UNIT- V

(CHAPTER-14)
Introduction and Typesof Spoilage
Points to be covered in this topic
1. INTRODUCTION

2.TYPES OF SPOILAGE
V Enzymatic spoilage:
Physical spoilage:
v Chemical spoilage:

3. SPOILAGE OF FRUITS AND VEGETABLES

4. FACTORS AFFECTING THE MICROBIAL

SPOILAGE OF PHARMACEUTICAL PRODUCTS

5. SOURCES AND TYPES OF MICROBIAL

CONTAMINANTS

6.PRESERVATION OF PHARMACEUTICAL
PRODUCTS USING ANTIMICROBIAL AGENTS

7.ASSESSMENT OF MICROBIAL
CONTAMINATIONAND SPOILAGE
OINTRODUCTION OF PHARMACEUTICAL SPOILAGE
Spoilage is waste or scrap arising from the
production process. The term is most commonly
applied to raw materials that have a short life
span, such as food used in the hospitality
industry.
Normal spoilage is the standard amount of waste or scrap that is
caused by production, and which is difficult to avoid. Abnormal
spoilage exceeds the normal or expected rate of spoilage.
For example, an overcooked meal cannot be served to a customer,
and so is instead classified as abnormal spoilage.
Hence, spoilage is a complex reaction in which a combination of
microbial and biochemical activities are interact. Legally they are
known as spoilage. These substandard drugs when proceed further
in order to be saleable as goods units are known as defectives.
Examples: Rancid meat, sour milk, moldy cheese etc.
The spoilage is mainly determined based on microbial colonization
form which is depends on the characteristics of products, method of
processing and the storage condition. Spoilage

TYPES OF SPOILAGE (Microbial spoilage) (Non- microbial spoilage

"Chemical Spoilage
Microbial Spoilage include the "Enzymatic soilage
contamination of Pharmaceutical "Physical spoilage
products with the microbes which lead to . Others spoilage
spoilage of the product affecting Drug safety
and quality, and is not intended for use.
Shortly Microbial Spoilage is defined as deterioration of pharmaceutical
products by the contaminant microbe.
 Enzymatic spoilage:
..M

It is based on enzymatic reactions

occurred in foods and changes in
chemical nature and resulted
rancidity.
This rancidity occurred by two types of chemical reactions hydrolytic and
oxidative rancidity.
For example: Triglyceride upon hydrolytic reaction in presence of lipase
enzyme forms glycerol and free fatty acid.
Further, Linolenic acid with various oxidative reactions forms 3-(E)
hexenal and 12-oxo-9 (Z) dodecenoic acid with the help of lipoxygenase
and hydroperoxide lyase.
ENZYMES FOODS SPOILAGE ACTION
Lipase Milk, oils Hydrolytic rancidity
Thiaminase Meat, fish Thiamine destruction
Peroxidases Fruits Browning
Proteases Egg, crab Reduction ofshelf life
Lipoxygenases Vegetables Destruction of vitamin A

Physical spoilage:
It occurs due to temperature, light, relative humidity and results in
mechanical damage of the food components.
For example: Oxidation of food occurs due to light and changes colour,
flavour and chemical nature, like greening of potatoes, sunlight flavour
in milk, loss of vitamin D, Eetc.
Chemical spoilage:
It is based on non-enzymatic chemical reaction occurred within the
foods and resulted change in flavor.
Chemical reactions in food are responsible for changes in the colour
and flavour of foods during processing and storage.
 Others spoilage: It occurs due to insects, rodents, birds, and other
animals and resulting in changes in colour, odour, and chemical nature.
Based on rate of spoilage, they are classified into three types like high
perishable, semi-perishable and stable or non-perishable spoilage.
() High perishable: Meat, fish, poultry, eggs, milk, fruits and vegetables
(ii) Semi-perishable: Potatoes, some apple varieties, nutmeats.
(iüi) Stable or non-perishable: Sugar, flour, dry beans.
Spoilage of fruits and vegetables
Fruits and vegetables are rich source of energy, body-building nutrients,
vitamins and minerals. Protected mechanically by the pectins which
constitute a protective gum between the cells and gives firmness.
Spoilage in fruits and vegetable starts with the hydrolysis of the pectin.
Once the pectinases have damage the structure of the fruit/vegetable, other
organisms start to contribute to the soft rot.
FOODS TYPE OF SPOILAGE MICROORGANISMS
SPOILAGE
Putrefaction Clostridium, Pseudomonas, Proteus
Fresh Meat
Souring Chromobacterium, Lactobacillus
Mouldy Penicillium, Aspergillus, Rhizopus
Cured Meat Souring Pseudomnonas, Micrococcus, Bacillus
Slimy Leuconostoc

Discolouration Pseudomonas
Fish
Putrefaction Chromobacterium, Halobacterium
Fresh Bacterial soft rot Pseudomonas spp.
vegetables and
fruits Gray mould rot Botryitis cinerea
Bitterness Pseudomonas spp.
Milk
Souring Lactobacillus thermophilus
Thermophilic Clostridium thermosaccrolyticum
acid
Canned food
sliminess Yeast, molds
* FACTORS AFFECTING THE MICROBIAL SPOILAGE OF
PHARMACEUTICALPRODUCTS
There are so many factors which affects Microbial spoilage. Some of these
factors reduce rate of spoilage where as some factors increases the rate
of spoilage.
These factors are related to nutritional requirement of micro
organisms, environment and nature of micro-organism
There factors must be studied to minimize the impact of spoilage.
Following factors affect the microbial spoilage of Pharmaceutical products.
pH
Extremes of pH prevent microbial attack. They grow at neutral pH,
therefore acidic or alkaline formulations are less susceptible to spoilage.
Around neutrality bacterial spoilage is more likely. with reports of
pseudomonas and related Gram-negative bacteria growing in antacid
mixtures, flavoured mouth washes and in distilled or demineralized
water. Above pH-8, the spoilage is rare for soap-based emulsions.
Products with low pH levels such as the fruit juice-flavoured syrups are
attacked by mould or yeast.
Yeasts are metabolizes of organic acids and raise the pH to levels where
secondary bacterial growth occurs. In food industry, low pH adjustment is
made to preserve foodstuffs only.
Storage Temperature
The actual storage temperature determines the spoilage by particular
types of microorganisms. Spoilage of pharmaceuticals occurs potentially
over the range of about 20°C to 60C.
Storage ina deep freeze at -20°C or lower is used
for long-term storage of foodstuffs and some 25°C
pharmaceutical raw materials, and dispensed total
parenteral nutrition feeds are stored in hospitals 5°C
for short periods at -20C to even further
mínimize the risk of spoilage.
A Nutritional Factors
Many spoilage microorganisms have simple nutritional requirements
and metabolic adaptability which enables them to utilize many
formulation components as substrates for biosynthesis, growth and also
trace materials contained in them.
The use of animal products and crude vegetable materials in a formulation
provides an additionally nutritious environment.
Demineralized water prepared by ion-exchange methods, contains
suffiient nutrients to allow significant growth of many water-bome
Gram-negative bacteria like Pseudomonas spp.
Water
It is the most important cause of the survival and growth of micro
organisms.
Some solute-rich medicines such as syrups appear to be 'wet', microbial
growth in them may be difficult since the microbes have to compete for
water molecules with the large numbers of sugar and other molecules of
the formulation which also interact with water via hydrogen bonding.
An estimate of the proportion of the non-complexed water in a
formulation available to equilibrate with any microbial contaminants and
facilitate growth can be obtained by measuring its water activity (Aw).
Vapour pressure of formulation
Ay =
Vapour press ure of water under similar condition

Redox Potential
The ability of microbes to grow in an environment is influenced by its
oxidation-reduction balance since they require compatible terminal
electron acceptors to permit function of their respiratory pathways.
The redox potential in viscous emulsion is high due to the high solubility
of oxygen ín most fats and oils.
It has a major influence on microbial stability of some formulations in
controlling the access of contaminants during both storage and use.
The most important dosage form such as parenteral drugs is protected
because of the high risks of infection by this route.
 Self-sealing rubber closures are used to prevent microbial entry into
multi-dose injection containers following withdrawals with a hypodermic
needle.
Wide-mouthed cream jars are replaced with narrow nozzle and flexible
screw capped tubes to remove the likelihood of operator-introduced
contamination during use.
Other factors affecting microbial spoilage of pharmaceutical products
include:
"Relative Humidity e Oxygen Availability
" Osmotic Pressure " Surface Tension

OsoURCES AND TYPES OF MICROBIAL CONTAMINANTSs

Flow of personnel Source and types of
Facility Material Waste microbial contaminants
HVAC
Microbes are very
Assembly
Equipments +Cleaning important part of our
Sources of Sterilazation environment.
Contamination
Personnel They are present almost
Water anywhere and
Utilities
Gases
everywhere.
Process Open vs Closed
Raw amterlal
Materials Reusable resin

Membrane Filter

So, contamination may occur to pharmaceutical products in large

scale manufacturing, in small scale hospital manufacturing or during
use by the patient.
Following are the different source of microbial contamination in
pharmaceutical products.
In large scale manufacturing: - In large scale manufacturing as well as
medium and small scale manufacturing contamination may occur from
following sources.
 Water
Water is a major source of contamination. Common water borne micro
organism like Pseudomonas, Achromo bacteria and other low demand
gram negative groups are present in portable water as well as in purified
water.
Ion-exchange column may be contaminated by water
source and micTO-Organism may multiply there to
contaminate purified water.
Raw materials
Pharmaceutical products are prepared from varieties of raw materials.
Clays and earth materials like Bentonite, kaolin ete may contain
anaerobia spores like Clostridium sp. Starch may contain coliform
batteria like E. Coli. Gums may contain actinomycetes.
Animal Products may contain a variety of bacterial like E. coli, Salmonella
sp ete.

Equipments
Equipments of manufacturing may contain microbes if it is not sterilized
properly. Grinder, blender, filter etc may contain non-speific and local
communities of micro-organism.
Containers
Containers may cause contamination if it is not sterile. In
hospital manufacturing -In hospital manufacturing water
and environment are the major source of contaminants.
In hospitals, water is stored in storage tank which may develop fungus,
bacteria and algi type of microbes.
Hospital air may be contaminated with pathogenic microorganism due to
the presences of infected patients and numerous visitors.
Herman source
Pharmaceutical products may be contaminated during use. Patient may
self contaminate his medicine.
Contaminants may travel to other patients through doctors nurses ete.
 OPRESERVATION OF PHARMACEUTICAL PRODUCTS
USING ANTIMICROBIALAGENTS
AntimicrTobial agents are those substances which can kill or inhibit
growth of micro-organism. These antimicrobial agents are included in
formulation in order to minimize levels of contaminated micro
organism. These antimicrobial agents are called preservatives. These
preservatives can generally prevent or kill low levels of contamination.
Preservatives are not used in those formulations which has low risk of
contamination and subsequent microbial growth.
Formulations which contains high level of acid, alkali, sugar etc may not
require preservative.
Formulations of antibiotics and other anti-microbial substances may not
require preservative.
& Airof the manufacturing area
Air is filled with billions of suspending particles and microbes. Fungus
spores, like Penicillium, mucor, Aspergillus ete.
Bacterial spores like Bacillus sp. etc are also present. These spores and
micro-organism may contaminate pharmaceutical products.
This type of contamination is minimized by practice of manufacturing in
clean room and in aseptic room under continuous flow of sterile air
through HEPA filter.
Personnel
Manufacturing staff may also contaminate

pharmaceutical products.
Personnel may be infected with various types of
infections like coliform bacteria, staphylococci,
strepto-cocci, Actino bacteria, Candida.
This types of contamination may be minimized by proper health check-up,
vaccination and hygiene of the personnel. Protective gear and pooper
training of the personnel may also minimize the contamination.
 3 TYPES OF MICROBIAL CONTAMINANT
Microbial contamination is broadly classiied in to direct contamination
and cross contamination (Flowchart 2).
(a) Direct contamination: Contamination occurred by microbial
components and poorly maintained heating, ventilation and air
conditioning system.
(b) Cross contamination: It is the process by which microbes are spread
indirectly from one to another through improper and unsterilized
equipment.
MicrobialContamination

Direct Cross
Contamination Contamination
-Physical -Physical
- Chemical - Chemical
- Biological -Biological
* DIRECT CONTAMINATION IS CLASSIEIED AS FOLLOWS
(a) Direct Physical contamination: Examples: Particles, fibres, metal parts
etc.

(b) Direct chemical contamination: Examples: Moisture, Gases, Vapors etc.

() Direct biological contamination: Examples: Microorganisms like
bacteria, viruses, molds, fungi etc.
* CROSS CONTAMINATION IS CLASSIEIED AS FOLLOWS:
(a) Physical cross contamination: Example: Leakage of oil seal from the
reactor.

(b) Chemical cross contamination: Examples: Moisture content is increased

when a product exposed to high relative humidity.
(c) Biological cross contamination: Example: Improper cleaning of
equipment, unclean equipments used for manufacturing process.
PHARMACEUTICAL PRODUCTS CONTAMINANTS
Plague vaccine Clostridium tetani
Serum vaccine Staphylococcus aureus
Thyroid tablets Salmonella muenchen

Antibiotic eye ointments Pseudomonas aeruginosa

Talcum powder Clostridium tetani
Saline solution Serratia marcescens
Antiseptic mouth wash Coliforms
Surgical dressings Clostridium species
Hand cream Klebsiella pneumoniae
ASSESSMENT OF MICROBIAL CONTAMINATION AND
SPOILAGE
The evaluation of a medicinal product's microbiological composition is
critical. A sterile product should be completely devoid of
microorganisms, as determined by a sterility test.
Non-sterile items, on the other hand, may include microorganisms. These
microorganisms have the potential to be both pathogenic and non
pathogenic.
Microorganisms like these can cause deterioration, which can pose
health risks. The overall number of microorganisms present in a product
must be minimaland below the allowable limit.
To detect the existence of certain bacteria, the types and characteristics
of the microbes should also be examined.
The evaluation of a medicinal product's microbiological composition is
critical. Asterile product should be completely devoid of microorganisms,
as determined by a sterility test.
Microorganisms like these can cause deterioration, which can pose health
risks.
 The overall number of microorganisms present in a product must be
minimal and below the allowable limit.
To detect the existence of certain bacteria, the types and characteristics of
the microbes should also be examined.
Non-sterile items, on the other hand, may include
microorganisms. These microorganisns have the
potential to be both pathogenic and non-pathogenic.
Tests to determine the total amount of microorganisms and types of
bacteria are called microbial limit tests.
The following tests are carried out to evaluate microbial contamination
and consequent deterioration.
Test of sterility -Some pharmaceutical items should be tested for sterility,
according to the Indian pharmacopoeia, the British pharmacopoeia, and
the United States pharmacopoeia. The procedures are comparable with
minor differences.
The tests prescribed by the Indian Pharmacopoeia are briefly reviewed.
There are two methods: direct inoculation and membrane filtering.
1. Direct inoculation -
In this procedure, a little amount of material is immediately introduced
to the pharmacopeia-specified culture medium. This inoculation
medium is then incubated for acertain amount of time.
The existence of growth implies the presence of a microorganism
derived from the sample. As a result, it is possible to determine that the
sample is not sterile. In the lack of any growth, a sample is said to be
sterile.

2. Membrane filtration
The material is filtered through a membrane filter and rinsed with diluting
solution in this procedure.
If microbes are present, they will be near the top of the filter paper. This
filter paper is now injected into the appropriate culture medium.
. If growth is detected, it shows that the product is not sterile.
 In both the above-mentioned methods, a positive and negative control
test must be performed.
Microbial limit test - The European Pharmacopoeia suggests assessing
microorganisms both qualitatively and quantitatively. The microbiological
Limit test is recommended by the United States Pharmacopoeia. It is
divided into two sections:
. () Total Aerobic Microbial Count
(ii) Test for Specific Microorganisms.
) Total Aerobic Microbial Count
In this approach, a defined amount of test sample (10gm) is combined
with a specific amount of peptone water (90ml).
This is called sample dilution. Dilutions of water-insoluble and fatty
compounds must follow precise procedures. The total microbial count is
determined using the following procedure:
To begin with, the sample is fltered through a membrane filter by mixing
10 mlof distilled water with 90 ml of peptone water.
After that, it is rinsed three times with
sterile peptone water. One filter paper
is incubated for 5 days at 30-350C in
Petri dish with Soyabean Caesin Digest
Agar medium. Colonies are counted to
ascertain bacterial count.
Another filter paper is immersed in Sabouraud Dextrose Agar Media and
incubated at 20-25°C for 5 days. The fungal count is then calculated.
Second, the sample plate count technique is examined. In this approach,
created dilution is directly transferred to four Petri dishes.
There are two for bacteria and two for fungus. 15 ml of Soyabean Caesin
Digest Agar medium is added to the first two Petri dishes. After 5 days of
incubation at 30-35°C, colonies are counted.
15 ml of Sabouraud Dextrose Agar Media was transferred to the
remaining two Petri dishes and incubated at 20-25°C. Colonies are tallied.
i) Test for Specific Microorganisms
Escherichia coli, Salmonella, Pseudomonas
aeruginea, and Staphylococcus aureus are idenified
using specific assays.
Mac-Conkey agar medium is used to culture E. coli.
Colonies are distinguished by their metallic gleam.
Salmonella colonies are detected as black or green on
bismuth sulphite agar medium.
Pseudomonas aeruginosa is recognized by growing it on cetrimide agar
medium. Colonies gradually turn a greenish colour.
Good Pharmaceutical Manufacturing Practice (GPMP):
Quality Control (QC) is that part of GPMP dealing with specification,
documentation and assessing conformance to specification.
Ahigh assurance of overall product quality is raised only from a detailed
specifcation, control and monitoring of all the stages that contribute to
the manufacturing process.
Parametric release is accepted as an operational rporer
alternative to routine sterility testing for batch
release of some finished sterile products where
the manufacturer can provide assurance that the
DGMP

product is of the stipulated quality, based on the

evidence of successful validation of the Moncd

manufacturing process and review of the

documentation on process monitoring carried
out during manufacturing.
º Quality Assurance (OA):
It is a combined scheme of management which embraces all the
procedures necessary to provide a high probability that a medicine will
conform consistently to a specified description of quality.
It includes formulation design and development (R&D), good
pharmaceutical manufacturing practice (GPMP), quality control (QC)
and post-marketing surveillance.
The risk of microbial infection and spoilage are raised from microbial
contamination during manufacture and storage and hence preservatives
are recommended further protection against environmental microbial
contaminants but it is relatively non-specific in their reactivity.
Laboratory tests are devised to challenge the product by used
'preservative challenge tests' where relatively large inocula of various
laboratory cultures are added to aliquots of the product and determine
their rate of inactivation by viable counting methods (single challenge
tests).
º Post-market Surveillance
It is most important stage to follow up a
medicine that is smooth floating in the Rkresuhanayala mey
nchenges
to the lFU.
market without any complain by the
customers.
A proper quality assurance system is included for monitoring in-use
performance and for responding to customer complaints.
These are constantly followed up in great detail in order to decide carefully
constructed and implemented schemes for product safety.
 UNIT- V
(CHAPTER- 15)
PRESERVATION OF PHARMACEUTICAL
PRODUCTS USING ANTIMICROBIAL AGENTS

Points to be covered in thistopic

1. INTRODUCTION
2. IDEAL PROPERTIES OF PRESERVATIVES

3. CLASSIFICATION OF PRESERVATIVES
CLASSIFICATION OF PRESERVATIVES BASED ON
MECHANISM OF ACTION

CLASSIFICATION BASED ON SOURCE

4.METHODS OF PRESERVATION
5. MICROBIAL STABILITY TEST

6. GROWTH OF ANIMAL CELLS IN CULTURE

7. TYPES OF ANIMAL CELL CULTURE

8. GENERAL PROCEDURE FOR CELL CULTURE

9. APPLICATION OF CELL CULTURES IN

PHARMACEUTICAL INDUSTRY AND
RESEARCH
OINTRODUCTION
Preservatives are the chemical substances used to improve or amplify
shelf life of drugs by decreasing or lowering the oxidation of active
ingredients and Excipients by reducing microbial production.
Preservatives are substances added to various pharmaceutical dosage
forms and cosmeticpreparations to prevent or inhibit microbial growth.
An ideal preservative would be effective at low concentrations against all
possible micro-organism, be nontoxic and compatible with other
constituent of the preparation and be stable for the shelf-life of the
preparation.

IDEAL PROPERTIES OF PRESERVATIVES

It should not be irritant.
It should not be toxic.
It should be physically and chemically stable.
It should be compatible with other ingredients used in formulation.
It should be act as good antimicrobial agent and should exert wide
spectrum of activity.
It should act in small concentrationi.e. it must be potent.
It should maintain activity throughout product manufacturing shelf life
and usage.
It must decrease the percentage of the microbes and prevent any re
growth They can be:
Microbiostatic& Microbiocidal in nature.
Some preservatives are ineffective with some microbial strains and should
be combined with others to be effective. Such as
Benzalkonium chloride, Organo-mercurial, Cetrimide, chlorhexidine and
3- cresol are combined

CLASSIFICATION OE Preservatives
Cationle Alcohols Phenolic Onganic aclds
PRESERVATIVES detergents compounds
I. Cationic detergents: Examples: Benzalkolium chloride, alkyl trimethyl
ammonium chloride.
II. Alcohols: Examples: Chlorbutanol, bronopol, phenyl and phenoxy
ethanol.
III. Phenolic compounds: Examples: chlorinated and isopropyl derivatives
of meta cresol.
IV. Organic acids: Examples: Salicylic acid, benzoic acid, acetic acid, lactic
acid, hydroxyl benzoicacid.
CLASSIFICATION OF PRESERVATIVES BASED ON MECHANISM OE
ACTION
1 Antioxidants:_The agents which prevent oxidation of active
pharmaceutical ingredient which otherwise undergo degradation due to
oxidation as they are sensitive to oxygen.
For example: Vitamin E,Vitamin C, Butylated-hydroxyanisole (BHA), Butylated
hydroxytoluene (BHT).
2. Antimicrobiall agents:They are the agents that are active against gram
positive and gram negative micro-organism which causes degradation of
pharmaceutical preparation, active in small inclusion level.
For example: Benzoates, Sodium benzoate, Sorbates etc.
3. Chelating agents:_They are the agents which form the complex with
pharmaceutical ingredient and prevent the degradation of pharmaceutical
formulation.
For example: Disodium ethylenediaminetetraacetic acid (EDTA),
Polyphosphates, Citric acid.
o CLASSIEICATION BASED ON SOURCE
i Natural Preservatives:_These preservatives are obtained by natural
sources that are plant, mineral and animal sources etc.
Example- Neem Oil, Salt (sodium chloride), Lemon, Honey.
ii. Artificial Preseryatives These preservative are man made by chemical
synthesis and active against various microorganisms in small concentration.
Example- Benzoates, Sodium benzoate, Sorbates, Propionets, nitrites.
* Eactoraffectingthe efficacyand availability ofpreservatives:
Temperature.
Chemical structure of preservatives
Capacity of preservatives.
Inoculum size.
Effect of pH.
Effect of containers and packaging.
Changes of concentration.
METHODS OF PRESERVATION
> Physical protection:
It is used for proper packaging of the pharmaceutical products under
aseptic condition or else there is a chance for microbial growth.
Operating persons are also an important factor for proper processing of
the products under aseptic environment.
> Preservative coating:
Aqueous raw materials used in the formulation
of paints and coatings create the perfect
environment for the growth of bacteria, fungi
and yeast. They can destroy valuable
pharmaceutical formulations.
Controlling these microorganisms helps increase efficiency, helps delivera
better end-use product and helps employees and consumers avoid contact
with spoilage microorganisms.
Biocides are necessary for protecting the integrity and functionality of
water-based paints and coatings from destruction by microbial
contamination as a result lengthening of product's shelf life and protect dry
film from algae, mold and mildew.
> Water proofprotection:
Packaging of the pharmaceutical products should be under water proof
protection because water favours the growth of microorganisms.
 Water vapour proofprotection:
This method is applicable for certain pharmaceutical products when they
are packing under proper care for minimizing microbial activity.
For 'dry' dosage forms with very low water activity (Aw) provides
protection against microbial attack. The moisture vapour properties of
packaging materials require careful examination.
Water vapour proofprotectionwith desiccant:
This method is also used for dry products that absorbed moisture from the
environment and is spoiled due to growth of microorganisms.
Packing should be proper with this method to minimize the microbial
growth and spoilage of the products.
Mode of Action: _Preservatives interfere with the growth,
multiplication and metabolism of the microorganisms by one or more
of the following mechanisms.
() Modifying the membrane permeability,
(ii) Denaturation of enzymes and other cellular proteins
(iii) Oxidation of cellular constituents
(iv) Hydrolysis.
For use in pharmaceutical products the antimicrobial preservatives
should be selected from those recommended in pharmacopoeias and
in minimum effective concentration.

Benzalkonium chloride Benzoic acid Benzyl alcohol

Butyl paraben Cetrimonium bromide Cetylpyridinium chloride
Chlorobutanol Chlorocresol Cresol

Ethyl paraben Methyi paraben Phenol

Phenoxyethanol Phenyl ethyl alcohol Phenylmercuric acetate

Phenylmercuric nitrate Potassium benzoate Potassium Sorbate

Propyl paraben Sodium Benzoate Sodium propionate

Sorbic acid Thimerosal Thymol
 Examples of antimicrobial preservatives commonly employed in
manufacturing of pharmaceutical products include: Methyl, ethyl,
propyl and butyl parabens; Sorbic acid, Na, K&Ca Sorbate; Benzoic acid,
Na, K and Ca benzoate; sodium metabisulfite, Propylene glycol, BHT
(butylhydroy toluene), BHA (Butylhydroxyanisol), benzaldehyde, essential
oils, phenol and mercury compounds. Parabens are among the most
commonly used antimicrobial preservatives.
MICROBIAL STABILITY TEST:
Microbial contaminants usually originate from two different sources during
production and filling and during the use of the cosmetics by the
Consumers.

It is necessary to carry out routine microbiologicalanalysis of each batch

of the finished product. The main potential pathogens in cosmetics are
Pseudomonas aeruginosa, Candida albicans, and Staphylococcus aureus.
These pathogens must not be detectable more than 0.1 g or 0.1 ml in a
cosmetic product.

SOME TESTS ARE AS FOLLOWS:

1. Screening test:
It is also known as plate count or dip slide method. This method is used to
detect aerobic bacteria in aqueous sample. Dip slide is coated on both
sides with a solid agar gel medium.
Asmall quantity of TTC (2,3,5-triphenyl tetrazolium chloride) is added
todetect aerobic bacteria in the sample.
The slide is dipped in to the aqueous solution for 10 seconds and excess
liquid is drained off from the slide. Then it is incubated at 35-37°C for 18
48 hours.
The colour appeared is compared with calibration chart. Aerobic bacteria
species grow on this medium and is detected by their ability to reduce TTC
dye to a red coloured Formosan dye. Developing bacterial colonies are
alter the TTC dye and are appeared as red spots
 Quantitative test:
Quantitative tests determine the actual count level of bacteria, molds
and yeasts in cosmetic products. This method is used for isolation of
microorganisms from cosmetic products include direct colony counts and
enrichment culturing.
Microbial stability studies are also carried out in Pharmaceutical products.
Raw materials play vital role in the product formulation. Pharmaceutical
raw material is defined as a substance that is used in the manufacturing of
pharmaceutical products.
During the manufacture of pharmaceuticals and cosmetics,
untreated raw materials are contaminated with
microorganisms beyond acceptable limits.
Hence it is necessary to control microbiological
contamination from raw materials under GMP regulations
in the pharmaceutical industry.
These microorganisms from plants (e-g. species of Erwinia,
Pseudomonas, Lactobacillus, Bacillus or Streptococcus)
such as gum acacia, Tragacanth, agar, powdered rhubarb, and
starches contains bacteria, which causes disease.
Water plays major role in product formulations because water is the
good media for microbial growth.
The water activity (Aw) of pharmaceutical and cosmetic products is the
measure of free water in the formulation. The amount of water that is in
free form is available to microorganisms for survival.
The measurement of Aw in pharmaceutical and cosmetic products predicts
the type and number of microorganisms responsible for product
degradation and maintains chemical stability.
Water activity is determined by dew point method and using electric
hygrometer (measure relative humidity).
 AntimicrobialEffectiveness Test
This test is used for estimating of preservative in a product. This test is
mainly used during development of formulation and in stability studies.
In this test, a product is inoculated with a controlled quantity of specific
microorganisms.
The test then compares the level of microorganisms found on a control
sample versus the test sample overa period of 28 days.
Test organisms are used for the purpose of challenging the preservative
system in a product. The suitable media are Soyabean Casein digest or
Sabouraud Dextrose Agar used for the test. The bacteria, yeast and mold
are used for this test.
A product is inoculated or contaminated with a number of organisms
between 1 * 105 (100,000) to 1 x 106 (1,000,000) colony forming units
(CFU) per ml of product. At various intervals, the product is tested to
determine its ability to control reproduction or destroy the
microorganisms.
Test and standard are incubated at 32.5 + 2.5C and 22.5 + 2.5°C for
bacteria and yeast respectively and the concentration of the
microorganisms in each of the standardized inoculum is determined by
plate count method.
Pharmaceutical Products: For testing purposes, the USP has divided
test articles into four separate categories:

Category 1 - Injections, other parenterals incuding emulsions, sterile nasal

products made with aqueous bases or vehicles.
Category 2 - Topicaly used products made with aqueous bases or vehicles,
non-sterile nasal products, and emulsions, incuding those
applied to mucous membranes.
Category 3 - Oral products other than antacids made with aqueous bases or
vehicles.

Category 4- Antacids made with an aqueous base.

 OGROWTH OF ANIMAL CELLS IN CULTURE
Cell culture is a technique which involves isolation of cells from animal/plant
body i.e. from their natural environment (in vivo) and practicing to grow
isolated cells in cellspecific media in plastic flask or petri dish in a controlled
environmental artificial condition (in vitro).
Cell culture means to keep cells alive and grow in an in vitro condition ina
nutritive media which are widely used for research and diagnosis of different
pathogens and to understand the function and mechanism of operation of
many cells.
Animal cell cultures are initiated by the dispersion of a piece of tissue
into a suspension of its component cells, which is then added to a culture
dish containing nutrient media.
In animal cells fibroblasts and epithelial cells are selected because these
cells grow very fast.
E
The plastic surface of dishes is used for cell culture. In 1907, Harrison
cultivated frog nerve cells in a lymph clot and observed the growth of
nerve fibres in-vitro for several weeks and hence he is recognized as the
father of cell culture.

$ TYPES OE ANIMAL CELL CULTURE:

Based on the number of cell divisions, cell culture is classifñed as primary
cell culture and cell lines. Cell lines can undergo finite or infinite cell
divisions.
(Animalcell culture

Primary cell Secondary cell

culture culture and cell line
Adherent Suspension Finite Continuous
cells cells cell lines cell lines
 Primary Cell Culture:
Thís cell culture is obtained from the cells of a host tissue. The cells
dissociated from the parental tissue are grown on a suitable container and
the culture thus obtained is called primary cell culture.
The culture is comprised of heterogeneous cells and most of the cells
divide only for a limited time. Based on their origin, primary cells grow
either as an adherent monolayer or in a suspension.
i Adherent Cells:
These cells are propagated as a monolayer and are anchorage
dependent. Monolayer cultures are defined as when the bottom of the
culture vessel is covered with a continuous layer of cells of one need to
be attached to a solid or semi-solid substrate for proliferation.
They adhere to the culture vessel with the use of an extracellular matrix
which is derived from tissues of organs that are immobile and embedded
as connective tissue.
For example: Fibroblasts and epithelial cells.
Majority of continuous cell lines grow as monolayers and such type of
cells are transferred directly to a cover slip for examination under
microscope.
Suspension Cells:
These types of cells do not attach to the surface of the culture vessels.
Hence, they are also kno wn as anchorage independent or non-adherent
cells which are grown in liquid culture medium.
Hematopoietic stem cells and tumor cells are grown in suspension much
faster which do not require frequent replacement of the medium. These
cultures have short lag period.
r Secondary Cell Culture and Cell Line
Secondary culture is the culture when a primary culture is sub-cultured. It
is also known as cell line or sub-clone.

The process involves removing the growth media and disassocating the
adhered cells by enzymatic treatment.
Sub-culturing of primary cells to different divisions leads to the generation
of cell lines. During growth period, cells with the highest growth capacity
predominate and result in genotypic and phenotypic uniformity in the
population.
Based on the life span of the culture cell lines are two types viz. Finite cell
lines and Continuous cell lines.

Finite cell lines

It is defined as the cell line that undergoes limited number of cell division
with a limited life span. The cells passage several times and then lose their
ability to proliferate, which is a genetically determined event knowm as
senescence. Cell lines derived from primary cultures of normal cells are
finite cell lines.

Continuous celllines
It is defined as a finite cell line which undergoes transformation and
acquires the ability to divide indefinitely. This transformation occurs
spontaneously or chemically or virally induced or from the establishment
of cell cultures from malignant tissue.
Prepared cell cultures are then sub-cultured and grown indefinitely as
permanent cell lines. These cells are less adherent and fast growing as a
result the cell density becomes higher and different in phenotypes from
the original tissue. They grow more in suspension medium.
Tissue of interest

Cellor tisue culture in vitro

Prlmary eulture
Isubh-eulkure
Secondary culture
sub-culkure
Cell line
Single cel lsolation Successve ub-culture

Immortallatien
Clonal cell line Senescence
Los of control
of cell growth
Immortalised Transfornmed cell line
cell line
(cancerons cells)
 OGENERAL PROCEDURE FOR CELL CULTURE
(a) Requirements:
Vertical aminar air flow, Incubator, Refrigerator, Microscope, Tissue
culture ware.

(b) Temperature:
. The temperature sets at as the same as body temperature of the host from
which cells are procured. Most animal cells required 36-37°C.
(c) Substrate:
Good compatible substrate is required for attachment and optimum
growth. Glass and specially treated plastics are commonly used as
substrate.
Thereafter attachment factor such as collagen, gelatin, laminin etc. are
used as substrate coating to improve growth and function of normal cels
derived from brain, blood vessels, kidney, liver, skin etc.
(d) Culture medium:
. t is an important and complex factor for the cell growth. The culture
medium is supplemented with various growth factors, pH and osmolality
regulator and provides essential gases like oxygen and carbon dioxide.
The medium is also supplemented with various nutrients like amino acids,
vitamins, minerals and carbohydrates which are essential for growth of cells
and provided energy for metabolism.
Choice of media is used based on the cells being
cultured. Generally, media like Dulbecco's modified
Eagle's medium (DMEM), Eagle's minimal essential
medium (EMEM), Glasgow Minimum Essential Medium (GMEM) are used
for cell culture. Prepared media is filtered and incubated at 4°C.

(e) Media and growth requirement:

Temperature should maintain at 37°C and optimum pH is 7.2 to 7.5. The
humidity is required to be maintained properly in the media with proper
gas phase ratio (Bicarbonate concentration and carbon dioxide in
equilibrium).
 For growth of cultured cells light intensity also plays a vital role. Inside
environment cells are cultured in dark because light induced production of
toxic compound.
Commonly used antibiotics are penicillin, streptomycin, Kanamycin etc.
Trace elements like iron, zinc, selenium, sugar, amino acids, vitamins,
choline, inositol etc.

() Selection of organ:
Different type of cells are grown in cultures including connective tissue
elements such as fibroblasts, skeletal tissue, cardiac, epithelial tissue (liver,
breast, skin, and kidney) and many different types of tumor cells.
On the basis of morphology (shape and appearance) or on the functional
characteristics of cells, they are divided into three types.
Epithelial like attached to a substrate and appears flattened and
polygonal in shape.
Lymphoblast like cells do not attach remain in suspension with a
spherical shape.
Fibroblast like - cells attached to an substrate appears elongated and
bipolar.
(g) Culturing of cel:
Cells are cultured as anchorage dependent or independent. Cell lines
derived from normal tissues are considered as anchorage-dependent which
grows only on a suitable substrate e.g. tissue cells.
Suspension cells are anchorage independent e.g. blood cells whereas
Transformed cell lines either grows as monolayer or as suspension.

Steps:
1. Use sterile technique:Tissue part is harvested and processed using
sterile equipment, reagents and techniques. Personal protective
equipments are used to avoid contamination. Al enzymes and reagents
are filtered sterile condition using a 0.22 micron membrane.
|2. Mincelcut tissue: Mince the tissue specimen into Small pieces (usually 2 x
4 mm) with sterile scissors or scalpel, and then placed the small pieces into
selected buffer, media or salt solution.
3. Washand addenzyme: Wash tissue two to three times to eliminate excess
blood proteins and then add enzyme(s) of choice, likely, collagenase,
protease, papain or trypsin.
Usually about 0.5 to 1.5 mg/ml of selected enzyme is sufficient.
4. Incubation: Further tissue specimen is incubated in optimum temperature
at 37°C for 30 to 90 minutes with periodical mixed the rock specimen.
5. Disperse and wash cells: Cells are dispersed by gentiy pipetted them and
then cell suspension is filtered using a fine mesh. The cells became settled
and decanted excess liquid containing enzymes. Further cells are washed
two to three times with Fetal Bovine Serum (FBS), Bovine Serum Albumin
(BSA) or other inhibitors can also be used to halt enzyme digestion.
6. Resuspension and measure cells: _Cells are resuspended in the correct
medium or buffer and then quantitatively determined the cell yield and
viability. This is an important step in the cell isolation process to evaluate
the result by dissociation technique. Most researchers are used a
haemocytometer for determining cellyield and trypan blue diazo dye to
measure cell viability.
NUMBEROF UNSTAINED CELLS X 100
%OF VA BLECELLS =
TOTAL NUMBER OF CELLS
Colls looolon by
onzymetlc and
mechanlcal
ction Cukure Prlmary
Conluent culture
culture
Organ
Sub-cu'turs

Socondery 8ub-culure
Cullun Cell dvision
prmery ontihuos upto
ol Wne finite divisions
Seoondary
Cells leoleted from or ooll line
lumor and aulured Tranolomed
Organ cells Sub-outure Cell ine
having Cel division
a tumor
continues up
to indeflinte division
Steps of animal cellculture
 > PRIMARY CUTURE
Cells when surgically or enzymatically are removed from the organism
and are placed in suitable culture environment and grown are called
primary culture.
They have finite life span and contain heterogeneous population of cells.
These cells upon subculture lead to generate cell lines which have
limited life span.
Lineage of cells are ornginated from the primary culture is known as cell
strain. Primary cultures are morphologically similar to the parent
tissues.

Individual
cells isolated
from tissue

Pnmary Contact inhibition

cell culture
Tissue
Primary Cell culture
ESTABLISHED CELL CULTURE
Primary cell culture when first subcultured is known as secondary cell
culture. Established or immortalized cell line is the ability to proliferate
indefinitely by random mutation and artificial modification such as
artificial expression ofthe telomerase gene.
Advantages:
Many kinds of cell lines.
Generally easy togrow and manipulate.
Proliferate indefinitely.
Contact inhibition.
Example: HeLa, Sf-9, Cervical cancer.
> Transformed Cell Culture
Transformation is a process of conversion of normal cell into a cell
having some or many of the attributes of different cell. When cell is
transformed, cells lose contact inhibition and become immortal.
 For exanmple: NIH 3T3 mouse cells are partially transformed and becarme
immortal but contact inhibited and grows in a mono layer.
Transformed cells lack contact inhibition of movement due to change of
cellsurface property and loss of many receptors.
They continue to grow and pile up on top of one another as they proliferate.
Transformed cells are also cultured through suspension medium where
cells do not attach to the surface of the culture vessels and are grown liquid
culture medium.
Hematopoietic stem cells and tumor cells are grown in suspension much
faster which do not require the frequent replacement of the medium.

O APPLICATION OF CELL CUITURES IN PHARMACEUTICAL

INDUSTRY AND RESEARCH

Cell culture is used in cellular and molecular biologY, providing excellent

model systems for studying the normal physiology and biochemistry of
cells (for example: metabolic studies, aging), the effects of drugs and toxic
compounds on the cells and mutagenesis and carcinogenesis.
Model System:_Cell culture is used as model system to study basic cell
biology and biochemistry. It is also used to study the interaction between
cell and disease causing agents like bacteria, virus. It helps to study the
effect of drugs and to study triggers for ageing.
Genetic Counseling: Fetal cell culture extracted from
pregnant women is used to study or examine the
abnormalities of chromosomes, genes using
karyotyping and these findings are used in early
detection of fetal disorders.

V Toxicity Testing: Animal cell culture is used to study

the effects of new drugs, cosmetics and chemicals and
growth of multiple cells, especially liver and kidney
cells. This technique is also used to determine the
maximum permissible dosage of new drugs.
 Cancer Research: _Cell culture is one of the most
important tools in cancer research. The basic
difference between normal cell and cancer cell can be
studied using animal cell culture technique. Normal
cells are induced cancer cells by using radiation,
chemicals, viruses and then cause of cancer is studied.
V Virologv: Animal cell cultures are used to
replicate the viruses instead of animals for the
production of vaccine. Cell culture can also be
used to detect and isolate viruses, and also to
study growth and development cycle of viruses.
Vaccine Production:_Cultured animal cells are
used for virus production and these viruses are
used to produce vaccines. For example: Vaccines
like polio, rabies, chicken pox, measles and hepatitis
Bare produced using animalcell culture.
Gene Therapy:_Cultured animal cells are
genetically altered and are used in gene therapy
technique. First cells are removed from the patient
lacking a functional gene or missing a functionall
gene. These genes are replaced by functional genes
and altered cells are culture and grown in
laboratory condition and are introduced into the
patient.

Drug Screening and Development: Animal cell

cultures are used to study the cytotoxicity of new drug.
This is also used to find out the efective and safe
dosage of new drugs. Cell-based assay plays an
important role in pharmaceutical industry.
 Geneticaly Engineered Protein: _Animal cell cultures are used to
produce commercially important genetically engineered proteins such as
monoclonal antibodies, insulin, hormones etc. Proteins extracted from
biological sources are important for substitution therapy. Interferon is
discovered in 1957 by cell cultured method by viral infection.
V Replacement Tissue or Organ:_Animal cell culture is used as
replacement tissue or organs. For example: artificial skin is produced to
treat patients with burns and ulcers. Recently artificial organ culture such
as liver, kidney and pancreas are successfully carried out for
transplantation.