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.
1
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
3
08/07/2020
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.
4
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,
7 and
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
8
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.
11
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.
08/07/2020
Mr.S.D.Mankar 08/07/202 0
Working Principle of Electron microscope
Electron microscopes use signals arising from the interaction of an
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,
transparent regions are brighter.
7. The electron beam coming out of the specimen passes to the objective lens, which
14
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)
15
08/07/2020
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.
08/07/2020
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.
17
08/07/2020
2) Scanning Electron Microscope (SEM)
18
08/07/2020
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
21
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
22
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.
24
08/07/2020
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.
25
This microscope uses reflected light instead of transmitted light used in
the ordinary light microscope.
