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PRESENTED
08-11-2020

GOVERNMENT
PHARMACY COLLEGE,
BRD MEDICAL COLLEGE
CAMPUS GORAKHPUR.
(AKTU CODE – 0937)
ASSIGNMENT-I
SUBJECT: PHARMACEUTICAL MICROBIOLOGY
SUBJECT CODE: BP303T
TOPIC: MICROBIAL SPOILAGE AND PRESERVATION OF
PHARMACEUTICAL PRODCUTS

SUBMITTED TO:
DR. AJAY KUMAR
ASSOCIATE PROFESSOR
PHARMACEUTICAL MICROBIOLOGY

SUBMITTED BY:
RUCHI SINGH
ROLL NO.1909370500040
B.PHARM(II)
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TABLE OF CONTENTS

TOPIC PAGE
INTRODUCTION iii
Types of microbial spoilage vi
Factors affecting microbial x
spoilage
Types of microbial contaminants xii
Sources of microbial contaminants xiii
Assessment of microbial xvi
contamination and spoilage
Preservation of xviii
pharmaceutical products
Evaluation of microbial stability xix
of formulation
References xxi
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INTRODUCTION
CONTAMINATION:

o Contamination is the introduction of microorganisms into the product

during manufacture or during subsequent uses of the product.

Medicines contain wide variety of ingredients mainly APIs and additives,

included to create formulations which are efficacious, stable and
sufficiently elegant to be acceptable by patients.

If microbial contaminants survive the manufacture or reach storage, they

are likely to meet conditions that are often conducive to their survival and
even reproduction of a large variety of non-fastidious bacteria, fungi and
yeast leading to microbial spoilage, unless measures are taken to monitor
them.

MICROBIAL SPOILAGE:

It is followed by the contamination of the product and describes the

processes and consequences of the microbial growth in it.

 Microbial contamination and spoilage of pharmaceuticals will not only

alter the aesthetic qualities of the product (color, smell, texture, and
so forth); such contamination may also render the product dangerous
to the user; or it may also nullify any intended therapeutic value of the
product.
 Pharmaceutical spoilage of pharmaceutical product is due to changes in
physical and chemical properties in their formulation which makes them
unfit for use.
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EFFECTS OF SPOILAGE ON PHARMACEUTICAL PRODUCTS

1. They become unfit for use; due to chemical and physic-chemical

deterioration of the formulation.
2. Active drug constituents may be metabolized to less potent or
chemically inactive forms.
3. Using contaminated products may lead to potential health hazard to
patients; resulting in outbreaks of medicament- related infections, and
contributing to the spread of disease.
4. Wastage of individual batches usually result in major financial problems
for the manufacturer.
5. The threat of damaging publicity due to recalls may have serious
economic implications for the manufacturer.
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MICROBIAL SPOILAGE

PHARMACEUTICAL PRODUCTS
PREVENTION
TREATMENT COMPONENTS/INGREDIENTS
DIAGNOSIS

GMP STABLE ACCEPTTANCE

SAFETY
EFFICIANCY
QUALITY

STERILITY
Acceptable microbial limits

SPOILAGE DETERIORATION

*PHYSICAL PROPERTY
*CHEMICAL PROPERTY

WASTAGE

MICROBIAL CONTAMINATION
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TYPES OF MICROBIAL SPOILAGE

o Microbial spoilage can be a result of one initial or

several sources of microorganisms; such as bacteria,
fungi and yeasts.
o Microbial spoilage may include:
1. Survival of low level of pathogenic microorganisms,
or survivals of high levels of opportunistic
pathogens.
2. The presence of toxic microbial metabolites.
3. Microbial growth and initiation of chemical and
physic-chemical deterioration of the formulation.

Types of spoilage:
 Acute human pathogens maybe present in the
finished product.

Examples:
a) Salmonella spp. In contaminated tablets and
capsules.
b) Cholera outbreak was traced to an oral
liquid medicine which was prepared with
contaminated water.
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c) Pseudomonas aeruginosa could contaminate

antiseptic solutions used in case of badly
burned patients.
d) Candida spp. In intravenous medicines.
e) Gram-negative bacteria in bladder washout
solutions.

 Chemical and physic-chemical deterioration of

pharmaceutical products.
o Microorganisms posses a wide variety of
degradative capabilities, which will cause
the deterioration of the products.

 The overall rate of deterioration of a

chemical will depend upon:
 Its chemical structure
 The physico-chemical properties of its
particular environment
 The type of the present microbes
 Whether the metabolites produced cans
serve as source of usable energy.
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 Pharmaceutical ingredients susceptible to

microbial attack.
1) Therapeutic agents: materials, such as alkaloids
(morphine and atropine), analgesics (aspirin, paracetamol),
thalidomide, barbiturates, steroid esters and mandelic acid,
can be metabolized and serve as substrates for growth of
microorganisms.

2) Surface-active agents: are generally stable due to

the slightly alkaline pH of the formulation, although readily
degraded once diluted into sewage.

3) Non-ionic surfactants: such as alkylpolyoxyethylene

alcohol emulsifiers, are readily metabolized by a wide
variety of microorganisms.

4) Organic polymers: many of the suspending agnts are

subject to microbial depolymeriation by extrcellular enymes
such as amylases(starches) pectinases(pectins),
cellulases(cellulose), dextranases(dextrans), and proteases
(proteins).

5) Humectants: low molecular wt. materials such as

glycerol and sorbitol are included in some products to
reduce water loss and may be readily metabolized unless
present in high concentration.
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6) Fats and oils: these hydrophobic materials are

usually attacked in aqueous formulations such as oil-in-
water emulsions.

7) Sweetening, flavoring and coloring agents: many of

these sugars and other agents used in pharmacy are ready
substrates for microbial growth.

8) Preservatives and disinfectants: gram negative

bacteria at concentration below their effective “use” levels
can metabolie many of these agents.

For eg. Pseudomonas spp. Could grow in stock solutions of

quaternary ammonium antiseptics and chlorohexidine.
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Factors affecting microbial spoilage of

pharmaceutical products
 Manipulating the design of the pharmaceutical products
can make it less favorable for microbiological growth
and spoilage, within the limitations of patient
acceptability and therapeutic efficacy.

Factors affecting microbial spoilage are:

1- Types and size of contaminant incoculum:

 Unexpected surge in the contaminant bioburden may
occur if:
A. Raw material were unusually contaminated
B. There was a lapse in th plant-cleaning protocol;
C. A biofilm detached itself fromwithin supplying
pipework;
D. Or the product has been grossly misused
during administration.
2- Nutritional factors:
 The use of crude vegetables or animal products in th e
formulation provides an additionally nutritious
environment.
 Most pathogens require specific growth factors, which
are normally absent in pharmaceutical formulations.
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They’re thus less likely to multiply but may remain

viable and infective for long time.
3- Moisture content: water activity (AW):
- Microorganism require water in appreciable
quanitites for growth.
4- Redox potential:
- The ability of microbes to grow ina n
environment is influenced by its oxidation-
redcution balance.
Vaccum packing/inclusion of o2 levels, reduces
attack by some spoilage bacteria but doesn’t
eliminate all.
5- Storage temperature.
- Spoilage of pharmaceuticals could occur over
the range of about -20oc to 60oc.
6- pH(potential of hydrogen)
- extreme pH prevent microbial attack.
Above pH 8.0 with soap- based emulsions,
spoilage is rare.
7- Packaging design :packaging can have a major
influence on microbial stability of some formulations
8- preservatives :microbes can be more resistant to
heat or desiccation in the presence of some
polymers such as starch, acacia or gelatin.
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SOURCES AND TYPES OF MICROBIAL CONTAMINANTS

Types of microbial contamination

TYPES OF MICROBIAL CONTAMINATION

BACTERIA FUNGI MOLD(FILAMENTOUS FUNGI)

GRAM(+)rods- environment,soil Yeast

ENVIRONMENT
GRAM(+)cocci- personnel Personnel
SOIL
GRAM(-)rods- water,soil

When investigating microbial contamination ina na industry,

it is important to have an idea of the possible
contaminants, likely sources of contaminants and stages in
the processing where contaminations may arise.

 There are three possible origins of microbial

contamination:

a) cross contaminants (biological product manufactured at

the same manufacturing facility.)

b) environmental contaminants(microorganisms present in

production facility.)

c)commonly known microbial conatminants (cause

contamination in biological products.)
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Sources of contamination;

 Microorganism will contaminate formulation from a

wide variety of sources. This is critical for sterile
products.

Sources of microbial contamination:

 The atmosphere
- Air is not a natural environment for microbial growth.
But organisms such as bacillus, clostridium, staph,
penicillin, aspergillus can survive
- degree of contamination depends on particle level
 Buildings
-potential mold contamination; nutrients come from plaster,
inadequate sealings.

 Water
- always concerned about pseudomonas
 Packaging
- mold spores, especially if any paper source
 People(!)
- greatest source of microbial contamination.
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SOURCES CONTAMINATION BY CONTROL

IN USE
*Human -Application by -Hand hygiene
hand -Use of suitable
applicators
-Individual packs
-Non-touch
techniques
*Environmental
-“splash-back” -Use of individual
mechanism supplies in small
-Air contamination quantities.
-Use of stock
solutions
*Equipment
-Re-use of -Use of disinfectants
applicataors -Use of diposable
-use of equipments.
contaminated
equipemnt

SOURCES CONTAMINATION BY CONTROL

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IN MANUFACTURE
*industry 1.raw materials -high microbiological
2.processing std maintenance
equipments -sterile environment
3.staff
4.packaging

*Hospital -Monitoring
1.water
-GMP
2.environment
-thorough washing
3.packaging
and drying


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ASSESSMENT OF MICROBIAL CONTAMINATION AND

SPOILAGE

Early indications of spoilage are often organoleptic,

including:
 Sickly tastes and smells.
 Discoloration induced by microbial pigments.
 Loss of viscosity
 Sedimentation of suspended ingredients
 Production of slimy, viscous masses in syrups.
Shampoos and creams.
 Fungal growth in creams resulting in gritty
structures.
 Changes in product pH.
production of gaseous metabolites seen as trapped
bubbles within viscous formulations.
Sterility testing
Pyrogen estimation
- Pyrogen a substance, typically produced by a
bacterium, which produces fever when introduced
into systemic circulation.
- To test pyrogen two tests areused:
 RP Test (rabbit pyrogen test)
 LAL test (limulus amoebocyte lysate test)
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Identification of e.coli,staph. Aureus,gram positive

coccci etc.
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PRESERVATION OF PHARMACEUTICAL PRODUCT

USING ANTIMICROBIAL AGENTS

 Antimicrobial preservatives may be included in the

formulation to minimize the risk of spilage and to kill
and anticipated low levels of contaminants introduced
during storage and usage.
 However, preservatives should never be added to mask
poor manufacturing processes.
 Preservative capacity is: the cumulative level of
contamination that preserved formulation can tolerate
before becoming so depleted as to become ineffective
 Ideally such preservatives should be:
- Having a broad spectrum of activity and rapid rate
of kill (aqueous solutions);
- Selective in reacting with the contaminants and
not the formulation ingredients (rarely found);
- Non-irritant and non-toxic to the patient
- Stable and effective throughout the life of the
product.
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EVALUATION OF MICROBIAL STABILITY OF

FORMULATIONS

o To challenge the efficiency of preservatives, the

“preservative challenge tests” are performed using
the following methods:

1. SINGLE CHALLENGE TEST

- A large inocula of various laboratory cultured
microorganisms are added to aliquots of medicine,
to determine their rate of inactivation, using the
viable bacterial counting methods (single challenge
test).
2. MULTIPLE CHAALLENGE TEST
- In another test, microorganism are re-inoculated
repeatedly at set intervals, to monitor the
efficiency of inactivation until the system fails
(multiple challenge test)

Good pharmaceutical manufacturing practice

(GPMP)
- A high assurance of overall product quality can only
come from a detailed specification, control
(documentation) and monitoring(assessing
conformance to specification) of all the stages
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that contribute to the manufacturing process.

Quality control procedures

- Among the recently used methods are:

1. Electrical impedance.
2. Use of fluorescent dyes and epifluorescence
3. The use of ‘vital’ stains
4. Estimating microbial adenosine riphosphate (ATP)
5. HPLC or GC characterization of volatile and
unpleasant- tasting metabolites
6. Laser scanning devices.

REFERNCES
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1. Hugo WB, Russell AD. Pharmaceutical microbiology. 6th ed.

Oxford, uk: Blackwell Scientific Publiciations;1998

2. Parker MS. Microbiological contamination and preservation of

pharmaceutical preparations. In: pharmaceutics: the science
of dosage form design 2nd edn china, Churchill livingstone,
2000; p220.

3. Shaikh D, Jamshed TA, Shaikh R. microbial contamination of

pharmaceutical preparations, Pak J pharm Sci 1988; 1:61-6.

4. Nigel, A.H. Microbial control of pharmaceuticals, old e –ept,

vol 1, 2002, 1753-1764.

5. Journal of pharmacy research vol. 3. Issue January 2010.