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1.
In a typical SEM, an electron beam is thermionically emitted from an electron gun fitted with
a tungsten filament cathode. Tungsten is normally used in thermionic electron guns because it
has the highest melting point and lowest vapor pressure of all metals, thereby allowing it to be
electrically heated for electron emission, and because of its low cost. Other types of electron
emitters include lanthanum hexaboride (LaB
6) cathodes, which can be used in a standard tungsten filament SEM if the vacuum system is
upgraded or field emission guns (FEG), which may be of the cold-cathode type using tungsten
single crystal emitters or the thermally assisted Schottky type, that use emitters of zirconium
oxide.
The electron beam, which typically has an energy ranging from 0.2 keV to 40 keV, is focused by
one or two condenser lenses to a spot about 0.4 nm to 5 nm in diameter. The beam passes
through pairs of scanning coils or pairs of deflector plates in the electron column, typically in the
final lens, which deflect the beam in the x and y axes so that it scans in a raster fashion over a
rectangular area of the sample surface.

Signals emitted from different parts of the interaction volume
Mechanisms of emission of secondary electrons, backscattered electrons, and characteristic X-
rays from atoms of the sample
When the primary electron beam interacts with the sample, the electrons lose energy by
repeated random scattering and absorption within a teardrop-shaped volume of the specimen

known as the interaction volume, which extends from less than 100 nm to approximately 5 µm
into the surface. The size of the interaction volume depends on the electron's landing energy,
the atomic number of the specimen and the specimen's density.
The energy exchange between the electron beam and the sample results in the reflection of
high-energy electrons by elastic scattering, emission of secondary electrons by inelastic
scattering and the emission of electromagnetic radiation, each of which can be detected by
specialized detectors. The beam current absorbed by the specimen can also be detected and
used to create images of the distribution of specimen current.
Electronic amplifiers of various types are used to amplify the signals, which are displayed as
variations in brightness on a computer monitor (or, for vintage models, on a cathode ray tube).
Each pixel of computer video memory is synchronized with the position of the beam on the
specimen in the microscope, and the resulting image is therefore a distribution map of the
intensity of the signal being emitted from the scanned area of the specimen. In older
microscopes images may be captured by photography from a high-resolution cathode ray tube,
but in modern machines they are digitised and saved as digital images.
PRINCIPLE OF SEM
The types of signals produced by an SEM include secondary electrons (SE), reflected or back-
scattered electrons (BSE), photons of characteristic X-rays and light (cathodoluminescence)
(CL), absorbed current (specimen current) and transmitted electrons. Secondary electron
detectors are standard equipment in all SEMs, but it is rare that a single machine would have
detectors for all other possible signals.
The signals result from interactions of the electron beam with atoms at various depths within the
sample. In the most common or standard detection mode, secondary electron imaging or SEI,
the secondary electrons are emitted from very close to the specimen surface. Consequently,
SEM can produce very high-resolution images of a sample surface, revealing details less than
1 nm in size. Back-scattered electrons (BSE) are beam electrons that are reflected from the
sample by elastic scattering. They emerge from deeper locations within the specimen and
consequently the resolution of BSE images is generally poorer than SE images. However, BSE
are often used in analytical SEM along with the spectra made from the characteristic X-rays,
because the intensity of the BSE signal is strongly related to the atomic number (Z) of the
specimen. BSE images can provide information about the distribution of different elements in
the sample. For the same reason, BSE imaging can image colloidal gold immuno-labels of 5 or
10 nm diameter, which would otherwise be difficult or impossible to detect in secondary electron
images in biological specimens. Characteristic X-rays are emitted when the electron beam

removes an inner shell electron from the sample, causing a higher-energy electron to fill the
shell and release energy. These characteristic X-rays are used to identify the composition and
measure the abundance of elements in the sample.
Due to the very narrow electron beam, SEM micrographs have a large depth of field yielding a
characteristic three-dimensional appearance useful for understanding the surface structure of a
sample. This is exemplified by the micrograph of pollen shown above. A wide range of
magnifications is possible, from about 10 times (about equivalent to that of a powerful hand-
lens) to more than 500,000 times, about 250 times the magnification limit of the best light
microscopes.
2.
Xerophiles:-
are extremophilic organisms that can grow and reproduce in conditions with a low availability of
water, also known as water activity. Water activity (aw) is a measure of the amount of water
within a substrate that an organism can use to support sexual growth. Xerophiles are often said
to be "xerotolerant", meaning tolerant of dry conditions. They can survive in environments with
water activity below 0.8. Endoliths and halophiles are often xerotolerant.
The common food preservation method of reducing water activities may not prevent the growth
of xerophilic organisms, often resulting in food spoilage. Many mold and yeast species are
xerophilic. Mold growth on bread is an example of food spoilage by xerophilic organisms. This
naming comes from the Greek xeros meaning dry, and philos meaning "loving."
Halophiles are organisms that thrive in high salt concentrations. They are a type of
extremophile organisms. The name comes from the Greek word for "salt-loving". While most
halophiles are classified into the Archaea domain, there are also bacterial halophiles and some
eukaryota, such as the alga Dunaliella salina or fungus Wallemia ichthyophaga.
A basophileisatype of white bloodcell.Basophilsare the least commonof the granulocytes,
representingabout0.5 to 1% of circulatingwhite bloodcells.[1] However,theyare the largesttype of
granulocyte.Theyare responsible forinflammatoryreactionsduringimmune response,aswell asinthe
formationof acute and chronicallergicdiseases,includinganaphylaxis,asthma,atopicdermatitisand

hay fever.[2] Theycanperformphagocytosis(cell eating),produce histamineandserotoninthatinduce
inflammation,andheparinthatpreventsbloodclotting.[3]
 Extremophilia, extremophile:Preference of livingextremalconditionsforsome microorganisms.
 Geophilia, geophilic,geophileReferringtoorganismsthatpreferthe soil.
 Halophilia,halophile:Attractiontosaltor salt-water.
 Heliophilia,heliophile:Attractiontosunlight.
 Hydrophilia:Attractiontowater.
 Hyperthermophilia, hyperthermophile,hyperthermophilic:Organismsthatthrive inextremely
hot environments.
 Acidophilia, acidophile: Preference of acidic conditions. Antonym: Acidophobia, acidophobe
 Alkaliphilia, alkaliphile: Preference of alkaline environments.
Hydrostatic pressure is an important parameter influencing the distribution of microbial life in
the ocean. In this study, the response of marine bacterial populations from surface waters to
pressures representative of those under deep-sea conditions was examined.
3. Short notes
Bacterial Transformation:-
Transformation is one of three processes by which exogenous genetic material may be
introduced into a bacterial cell; the other two being conjugation (transfer of genetic material
between two bacterial cells in direct contact), and transduction (injection of foreign DNA by a
bacteriophage virus into the host bacterium).
Horizontal Genetic Transfer:-
Horizontal gene transfer (HGT) is the movement of genetic material between unicellular and/or
multicellular organisms other than via vertical transmission (the transmission of DNA from
parent to offspring.) HGT is synonymous with lateral gene transfer (LGT) and the terms are
interchangeable.
Vertical Genetic Transfer:-
vertical transfer, the transmission of genes from the parental generation to offspring via sexual
or asexual reproduction.
the insertion of copies of a gene into living cells in order to induce synthesis of the gene's
product: the desired gene may be microinjected directly into the cell or it may be inserted into

the core of a virus by gene splicing and the virus allowed to infect the cell for replication of the
gene in the cell's DNA.
Parthenogenesis:-
Parthenogenesis is most simply defined as reproduction without fertilization. More specifically, it
occurs when a female gamete develops a new individual without being fertilized by a male
gamete. It is often called a form of "asexual reproduction," but it is more accuratley defined as
an incomplete form of sexual reproduction. This is because it involves the production, activation,
and development of a female egg which is a specialized reproductive cell (Kaufman, 1983).
Parthenogenesis is not to be confused with hermaphroditic species which can also reproduce
by themselves. Hermaphroditic species reproduce by themselves because an organism can
produce both the male and female gamete.
4.Biochemical test for identification of bacteria, namely:-
Urease Test
Urease broth is a differential medium that tests the ability of an organism to produce an exoenzyme,
called urease, that hydrolyzes urea to ammonia and carbon dioxide. The broth contains two pH buffers,
urea, a very small amount of nutrients for the bacteria, and the pH indicator phenol red.
Catalase Test
The catalase test is used to differentiate staphylococci (catalase-positive) from streptococci (catalase-
negative). The enzyme, catalase, is produced by bacteria that respire using oxygen, and protects them
from the toxic by-products of oxygen metabolism.

Catalase is a common enzyme found in nearly all living organisms exposed to oxygen (such as bacteria,
plants, and animals). It catalyzes the decomposition of hydrogen peroxide to water and oxygen. It is a
very important enzyme in protecting the cell from oxidative damage by reactive oxygen species (ROS).
Starch Hydrolysis
The purpose is to see if the microbe can use starch, a complex carbohydrate made from glucose, as a
source of carbon and energy for growth. Use of starch is accomplished by an enzyme called alpha-
amylase.
Alpha-amylase: A medium containing starch is used. After inoculation and overnight incubation, iodine
reagent is added to detect the presence of starch. Iodine reagent complexes with starch to form a blue-
black color in the culture medium. Clear halos surrounding colonies is indicative of their ability to digest
the starch in the medium due to the presence of alpha-amylase.
Carbohydrate Utilization/Test
To test cells for the ability of an organism to utilize and digest several sugars or small carbohydrates. It is
useful in identifying Gram negative bacilli, especially Enterobacteriaceae, though it may aid in identifying
many other species as well.
The Carbohydrate Utilization test uses Phenol Red Broth (or Purple Broth) to test for the utilization of
different sugars. Phenol Red Broth is a differential media that includes the pH indicator Phenol Red,
peptones, and a series of tubes each with a different sugar. Different sugars and polyhydric alcohols may
be used, but glucose, sucrose, lactose, and mannitol are often studied among others. One inoculates
bacteria into each tube, if the strain of bacteria utilizes that sugar, an acid will form changing the color of
Phenol Red from reddish-orange to yellow.
Gelatin Utilization /Test
Nutrient gelatin is a differential medium that tests the ability of an organism to produce an exoenzyme,
called gelatinase, that hydrolyzes gelatin. Gelatin is commonly known as a component of gelled salads
and some desserts, but it's actually a protein derived from connective tissue.
At temperatures above 32°C, it is a viscous liquid. Gelatinase allows the organisms that produce it to
break down gelatin into smaller polypeptides, peptides, and amino acids that can cross the cell
membrane and be utilized by the organism.
5. Endotoxin and exotoxin properties and differences.
Exotoxins:
Definition: Toxins that are released extracellularly as the organism grows are called exotoxins. Exotoxins
may travel from a focus of infection to distant part of the body and cause damage. E.g. Neurotoxin
(botulinum toxin, tetanus toxin), Enterotoxin (cholera toxin), Cytotoxin

Endotoxins:
Definition: Endotoxins are lipopolysaccharides toxin produced by Gram negative bacteria. The name
endotoxin is derived from the fact that these toxins are generally cell bound and released only when the
cell lyses.
Basic properties and differences between Exotoxins and Endotoxins
Property Exotoxins Endotoxins
Biomolecule/Chemistry Proteins
Lipopolysaccharide-Lipoprotein
complex
Location of genes Plasmid or bacteriophage Bacterial chromosome
Source
Excreted by certain gram positive or gram
negative bacteria
Cell wall of Gram Negative bacteria,
released only after lysis of cells
Heat Stability
Destroyed rapidly at 60oC (except
staphylococcal enterotoxin) Stable at 100oC for one hour
Mode of Action
(Symptoms)
Specific. Either cytotoxin, enterotoxin or
neurotoxin with defined action on cells or
tissues General. fever, diarrhea, vomiting
Toxicity
Highly toxic, often fatal (fatal dose on the
order of 1 µg)
Weakly toxic, rarely fatal (fatal dose
on the order of hundreds of
micrograms)
Immunogenicity
Highly immunogenic, stimulate the
production of neutralizing antibody
(antitoxins) Relatively poor immunogenicity
Toxoid
potential/Vaccines
Treatment of toxin with formaldehyde will
destroy toxicity, but treated toxins remain
immunogenic.
Toxids used as vaccines
No toxoid formed and no vaccine
available
Typical disease Tetanus, diphtheria, botulism
Meningococcemia, sepsis by gram
negative rods

S.N. Exotoxins Endotoxins
1 Excreted by organisms, living cell Integral part of cell wall
2
Found in both Gram positive and Gram
Negative bacteria
Found mostly in Gram Negative Bacteria
3 It is polypeptide It is lipopolysaccharide complex.
4 Relatively unstable, heat labile (60°C) Relatively stable, heat tolerant
5 Highly antigenic Weakly immunogenic
6 Toxoids can be madeby treating with
formalin
Toxoids cannot be made
7 Highly toxic, fatal in µg quantities Moderately toxic
8 Usually binds to specific receptors Specific receptors not found
9 Not pyrogenic usually, Toxin Specific
Fever by induction of interleukin 1 (IL-1)
production, Shock
10 Located on extrachromosomal genes
(e.g. plasmids)
Located on chromosomal genes
11 Filterable Not so
12 It has no enzymatic activity It has mostly enzymatic activity
13 Its molecular weight is 10KDa Its molecular weight is 50-1000KDa
14 On boiling it get denatured. On boiling it cannot be denatured.
15 Detected by many tests (neutralization,
precipitation, etc)
Detected by Limulus lysate assay
16
Examples: Toxins produced
by Staphylococcus aureus, Bacillus
cereus, Streptococcus pyogenes,
Bacillus anthrcis(Alpha-toxin, also
known as alpha-hemolysin (Hla))
Examples: Toxins produced by E.coli,
Salmonella Typhi, Shigella, Vibrio
cholera(Cholera toxin- also known as
choleragen)
17 Diseases: Tetanus, diphtheria, botulism
Diseases: Meningococcemia, sepsis by
gram negative rods

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general microbiology

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