oxygen level:
The importance of oxygen to the microbes correlates with its metabolism.
• Aerobes: an microbe able to grow in oxygen (atmospheric O2)
• Anaerobes: an microbe that can grow in the absence of oxygen

Oxygen is easily reduced to toxic products:

o Superoxide radicals
o Hydrogen peroxide
o Hydroxyl radical
o They damage proteins and DNA

Aerobes produce protective enzymes;

1. Superoxide dismutase
2. Catalase

Growing Anaerobic Microbes: Gas-Pak: Produces H2 that forms H2O in presence of oxygen and thus,
helps to remove oxygen to promote growth of anaerobic microbes
 e. UV radiation:
UV light:
o Causes DNA mutations leading to death because of formation of thymine dimers in DNA which in
turn causes DNA damage
o Bacterial pigments produced by some bacteria gives protection from UV light

NUTRITIONAL REQUIREMENTS FOR BACTERIA

Process by which chemicals (nutrients) are acquired from the environment and used for cellular activities

All living organisms needs nutrients for growth

All bacterial requires two things for growth and life:

-A source of energy and electron source for metabolism

-A source of matter like C, O, H, N, S, P, Trace minerals for building cells

Elemental analysis of E. coli (dry weight) showed following:

o Carbon – 50%
o Oxygen – 20%
o Nitrogen – 14%
o Hydrogen – 8%
o Phosphorus – 3%
o Sulfur – 2%
o Potasssium – 2%
o Ca,Mg – 0.5%
o Iron – 0.2%
o Trace elements – 0.3%

Following are the nutritional requirements of bacteria

1)Source of energy:

a)phototrophs: utilizes light as source of energy

b)Chemotrophs: utilizes chemicals as source of energy

2) Electron Source:

a)Lithotrophs : utilizes inorganic compounds as electron source

b)Organotrophs : utilizes organic compounds as electron source

3) Carbon source :

a)Autotrophs : utilizes CO2 as carbon source

b)Heterotrophs : utilizes organic compounds as carbon source

4) Nitrogen source: for synthesis of proteins (amino acids), DNA, RNA, ATP etc. Nitrogen, nitrates, ammonia or
organic nitrogen (amino acids) compounds are used as nitrogen source for bacteria
5) Oxygen: Source may be water, molecular oxygen or from other oxygen containing nutrients

6) Minerals (Inorganic salts):

a)Sulfur : Utilized for sulfur containing amino acids and some vitamins. Sulfates, H2S, Sulfur containing amino acids
used as sulfur source

b)Phosphorus: Phosphorus needed for nucleic acid synthesis and formation of phospholipids, DNA, RNA, ATP etc.
Inorganic phosphate are primary source of phosphorus for bacteria

c) Trace elements : for growth of bacteria metal ions like K, Mg, Ca, Fe are utilized as co-factors in enzymatic
reactions

d) Other ions are also needed in very low concentration like Cu, Mn, Co, Zn etc

7) Vitamins : Functions as co-enzymes or building blocks for enzyme. Some bacteria synthesize vitamins, others
depends on the host for vitamin requirements

8) Water : Major essential nutrient as it accounts for about 80 – 90% of total weight of cells. Hydrogen and Oxygen
derived metabolically from water.

CLASSIFICATION:

A)On basis of Electron Source:

a)Lithotrophs

b)Organotrophs

B) On basis of Energy Source:

a) Chemotrophs: further classified

I) On basis of Electron source: II) On the basis of carbon source:

i) Chemolithotrophs i) Chemoautotrophs

ii) Chemoorganotrophs ii) Chemoheterotrophs

b) Phototrophs: further classified

I) On basis of Electron source: II) On basis of Carbon source:

i) Photolithotrophs i) Photoautotrophs

ii)Photoorganotrophs ii)Photoheterotrophs
 BACTERIAL METABOLISM
-The bacterial metabolism is dependent on whether they are aerobic or anaerobic.

-Aerobic bacteria utilize glucose by OXIDATION.

-Anaerobic bacteria utilizes glucose by FERMENTATION.

FERMENTATION:

-Utilization of glucose under anaerobic condition is called fermentation. Bacterial fermentation occurs via three
pathways:

1)Glycolysis (also called EMP or Embden-Meyerhof Parnas pathway): Glucose is converted to pyruvate; seen in
most of the bacteria.

2)Entner- Doudoroff (ED) pathway: It is rarely seen in few bacteria such as Pseudomonas. Here, the glucose is
converted to KDPG (keto-deoxy-phosphogluconate) which is then futher converted into pyruvate.

3)Pentose phosphate pathway: It seen in most of the bacteria; glucose is converted to pentose sugars. such as
xylulose phosphate and ribulose phosphate which are used in the biosynthesis of aromatic amino acids, vitamin B6
and ribose 5-phosphate, which is a major component of nucleic acids.

Utilization of Pyruvate:

-Pyruvate produced at the end of Glycolysis pathways are further utilized in many ways (as given below) to
produce acids (lactic acid, formic acid, pyruvic acid), gas (hydrogen, carbon dioxide) and alcohols.

-Homolactic fermentation produces lactic acid. This is seen in enterococci, streptococci and lactobacilli.

-Heterolactic fermentation produces ethanol. This is seen in yeast (Saccharomyces cerevisiae)

-Mixed acid fermentation: Pyruvate is metabolized to produce a number of different products, such as acetic acid,
ethanol, succinic acid and formic acid. The nature and amount of acid production depends on the organism

-Butanediol fermentation: Pyruvate is metabolized to acetoin, which is further converted to butanediol. This is
produced by Klebsiella aerogenes (previously known as Enterobacter aerogenes) and few other bacteria.
Production of acetoin is detected by an important biochemical test called Voges-Proskauer reaction. (VP test)

-Butanol formation: It is seen in anaerobic bacteria Clostridium

-Propionic acid formation: This is observed in anaerobic bacteria, such as Bacteroides, and Propionibacterium

-During fermentation, energy rich phosphate molecules are produced as by-products which convert adenosine
diphosphate (ADP) to adenosine triphosphate (ATP). This process is known as substrate-level phosphorylation.

-During glycolysis, net of 2 ATP molecules per mole of glucose are produced.

-Fermentation is carried out by both obligate and facultative anaerobes.

OXIDATION :

-Oxidation refers to oxidative utilization of glucose (by Krebs cycle) followed by production of ATP via oxidative
phosphorylation and transfer of electrons in electron transport system.

-Kerbs cycle:

 The pyruvic acid produced at the end of glycolysis and ED pathway undergoes a series of oxidative
decarboxylation steps to produce a number of high energy compounds, such as nicotinamide adenine
dinucleotide phosphate (NADPH) and flavin adenine dinucleotide (FADH) which finally enter the electron
transport system.

Electron transport system:

 It is located in cell membrane of bacteria, in contrast to mitochondria of eukaryotes.

 It consists of alternating hydrogen and electron carriers located in a sequence across the cell membrane.
 Transfer of protons down the electron transport system creates a membrane potential. The energy of this
potential is harnessed by ATP synthase which catalyses the formation of ATP from ADP.
 Krebs cycle results in a net gain of 38 ATP molecules per mole of glucose.

BACTERIA PRESERVATIVES
INTRODUCTION:

-Bacterial species vary greatly in the ability of their cultures to remain alive after the completion of growth(e.g.
after 24 hr at 37°C)

-Problem maintaining them for various periods in a viable condition

-Necessary to eliminate genetic instability, protection against contaminants and retain their original characters

-Most laboratories maintain a large collection of pure cultures, frequently referred to as the stock culture
collection

-Some species such as Neisseria are poorly viable and their cultures usually die out within few days in routine
conditions, must be subcultured regularly( 2-4 days).

-Culture collections of microorganisms are valuable resources for and scientific research in microbial diversity and
evolution, patient care management, epidemiological investigations, and educational purposes .

-Preserved individual strains of microorganisms serve as permanent records of microorganisms’ unique phenotypic
profiles and provide the material for further genotypic characterizations .
-Effective storage is defined by the ability to maintain an organism in a viable state free of contamination and
without changes in its genotypic or phenotypic characteristics.

-Microbial preservation methods have been evaluated extensively over the past 50 years, and often, optimal
methods for preservation depend on a microorganism’s taxonomic classification.

MAINTENNANCE METHODS:

 Pure culture- should be free from contaminants, kept pure

 Frequent way of erroneous results and conclusions if faulty or contaminated cultures
 Stock cultures- organisms of interest maintained in laboratory as reference
 Necessary to prevent contamination, retain viability and remain true to type
 Two methods of maintenance of cultures
o Agar Slants
o Agar Stabs

AGAR SLANTS:

-Agar slants are a form of solid media generated from the addition of a gelling agent, such as agar, to a broth
culture .

-Inoculated and incubated until reasonable growth – placed in a refrigerator after well sealing done to prevent
drying .

-Hardy organisms(Spore formers, GPCs, Yeasts and Fungi)- remain viable for weeks to months.

-Regular transfer to fresh slants necessary.

AGAR STABS:

-An inoculation technique used when inoculating semi-solid medium for the analysis of motility or oxygen usage,
or when inoculating certain types of solid medium.

-Inoculation is done with a straight needle having a culture – deep all the way to the bottom.

-Culture grows as a thin zone along the inoculation line.

-Acid produced as waste is diluted along the agar.

-Preserve upto 2 weeks.

SHORT TERM PRESERVATION METHODS: LONG TERM PRESERVATION METHODS:

A) Frequency of transfer A) Ultra low temperature freezing

B) Immersion in oil B) Freeze-drying (lyophilization)

C) Freezing at -20°C

D) Drying

E) Storage in distilled water

SHORT TERM PRESERVATION METHODS:

A)Direct Transfer to Subculture :

-Simplest method for maintaining the short-term viability of microorganisms : saved for more than 1 week .

-Increases the likelihood of mutation with undesirable changes .

-Maintenance Medium- support the survival of the microorganism but minimize its metabolic processes and slow
its rate of growth; distilled water, tryptic soy broth, and nutrient broths with or without cryopreservatives .

-Transfer organisms into screw-top test tubes and to store them in an organized location away from light and
significant temperature changes; storage at lower temperatures (5 to 8C) .

-No set protocol for the frequency of transfer since storage conditions, media used and types of microorganisms
vary among laboratories.

B) Immersion in Oil

-An alternative to capping tubes- add a layer of mineral oil to the top of the specimen .

-Many bacteria and fungi can be stored for periods of up to 2 to 3 years by this method, and transfers are not
needed as frequently.

-Microorganisms are still metabolically active in this environment and mutations can still occur.

-Mineral oil should be medicinal-grade oil with specific gravity of 0.865 to 0.890 and heated to 170C for 1 to 2 h in
an oven : sterilization .

-An inoculum of 5 to 10 colonies of the microorganism should be placed on an agar slant or in tubed broth media .

-A layer of mineral oil at least 1 to 2 cm deep is added, and the agar must not be exposed to air

C) Freezing at -20°C

-Refrigeration or freezing in ordinary freezers at -20°C may be used to preserve microorganisms for periods longer
than those that can be accomplished by repeated transfers .

-Viability may be maintained for as long as 1 to 2 years for specific microorganisms, but overall, damage caused by
ice crystal formation and electrolyte fluctuations results in poor long-term survival .

-Modern self-defrosting freezers with freeze-thaw cycles must be avoided because cyclic temperature fluctuation
will destroy the microorganism.

D) Drying

-Molds and some spore-forming bacteria may be dried and stored for prolonged periods.

-Soil should be autoclaved for several hours on two successive days. A 1-ml suspension of the microorganism is
inoculated into the sterile glass tube, and the tube is left open to air dry before being closed with a sterile stopper
and sample is stored in a refrigerator.

-Instead, commercial silica gel can be used in small cotton-plugged tubes after being heated in an oven to 175C for
1.5 to 2 h, with moderately successful recovery of fungi .
-Gelatin discs: a thick suspension of bacteria is prepared and added to nutrient gelatin . Drops of the bacterial
suspension in gelatin are placed on sterile waxed paper or on a plastic petridish and then dried off over P2O5
under vaccum .

-Alternatively, a suspension of 10ˆ8 microorganisms can be inoculated onto sterile filter paper strips or disks’. The
paper is dried (over phosphorus pentoxide in a desiccator under vacuum )in air or under a vacuum and is placed in
sterile vials. These vials can be stored in the refrigerator for up to 4 years, and then single strips or disks can be
removed as needed .

-Commonly used for quality control organisms.

-L- drying:

 described by Lapage et al .
 bacteria in small ampoules are dried from the liquid state using a vacuum pump and desiccant and a
water bath to control the temperature.

E) Storage in Distilled Water :

-Most organisms grow poorly in distilled water, but some survive for prolonged periods .

-Many fungi and Pseudomonas sp. survive for several years in distilled water at room temperature.

LONG TERM PRESERVATION METHODS:

A)Ultra low temperature freezing:

 Maintained at temperatures of -70°C or lower for prolonged periods using ultralow-temperature electric
freezers and liquid nitrogen storage units
 Presence of a cryopreservatives such as glycerol may reduce the risk to microorganisms upon short
exposure to higher temperatures
 Storage Vials – Plastic (polypropylene) or glass (borosilicate) tubes- able to withstand very low
temperatures and maintain a seal for their contents
 Cryoprotective Agents – To protect microorganisms from damage during the freezing process, during
storage, and during thawing = are added to the culture suspension

B)freeze-drying (lyophilization):

 Methods are less labor-intensive over time, require less laboratory space (e.g., a cryovial versus broth or
agar media), and reduce the chances of mutation events
 Of course mutation still occur (e.g. mecA gene lost during long term preservation at -80°c)
 Prolonged incubation requires-
o Drying of the culture prevented by hermetic sealing
o Stored in a dark, either at RT or 4°C but not at 37°C
o Suitable preservation media
THERE ARE 2 TYPES OF CRYOPROTECTIVE AGENTS:

(Cryoprotective agents are used to prevent damage to the biological cells or tissues caused by ice crystals during
freezing.)

1) Those that enter the cell and protect the intracellular environment and (e.g. Glycerol and dimethyl sulfoxide
(DMSO),

2)Others that protect the external milieu of the organism(e.g. sucrose, lactose, glucose, mannitol, sorbitol,
dextran, polyvinylpyrrolidone, polyglycol, and skim milk )

-Universal cryoprotectant is DMSO 5% (V/V)

-Glycerol 10% (V/V) is the best preservative for bacteria

PREPARATION OF MICROORGANISMS FOR FREEZING:

-Cultures are allowed to mature to the late growth or stationary phase before being harvested .

-Broth specimens are centrifuged (low revolution) to create a pellet of microorganisms and resuspended in 2 to 5
ml of broth with the appropriate concentration of cryoprotectant additive .

-Volume of the aliquots to be frozen is typically 0.2 to 0.5 ml.

FREEZING METHODS:

-American Type Culture Collection (ATCC) recommends slow, controlled-rate freezing at a rate of 1°C per min until
the vials cool to a temperature of at least -30°C, followed by more rapid cooling until the final storage temperature
is achieved .

-When organisms are stored in liquid nitrogen, however, it is still recommended that vials be placed initially in a -
60°C freezer for 1 h and then transferred into the liquid nitrogen (-196°C).

-Small glass beads or plastic beads can also be added to storage vials before freezing- culture suspension will coat
the beads, and then individual beads can be removed from storage for reconstitution without thawing the entire
sample.

THAWING:

-Stored culture vials should be warmed rapidly in a 35°C water bath until all ice has disappeared.

-Once a vial is thawed, it should be opened and the organism should be transferred to an appropriate growth
medium immediately .

-Great care must be exercised during the thawing phase since rapid temperature changes and resulting air
pressure changes inside vials can cause the vials to explode .

-So that protective clothing and eye wear must be worn during this process.
FREEZE-DRYING (LYOPHILIZATION):

DEFINITION:

 Freeze drying is a stabilizing process in which a substance is first frozen and then the quantity of the
solvent is reduced, first by sublimation (primary drying stage) and then desorption (secondary drying
stage) to values that will no longer support biological activity or chemical reactions.
 or it is a process by which a solvent (usually water) is removed from a frozen foodstuff or a frozen solution
by sublimation of the solvent and by desorption of the sorbed solvent (nonfrozen solvent), generally
under reduced pressure.

-Better preservation occurs with freeze-drying than with other methods because freeze-drying reduces the risk of
intracellular ice crystallization that compromises viability .

-Removal of water from the specimen effectively prevents this damage .

-Lyophilization is greatest with gram-positive bacteria (spore formers in particular) and decreases with gram-
negative bacteria, but overall, the viability of bacteria can be maintained for as long as 30 years.

-Large numbers of vials of dried microorganisms can be stored with limited space, and organisms can be easily
transported long distances at room temperature .

-The process combines freezing and dehydration- Organisms are initially frozen and then dried by lowering the
atmospheric pressure with a vacuum apparatus.

OBJECTIVES OF LYOPHILIZATION PROCESS:

 To preserve the biological activity of a product.

 To reduce the product weight to lower the transportation cost.
 To extend the shelf life or stability.
 To dry thermolabile materials.
 To eliminate the need for refrigerated storage.

PRINCIPLE:

-Lyophilization is carried out using a simple principle of physics , sublimation. Sublimation is the transition of a
substance from the solid to the vapour state, without passing through an intermediate liquid phase.

-Lyophilization is performed at temperature and pressure conditions below the triple point (4.58 mm Hg, 0 ºC), to
enable sublimation of ice.

-The entire process is performed at low temperature and pressure by applying vacuum, hence is suited for drying
of thermolabile compounds.

TRIPLE POINT:Triple point of a substance is the temperature and pressure at which three phases (gas, liquid, and
solid) of that substance may coexist in thermodynamic equilibrium. The triple point of pure water is at 0.01°C
(273.16K, 32.01°F) and 4.58 mm (611.2Pa) of mercury and is used to calibrate thermometers.
STEPS INVOLVED IN LYOPHILIZATION:

A)FREEZING STAGE:

 Freezing the product solution to a temperature below its eutectic temperature.

 Decrease the shelf temperature to -50 C.
 Low temperature and low atmospheric pressure are maintained.

B)PRIMARY DRYING STAGE(SUBLIMATION):

 After freezing, the product is placed under vacuum. This enables the frozen solvent in the product to
vaporize without passing through liquid phase, a process known as SUBLIMATION.
 Heat is introduced from shelf to the product under graded control by electrical resistance coils or
circulating silicone.
 The temperature and pressure should be below the triple point of water i.e., 0.0098°C and 4.58mmHg.
 Easily removes moisture up to 98% to 99%.

C)SECONDARY DRYING STAGE (DESORPTION):

 Heat is applied to the frozen product to accelerate sublimation .

 The temperature is raised to 50°C – 60°C and vacuum is lowered about 50mmHg.
 Bound water is removed.
 Rate of drying is low.
 It takes about 10-20 hrs.

D)PACKING:

 Ampoules are sealed by either tip sealing or pull sealing method .

 Vials and bottles are sealed with rubber closures and aluminum caps.

STORAGE VITALS:

 Glass vials are used for all freeze-dried specimens .

 Cryoprotective Agents – Two most commonly used agents are 1. Skim milk, and 2. Sucrose.

PREPARATION OF MICROORGANISMS FOR FREEZING:

-As with simple freezing, maximum recovery of organisms is achieved by using microorganisms in the late growth
or stationary phase from the growth of an inoculum in an appropriate growth medium.

-High concentrations of microorganisms are considered to be important- ATCC concentration of at least 10ˆ8
CFU/ml but Heckly suggests a concentration of 10^10 CFU/ml or higher.

FREEZE DRY PRODUCT CHARACTERISTICS:

 Sufficient strength
 Uniform color
 Sufficiently dry
 Sufficiently porous
 Sterile
 Free of pyrogens and particulates
 Chemically stable both in dry state and reconstitution
STORAGE:

-Storage at room temperature does not maintain viability and is not recommended

-Storage at 4°C in an ordinary refrigerator is acceptable, but survival may be improved at temperatures of -30 to -
60°C

-Reconstitution:

 The surface of the vial should be wiped with 70% alcohol, and then the top of the glass vial can be scored
and broken off or punctured with a hot needle.
 A small amount (0.1 to 0.4 ml) of growth medium is injected into the vial with a needle and syringe or a
Pasteur pipette, the contents are stirred until the specimen is dissolved, and then the entire contents are
transferred with the same syringe or a pipette to appropriate broth or agar media.

ADVANTAGES OF LYOPHILIZATION:

 Removal of water at low temperature .

 Thermolabile materials can be dried.
 Sterility can be maintained.
 Reconstitution is easy.

DISADVANTAGES OF LYOPHILIZATION:

 Many biological molecules are damaged by the stress associated with freezing, freeze-drying, or both. –
E.g. the process of drying causes extensive damage to molds, protozoa, and most viruses – Hence, these
microorganisms can not be stored by this method .
 The product is prone to oxidation, due to high porosity and large surface area. Therefore the product
should be packed in vacuum or using inert gas .
 Cost may be an issue, depending on the product.

COMMON LYOPHILIZED PRODUCTS:

o Bacteria
o Viruses
o Vaccines
o Plasma
o Pharmaceuticals
 VIRUS MORPHOLOGY AND CLASSIFICATION
-Not cells

-Smallest infectious agent (20nm to 300 nm)

 smallest RNA virus Picornavirus

 smallest DNA virus Parvovirus
 largest virus Poxvirus

-Most have spherical shapes EXCEPT

 Rhabdovirus bullet-shaped
 Poxvirus brick-shaped
 Bacteriophage tadpole-shaped
 Tobacco Mosaic virus

-Composed of only one type of nucleic acid.

-Obligate intracellular parasites inert in the extracellular environment multiply only in living cells parasites at
the genetic levels.

-Do not undergo mitosis or binary fission.

-Do not have cell wall & ribosome.

-Sensitive to interferon.

STRUCTURE:

1)VIRAL NUCLEIC ACID (GENOME):

 contain the genetic material necessary for replication.

 either a DNA or RNA.
 maybe single-stranded or double-stranded.
 can be linear or circular.
 Segmented or non-segmented.
 haploid contain only one copy of their genes EXCEPT Retroviruses.
 used as major criterion in the classification of viruses.

2) CAPSID:

-protein coat made up of repeating subunits CAPSOMERES (gives the virus its geometric symmetry)

-2 forms of symmetry:

1. icosahedral – capsomeres arranged in 20 triangles

2. helical – arranged in a hollow coil that appears

rod-shaped
-Functions of the viral capsid:

 protect the genetic material

 site of receptors =>mediate attachment
 serve as antigenic determinants
 induce antibody production
 determinants of type specificity
 Provide the structural symmetry of the virus

3)ENVELOPE:

-a lipoprotein derived from the host cell membrane & virus specific protein.

-composed of glycoproteins (peplomers) that appear as spikes.

-Acquired through budding from the host’s cell membrane during maturation.

-Confers instability to the virus because of the loss of infectivity due to disruption or loss of lipid due to which it
is more sensitive to heat, detergents & lipid solvents.

Functions:

 site of receptors attach to host cell receptors & membrane fusion

 antigenic determinants
 stimulate antibody production
 determinants of type specificity

TERMINOLOGIES:

VIRION – a complete viral particle

- in naked viruses virion is identical to the nucleocapsid

- in enveloped viruses must acquire envelop before it is considered a virion

NUCLEOPCAPSID – a protein-nucleic acid complex

ATYPICAL VIRUS LIKE AGENTS:

DEFECTIVE VIRUSES – composed of nucleic acid & proteins but cannot replicate without a helper virus

PSEUDOVIRION – contain host cell DNA instead of viral DNA within the capsid

VIROIDS – consist solely of a single molecule of circular RNA without a protein coat or envelope

PRIONS –infectious protein particles composed solely of proteins

VIRUS CLASSIFICATION:

1. Virion morphology - size, shape, symmetry, +/- of peplomers, +/- of membrane

2. Virus genome – DNA or RNA, size, strandedness, segments
3. Physicochemical properties of the virion – molecular mass, buoyant density, stability & susceptibility
4. Antigenic properties
5. Virus protein properties – number, size, activities
6. Biological properties – host range, MOT, vector relationships, pathogenicity, tissue tropism & pathology
GENERAL PRINCIPLES:

DNA VIRUSES:

 Double stranded DNA except Parvovirus

 Naked except Herpes, Hepadna & Poxviruses
 Icosahedral symmetry except Poxviruses
 Replicate in the nucleus except Poxviruses

RNA VIRUSES:

 Single stranded RNA except Reovirus

 Enveloped except Reovirus & Picornavirus
 Helical symmetry except Reovirus, Picornavirus & Togavirus
 Replicate in the cytoplasm except Orthomyxovirus & retroviruses

CLASSIFICATION OF PARASITES

Classification of medically important parasites

–Helminthology.

–Protozoology.

–Entomology.

Types of medically important helminths

–Trematodes

–Cestodes

–Nematodes

 Intestinal nematodes
 Tissue nematodes

A-Trematodes (Flukes) :

1-General morphological characters:

•Hermaphrodite except blood flukes (Unisexual)

•Leaf-like, flattened and bilaterally symmetrical

•There is no body cavity.

2-Organs of fixation:

Almost all have 2 suckers, oral (at the anterior end, around the mouth) and ventral (on the ventral surface).
3- Habitat:

They have different habitat; intestinal, hepatic, blood and pulmonary flukes.

4-General outline of the life cycle:

•Adult → egg → miracidium → sporocyst→ redia→ cercaria → metacercaria→ adult.

•Egg is operculated, and should reach to a water source to hatch.

•The first intermediate host is snail, and the second (if present) is marine creature.

•Except for Schistosoma, egg has a spine, no redia and no metacercaria.

5-Important stages in the life cycle:

•The diagnostic stage is the egg.

•The infective stage is the encysted metacercaria except in Schistosoma, it is the cercaria.

•The mode of infection is by ingestion of food contaminated with the infective stage except in Schistosoma, it is by
skin penetration of the cercaria.

B-Cestodes(tapeworms)

1-General morphological characters:

• Hermaphrodite.

• Ribbon-like flattened and bilaterally symmetrical.

• There is neither body cavity nor digestive tract.

• The body is formed of 3 parts:

- Head (Scolex) which is provided by organs of attachment.

- Neck is composed of actively dividing cells (stem cells) and it is responsible for giving rise to new segments.

- Body is composed of several segments (Immature, Mature and Gravid segments).

2-Organs of fixation:

2 types:

 Suckers (4) with or without hooks that are arranged in one or more circles on the apex of the scolex called
“rostellum”.
 Bothria (2) that take the form of grooves.

3-Habitat:

 They all (without exception) are intestinal parasites.

4-General outline of the life cycle.

 Adult → egg → cysticercus (the larval stage) → adult, Except for D. latum, give oval operculated egg
containing hexacanthembryo (coracidium).
  All cestodes require one intermediate host (usually vertebrate), except

D. latum requires two intermediate hosts.

H. nana does not require an intermediate host.

5-Important stages in the life cycle.

 The diagnostic stage is the egg.

 The infective stage is the last larval stage which is different according to:
 No intermediate host (H. nana): Egg.
 1 intermediate host (Other cestodes): Cysticercus
 2 intermediate hosts (D. latum): Pleurocercoid.
 The mode of infection is by ingestion of food contaminated with the infective stage.

C-Nematodes (cylindrical worms)

1-General morphological characters

 Bilateral symmetrical
 Has body cavity
 Separate sexes
 Elongate and cylindrical

2-Organs of fixation are present in the mouth and buccal cavity as lancets and teeth.

3-Habitat: Intestinal (small and large) and tissue nematodes.

4-General outline of the life cycle.

•Adult → egg → larva → adult.

•Tissue nematodes are viviparous (lay larvae). Larva molts its cuticle 3 times .
 PROTOZOA:

General characters of protozoa

•Body is formed of cytoplasm and nucleus.

•Cytoplasm is differentiated into ectoplasm and endoplasm.

•Nucleus contains karyosome and peripheral chromatin.

Types of medically important protozoa

1. Pseudopodia

Locomotory organs: Pseudopodia

Habitat: Intestinal and free living

General outline of the life cycle and important stages: Trophozoite and cyst

2. Ciliates

Locomotory organs: Cilia.

Habitat: Intestinal

General outline of the life cycle and important stages: Trophozoite and cyst

3- Flagellates

General morphological characters : Flagella as Locomotory organs

Habitat: Intestinal, urogenital and, blood

General outline of the life cycle and important stages:

Intestinal and urogenital flagellates: Trophozoite and cyst.

Blood flagellates:

•Leishmania: Amastigotes (in human) and promastigotes (Vector).

• Trypanosoma: Trypomastigotes (human) and Epimastigotes (Vector).

4-Coccidia

Locomotory organs: They are sporulated

They are members of Apicomplexa

Habitat: Intestinal, tissue and blood.

General outline of the life cycle: Gametogony (Sexual reproduction).

Important stages in the life cycle: Sporozoite, Schizont, merozoite, gametocytes, oocyst
 CLASSIFICATION OF FUNGI
ON THE BASIS OF MORPHOLOGICAL CLASSIFICATION:

1. Yeast: C.neoformans
2. Yeast –like fungi: C.albicans
3. Moulds- Dermatophytes
4. Dimorphic fungi: Most of fungi causing systemic infections.

1.YEAST AND YEAST LIKE FUNGI:

 Yeasts are unicellular which are round to oval .

 Size range from 2 to 60µm.
 Reproduce asexually by blastoconidia formation (budding) which is the characteristic of the yeast
.Sexually by the production of ascospores or basidiospores.
 If there is no constriction at its base then it is termed as germ tube.
 Pseudohyphae represent elongated buds that have failed to separate and are connected to one another
to create a “link of sausage” appearance.
 Usually size, shape, presence or absence of capsule are helpful in the separation of Cryptococcus species
from Candida species.

2.MOULDS:

 The basic structural units of the molds are tube like projections known as hyphae.
 As the hyphae grow, they become intertwined to form a loose network, the mycelium.
 They penetrate the substrate from which it obtains the necessary nutrients for the growth.
 Hyphae usually have cross walls that divide them into numerous cells. These cross walls, called septa have
small pores through which cytoplasm is continuous throughout the hyphae.

Mycelium are of three kinds:

1. Vegetative mycelium are those that penetrates the surface of the medium and absorbs nutrients.
2. Aerial mycelium are those that grow above the agar surface
3. Fertile mycelium are aerial hyphae that bear reproductive structures such as conidia or sporangia
3.DIMORPHIC FUNGI:

They have two morphology at two different temperatures, usually they are pathogenic.

 yeast form or spherule : In body temperature i.e., 37ºC

 Filamentous form: at room temperature i.e., 25ºC

ON THE BASIS OF SYSTEMIC CLASSIFICATION:

SCOPES OF MICROBIOLOGY:

IMPORTANCE OF PHARMACEUTICAL MICROBIOLOGY:

o Evaluation of bio burden in production areas

o Removal of microbial contamination by sterilization techniques
o Sterility Test
o Bacterial Endotoxin test e.g. LAL Test for parenterals
o Evaluation of disinfectants by Phenol Coefficient test
o Efficacy test for preservatives
o Assay of Antibiotics, Vitamins etc.
o Production of Antimicrobial Agents e.g. Penicillin, Streptomycin etc.
o Production of Vaccines e.g. DPT, Tetanus, BCG.
o Production of Vitamins B12
o Production of Enzymes like amylase, protease, lipase
o Production of steroids like testosterone, progesterone

SCOPES OF PHARMACEUTICAL MICROBIOLOGY:

o Hospitals
o Research and Development
o Pharmaceutical and vaccine production
o Pathology Laboratories
o Quality control of drugs
o Food and Dairy industry