A phase-contrast microscope is a type of light microscopy that intensifies contrasts of

transparent and colourless objects by influencing the optical path of light. The phase-
contrast microscope has the ability to show components in a cell or bacteria, which would
otherwise be very difficult to see in a basic light microscope.

The idea of phase-contrast microscopy was first described in 1934 by Dutch physicist Frits
Zernike. Upon realization of the importance of the phase-contrast microscope, Zernike was
awarded the Nobel Prize in Physics in 1953.

The phase-contrast microscope operates on the basis of the fact that when light passes via a
transparent part of a specimen, it travels slower and tends to be shifted compared to the
uninfluenced light.

However, this difference in phase is not visible to the human eye. A transparent phase-plate
in the phase-contrast microscope assists in increasing the change in phase to half a
wavelength, thus causing a difference in brightness, and leading to the transparent object
shining out in contrast to its surroundings.

In biological and medical research, transparent and colorless components in a cell can be
clearly studied by using the phase-contrast microscope. Cell division is no longer a mystery,
as the phase-contrast microscope can be used to examine the whole process in detail.

cilia and flagella can be viewed with bright field microscopy, but the sharp contrast can only
be seen via a phase-contrast microscope. Likewise, clear details of the amoebae can be seen
via a phase-contrast microscope.

High-contrast images of transparent specimens, such as micro-organisms, thin tissue slices,

living cells in culture, latex dispersions, lithographic patterns, glass fragments, and sub-
cellular particles, like nuclei and organelles, can be viewed in detail.

Dark-field microscopy is ideally used to illuminate unstained samples causing them to appear
brightly lit against a dark background. This type of microscope contains a special condenser
that scatters light and causes it to reflect off the specimen at an angle. Rather than
illuminating the sample with a filled cone of light, the condenser is designed to form a hollow
cone of light. The light at the apex of the cone is focused at the plane of the specimen; as this
light moves past the specimen plane it spreads again into a hollow cone. The objective lens
sits in the dark hollow of this cone; although the light travels around and past the objective
lens, no rays enter it.

The entire field appears dark when there is no sample on the microscope stage; thus the name
dark-field microscopy. When a sample is on the stage, the light at the apex of the cone strikes
it. The rays scattered by the sample and captured in the objective lens thus make the image.

Samples observed under dark-field microscopy should be carefully prepared since dust and
other particles also scatter the light and are easily detected. Glass slides need to be thoroughly
cleaned of extraneous dust and dirt. It may be necessary to filter sample media (agar, water,
saline) to exclude confusing contaminants. Sample materials need to be spread thinly; too
much material on the slide creates many overlapping layers and edges, making it difficult to
interpret structures.

Dark-field microscopy has many applications in microbiology. It allows the visualization of

live bacteria, and distinguishes some structure (rods, curved rods, spirals, or cocci) and
movement.

Electron Microscopy

Electron microscopy uses magnetic coils to direct a beam of electrons from a tungsten
filament through a specimen and onto a monitor.

Electron microscopy uses a beam of electrons as an energy source. An electron beam has an
exceptionally short wavelength and can hit most objects in its path, increasing the resolution
of the final image captured. The electron beam is designed to travel in a vacuum to limit
interference by air molecules. Magnets are used to focus the electrons on the object viewed.

There are two types of electron microscopes. The more traditional form is the transmission
electron microscope (TEM). To use this instrument, ultra-thin slices of microorganisms or
viruses are placed on a wire grid and then stained with gold or palladium before viewing, to
create contrast. The densely coated parts of the specimen deflect the electron beam and both
dark and light areas show up on the image. TEM can project images in a much higher
resolution—up to the atomic level of thinner objects.
 The second and most contemporary form is the scanning electron microscope (SEM). It
allows the visualization of microorganisms in three dimensions as the electrons are reflected
when passed over the specimen. The same gold or palladium staining is employed.

Electron microscopy has multiple applications. It is ideal to:

 explore the in vivo molecular mechanisms of disease;

 visualize the three dimensional architecture of tissues and cells;

 determine the conformation of flexible protein structures and complexes;

 observe individual viruses and macromolecular complexes in their natural biological

context.

There are several kinds of sterilization equipment using different technology and
processes, according to the application.

 Steam Sterilizer.
 Dry Heat Sterilizer.
 ETO Sterilizer.
 Sterilizing Tunnel.
 CIP System.
 SIP System.

Steam sterilizers (also referred to as autoclaves) are an essential part of the decontamination
and sterilization process performed by sterile processing departments (SPD) in healthcare
facilities.

Dry heat sterilization (killing or removal of all microorganisms, including bacterial spores)
technique requires longer exposure time (1.5 to 3 hours) and higher temperatures than moist
heat sterilization. Various available methods of dry heat sterilization are; hot air
oven, incineration, flaming (wire loop) etc.
Sterilizing by dry heat is accomplished by conduction. The heat is absorbed by the outside
surface of the item, then passes towards the centre of the item, layer by layer. The entire item
will eventually reach the temperature required for sterilization to take place.

Ethylene Oxide (EtO) is a common gas used for low temperature sterilization. It is a
colorless, poisonous gas that attacks the cellular proteins and nucleic acids of
microorganisms. It is most commonly used to sterilize instruments with long lumens such as
endoscopes and all materials that have to be sterilized but cannot withstand higher
temperature. EtO process temperatures from 25 - 55°C are used. A lower temperature results
in a less efficient process which leads to a longer exposure time.

A sterilizing tunnel for pharmaceutical containers has an entry zone, a heating zone, and two
cooling zones. The individual zones contain ventilating fan units that generate air currents,
which serve to heat and cool the containers that a transport apparatus conveys through the
sterilizing tunnel. The heating and cooling units, which operate using the principle of heated
and cooled air flow, is supplemented with radiant heat-generating unit. The sterilizing tunnel
makes it possible to achieve a uniform heating and cooling of the containers, preventing
stress cracks in the containers and permitting intentional heating that makes it possible, for
example, for the containers to be coated with a substance.

The cleaning requirements are best met with Cleaning-in-Place (CIP) systems.

CIP systems offer fast, efficient and reliable cleaning of all types of process plant. It's a
method which cleans complete items of plant equipment or pipelines circuits without
dismantling the equipment.

CIP systems are divided in different operations:

1- Flushing in order to eliminate residues

2- Alkaline cleaning operation: alkaline detergents dissolve fat and proteins, and cleaning
where harder deposits have occured

3- Intermediate water rinse

4- Acidic cleaning operation: for neutralising the caustic remaining on the surfaces of the
plant. The acidic detergents remove mineral deposits in the equipment (especially warm areas
like in the pasteurizer)

5- Final water rinse: Cold water purges out the residual acid solution
CIP is a closed system where recirculating cleaning solution is applied (often with nozzles)
that cleans, rinses and sanitises equipment. The CIP system is usually automatically
controlled and the cleaning sequences are given the optimum timing for efficient cleaning of
all parts of the plants.

Sterilization-In-Place (SIP) are systems designed for automatic cleaning and disinfecting
without major disassembly and assembly work. We design, develop, manufacture, supply and
install Mobile and Fixed CIP & SIP Units for sanitization and sterilization. The units are
custom made, modular, skidded in automated or semi-automated Models as per the required
time cycle for cleaning and sterilization as a part of cGMP requirements from portable to
large fixed Multi-Tank system.

Disinfectants: Disinfectants are antimicrobial agents that are applied to non-living objects to
destroy microorganisms. The process of killing the microbes is called disinfection. It may be
defined as “cleaning of an article of some or all of the pathogenic organisms that cause
infection.

Antiseptics: Antiseptics (from Greek anti- against + septikos – putrefactive) are

antimicrobial substances that are applied to living tissue such as skin to reduce the possibility
of infection, sepsis, or putrefaction. Some antiseptics are true germicides, capable of
destroying microbes (bactericidal), whilst others are bacteriostatic and only prevent or inhibit
their growth.

Properties of Disinfectant and Antiseptic:

A perfect disinfectant would offer complete and full sterilization, without harming other
forms of life. It should not be expensive, and non-corrosive. Most disinfectants are also, by
nature are potentially harmful (even toxic) to humans or animals.

Disinfectants are frequently used in hospitals, dental surgeries, kitchens and bathrooms to kill
infectious organisms.

The disinfectant should be broad spectrum (eliminates bacteria, viruses, protozoa, fungi and
spores), non-irritating, non-toxic, non-corrosive and inexpensive. Selection should be based
on effectiveness against the potential pathogenic agent, safety to people, impact on
equipment, environment and expenses.
Evaluation of Disinfectant:
Evaluation of disinfectant by Phenol coefficient method

I. Sterility Test:
Sterility testing of pharmaceutical products is carried out during the sterilization-validation
process

1. Sterility Test Conditions:

The following sterility test conditions are required:

(i) Environmental Conditions:
It should be designated to provide environmental conditions which avoid accidental
contamination of the product during the test, i.e. conditions equivalent to those for the aseptic
preparation of pharmaceutical products. A suitable environment is a class A laminar
airflow cabinet located in a class B room. Regular microbiological monitoring of the
working area with contact swabs and settle plates should be carried out.

(ii) Culture Conditions:

Sterility testing involves testing of viable microorganisms on culture media. It follows
appropriate conditions for growth microorganism of on culture media. Factors affecting the
growth of microorganisms indicate the importance of selecting the most appropriate culture
conditions for sterility testing.

(iii) Factors affecting growth of bacteria and fungi:

A number of factors are responsible for growth of microorganisms. The major factors
affecting growth are nutrition, moisture, air, temperature, pH, light, osmotic pressure and the
presence of growth inhibitors which affect the growth of bacteria and fungi.

2. Culture Media used for Sterility Testing:

Culture media suitable for sterility testing must be capable of initiating and maintaining the
vigorous growth of a small number of organisms. The organism may be aerobic and
anaerobic bacteria and the lower fungi.

3. Sterility of the Media:

The media for sterility test must be sterile in order to eliminate the possibility of false
positives results obtained from contaminated media. This assurance is obtained by incubating
portions of the media at required temperature for 14 days. Media intended for detection of
bacteria are incubated at 30-35°C and for fungi at 20-25°C. Sterility is confirmed by the
absence of microbial growth.

Antibiotic Assay:
Microbial growth inhibition under standard conditions may be utilized for
demonstrating the therapeutic efficacy of antibiotics. Any subtle change in the antibiotic
molecule which may not be detected by chemical methods will be revealed by a change in the
antimicrobial activity.

There are two methods usually employed for antibiotic assay:

i. Cup-plate (or cylinder-plate) method

ii. Turbidimetric (or tube assay) method

II. Microbial Limit Tests (MLT):

Microbial quality of non-sterile pharmaceutical products can be controlled by the adopting of
one or two types of standard. The first is a limit of the total numbers of viable organisms in
a given weight or volume of liquid. It is known as total viable count (TVC) and another
is the total exclusion of specific pathogens.

Bactericidal

The main defining feature of a bactericidal substance is that these

antimicrobial treatments directly kill bacteria.

These agents “attack” microbes by affecting the cell wall, lipids, enzymes, or
protein synthesis within the cell – sometimes even completing a combination
of these mechanisms. By disrupting the cell wall str ucture of existing cells
and inhibiting the formation of new cells, bactericidal substances cause
bacterial cells to die off, therefore decreasing the amount found in the
individual affected.

Bacteriostatic: Bacteriostatic agents can achieve this by obstructing the

metabolic mechanisms of the bacterial cell, in most cases targeting the
protein synthesis. While doing this does not cause outright cell death, it
does effectively inhibit further growth and DNA replication of th e
bacterial cells.

When bacteriostatic agents are utilized, the treatment will regulate the number
of bacterial cells. While the bacteria will not be eliminated, their numbers
will not increase.