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B.Pharm Microbiology unit:- 1 (Hand Written Notes)

Description

Introduction to pharmaceutical
microbiology
Introduction
Microbiology is a biological science involved with the study of microscopic organisms. Microbiology is made up of several sub-disciplines, including: bacteriology (the study of bacteria), mycology (the study of fungi), phycology (the study of algae), parasitology (the study of parasites), and virology (the study of viruses, and how they function
inside cells) [1]. These broad areas encompass a number of specific fields. These fields include: immunology (the study of the immune system and how it works to protect us from harmful organisms and harmful substances produced by them); pathogenic micro biology (the study of disease-causing microorganisms and the disease process (epidemiology and etiology)); microbial genetics (which is linked to molecular biology); food microbiology (studying the effects of food spoilage); and so on [2]. The microbiological discipline of relevance here is pharmaceutical microbiology, an applied branch of microbiology (once considered as an off-shoot of industrial microbiology but now a distinct field). Pharmaceutical microbiology is concerned with the study of microorganisms associated with the manufacture of pharmaceuticals. This is with either using microorganisms to help to produce pharmaceuticals or with controlling the numbers in a process environment. This latter concern is about ensuring that the finished product is either sterile or free from those specific strains that are regarded as objectionable. This extends through the manufacturing process, encom passing starting materials, and water. Pharmaceutical microbiologists are additionally interested in toxins (microbial by-products like endotoxins and pyrogens), particularly with ensuring that these and other “vestiges” of microorganisms (which may elicit
adverse patient responses) are absent from products. Microbiological contamination becomes a problem when it results in unwanted effects occurring in pharmaceutical preparations. In drawing from risk assessment terminology, pharmaceutical microbiology centers on understanding the likelihood of product contamination arising; understanding the severity of such contamination; considering ways to minimize contamination; and, where contamination cannot be satisfactorily mitigated, using established and developing new methods to detect
contamination. To understand the severity, it is necessary to understand the type of product, its in tended use, and the nature and numbers of contaminants. Microbial contamination of sterile injectable products (parenterals) presents the greatest risk, for this may lead to death of the patient, whereas with other products, aromas, off-flavors, or discolorations, caused by microorganisms, may have fewer adverse effects. Therefore, with respect to sterile products, the main concern is with any potential microbial contamination. With

Introduction, history of microbiology, its branches, scope and its importance. Introduction to Prokaryotes and Eukaryotes : Study of ultra-structure and morphological classification of bacteria, nutritional requirements, raw materials used for culture media and physical parameters for growth, growth curve, isolation and preservation methods for pure cultures, cultivation of anaerobes, quantitative measurement of bacterial growth (total & viable count). Study of different types of phase constrast microscopy, dark field microscopy and electron microscopy

Phase contrast microscopy Definition:-Unstained living cells absorb practically no light. Poor light absorption results in extremely small differences in the intensity distribution in the image. This makes the cells barely, or not at all, visible in a bright field microscope.
Phase2 -contrast microscopy is an optical microscopy technique that converts phase shifts in the light passing through a transparent specimen to brightness changes in the image. It was first described in 1934 by Dutch physicist Frits Zernike When light passes through cells, small phase which are invisible to the human eye. In a phase-contrast microscope, these phase shifts are converted into changes in amplitude, which can be observed as
differences in image contrast.

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Microscopy

Phase contrast microscopy

Definition:-Unstained living cells absorb practically no light. Poor light

absorption results in extremely small differences in the intensity

distribution in the image. This makes the cells barely, or not at all, visible

in a bright field microscope.

Phase2 -contrast microscopy is an optical microscopy technique that

converts phase shifts in the light passing through a transparent

specimen to brightness changes in the image.

It was first described in 1934 by Dutch physicist Frits Zernike.

Principle of Phase contrast Microscopy

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When light passes through cells, small phase

which are invisible to the human eye.

In a phase-contrast microscope, these phase shifts are

converted into changes in amplitude, which can be observed as

differences in image contrast.

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Applications of Phase contrast Microscopy

To produce high-contrast images of transparent specimens, such as

living cells (usually in culture),

Microorganisms,

Thin tissue slices,

lithographic patterns,

fibers,

latex dispersions,

glass fragments,

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subcelluar particles (including nuclei and other organelles).

Applications of phase-contrast microscopy in biological research are numerous.

Advantages

Living cells can be observed in their natural state without previous

fixation or labeling.

It makes a highly transparent object more visible.

No special preparation of fixation or staining etc. is needed to study an

object under a phase-contrast microscope which saves a lot of time.

Examining intracellular components of living cells at relatively high

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resolution. eg: The dynamic motility of mitochondria, mitotic

chromosomes & vacuoles.

Limitations

Phase-contrast condensers and objective lenses add considerable cost to a

microscope, and so phase contrast is often not used in teaching labs except

perhaps in classes in the health professions.

Electron microscope definition

An electron microscope is a microscope that uses a beam of

accelerated electrons as a source of illumination.

It is a special type of microscope having a high resolution of images, able to

magnify objects in nanometers, which are formed by controlled use of electrons

in vacuum captured on a phosphorescent screen.

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Ernst Ruska (1906-1988), a German engineer and academic professor, built the

first Electron Microscope in 1931, and the same principles behind his prototype

still govern modern EMs.

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Mr.S.D.Mankar 08/07/202 0

Working Principle of Electron microscope

Electron microscopes use signals arising from the interaction of an

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electron beam with the sample to obtain information about structure,

morphology, and composition.

1. The electron gun generates electrons.

2. Two sets of condenser lenses focus the electron beam on the

specimen and then into a thin tight beam.

3. To move electrons down the column, an accelerating voltage (mostly

between 100 kV-1000 kV) is applied between tungsten filament and

anode.

4. The specimen to be examined is made extremely thin, at least 200 times thinner

than those used in the optical microscope. Ultra-thin sections of 20-100 nm are cut

which is already placed on the specimen holder.

5. The electronic beam passes through the specimen and electrons are scattered

depending upon the thickness or refractive index of different parts of the specimen.

6. The denser regions in the specimen scatter more electrons and therefore appear

darker in the image since fewer electrons strike that area of the screen. In contrast,

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transparent regions are brighter.

7. The electron beam coming out of the specimen passes to the objective lens, which

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has high power and forms the intermediate magnified image.

8. The ocular lenses then produce the final further magnified image.

Types of Electron microscope

1. Transmission Electron Microscope(TEM)

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In TEM a beam of electron is projected from electron gun and pass

through a series of electromagnetic lenses.

They get scattered and transmitted through the object and pass through

objective lens which magnifies image of object.

The projection lens further magnifies the image and project it on

fluor1e6 scent screen.

The electron image is converted into visible form by projecting of

fluorescent screen.

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An electron beam has low penetration power through solid matter.

Hence very thin section of specimen is required.

Application:-

TEM is useful in shadow casting, ultra thin sectioning, localization of

cells constituents & enzymes & autoradiography.

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2) Scanning Electron Microscope (SEM)

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In SEM the specimen is subjected to a narrow electron beam which rapidly

moves over the surface of specimen. ( Scann).

These causes the release of secondary electrons from the specimen surface.

The intensity of secondary electrons is depends on shape and chemical

composition of the object.

The secondary electrons are collected by detector which generates the

electron signals.

These signals are then scanned in the manner of a television system to

produce an image on cathode ray tube.

Applications

Electron microscopes are used to investigate the ultrastructure of a wide range of biological

and inorganic specimens including microorganisms, cells, large molecules, biopsy samples,

metals, and crystals.

Industrially, electron microscopes are often used for quality control and failure analysis.

Mode2r0n electron microscopes produce electron micrographs using specialized digital

cameras and frame grabbers to capture the images.

Science of microbiology owes its development to the electron microscope. Study of

microorganisms like bacteria, virus and other pathogens have made the treatment of

diseases very effective.

Advantages

Very high magnification

Incredibly high resolution

Material rarely distorted by preparation

It is possible to investigate a greater depth of field

Diverse applications

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Limitations

As the penetration power of the electron beam is very low, the object should be

ultra-thin. For this, the specimen is dried and cut into ultra-thin sections before

observation.

As the EM works in a vacuum, the specimen should be completely dry.

Expensive to build and maintain

Requiring researcher training

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Image artifacts resulting from specimen preparation.

This type of microscope is a large, cumbersome extremely sensitive to vibration and

external magnetic fields.

Dark Field Microscopy:

A dark field microscope is arranged so that the light source is

blocked off, causing light to scatter as it hits the specimen.

This is ideal for making objects with refractive values similar to the

background appear bright against a dark background.

When light hits an object, rays are scattered in all dire2c3tions. The

design of the dark field microscope is such that it removes the

dispersed light, or zeroth order, so that only the scattered beams hit

the sample.

The introduction of a condenser and/or stop below the stage ensures

that these light rays will hit the specimen at different angles, rather than

as a direct light source above/below the object.

The result is a “cone of light” where rays are diffracted, reflected and/or

refracted off the object, ultimately, allowing the individual to view a

specimen in dark field.

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The dark-ground microscopy makes use of the dark-ground microscope,

a special type of compound light microscope.

The dark-field condenser with a central circular stop, which illuminates

the object with a cone of light, is the most essential part of the dark-

ground microscope.

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This microscope uses reflected light instead of transmitted light used in

the ordinary light microscope.

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