Microbiology, The Science The biology comes from bios referring to living

organisms and logy means the study of, thus biology is the study of living
organisms.Micro means very small, viewed by microscope Microbiology is the
study of very small living organisms called microorganisms or microbes, these
include bacteria, algea, protozoa, fungi and viruses.Because many scientist do
not consider viruses as living organisms, the terms infectious agents or infectious
particles are often used in reference to viruses. A disease causing
microorganisms called pathoger (3% of all known microbes.The others are non
pathogenic (97%), the beneficial microbes are 87%.Microbes live on and in our
bodies e.g. skin, in the mouth and intestine are known as indigenous microflora
(or indigenous microbiota). Some of them cause disease accidentally and known
as opportunistic pathogens (10%).Diseases caused by microbes are called
infectious disease.Ubiquitous microbes means that they are virtually found every
where in or on the body and in different environment of the globe.Many bacteria
and fungi are Saprophytes, which ai fertilization by returning inorganic nutrients
to the st Saprophytes break down dead and dying organic materials (plants and
animals) into nitrates, phosphates, carbon dioxide, water and other chemicals
necessary for plant growth.Saprophytes also destroy papers, feces and other
biodegradable matters, although they cannot break down most plastics or
glass.Nitrogen-fixing bacteria, that live in the root nodules of certain plants
called legumes are able to return nitrogen from the air to the soil in the form of
ammonia for use by other plants Introduction to Microbiology.The spread of
certain diseases from one person to another long ago suggested the existence of
invisible, transmissible agent of infection. Microscopic organisms (microbes) were
not seen, however, until Antony Van Leeuwenhock (1632-1723) made
microscopes with sufficient magnification, then after, the science of microbiology
began.Leeuwenhock observed motile microorganisms. ជ from a decayed tooth
under the microscope, ne observed major morphological classes of bacteria i.e.
spheres, rods and spirals as well as large microbes i.e. protozoa, algae and yeast.
In 1767 and after the convention of compound microscope, Linnaeus
distinguished 6 species of microbes assigned to one class, and 600 types were
figured in 1838. A growth medium or culture medium is a solid, liquid, or
semi-solid designed to support the growth of a population of microorganisms or
cells via the process of cell proliferation or small plants like the moss
Physcomitrella patens. Different types of media are used for growing different
types of cells.Liquid media are also called broths, they allow for uniform and
turbid growth of bacterial strains when incubated at 37ºC for 24hrs. The media is
used for the profuse growth of microorganisms and fermentation studies.
Examples include Tryptic soy broth, phenol red carbohydrate broth, MR-VP broth,
and nutrient broth.Culture media is a gel or liquid that contains nutrients and is
used to grow bacteria or microorganisms. They are also termed growth media.
Different cell types are grown in various types of medium. Nutrient broths and
agar plates are the most typical growth media for microorganisms.Media that
support the growth of many different microorganisms without distinguishing
genera or species are nutritive. In contrast, differential media allow several
different types of bacteria to grow, but also contain compounds that allow
microbial genera (or even species) to be visually differentiated. Viruses are
strict intracellular parasites of other living cells, not only of mammalian and plant
cells, but also of simple unicellular organisms, including bacteria (the
bacteriophages).Viruses are simple forms of replicating, biologically active
particles that carry genetic information in either DNA or RNA molecules enclosed
in a protein coat or capsid.Proteins frequently glycoproteins in the capsid
determine the specificity of interaction of a virus with its host cell.The capsid
protects the nucleic acid and facilitates attachment and penetration of the
host cell by the virus. Inside the cell, viral nucleic acid redirects the host's
enzymatic machinery to functions associated with replication of the virus.
In some cases, genetic information from the virus can be incorporated as
DNA into a host chromosome.In other instances, the viral genetic
information can serve as a basis for cellular manufacture and release of
copies of the virus. This process calls for replication of the viral nucleic
acid and production of specific viral proteins.
Spirochetes, also known as spirochaetes, are a distinct double-membrane,
Gram-negative bacteria that have spiralled or helically-coiled cells.
Spirochaetaceae family includes four genera: Spirochaeta, Cristispira,
Treponema, and Borrelia. Two of these genera—Spirochaeta and Cristispira—are
considered free-living and commensal, respectively. The other three genera—
Treponema, Borrelia, and Leptospira—contain pathogenic species.Spirochete
taxonomy defines three families. Family Spirochaetaceae includes a diverse
group of organisms that have adapted to a range of niches. At one extreme,
there are a large number of free-living Spirochaeta species.Spirochetes are the
etiological agents of several important animal and human diseases, such as
syphilis, Lyme disease and leptospirosis. Bacteria are the smallest (0.1 to 10 µm)
living cells.They have a cytoplasmic membrane surrounded by a cell wall; a
unique interlinking polymer called peptidoglycan makes the wall rigid.The simple
prokaryotic cell plan includes ncludes no no mitochondria, lysosomes,
endoplasmic reticulum, or other organellesIn fact, most bacteria are about the
size of mitochondria. Their cytoplasm contains only ribosomes and a single,
double-stranded DNA chromosome.Bacteria have no nucleus, but all the
chemical elements of nucleic acid and protein synthesis are present. Although
their nutritional requirements vary greatly, most bacteria are free-living if given
an appropriate energy source. They divide by binary fission and can be grown in
artificial culture, often in less than 1 day. Archaebacteria differ radically from
other bacteria in structure and metabolic processes; they live in environments
humans consider hostile (eg, hot springs, high salt areas) but are not associated
with disease. Ultrastructure of a Bacteria Cell Bacteria are single-celled
microorganisms with the absence of the nucleus and other cell organelles;
hence, they are classified as prokaryotic organisms. They are also very versatile
organisms, surviving in extremely inhospitable conditions. Such organisms are
called extremophiles.Bacteria are unicellular organisms that have a variety of
sizes, shape, and envelope structures. The minimal requirements are cytoplasm,
a cell membrane that sur- rounds the cytoplasm, and a DNA chromosome. A few
have internal structures such as vacuoles and storage bodies but none have true
organelles. nutritional requirements of bacteria .The bacterial cell contains
water (80% of total weight), proteins, polysaccharides, lipids, nucleic acids,
mucopeptides and low molecular weight compounds. For growth and nutrition of
bacteria, the minimum nutritional requirements are water, a source of carbon, a
source of nitrogen and some inorganic salts. Growth and cultivation of
bacteria Bacteria cultivation is a biological activity wherein microorganisms
multiply themselves in a predetermined culture media under laboratory
conditions. Whereas, microbial culture media are used to identify the types of
microorganisms and their abundance in culture media are being tested precisely.
Phases Bacterial colonies progress through four phases of growth: the lag phase,
the log phase, the stationary phase, and the death phase.Bacterial growth is
proliferation of bacterium into two daughter cells, in a process called binary
fission. Providing no mutation event occurs, the resulting daughter cells are
genetically identical to the original cell. Aerobic bacteria are those that require
oxygen to carry out metabolisms. They are in a large majority and make up 75%
of all the known types of bacteria. Most of them live on or near the surfaces of
plant or animal life. Symbiosis The relationship between aerobic bacteria and its
host is symbiotic, as it provides both populations with benefits such as defense
against other harmful organisms or nutrients for metabolism. This is called
mutualism and for aerobic bacteria, nutrients could include sugars, amino acids,
vitamins or even oxygen. Some aerobic bacteria can be found in animals such
as intestinal flora in humans or animals like cows and pigs. Ex Bacillus subtilis,
Bacillus fusiformis and Bacillus tenuis. Aerobic bacteria provide many benefits to
its host such as defense against other harmful organisms or nutrients for
metabolism. They can be found in the intestines of animals in cow’s and pigs.
One example is the acidophilians which provides the immune system with an
alternate pathway for defense against pathogenic organisms by breaking down
cell walls of microbes that can cause disease. Anaerobic bacteria are those that
do not require oxygen to carry out metabolisms. They are found in the extreme
environment, such as hot springs or sink holes like the Maracas, or anaerobic
environments where there is no oxygen such as in caves and crevices, often
created by petroleum. They break down other organic matter like dead insects
and animals and feed off it with hydrogen sulfide around them or produce it
themselves. Symbiosis The relationship between anaerobic bacteria and its host
is parasitic in nature, as it only provides benefits to the symbiont and provides no
benefits to its host. Ex Prevotella copri and Desulfovibrio vulgaris In these
conditions, the host does not contribute in any way to the symbiotic relationship.
This means that anaerobic bacteria do not provide any benefits to their hosts.
Difference Aerobic and anaerobic bacteria use fermentation to produce ATP
which is a major energy source for all living cells. The end result is carbon
dioxide plus hydrogen sulfide. The difference between aerobic and
anaerobic bacteria is similar to the difference between symbiotic and parasitic
relationships. Anaerobic bacteria provide benefits to their host in an environment
with low oxygen, such as hot springs or sinkholes. Aerobic bacteria on the other
hand are found in a wide range of habitats from soil to water; they have been
found even in the human intestines. ENRICHMENT MEDIA Basic principle is to
control the nutrients and culture conditions in such a way that it suits mainly to a
specific species y temperature, air supply, light, pH | When we assume low
amount of potential pathogens being present in the specimen, they have to be
enriched first, to multiply up the low number – e.g. serum bouillon, dextrose
bouillon, chopped meat bouillon | Promotes the growth of a particular organism
by providing it with the essential nutrients, and rarely contains inhibitory
substances to prevent the growth of normal competitors SELECTIVE MEDIA |
Used for growth of only selected microorganisms. Selection by y Adding
antibiotics, prevents the growth of other cells y Lacking amino acids y May
contain stains and color indicators (EMB) Eosin-methylene blue agar (EMB)
Contains methylene blue, toxic to Gram + bacteria, allowing only the growth of
Gram – bacteria |MacConkey agar (MCK) For Gram – bacteria | Buffered charcoal
yeast extract agar (BCYE) Selective for certain Gram - , for example Legionella. |
Mannitol salt agar (MSA) Selective for Gram + bacteria | Hektoen enteric agar
(HE) Shigella, Salmonella | Thiosulfate citrate bile sucrose (TCBS) Vibrio cholerae
DIFFERENTIAL MEDIA | Distinguishes one microorganism type from another
growing on the same media on a difference in the colony appearance y Color,
shape, growth pattern y Dyes in the medium, pH indicators | Eosin-methylene
blue agar (EMB) y Differential for lactose and sucrose fermentation | MacConkey
agar (MCK) Differential for lactose fermentation.Mannitol salt agar
(MSA) .Differential for mannitol fermentation Bacterial Isolation Introduction
There are approximately 10,000 named species of microbes. It is estimated that
there are between 10,000 and 100,000 more unidentified species for every
identified one. Not only are there many types of bacteria, there are a lot of
individual bacteria. A single spoonful of soil can have 100 million individual
bacteria. A scraping of your gums can yield 1 million bacteria per cm 2 (a cm2 is
about the size of your little fingernail). The bacteria in and on our bodies makes
up about 10% of our dry body weight.Most of the currently known species of
bacteria have been identified using traditional microbiological techniques such
as the gram stain reaction, morphology, and metabolic reactions. Bacteria rarely
live alone but in communities with other bacteria. This is true both in the
environment and in and on our bodies. This class focuses on the role of bacteria
in disease. Isolating a single bacterium species is the first step in identifying the
bacteria possibly responsible for a disease process.The first requirement for
physically isolating a bacterium is that it can be cultured in the laboratory. This
requires knowledge of optimal temperature for growth, optimal oxygen
requirements, and optimal nutritional needs. We work with a very limited
number of bacteria in this course. The bacteria we work with are also very easy
to culture in the lab. Most bacteria are not this agreeable!There are two main
ways to isolate organisms.1Streaking for isolation on an agar plate 2 The pour
plate method1 Streaking for isolation on an agar plate involves the successive
dilution of organisms until you have the cells at a low enough density that single
cells are physically isolated spatially to give rise to recognizable individual
colonies. In the pour plate method, you dilute your sample sufficiently before you
add it to molten cooled agar and then pour this mixture in a dish. The isolated
cells give rise to individual colonies growing in the agar itself. This technique can
be a little tricky. If the melted agar is too hot you kill all the bacteria. If the
melted agar is too cool you end up with a big lump in your Petri dish. The
streaking method yields individual colonies on the surface of the agar. This
technique is much faster and easier to master.Procedure . There are several
methods of streaking for isolation. The vast majority of our students have been
most successful with the quadrant method of streaking which is described below
1 Label your plate with your name, date, section, and organism.2
Use BSL2 procedures to obtain a loop-full of organisms from your tryptic soy
broth (TSB) tube. Refer to the aseptic technique protocol.Be sure that you have
adequately mixed your broth tube so the organisms are uniformly suspended in
the broth3 Recap your TSB tube 4 You can do the next part with your plate on
the lab benchmor holding it in your hand. You decide which works best. Lightly
drag your loop back and forth across the surface of the agar.The more you drag
the more bacteria you deposit.The general idea is to decrease the bacterial
concentration with each swipe.Four to five zigzags seems to work
well.Experiment with your different plates. Be sure to keep track of what you did
on each plate. 5 If you are using an incinerator, sterilize your loop. If you are
using plastic loops, discard your used loop in the cavicide container and obtain a
new sterile plastic loop.6 Do not go back into the original broth tube.7Touch your
loop to the agar surface against the far end of your first streak. Repeat by
dragging back and forth.Do not drag into the center of your plate.You should be
able to see the faint indentations of your streaking line on the agar
surface.8Using a sterile loop, repeat the procedure on your second streak.9Using
a sterile loop, repeat the procedure on your third streak. Zigzag the last part into
the center of the plate. You should end up with isolated colonies somewhere in
your last streak.9If you are using an incinerator, sterilize your loop. If you are
using plastic loops, discard your used loop in the cavicide container.10 Replace
the lid on your plate.11 Place your completed plates agar side up on the
incubation rack on the front desk in the incubate section. Staining techniques
Simple stain: Basic dyes (methylene blue or basic fuchsin) are used to provide
the color contrast, but imparts the same color to all the bacteria in a
smear.Negative staining: A drop of bacterial suspension is mixed with dyes
(India ink or Nigrosin). The background gets stained black where as unstained
bacterial/yeast capsule stand out in contrast.Impregnation methods: Bacterial
cells and structures are thickened by impregnation of silver salts on their surface
to make them visible, e.g. for demonstration of bacterial flagella and spirochetes
Differential stain: Two stains are used which impart different colors to different
bacteria or bacterial structures, which help in differentiating bacteria. Most
commonly employed differential stains are: Gram stain: Differentiates bacteria
into gram-positive and gram-negative groups.Acid-fast stain: Differentiates
bacteria into acid fast and non acid-fast groups. Albert stain: differentiates
bacteria having metachromatic granules from other bacteria that do not have
them gram srain Originally developed by Hans Christian Gram (1884).Gram
stain still remains the most widely used test in diagnostic bacteriology. Gram-
positive bacteria resist decolorization and retain the color of primary stain i.e.
violet.Gram-negative bacteria are decolorized and, therefore, take counterstain
and appear pink. Uses Identification of Gram-positive and Gram-negative.For
identification of Gram staining from bacterial culture helps in further
identification of bacteria.Helps in initiation of empirical treatment with broad
spectrum antibiotics can be started early before the culture report is
available.Gram stain helps in early presumptive identification of fastidious
organisms, such as Haemophilus.Gram stain gives a preliminary clue in
anaerobic culture (Clostridium).Helps in identification of Candida and
Cryptococcus spp Acid-fast stain Discovered by Paul Ehrlich and subsequently
modified by Ziehl and Neelsen. Acid fastness is due to presence of mycolic acid
in the cell wall. Methods Step1 (primary stain) - Carbol fuchsin (1%) for 5
minutes. Intermittent heating is done by flaming until the vaporize.Step2
(decolorization) - 25% sulfuric acid for 2–4minutes. Step 3 (counter staining) -
0.1% methylene blue slide for 30 seconds Viable Bacteria Count: Determines
the number of living bacteria only. A viable cell is defined as a cell which is able
to divide and form a colony. Common methods:1. Pour Plate Method2.Spread
plate Method3. Membrane Technique.Principle Viable Bacterial Count is one of
the most common methods, for the enumeration of bacteria. Serial dilutions of
bacteria are plated onto an agar plate either by spread plate method or pour
plate method. The plates. are incubated so that colonies are formed.
Multiplication of a bacterium on solid media results in the formation of a
macroscopic colony visible to the naked eye. The total number of colonies is
counted and this number multiplied by the dilution factor to find out the
concentration of cells in the original sample. Counting plates should have 30-300
colonies at least. 1.Spread Plate Technique Make a dilution series (a series of
sequential dilution) from a sample.Pipette out 0.1 ml onto the center of the
surface of an agar plate.Dip the L-shaped glass spreader into alcohol.Spread the
sample evenly over the surface of agar using the sterile glass spreader, carefully
rotating the Petri dish underneath at the same time.Incubate the plates at 37°C
until bacterial colonies grow.Calculate the CFU value of the sample. Once you
count the colonies, multiply by the appropriate dilution factor to determine the
number of CFU/ml in the original sample. Only plates with 30- 300 colonies are
statistical.2. Pour Plate Technique In this method, a fixed amount of inoculum
(generally 1 ml) from a broth/sample is further inoculated in the warm agar
medium. Warm agar (approx. 15mL) is then poured into the plate and mixed
well. After the solidification of the agar, the plate incubated at 37°C for 24-48
hours.Microorganisms will grow on the surface.Each (both large and small)
colony is carefully counted (using magnifying colony counter if needed). Each
colony represents a "colony-forming unit" (CFU). The number of microorganisms
present in the particular test sample is determined using the formula:CFU/mL
CFU x dilution factor /volume 3. Membrane filter technique Bacteria from
aquatic samples are trapped on membranes. Membrane filter placed on
appropriate nutrient medium.Incubate for 24-48 hours so that colonies grow on
membrane. Colony count determines the number of bacteria in the sample.
Sterilization is a meticulous and critical process employed in healthcare
facilities to completely eradicate all forms of life, particularly microorganisms like
bacteria, fungi, spores, and even viruses, from various surfaces, objects, or
liquids. This comprehensive procedure is achieved through a range of methods,
including the application of heat, chemicals, irradiation, high pressure, or
filtration. It is essential to distinguish sterilization from other related terms like
disinfection, sanitization, and pasteurization. Unlike these methods, which aim to
reduce but not entirely eliminate harmful microorganisms, sterilization ensures
that once an item has undergone this process, it is rendered entirely free from
any potentially hazardous biological agents, making it sterile or aseptic.
Sterilization is not to be confused with disinfection, a process that involves the
removal or killing of organisms that are capable of causing infections, although it
may not necessarily result in complete sterilization. Common disinfectants
include substances like phenol, formaldehyde, chlorine, and iodine. Sterilization,
on the other hand, goes the extra mile by achieving the total removal of all types
of microorganisms, including both vegetative and spore forms. This is
accomplished through a combination of physical and chemical techniques,
resulting in the elimination of approximately 106 log colonyforming units
Method of sterilization 1) physical method of microbial control aim to
eliminate or neutralize microorganisms through the use of extreme
temperatures, removal of water, radiation, sound waves, or filtration. These
techniques exploit the vulnerability of microorganisms to temperature variations,
desiccation, genetic mutations from radiation, cell breakdown through
sonication, and filtration to block their passage. Sterilization is the complete
eradication of all forms of microorganisms, while disinfection reduces them but
may not achieve complete sterility. Medical devices that contact sterile body
tissues or fluids must be sterilized to prevent disease transmission, with steam
sterilization being the preferred method for heat-resistant items. Understanding
these techniques is crucial in preventing the spread of pathogens in healthcare
settings. Sterilization removes or deactivates all life forms, while disinfection and
sanitization reduce them, and pasteurization only partially eliminates
microorganisms.two methods involve in physical method Dry heat
sterilization in microbiology is a method that relies on elevated temperatures,
typically exceeding 356°F (180°C), and does not involve moisture. This high
temperature effectively eliminates microorganisms through a process known as
destructive oxidation, which targets and breaks down essential cell components,
particularly proteins. This results in the death of the microorganisms. Maintaining
this temperature for an extended period, often around an hour, ensures the
destruction of even the most resilient bacterial spores Moist heat
sterilization, using high-pressure steam in devices like autoclaves, is a cost-
effective and fast method to eliminate harmful microorganisms on objects
without toxic chemicals. This widely used technique is crucial in healthcare for
sterilizing equipment, such as surgical tools, and relies on pure steam and
efficient air removal. Monitoring temperature and using indicators like Bowie-Dick
tape ensure its effectiveness. It's the go-to method for medical device
sterilization, known as steam sterilization. 2 Chemical sterilization is a vital
method used across various industries, including healthcare, food production,
and agriculture, to eliminate harmful microorganisms such as bacteria, fungi, and
viruses. This technique is particularly advantageous when other sterilization
methods like heat, high pressure, irradiation, or filtration are unsuitable for the
materials involved. Chemical sterilization offers several benefits, including
affordability, speed, efficiency in eradicating target microorganisms, and minimal
reliance on complex equipment. Chemical sterilization methods can be
categorized as liquid or gaseous. The choice between them depends on factors
like material type and heat sensitivity. The Food and Drug Administration (FDA)
regulates the chemicals employed in sterilization processes and tends to
recommend gaseous techniques for enhanced sterility assurance Radiation is a
non-thermal sterilization technique that eradicates microorganisms in various
products using gamma radiation, electron beams (beta particles), or ultraviolet
light. Unlike many sterilization methods, it doesn't rely on high temperatures.
This method has gained popularity since the late 1950s and is especially
valuable in medicine and healthcare, as it can effectively sterilize products like
tissue allografts, pharmaceutical packaging, and medical devices. two main
types of radiation 1) Ionizing radiation, such as gamma or X-rays, uses short-
wavelength, high-intensity radiation to damage microorganisms' DNA. Non-
ionizing radiation, like ultraviolet light, has longer wavelengths and lower energy,
making it suitable for surface sterilization. 2) Non-ionizing radiation can also
break down ozone in water to kill bacteria, though it requires subsequent
treatment to make the water safe for use. In medical device sterilization, ionizing
radiation is often used, with gamma rays from a cobalt 60 isotope source or
machine-generated accelerated electrons. Gamma irradiation is the preferred
choice when materials are sensitive to high temperatures but compatible with
ionizing radiation. It exposes packages to a 60Co source for a specified time,
typically at a dose of 25 kGy. 4 The mechanical method of sterilization, also
known as the filtration method in microbiology, involves the use of membranous
filters with small pores to effectively sterilize liquids or gases by excluding
particulate matter and microorganisms based on their size. This process consists
of three essential steps: sieving, adsorption, and trapping. 1. Sieving: Filtration
works like a sieve, where a porous membrane filter with specific pore sizes is
used. These pores act as a physical barrier, preventing particles and
microorganisms larger than the filter's pore size from passing through 2.
Adsorption: Contaminants, such as microorganisms and particles, are not just
physically blocked by the filter; they can also be adsorbed or attracted to the
filter material's surface. This helps to capture and immobilize the contaminants
3. Trapping: The matrix of the filter material itself plays a role in trapping
contaminants. Microorganisms and particles that are too large to pass through
the pores or have been adsorbed are effectively trapped within the filter
material, ensuring they do not pass through.

The validation process is the documented evidence that provides a high

degree of assurance of a desired result with predetermined compliance. The
term validation is widely used in pharmaceutical industries. This term comes
from the word “valid or validity, " meaning “legally defined”. The Food and Drug
Administration (FAD) first proposed the validation concept in the mid-1970s to
improve the quality of pharmaceutical products. Since a wide variety of
procedures, methods, or activities are validated to check and improve
their quality. 1. Analytical method validation: The purpose of analytical
validation is to verify that the selected analytical procedure will give reliable
results that are adequate for the intended purpose.There are different
parameters that come under analytical method
validation .Precision .Accuracy. .Repeatability .Reproducibility .Specification .Line
arity. Range. Detection limit 2. Process validation:This type of validation
demonstrates documented proof, which carries a higher degree of surety that
the process will consistently produce a product that meets all the predetermined
quality characteristics and specifications. The process validation also assures the
repeatability of the process and decreases the risk of manufacturing problems,
leading to an increase in output of predetermined quality. On the bases of the
stage of production under process validation, 3. Cleaning validation provides
documented setup with a high degree of surety that particular
system/equipment or part of the equipment is consistently clean-up to
predetermined quality and acceptable limits. Pharmaceutical products are
contaminated by a variety of substances such as lubricants, airborne materials,
prepared product residues, and microbes. Hence, an adequate cleaning
procedure plays an important role to prevent contamination and cross-
contamination. 4. Equipment validation is an established documented setup
that proves any equipment works correctly and leads to accepted and accurate
results (predetermined results). The process of equipment validation is based on
the principle that equipment must be designed, constructed, maintained, and
adapted to perform the operations which are to be carried out. Equipment is the
basic component of pharma industries; therefore, before performing a process in
pharma industries, it becomes primarily important to issue equipment validation
(documented evidence of equipment). Water disinfection is a technique
designed to destroy or inactivate most microorganisms in wastewater.The
technique of disinfection is a last step in water treatment, which is enforced
directly to waste water for the control of many pathogens which cause harm to
all living organisms. From many years chlorine and its constituents were used as
disinfectants to control odors and various diseases in wastewater. Application of
disinfectants inactivates the pathogens by destroying the cellular structure of
microbes or disrupts their metabolism and makes them unable to grow or
reproduce. In case of bacteria, inactivation means the inability of the bacteria to
divide and form colonies. Inactivation makes viruses unable to form plaques in
host cells. Protozoan Cryptosporidium oocysts become unable to multiply and
thus prevent the spread of infection in a host cell.There are two kinds of
disinfection: Primary disinfection: This kind of disinfection achieves the
desired level of microorganism kill or inactivation .Secondary disinfection: This
kind of disinfection not only achieves the desired level of disinfection but,
maintains a residual disinfectant concentration in the finished water that
prevents the re-growth of microorganisms. Disinfectants are the substances
which are applied to biotic or abiotic surfaces such as directly on the skin, in
bathrooms, kitchens, or in production facilities, but may also be added to for
example drinking water or swimming pool water.Disinfecting agents are
registered by the Environmental Protection Agency (EPA) as “antimicrobials” and
are substances used to control, prevent, or destroy harmful microorganisms (i.e.,
bacteria, viruses, or fungi) and must provide a 99.9 percent inactivation of
microbes, their cysts and enteric viruses to protect health. When a killing action
is implied, the suffix –cide (e.g. biocide, bactericide, virucide, sporicide) is used,
while –static (e.g. bacteriostatic, virostatic, sporostatic) is added when an
organism’s growth is merely inhibited or it is prevented from multiplying.
Properties of Disinfectants Disinfectants should have wide spectrum of
activity, competent to destroy microbes, should be operating in the presence of
organic matter, effective in any pH, have high penetrating power, non-toxic, non-
allergenic, non-irritative or non-corrosive, should not leave non-volatile residue
or stain, efficacy should not be lost on reasonable dilution, should not be
expensive and must be available easily. High-Level Disinfectants – These
types of disinfectants kills vegetative micro organisms and inactivates viruses,
but not necessarily high numbers of bacterial spores. Such disinfectants are
capable of sterilization when the contact time is relatively long (6 to 10 hours).
As high-level disinfectants, they are used for relatively short periods of time (10
to 30 minutes). These chemical germicides are potent sporicides and in the
United States, are classified by the FDA as sterilant/disinfectants. They are
formulated for use on medical devices, but not on environmental surfaces such
as laboratory benches or floors.Intermediate-Level Disinfectants – These
disinfectants kill vegetative microorganisms, including Mycobacterium
tuberculosis, all fungi, and inactivate most viruses. Chemical germicides used in
this procedure often correspond to Environmental Protection Agency (EPA-
approved “hospital disinfectants” that are also “tuberculocidal.” They are used
commonly in laboratories for disinfection of laboratory benches and as part of
detergent germicides used for housekeeping purposes.Low-Level
Disinfectants – These types of disinfectants kill most vegetative bacteria
except M. tuberculosis, some fungi, and inactivates some viruses. The EPA
approves chemical germicides used in this procedure in the US as “hospital
disinfectants” or “sanitizers. Immunity can be defined as the body’s ability to
defend against infections. Its a way of protecting the body against an infectious
disease. As immune system recognizes an antigen, it attacks it. The phrase
‘immunity’ derives from the Latin immunitas, the felony fame of Roman city-states
granted immunity from paying tributes to Rome or to people free of municipal
duties; the foundation munis relating to alternate and (ex)changeable goods. This
is the direct starting place of the criminal which means ‘immunity from
prosecution’. However, withinside the first century, Lucan (De Bello Civile) had
already used the phrase metaphorically to explain the Psylli of North Africa as
resistant to the bites of venomous snakes. Biological immunity can check with
constitutive bodily innate mechanisms, which include the bodily safety afforded
towards contamination via way of means of the skin. The interest of herbal killer
(NK) cells towards virus-inflamed cells or the herbal resistance of mice to diphtheria
toxin due to the absence of a receptor for that toxin. Immunity also can be innate
however inducible, as withinside the antiviral country prompted via way of means
of publicity to double-stranded RNA (dsRNA). Finally, immunity to precise microbes
may be obtained throughout the life of the person via way of means of
contamination or vaccination. Innate immunity: Everyone is born with innate
(or natural) immunity, a type of general protection. For example, the skin acts as
a barrier to block germs from entering the body. And the immune system
recognizes when certain invaders are foreign and could be dangerous.Adaptive
immunity: Adaptive (or active) immunity develops throughout our lives. We
develop adaptive immunity when we’re exposed to diseases or when we’re
immunized against them with vaccines.Passive immunity: Passive immunity is
“borrowed” from another source and it lasts for a short time. For example,
antibodies in a mother’s breast milk give a baby temporary immunity to diseases
the mother has been exposed to.The immune system takes a while to develop and
needs help from vaccines. By getting all your child’s recommended vaccines on
time, you can help keep your child as healthy as possible Function Passive and
Active Immunity Immunity to a disease is achieved through the presence of
antibodies to that disease in a person’s system. Antibodies are proteins produced
by the body to neutralize or destroy toxins or disease-carrying organisms.
Antibodies are disease-specific. For example, a measles antibody will protect a
person who is exposed to measles disease but will have no effect if he or she is
exposed to mumps. Active Immunity results when exposure to a disease
organism triggers the immune system to produce antibodies to that disease. It can
be acquired through natural immunity or vaccine-induced immunity.Natural
immunity is acquired from exposure to the disease organism through infection with
the actual disease. Vaccine-induced immunity is acquired through the introduction
of a killed or weakened form of the disease organism through vaccination. Either
way, if an immune person comes into contact with that disease in the future, their
immune system will recognize it and immediately produce the antibodies needed
to fight it. Active immunity is long-lasting, and sometimes life-long. Passive
immunity is provided when a person is given antibodies to a disease rather than
producing them through his or her own immune system. A newborn baby acquires
passive immunity from its mother through the placenta. People can also get
passive immunity through antibody-containing blood products such as immune
globulin, which may be given when immediate protection from a specific disease is
needed. The major advantage of passive immunity is that protection is immediate,
whereas active immunity takes time (usually several weeks) to develop. However,
passive immunity lasts only for a few weeks or months. Only active
immunity is long-lasting. endotoxins are located in the outer membrane of the
bacteria. It is recommended as a cell-associated substance responsible for the
structure of bacteria. Endotoxins are also known as Lipopolysaccharides or LPS.
The LPS are found on the outer surface of the Gram-negative bacteria. The LPS
discharges at the bacterial cell death. In this, the toxicity is related to the lipid
component, on the other side immunogenicity is related to polysaccharide
components. The Lipopolysaccharides gives rise to various inflammation and
activates complement by the alternative pathway. During the growth of the Gram-
negative bacteria, it produces a small amount of endotoxin. Endotoxins play an
important role in increasing the natural immunity. Exotoxin Exotoxins are proteins
or polypeptides. It acts at tissue which is away from the original point of bacterial
growth. Exotoxins are generally produced by bacteria or with the action of the cell.
It is released by bacteria into the surrounding. Usually, the exotoxins are produced
at the growth of the cells of bacteria. The toxin production is specific to a certain
family of bacteria that are responsible to produce disease. For example,
Corynebacterium diphtheria is responsible to produce diphtheria toxin, Clostridium
tetani are responsible to produce tetanus toxins.These are the poisonous strains of
the bacteria responsible for the production of toxins, on the other side
nonpoisonous strains are not at all responsible for such productions. Exotoxins are
considered to be the most dangerous and toxic substances. A booster dose is an
extra administration of a vaccine after an earlier (primer) dose. After
initial immunization, a booster provides a re-exposure to the
immunizing antigen. It is intended to increase immunity against that antigen
back to protective levels after memory against that antigen has declined through
time. For example, tetanus shot boosters are often recommended every 10
years, by which point memory cells specific against tetanus lose their function or
undergo apoptosis. The need for a booster dose following a primary vaccination
is evaluated in several ways. One way is to measure the level
of antibodies specific against a disease a few years after the primary dose is
given. Anamnestic response, the rapid production of antibodies after a stimulus
of an antigen, is a typical way to measure the need for a booster dose of a
certain vaccine. If the anamnestic response is high after receiving a primary
vaccine many years ago, there is most likely little
1. Schick test The scientific principle behind the Schick test is based on
antibody-antigen reactions .The bacteria is placed under the patient's
skin in a small amount of liquid during the procedure. This is called an
intradermal injection. The patient's body is watched to see if it makes
antibodies against the bacteria.If so,the patient is considered
immunized and said to have a"positive" result’ On the other hand, the
patient is said to have a "negative" result if there is no reaction. A
sterilized dose of diphtheria toxin (0.1 ml) is injected intradermally
along the forearm of the patient.The reaction of the injected toxin on
the cutaneous tissue is then examined for any changes or signs of
sensitivity. In case of a positive reaction, a follow-up vaccination is
done.As per the standard guidelines , the patient should not leave the
test site for around 15 minutes after the test. Schick test is done
for observing the initial reaction of the body to the toxin. When the test
is complete, the test results are recorded. The Schick test is an
antibody test. It is widely used to detect bacterial infections in an early
stage and in an uncomplicated manner. Here are the implications of the
Schick Test: Speed: The Schick test has the potential to offer a rapid
diagnosis. It also enables the initiation of timely treatment for
diphtheria and other bacterial infections.Accuracy: By detecting the
presence of diphtheria toxin in the patient’s body, it can accurately rule
out other infections and, thus, prevent delayed treatment or
misdiagnosis. Simplicity: The Schick Test is uncomplicated. It doesn’t
require complex procedures or sophisticated equipment to be
performed Accessibility We are often limited by resources. But this
inexpensive and accessible test helps us to overcome these
obstacles.Detection: The Schick test can detect diphtheria toxin even
at extremely low concentrations. It can successfully identify the risk of
other infections . Southern blots are used to determine the identity, size,
and abundance of specific DNA sequences. The southern blot protocol
begins with DNA extraction from the cells or tissues, which is then
enzymatically digested to produce DNA fragments. The fragments are
separated by size on an agarose or polyacrylamide gel via electrophoresis.
Smaller fragments will migrate farther on the gel than larger ones.
Following electrophoresis, the DNA on the gel is transferred to a nylon
membrane. The membrane is incubated with a nucleic acid probe that has
a sequence homologous to the target sequence and is labeled with
radioactivity, fluorescent dye, or an enzyme capable of generating a
chemiluminescent signal. Hybridization of complementary sequences
occurs during incubation, and the unhybridized probe is removed by
washing with buffer. The fully hybridized labeled probe molecules will
remain bound to the blot. Detection methods differ based on the probe
label; radiolabeled probes are visualized with X-ray film or
phosphorimaging, and enzymatically labeled probes are visualized with
chemiluminescent substrate. 1DNA isolation 2 Restriction digestion 3 Gel
electrophoresis 4 Transfer 5 Pre-hybridization 6 Hybridization: Western
blots are used to determine the identity, size, and abundance of specific
proteins within a sample. The western blot protocol begins with sample
lysate preparation from tissue or cell culture and separation on a
polyacrylamide gel via electrophoresis. The separated proteins are then
transferred to a nitrocellulose or polyvinylidene difluoride (PVDF)
membrane. The membrane is incubated with a blocking agent to prevent
nonspecific binding, followed by incubation with a primary antibody to
bind the protein of interest. There are two detection methods, direct and
indirect. Direct detection (Figure 2) relies on a labeled primary antibody,
whereas indirect detection requires a primary antibody directed against
the target protein, and a secondary antibody directed against the
immunoglobin class or subclass of the primary antibody’s species (Figure
3). Visualization methods include colorimetric assays in which a colored
precipitate is produced, chemiluminescence, and fluorescence widal
Donor sera were screened by slide agglutination with Salmonella
enterica serotype Typhi O and H antigens (Difco). The positive donor sera
and all patients' sera were serially diluted in tubes with 08.5% NaCl from
1/50 to 1/6,400, and antigens (H and O) were added. The tubes were
incubated at 37°C for 2 h and then at room temperature overnight and
examined for agglutination by an agglutinoscope. The Widal test was
performed for group 1,
Microbial Assay Methods There are several methods of performing a microbial
assay. These include the disc diffusion method (otherwise known as the
cylindrical cup plate method), the tube assay (or turbidimetric) method, the
urease assay, and the luciferase assay. These methods have different uses. The
cylindrical cup plate method involves using circular plates of nutrient agar
inoculated with a susceptible test organism. Some characteristics of the cup
plate method include the selection of inoculant concentration to obtain suitable
dose-response and sharp inhibition zones at different concentrations of the
standard., keeping plates on a flat surface to ensure equal distribution of
inoculant, and incubating plates at a suitable temperature over a set
time.Turbidimetric methods differ from the commonly used cylindrical cup plate
assay by using tubes. Controls that are not inoculated with the target compound
(usually antibiotics) are used to set the optical apparatus used to measure
growth. When the tubes have been incubated, growth is arrested by adding
formaldehyde R, or the opacity of tubes can be measured at a set incubation
time. Appropriate statistical methods are used to calculate potency. Culture
Media Used Different culture media can be used in a microbial assay. The type
of media selected depends on the microbe being used in the assay, for example,
anaerobic bacteria, aerobic bacteria, or fungi. A fluid thioglycollate medium is
used for anaerobic bacteria but can also be used for aerobic bacteria. Soyabean-
casein digest is a culture medium for aerobic bacteria and fungi. Additionally,
alternative thioglycolate medium can be used for viscous and turbid products as
well as in devices that possess tubes with small lumina. Culture media must be
produced under strict conditions to ensure sterility so that the results obtained
by the microbial assay on the potency of target compounds are error-free .
Vaccines and serum therapy have led to a significant decrease and control of
infectious diseases, but effective immunization remains elusive. Traditional and
cutting-edge methods, as well as various platforms, for producing vaccines, have
been used. The urgent need for vaccination has been brought home once more
by the recent global spread of highly contagious diseases like Ebola, MERS, and
SARS-CoV-2. Due to the complexity of the immunology of infectious diseases and
a lack of knowledge, therapeutic vaccines for certain diseases are not yet
available. However, next-generation platforms like DNA-derived vaccines,
messenger RNA, virus-like particles, and recombinant technology offer an
exciting and promising way to make vaccines, as they are cheap, safe, and
effective and can be used by a large number of people quickly. These platforms,
in contrast to conventionally derived vaccines, are likely to provide effective
solutions for some infectious but noncurable disease like human
immunodeficiency virus and noninfectious diseases like cancer. To combat the
existing and emerging public health threats, increased funding and careful
monitoring of new data are needed. The World Health Organization brings
together international experts in specific fields through its biological
standardization programme to develop and revise specific recommendations for
the production and quality control of vaccines of major international public health
importance. Authoritative, harmonized guidelines and recommendations, for use
by manufacturers and regulatory authorities, are published in the reports of ECBS
meetings in the WHO Technical Report Series. B12 Microbiological assay has
been the most commonly used assay technique for foods and has been used for
the majority of available food table data. It relies on the specific requirement for
vitamin B12 by certain bacterial organisms (e.g. Lactobacillus delbreueckii) to
enable their growth in a supporting medium.Large scale industrial production of
vitamin B12 occurs via microbial fermentation, predominantly utilizing
Pseudomonas denitrificans, Propionibacterium shermanii, or Sinorhizobium
meliloti .The turbidimetric methods in routine use at two laboratories for the
microbiological assay of vitamin B12 have been compared. Attempts were made
to standardize some major parts of the method, i.e., assay design, test strain
(Lactobacillus leichmannii), test medium, and reference standard. B2 The
microbial source for Vitamin B2 is Ashbya gossypii, Bacillus subtilis and Candida
spp. The microbial source for Vitamin C is Aspergillus niger. Explanation: Vitamins
are the organic compounds required in trace quantities Vitamin B2, also called
riboflavin, is one of 8 B vitamins. All B vitamins help the body to convert food
hese B vitamins, often referred to as B-complex vitamins, also help the body
metabolize fats and protein.
Typhoid fever is a life-threatening infection caused by the bacterium Salmonella Typhi. It is usually
spread through contaminated food or water. Once Salmonella Typhi bacteria are ingested, they
multiply and spread into the bloodstream. symptoms Fever that starts low and increases throughout
the day, possibly reaching as high as 104 degrees Fahrenheit (40 degrees Celsius).Chills.Headache.
Weakness and fatigue.Muscle aches.Stomach pain.Diarrhea or constipation. Rash. Typhoid is caused
by the bacteria S. typhi. It spreads through food, drinks, and drinking water that are contaminated
with infected fecal matter. Washing fruit and vegetables can spread it as well if the water is
contaminated. Some people have typhoid without experiencing any symptoms. Others continue to
harbor the bacteria after their symptoms have gone. Sometimes, the disease can appear again.
People who test positive for typhoid may not be allowed to work with children or older adults until
medical tests are negative. Treatment Typhoid fever can usually be treated successfully with a course
of antibiotic medicine. The infection can usually be treated at home, but you may need to be
admitted to hospital if it's severe. Antibiotics that may be given for typhoid fever are:
Fluoroquinolones. These antibiotics, including ciprofloxacin (Cipro), may be a first choice. They stop
bacteria from copying themselves. Tuberculosis (TB) is a serious illness that mainly affects the lungs.
The germs that cause tuberculosis are a type of bacteria. Tuberculosis can spread when a person
with the illness coughs, sneezes or sings. This can put tiny droplets with the germs into the air.
Common symptoms of TB•Prolonged cough• Chest pain• Weakness or fatigue• Weight loss• Fever•
Night sweats.Often, these symptoms will be mild for many months, thus leading to delays in seeking
care and increasing the risk of spreading the infection to others. Causes Tuberculosis bacteria spread
through the air, just like a cold or the flu. You can get TB only if you come into contact with people
who have it. When someone who has TB coughs, sneezes, talks, laughs, or sings, they release tiny
droplets that contain the germs. If you breathe in these germs, you can get the disease. This is why
people who have active tuberculosis in their lungs or throat are more likely to infect others. You
usually can't spread TB if you have it in other areas of your body. TB is not easy to catch. You’re most
likely to get it from co-workers, friends, or family members with whom you spend lots of time
indoors. Tuberculosis germs don’t thrive on surfaces. You can’t get it from: Shaking
hands ,Kissing,Sharing food or drink,Sharing bed sheets, towels, or toothbrushes Toilet seats
treatment TB disease is curable. It is treated by standard 6 month course of 4 antibiotics. Common
drugs include rifampicin and isoniazid. In some cases the TB bacteria does not respond to the
standard drugs. In this case, the patient has drug-resistant TB. Treatment for drug-resistant TB is
longer and more complex.The course of TB drugs is provided to the patient with information,
supervision and support by a health worker or trained volunteer. Without such support, treatment
adherence can be difficult. If the treatment is not properly completed, the disease can become drug-
resistant and can spread. In the case of TB infection (where the patient is infected with TB bacteria
but not ill), TB preventive treatment can be given to stop the onset of disease. This treatment uses
the same drugs for a shorter time. Recent treatment options have shortened the duration to
treatment to only 1 or 3 months, as compared to 6 months in the past. Cholera is an acute, diarrheal
illness caused by infection of the intestine with the toxigenic bacterium Vibrio cholerae serogroup O1
or O139. An estimated 1.3 to 4 million people around the world get cholera each year and 21,000 to
143,000 people die from it. Caused Cholera is an acute diarrheal illness caused by infection of the
intestine with Vibrio cholerae bacteria. People can get sick when they swallow food or water
contaminated with cholera bacteria. Symptoms Cholera is an extremely virulent disease transmitted
through the ingestion of contaminated food or water (2). Cholera can cause severe acute watery
diarrhoea and the severe forms of the disease can kill within hours if left untreated. Treatment Oral
or intravenous hydration is the primary treatment for cholera. In conjunction with hydration,
treatment with antibiotics is recommended for severely ill patients. It is also recommended for
patients who have severe or some dehydration and continue to pass a large volume of stool during
rehydration treatment.

Hepatitis is a general term used to describe inflammation of the liver. Liver inflammation can be
caused by several viruses (viral hepatitis), chemicals, drugs, alcohol, certain genetic disorders or by
an overactive immune system that mistakenly attacks the liver, called autoimmune hepatitis. Causes
Heavy alcohol use, toxins, some medications, and certain medical conditions can cause hepatitis.
However, hepatitis is often caused by a virus. In the United States, the most common types of viral
hepatitis are hepatitis A, hepatitis B, and hepatitis C. symptoms Some people with hepatitis do not
have symptoms and do not know they are infected. If you do have symptoms, they may include:Fever
,Fatigue,Loss of appetite,Nausea and/or vomiting,Abdominal pain,Dark urine ,Clay-colored bowel
movements,Joint pain,Jaundice, yellowing of your skin and eyes Treatment for hepatitis depends on
which type you have and whether it is acute or chronic. Acute viral hepatitis often goes away on its
own. To feel better, you may just need to rest and get enough fluids. But in some cases, it may be
more serious. You might even need treatment in a hospital. There are different medicines to treat
the different chronic types of hepatitis. Possible other treatments may include surgery and other
medical procedures. People who have alcoholic hepatitis need to stop drinking. If your chronic
hepatitis leads to liver failure or liver cancer, you may need a liver transplant. Syphilis is a sexually
transmitted infection (STI) that spreads when you have vaginal, anal or oral sex with someone who
has the infection. A bacteria causes it. Antibiotic medication treats syphilis. Untreated syphilis can
lead to serious health problems, including blindness and damage to your brain, heart, eyes and
nervous system. symptoms vary depending on the stage of the infection. You’re most contagious in
the early stages, when you’re most likely to notice symptoms. During the first stage, one or more
sores develop on your genitals. You may not notice them or mistake them for a pimple or other skin
lesion.During the second stage, you may get a rash and experience flu-like symptoms, such as
fatigue, fever, sore throat and muscles aches.After the second stage, the symptoms of syphilis are
hidden (latent stage). Just because you don’t have symptoms doesn’t mean the infection is gone. The
only thing that cures the infection and prevents it from progressing is treatment with medication.
Causes The bacteria Treponema pallidum causes syphilis. An infected person spreads the bacteria
through vaginal, anal or oral sex. The bacteria can enter your body through your anus, vagina, penis,
mouth or broken skin. The bacteria continues to spread throughout your body, which can eventually
damage certain organs. Treatment Your healthcare provider treats syphilis with antibiotics.
Antibiotics are a type of medication that treats bacterial infections. Penicillin is the most commonly
used medication for syphilis. How much medication you need and how long you take it depends on
your syphilis stage and symptoms.You must finish all your antibiotics even if the sore or rash goes
away. It’s important to contact anyone you’ve had sex with within the last two years and let them
know they should be tested. Your healthcare provider will test your blood after syphilis treatment to
make sure the infection is gone. You can get syphilis again after getting treated, so be sure to practice
safe sex and get tested regularly if you have an increased risk of syphilis. HIV stands for human
immunodeficiency virus. HIV harms your immune system by destroying a type of white blood cell
that helps your body fight infection. This puts you at risk for other infections and diseases.Symptoms
he first signs of HIV infection may be flu-like symptomsFever.Chills.Rash.Night sweats (heavy
sweating during sleep).Muscle aches Sore throat.Fatigue. Swollen lymph nodes.Mouth ulcers.These
symptoms may come and go within two to four weeks. This stage is called acute HIV infection.
Treatment There is no cure for HIV infection, but it can be treated with medicines. This is called
antiretroviral therapy (ART). ART can make HIV infection a manageable chronic condition. It also
reduces the risk of spreading the virus to others. Most people with HIV live long and healthy lives if
they get ART as soon as possible and stay on it. It's also important to take care of yourself. Making
sure that you have the support you need, living a healthy lifestyle, and getting regular medical care
can help you enjoy a better quality of life.