1.
PHARMACEUTICAL MICROBIOLOGY
(BP303T)
Unit-V
Part-1
Types of spoilage, factors affecting the microbial
spoilage of pharmaceutical products, source and type
of contaminants.
Name: Ms. Pooja Deepak Bhandare
Assistant Professor
G H RAISONI UNIVERSITY
SCHOOL OF PHARMACY

2.
Introduction:
• In our environment microbes are present everywhere i. Air, water, soil
etc.
• These microbes are to cause spoilage of not only food products but
also pharmaceutical products.
• The spoilage of pharmaceutical products can result in serious health
hazards to the consumers as well as cause financial problems to the
manufacturers.

3.
• Definition: Pharmaceutical products are considered
microbiologically spoiled when even low levels of pathogenic microbes
or toxic microbial metabolites are present and detectable physical or
chemical change have been noted in the product.
• In short it can be defined as deterioration of pharmaceutical products
due to microbial contamination

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Types of Microbial Spoilage:
1. Infection induced due to contaminated pharmaceutical products:
• Pharmaceutical products may get infected from many sources like raw material,
manufacturing, storage etc.
• Such infected products when consumed causes the diseases.
• E.g. Cholera, Pseudomonas infections, Botulism etc.
• The common infections found in dosage form are summarized in Table no. 1.1.

5.
Dosage Form Contaminating
Microbes
Outcome
Tablets / Capsules SAmonella species Salmonella Infection
Antiseptic Solutions Pseudomonas species Septicaemia.
Eye products Pseudomonas
aeruginosa
Eye infections.
Ointments and Creams Gram negative
organisms
Dermal infections
IV medicines Candidia species Septicaemia.
Table no. 1.1 Common pathogens spoiling pharmaceutical products:

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2. Physicochemical spoilage –
This type of spoilage may change physico-chemical properties of product.
Following types of changes may occur.
i) Viable growth - Viable growth of microbes may occur inside the
container. These growth may be visible in the form of floating layers,
turbidity, hemps, ete. Contamination of products by fungus species like
Aspergillus sp. may cause this type spoilage. Some bacterial species may
also show viable growth.

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ii) Gas production -Some microbes may produce gas inside the containers.
These gases may be visible in the form of bubbles, floccules etc. Contamination
of products with bacteria like E. coli may produce gas if it contains sugars
iii) Colouration / Decolouration -Some microbes may decolourise formulation
or it may produce unique colour which is different from the normal colour of the
product.
iv) Odour formation-Microbial growth in the finished product may produce
bad odour or characteristic odour.It may produce a characteristic rotten smell.
v) Taste change -Microbial spoilage may change the taste of the oral
formulations. It may impart bitter or obnoxious taste to the oral formulatio

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3. Physical Spoilage:
• These changes in physical appearance are caused by microbial activities in
the formulation.
• Cracking of emulsion: Microbes eat off the emulsifying agent causing instability
of emulsions resulting in separation of phases.
• Odor changes: Microbes due to their activity in the pharmaceutical product impart
smell.
• e.g. Toluene smells due to spoilage of Balsam of Tolu by Penicillium species.
• Smell of geosmin to water phases by Actinomycetes contamination.

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4. Biological spoilage:
• Microbial contamination of the products result in formation of chemicals in the
products due to microbial metabolic activity, this is terms as Biological spoilage.
• Mainly two types of chemicals are produced by microbes,
• Microbial Toxins.
• Microbial Metabolites.
• Microbial Toxins: Endotoxins produced by some gram –ve bacteria like E.coli.
• Microbial Metabolites: Different types of amines and organic acids.

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5. Chemical spoilage:
• This occurs due to various types of chemical reactions, mediated by
contaminating microorganisms.
• The ingredients in formulation that are susceptible for chemical
damage by microbial actions are tabulated in Table 1.2.

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Sr No. Ingredient Microorganism / Microbial Enzyme Action
1. Active Ingredient:
1.Atropin Pseudomonas Hydrolysis
2.Aspirin Clostridium / Bacillus Hydrolysis
2. Diluents
1.Starch Bacterial Amylase Depolymerization
2.Cellulose Bacterial Cellulase Depolymerization
3. Preservatives like hydroxy
benzoate ester
Pseudomonas species
Degradation
Table 1.2 Susceptibility of pharmaceutical ingredients to microbial contamination

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 Factors affecting microbial spoilage:
• By understanding various factors promoting growth of microbes in the formulation it is possible to manipulate
the formulations and to avoid their microbiological spoilage without affecting the patient acceptance and
therapeutic efficacy of the formulation.
• Some identified factors that affect microbiological spoilage of pharmaceutical products are as follows,
• Size of contaminant inoculum
• Nutritional factors
• Moisture content
• pH
• Storage temperature
• Redox potential
• Packaging design

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1. Size of contaminant inoculum:
• The rate of deterioration of a pharmaceutical product depends on the size of
microbial population in the product.
• Lower microbial population size shows lesser degradation changes as compared
to large size populations.
• However, inoculum size alone is not always a reliable indicator of likely
spoilage potential.
• A very low level of pseudomonas in a weakly preserved solution may suggest a
greater risk than tablets containing high numbers of fungal and bacterial spores.

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2. Nutritional factors:
• Most of the organic and inorganic ingredients act as potential carbon or
nitrogen substrates for microbial growth.
• The complexity of many formulations offers considerable nutritional variety for
a wide range of microorganisms.
• The use of crude vegetable or animal products in a formulation provides an
additionally nutritious environment.
• Even demineralised water prepared by good ion-exchange methods will
normally contain sufficient nutrients to allow significant growth of many
waterborne Gram-negative bacteria such as Pseudomonas spp.

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3. Moisture content:
• By measuring a product’s water activity (Aw ), it is possible to obtain an
estimate of the proportion of uncomplexed water that is available in the
formulation to support microbial growth.
• Most microorganisms grow best at high water activity.
• Hence, formulation can be protected from microbial attack by lowering
their water activity with addition of suitable levels of sugars, polyethylene
glycols or sodium chloride or by drying.

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4. pH:
• Extremes of pH prevent microbial attack.
• Around neutrality, bacterial spoilage is more likely, with reports of
Pseudomonads and Gram-negative bacteria growing in antacid mixtures,
flavoured mouthwashes and in distilled or demineralised water.
• Above pH 8 (e.g. with soap-based emulsions) spoilage is rare.
• In products with low pH levels (e.g. fruit juice-flavoured syrups with a pH 3–4),
mould or yeast attack is more likely.
• Yeasts can metabolize organic acids and raise the pH to levels where secondary
bacterial growth can occur.

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5. Storage temperature:
• Spoilage of pharmaceuticals could occur potentially over the range of about -20°C to
60°C •
• The particular storage temperature may selectively determine the types of
microorganisms involved in spoilage. •
• A deep freeze at -20°C or lower is used for long-term storage of some pharmaceutical
raw materials and short-term storage of dispensed total parenteral nutrition (TPN)
feeds prepared in hospitals. •
• Reconstituted syrups and multi-dose eye-drop packs are sometimes dispensed with the
instruction to ‘store in a cool place’ such as a domestic fridge (8°–12°C), partly to
reduce the risk of growth of contaminants inadvertently introduced during use.

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6. Redox potential:
• The ability of microbes to grow in an environment is influenced by its
oxidation-reduction balance (redox potential), as they will require
compatible terminal electron acceptors to permit their respiratory
pathways to function.
• The redox potential even in fairly viscous emulsions may be quite high
due to the appreciable solubility of oxygen in most fats and oils.

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7. Packaging design:
• Packaging can have a major influence on microbial stability of some formulations in controlling the
entry of contaminants during both storage and use.
• Considerable thought has gone into the design of containers to prevent the ingress of contaminants
into medicines for parenteral administration, owing to the high risks of infection by this route.
• Self-sealing rubber wads must be used to prevent microbial entry into multi-dose injection containers
following withdrawals with a hypodermic needle.
• Where medicines rely on their low Aw to prevent spoilage, packaging such as strip foils must be of
water vapour-proof materials with fully efficient seals.
• Cardboard outer packaging and labels themselves can become substrates for microbial attack under
humid conditions, and preservatives are often included to reduce the risk of damage.

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Sources and Types Of Contamination:
• Contaminants can gain entry into a production process stream from several
sources such as,
• Personnel,
• Poor facility design,
• Incoming ventilation air,
• Machinery and other equipment for production,
• Raw material and semi-finished material,
• Packaging material,
• Utilities,
• Different media used in the production process as well as for cleaning and Cleanroom
clothing.

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1. Personnel:
• Personnel who are supervising or performing drug manufacturing or control can
be a potential source of microbiological contamination and a vector for other
contaminants like,
• Natural skin flora of microorganism can be a main source of contamination e.g.
Staphylococcus aureus, Sarcina spp., Alcaligenes spp. Etc, various dermatophytic fungi
viz. Epidermophyton spp., Microsporum spp. etc.
• If persons having wound they may support organisms like, Staphylococcus aureus,
Staphylococcus pyogenes, Proteus spp., Pseudomonas aeruginosa etc.
• The respiratory discharge of the personnel may include organisms like, Staphylococcus
aureus, Staphylococcus salivarius, Neisseria pharyngis, Klebsiella pneumoniae etc.

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The main reasons for contamination from the personnel include:
Lack of training
Direct contact between the operator’s hands and starting
materials, primary packaging materials and intermediate or bulk product
Inadequate personnel cleanliness
Access of unauthorized personnel into production, storage, and product control areas
Inadequate gowning and personnel protective equipment, and
Malpractices like eating food, drinking beverages, or using tobacco in the storage and
processing areas.

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2. Working Environment:
• Air is not a natural environment for microbes to grow as it does not contain water and nutrients needed
for the growth.
• However the microbes are carried into the atmosphere and remain suspended on particles of dust, skin,
clothing, moisture or nasal droplets, sputum following sneezing or coughing.
• The microbial load of the air may get increased during handling of contaminated materials during
dispensing, blending and formulation processes.
• Use of starch and sugars in dry state may increase fungal counts.
• Settle plate and different air sampling methods are commonly used for isolation and identification of
microbes present in the working environment.
• Microbes which are commonly isolated from atmosphere are, Staphylococcus species, Streptococcus
spp., Bacillus spp., Microsporum spp., Aspergillus spp., Penicillium spp. etc.

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3. Buildings and Facility:
• The buildings and manufacturing facilities may also contribute to the
contamination.
• Walls and ceilings of building are sources commonly for Aspergillus spp.,
Cladosporium spp., Penicillium spp., Bacillus spp. etc. They are mainly
found in poorly ventilated buildings.
• These microorganisms utilize the contents of the wall paint as nutrients.

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• The other reasons of contamination due to facility issues include:
• Inadequate filth and pest controls.
• Rough floors, walls and ceilings.
• Lack of air filtration systems.
• Improper lighting and ventilation.
• Poorly located vents, and drains.
• Inadequate washing, cleaning, toilet, and locker facilities to allow for sanitary operation,
cleaning of facilities, equipment, and utensils; and personal cleanliness.

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4. Equipments:
• The equipment and utensils used in processing, holding, transferring and packaging are the common
source of pharmaceutical contamination.
• The main reasons for contamination from the equipment include:
• Inappropriate design, size, material leading to corrosion and accumulation of static material and/or
adulteration with lubricants, coolants, dirt, and sanitizing agents.
• Improper cleaning and sanitization.
• Design prevents proper cleaning and maintenance.
• Improper calibration and irregular service, and
• Deliberate use of defective equipment.

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5. Materials:
• The raw materials used for production can be a potential source of
contamination.
• Animal origin raw materials like gelatin, thyroid, pancreas and Plant origin
products like starch, acacia tragacanth etc. are known sources of microbial
contamination.
• Raw materials of mineral origin are usually free from microbes while
some like zinc oxide, calamine, bentonite may contain spores of
Clostridium spp.

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• The main reasons for contamination from the raw materials include:
• Storage and handling mistakes causing mix-ups or selection errors.
• Contamination with microorganisms or other chemicals.
• Degradation from exposure to excessive environmental conditions such as heat, cold,
sunlight, moisture, etc.
• Improper labelling.
• Improper sampling and testing, and
• Use of materials that fail to meet acceptance specifications.

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6. Manufacturing Process:
• There are various opportunities for contamination of raw material, intermediates or
packaging materials throughout the manufacturing process.
• The main reasons for contamination during manufacturing process include:
• Lack of dedicated facilities to manufacture a single product
• Inappropriate cleaning in-between batches to minimize the amount of product changeovers
• Use of an open manufacturing system exposing the product to the immediate room environment.
• Inappropriate zoning.
• Absence of an area line clearance according to approved procedures following each cleaning process
and between each batch, and
• Lack of cleaning status labelling on all equipment and materials used within the manufacturing
facility.

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7. Water:
• Water is one of the main constituents of many preparations and is also needed
for washing and many processes.
• Fresh water contains many microbes, while water contaminated with sewage
waste contains microbes of Proteus spp., Escheresia spp., Clostridium spp.,
Streptococcus species, Flavobacterium spp. etc.
• Softened water is commonly used for washing purposes in industry, microbes
from Bacillus and Staphylococcus spp. May come in during the process.
• Water for industrial use may be treated with filtration or uv or stored at elevated
temp. To discourage microbial growth in it.

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8. Packaging:
• The packaging of pharmaceuticals plays a dual role; it holds the product
and also prevents it from spoilage.
• The materials used for pharmaceutical labelling and packaging contains
many species of microbes like Bacillus, Clostridium, Penicillium etc.
• The containers and closures are needed to be sterilized before use.