Unit 5 Microbiology

Unit5 Microbiology
S.Y.B.Pharm Sem 3
- Dhruvi Jain
Spoilage of Pharmaceutical Products
• Pharmaceutical Product: Active drug + Formulation Additive

• Spoilage: chemical or physico-chemical degradation of product
making it unsuitable for use
- change in their efficacy, action, etc: makes it ineffective
Huge loss to the Attract litigation

manufacturer from consumers
Potential health
hazard to patients
Types of Spoilage
Physical Chemical Microbial
Chemical Contamination
Physical factors reactions by
microorganism
Eg: pH, Eg: oxidation, Eg: fungi,

temperature, reduction, bacteria,
moisture hydrolysis, moulds
photolysis,
Types of Microbial Spoilage
• It includes contamination of the product with microbes

which affects the drug safety and quality and is not fit for use
Physico-chemical Chemical Spoilage Biological Spoilage

spoilage
• Viable Growth • Hydrolysis • Release of toxins

• Gas production • Acetylation • Microbial
• Coloration • Depolymerization metabolites
• Odor • Degradation
• Taste
1) Physico-chemical Spoilage:
- In this spoilage the microorganisms induce chemical changes that
deteriorates or alters the physical properties of the product
a) Viable Growth:
- Visible growth of microorganisms occur on the surface of formulation
- layer of moulds over syrups, turbidity by bacterial growth in creams
- Eg: Aspergillus niger
b) Gas Production:
- Some organisms produce gas by metabolic activities and form bubbles,
floccules or foam over the formulation
- Formulations containing starch and carbohydrates are more susceptible to gas
production
- Example:
Moulds and yeast – produce CO2 in syrups
E. coli and Klebsiella – produce gas in creams containing vitamins
Desulfovibrio – produce H2S gas in suspensions
c) Coloration/Decoloration:
- Color changes occur due to alteration of chemical nature, change in pH,
redox or production of other metabolite
- Example:
Pseudomonas aeruginosa: blue-green, brown
Moulds: surface decoloration of tablets
d) Odor and taste:

- Microbial growth in the finished product may produce bad smell or
characteristic odour
- sour - fatty acids, fishy - amines, bad eggs, bitter, earthy
- Example:
Actinomycetes – produce geosmin in water phase of formulation responsible
for petrichor smell
2) Chemical Spoilage:
- In this spoilage the microorganisms induce chemical reactions
that deteriorates chemical nature of the drug or excipient
a) Hydrolysis:
- Microbes produce enzymes that catalyse hydrolysis of pharmaceutical
product
- Example:
Bacillus – gelatinase hydrolyses gelatin
E.coli, Bacillus, Aspergillus – esterase that hydrolyse aspirin
b) Acetylation:
- some organisms produce enzymes that cause acetylation of drug and loses its
activity
- Example:
Staphylococci and Streptococci: produce chloramphenicol acetyl transferase
that causes acetylation of chloramphenicol
c)Polymerization/Depolymerization:
- Depolymerization: process of degrading polymers to monomers
- Diluents, binders, thickening agents are made of polymers
- Result of depolymerization: loss of viscosity, and sedimentation of suspended
ingredients
- Example:
amylase: depolymerize starch
pectinase: depolymerize pectin
cellulase: depolymerize cellulose
c) Polymerization/Depolymerization:
- polymerization of sugars and surfactant molecules can produce:
slimy, viscous masses in syrups, shampoos and creams,
fungal growth in creams has produced ‘gritty’ textures
d) Degradation/metabolization:
- Some microorganisms cause degradation or metabolization of active
therapeutic agent or formulation
- Example:
Penicillin is degraded by b-lactamase producing bacteria
Prednisone is degraded by Aspergillus sp.
3) Biological spoilage:
- In this spoilage as a result of metabolic activity the microorganism
produce undesirable chemicals which have harmful biological effect
- toxins, pyrogens or other harmful metabolites
- Microbial toxins:
Endotoxin – LPS: Gram negative E. coli
Exotoxin – toxin botulinum: Clostridium botulinum
- Microbial metabolites:
- Biosynthetic products of bacterial metabolism which are toxic
- Examples: organic acids, amines
Type and degree of Microbial Spoilage
True Opportunist
Pathogen Pathogen
➢ Eg: Ps. aeruginosa, Klebsiella, Serratia

➢ Eg: Clostridium tetani
➢ Pathogenic when given the opportunity
Salmonella spp.
➢ Simple nutritional requirement –
➢ Talcum powder containing Cl. Tetani – 106–107CFU/g or CFU/ml
wound infections and neonatal deaths
➢ Can survive in disinfectants and
antiseptic solutions
➢ Thyroid and pancreatic powders
containing Salmonella – outbreak of ➢ Compromised hospital patients, i.e. the
Salmonellosis elderly, burned, traumatized or
immunosuppressed – at high risk
Factors Affecting Microbial Spoilage
No. and
type of
microbes
Presence of Nutritional
protection factors
materials
Factors
Packaging Moisture
design content
Storage Redox
temp. Potential
1) Number and type of microorganisms:

➢ Low numbers: may not cause spoilage if it is unable to replicate
- may cause spoilage during long term storage
➢ High numbers: present unacceptable challenge

- product will be damaged quickly
• However, inoculum size is not always a reliable indictaor of likely spoilage

potential
➢ Low levels of aggressive pseudomonads in a weakly preserved solution may

suggest a greater risk than tablets containing fairly high numbers of fungal
and bacterial spores.
2) Nutritional Factors:
➢ Organic and inorganic components: carbon and nitrogen source for
microorganisms
➢ Formulations contain: crude vegetable, animal and microbial products
➢ Eg: additives like sugar, amino acids, alcohols – microbial nutrients
➢ Demineralized water produce by ion-exchange resins may contain nutrients
➢ Primary contaminants may produce metabolites which could be used by

aggressive microbes as nutrient.
3) Moisture content (Water activity):

- Amount of water available for growth of microorganism
Water Activity Mositure / Solute / sugar Microbial
water growth
High High Low High
Low Low High Low
Organism Water activity

Gram negative, E. coli 0.95
Staphylococci 0.9
Yeasts 0.88
Osmotolerant yeast 0.73
Aspergillus glaucus 0.61
3) Moisture content (Water activity):

➢ Amount of water available for growth of microorganism
➢ Reduce Aw: adding sugars or polyethylene glycols - resist microbial attack

• Syrup BP (67% sucrose; Aw = 0.86): attack by osmotolerant yeast
➢ Reduce Aw: drying (tablets, capsules, powders)

➢ Tablet film coatings and foil strip packing: greatly reduce Aw
• Condensed water films can accumulate on the surface of dry products: high
localized Aw – fungal growth
4) Redox Potential:
- Microorganisms require terminal electorn acceptor for respiration
- ability of microbes to grow in an environment is thus influenced by its

oxidation-reduction balance
- Presence or absence of oxygen
- Presence of dissolved oxygen increases redox potential of the product

thereby promote microbial growth and spoilage: viscous emulsions, fats and
oils
5) Storage temperature:
- Great controller of microbial spoilage
- The storage temperature will selectively determine the microbe involved in
spoilage
- Microbial growth can occur at range of -20℃ to 60℃
➢ Long term & short term storage of pharmaceutical products: -20℃ or below
➢ Reconstituted syrups and multi-dose eye-drop packs: domestic fridge (8°–

12°C)
➢ Water for Injections (EP): 80°C or above after distillation and before packing
and sterilization to prevent possible regrowth of Gram negative bacteria and
the release of endotoxins.
6) pH:
➢ Microbes grow best at neutral pH
➢ Pseudomonads and Gram negative spoilage: mouthwashes, antacids,

demineralised waters at neutral pH
➢ Above pH 8 Eg: soap-based emulsions – spoilage is rare
➢ Below pH 6 Eg: 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
➢ Low pH is used to preserve food stuffs - Eg: Pickles, yogurts

7) Packaging Design:
➢ Single-dose ampoules and vials: contamination is negligible
➢ Multi-dose containers: contamination by user itself
➢ Change in designs of these containers may minimize contamination and

spoilage:
• Wide mouthed containers replaced with narrow mouthed tubes with screw
cap closures.
• Self sealing rubber wads: prevent entry of oxygen (redox potential) and
water (water activity)
• Preservatives are often included to reduce risk of damage
8) Protection of microorganisms within pharmaceutical products:

➢ Some materials in formulation may protect microbes during sterilisation
process.
• Polymers such as starch, acacia, gelatin – makes microbes resistant to heat

and dessication
• Adsorption to particulate matter – aid in survival
• suspended particles such as kaolin, magnesium trisilicate or aluminium

hydroxide gel, surfactants, proteins – increase longevity of contaminants and
resistance to preservatives
Assessment of Microbial Spoilage
Microbial Limit Test
Total Aerobic Viable Test for specified

Count Microorganism
Total Aerobic Microbial Total Combined Yeast

Count and mold count
(TAMC) (TYMC)
• Soyabean-Casein Digest Agar • Sabourauds Dextrose Agar

• Sample Preparation:
- Weigh and transfer 10g or 10ml of sample to 90ml SCDM or peptone water (10-1
dilution)
- If the sample contains antimicrobial activity it may be neutralized by adding

neutralizing agents
Type of antimicrobial Inactivator

Phenolics, parabens Polysorbate 80
Iodine, Quaternary ammonium Lecithin, Sodium Lauryl

compounds (QACs) sulphate
Alcohols, aldehydes, sorbates Dilution
Mercurials, halogens Sodium Thiosulphate

Pour Plate Technique

Total Aerobic Microbial Count Total Yeast and mold count
1) Transfer 1mL of sample dilution 1) Transfer 1mL of sample dilution
to 20mL of Soyabean-casein to 20mL of Sabourauds
digest agar. Dextrose agar
2) Pour the mixture into2 separate 2) Pour the mixture into2 separate
sterile petri dish sterile petri dish
3) Incubate at 30-35℃ for 3-5 3) Incubate at 20-25℃ for 5-7

days days
4) Count the number of colonies, 4) Count the number of colonies,

calculate average * Dilution calculate average * Dilution
Factor = CFU/ml Factor = CFU/ml
Spread Plate Technique

Total Aerobic Microbial Count Total Yeast and mold count
1) Transfer 0.1mL of sample 1) Transfer 0.1mL of sample
dilution to solidified Soyabean- dilution to solidified Sabourauds
Casein Digest agar Plate Dextrose agar plate
2) Spread the sample on the 2) Spread the sample on the

surface of the plate surface of the plate
3) Incubate at 30-35℃ for 3-5 3) Incubate at 20-25℃ for 5-7

days days
4) Count the number of colonies, 4) Count the number of colonies,

calculate average * Dilution calculate average * Dilution
Factor = CFU/ml factor = CFU/ml
• Test for specified Microorganisms
a) For E. coli : - Identification: Indole test
- Agar: MacConkey Agar • ability of organism to produce
tryptophanase enzyme which
Brick red colony
degrades tryptophan to Indole
• Presence of Indole is detected by
adding Kovacs reagent which turn to
cherry red ring
b) For Staphylococcus aureus :
Sr. No. Media Colony
1. Vogel Johnson Agar Black surrounded by yellow zone
2. Salt Mannitol Agar Yellow colonies
3. Baird Parker Agar Black colony surrounded by zone of clearance
b) For Staphylococcus aureus :

• Identification test: Coagulase test
- Transfer loopful of sample in 5ml

of mammalian plasma
- Incubate at 30℃ for 3 hrs
- Production of coagulase enzyme

causes coagulation of plasma
- Coagulation is positive for S. aureus

c) For Pseudomonas :
Media Colony
Cetrimide Agar Greenish
Pseudomonas agar Fluorescein – yellowish
Pyocyanin – greenish blue
c) For Pseudomonas:
• Identification: Oxidase Test
- Cytochrome oxidase enzyme transfers

electron from donor to acceptor
- Aerobic organisms use oxygen as e- acceptor
- The test reagent, N, N, N’, N’-tetramethyl-p-

phenylenediamine dihydrochloride acts as
an artificial electron acceptor for the enzyme
oxidase
- The dye is reduced to deep purple color

d) For Salmonella :
Media Colony
Bismuth Sulphite Agar Black or green
Xylose-Lysine-Deoxycholate Red/Pink with or without
Agar black centre
d) For Salmonella
• Identification: Triple sugar ion slant
- Salmonella ferments glucose and

decarboxylates lysine producing alkaline
products – slant turns pink
- If sulfate is reduced – deep tube black
- If sulfate not reduced – deep tube yellow

Organism Selective Medium Colony Identification Result

morphology test
E. coli MacConkeys Pink Indole Red Ring
S. aureus •Vogel Johnson •Black Coagulase Coagulation

•Salt Mannitol •Yellow
•Baird Parker •Black
Pseudomonas Cetrimide Bluish-green Oxidase Purple colour
Salmonella •Bismuth Sulphite •Black or Triple sugar Red slant, yellow

•Xylose Lysine green ion deep tube with
Deoxycholate •Red with or or without
without black blackening
centre
Preservation of Pharmaceutical
Products
Preservatives
• A preservative is a natural or synthetic chemical that is added to products to

prevent decomposition by microbial growth or by undesirable chemical
changes
✓ It should not mask any deficiency in the manufacturing procedure

✓ It should be an integral part of the formulation
• A single preservative is not suitable for preservation of all pharmaceutical

preparation
• Combination of two or more preservatives is used to extend the range and
spectrum of preservation
✓ Tablets: Germall 115 + Paraben (antibacterial + antifungal)

✓ Eyedrops: Phenylethyl alcohol + phenoxetol + benzalkonium chloride
Need for Preservatives
To protect To enhance
drug from activity and
microbial efficacy of
attack drug
To stabilize To increase
our product shelf life of
our product
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 act as good antimicrobial agent
➢ It must be potent
➢ It should maintain activity throughout product manufacturing, shelf

life and usage
Performance Requirements
Antimicrobial • Active against microbes at low

Activity concentration
Aqueous Solubility • Should be soluble to reach MIC
• Stable during and at the end of

Stability Properties manufacturing
• Remain in continuous phase in multiphase

Partitioning behavior products
Organoleptic • Acceptable odor and taste during

Properties administration of product
Classification based on mode of action
1) Antioxidants:
The agent which prevent oxidation of API which otherwise undergi
degradation due to oxidation as they are sensitive to oxygen
- Eg: Vitamin E, C, Butylate hydroxy anisole (BHA), Butylate hydroxy toluene
(BHT)
2) Antimicrobial agents:
The agent which is active against Gram +ve and Gram –ve microorganisms
which cause degradation of pharmaceutical preparation
- Eg: Benzoates, Sodium Benzoate, Sorbates
3) Chelating Agents:
The agents which form the complex with pharmaceutical ingredient and
prevent the degradation of formulation
Eg: EDTA, polyphosphates, citric acid
Preservatives used in Pharmaceutical
formulations
Formulation Chemical Preservative

Tablets Methyl paraben
Injections Phenol
Benzyl alcohol
Methyl hydroxy benzoate
Eye drops Benzalkonium chloride
Liquids/mixtures Alcohols
Benzalkonium chloride
Methyl paraben
Chloroform
Semisolids Chlorocresol
Dichlorobenzyl alcohol
Evaluation of Preservatives
Preservative Efficacy Test (PET) or Antimicrobial Effectiveness Test

(AET)
➢ Why needed?
✓ To determine the efficacy of preservative
✓ To Guarantee that no inappropriate organisms are able to sustain themselves
within the product or its packaging
✓ To ensure that the preservative maintains its ability to inhibit microorganism
Challenging the Monitoring

product with kill/survival Interpreting
defined CFU rate at defined the efficacy
time
Product Test Culture

Category Organism Media
Procedure Interpretation
• Test Organism: • Media:

➢ Staphylococcus aureus
➢ Pseudomonas aeruginosa ➢ Soybean Casein Digest Medium
➢ E. coli
➢ Fungi/mold Aspergillus
niger ➢ Sabourauds Dextrose Agar
➢ Yeast Candida albicans
• Procedure:
Determine initial
Inoculate product 1105 – 1*106 concentration by
with specific test microbes/ml of plate count method
organism product
Log (A)
Determine viable
Calculate Log count at 7, 14, 21
reduction = and 28 days of Incubate at 20-
inoculation 25℃
log (A) – log (B)
Log (B)
For Category 1 – Ophthalmic and parenteral preparations
Bacteria 3 log reduction in 14 days, no increase in 28 days
Fungi No increase from initial count at 14 and 28 days
For Category 2 – Topical formulations
Bacteria 2 log reduction in 14 days, no increase up to 28 days
For Category 3 - Oral products other than antacids
Bacteria 1 log reduction in 14 days, no increase up to 28 days
For Category 4 - Antacids
Bacteria No increase from initial count at 14 and 28 days
Cell Culture
Cell Culture
• What is Cell culture?

• Types of cell culture
• Requirements for cell culture
• Procedure of cell cultrue
• Applications of cell culture
Cell Culture
What is Cell Culture
In-vivo In-vitro
What is Cell 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 cell specific media in
plastic flask or petri dish in a controlled environmental artificial
condition (in vitro).
Types of Cell Culture
• Types of culture:
➢ Organ culture: Entire organ or embryo are excised from body

and cultured
– Eg: Heart
➢ Tissue culture: Fragments of excised tissue are cultured

– Eg: muscle tissue
➢ Cell culture: Cells from tissue is dispersed and cultured

– Eg: epithelial cells
1) Primary culture: Primary culture is the cell culture system that

is formed by culture cells directly obtained from tissue and
proliferate them until they occupy all of the available
substrate (i.e. reach Confluence).
a) Adherent: remain attached to solid support

b) Suspension: suspended in liquid media
Heterogenous Finite life

Subculture
cells span
Confluency
1) Primary Cell Culture:
a) Mechanical Disaggregation:
- For the disaggregation of soft tissues: e.g. spleen, brain
- chopping or slicing of tissue into pieces and collection of spill out cells.
b) Enzymatic Disaggregation:
i. Trypsin: Trypsinization – Cold and warm
ii. Collagenase
iii. Other enzymes: bacterial protease
c) Primary Explant Technique:

- Tissue spread on growth surface: outgrowth of cells (explant) – removed
and cultured
2) Secondary culture: When cells from primary culture on

confluency are isolated and cultured in new media, it is called
as secondary culture.
• It is also known as sub-culture, splitting of cells or passage
• Subculture allows fresh nutrients and more space for the
expansion of the cells.
• Cells from primary culture are splits by trypsin/EDTA
treatment.
Homogenous Finite life Contact
cells span inhibition
3) Cell Line:
• After first sub-culture the primary cell culture is known as

cell line.
a) Finite cell line: Normally, cells divide to a limited times, after

that loose their proliferation ability (senescence)
b) Continuous cell line: Cell line which are chemically or

virally transformed (transformation) acquires the ability to
proliferate indefinitely Eg- cancerous cell line
Finite cell line Continuous cell line

• Cells divide to a limited • Transformed to proliferate
times indefinitely
• Contact inhibition • Absence of contact
inhibition
• Anchorage dependence
• Absence of anchorage
• Low growth rate dependence
• Monolayer • High growth rate
• Monolayer or suspension
culture
Types
Primary Secondary Cell line

cell culture cell culture
Adherent Suspension Finite Continuous

Procedure of Cell Culture
Requirements for Cell Culture
https://www.youtube.com/watch?v=CMRKKl9XSDU
Requirements for Cell Culture
Applications of Cell Culture
1) Model system
• interaction between bacteria and virus
• Process of aging
• Effect of pharmaceutical
2) Virology
• Identify, isolate and reproduce viruses
• Study viral infection
3) Vaccine Production
• Viruses produced for making vaccines
• Eg: polio, rabies, measels
4) Cancer research
• Fundamental difference between normal and malignant cell can be
studied
• Mechanism and cause of cancer can be studied
• Identify most effective medication for killing just cancerous cells
5) Drug Screening and toxicity testing
• determine the cytotoxicity and safe dose of drug using cell-based assays
• Study the impact of novel products on cell survival and proliferation
• Liver and kidney cells
6) Genetic Engineering
• Introduce new DNA or RNA into the cell
• Study the expression of new genes and its effect on health of cell
7) Genetically engineered protein
• Produce commercially important proteins such as monoclonal antibody,
insulin, hormones, etc
8) Gene Therapy
• Cells removed from patient lacking a gene
• Missing gene is replaced in cell culture – cells are grown
• Altered cells administered to patients
• Another method using viral vector
9) Genetic counseling
• Fetal cell culture extracted from pregnant women – examine
abnormalities using karyotyping
• Helps in early diagnosis of fetal disorders
10) Replacement tissue or organ

• Regeneration of tissue or organ can be accomplished using cell culture
• For Eg: artificial skin for burns and ulcers
• Research going on for artificial organ Eg: kidney, liver, pancreas

Unit 5 Microbiology

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Unit 5 Microbiology

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Unit5 Microbiology

• Pharmaceutical Product: Active drug + Formulation Additive

Huge loss to the Attract litigation

Physical Chemical Microbial

Eg: pH, Eg: oxidation, Eg: fungi,

• It includes contamination of the product with microbes

Physico-chemical Chemical Spoilage Biological Spoilage

• Viable Growth • Hydrolysis • Release of toxins

d) Odor and taste:

➢ Eg: Ps. aeruginosa, Klebsiella, Serratia

1) Number and type of microorganisms:

➢ High numbers: present unacceptable challenge

• However, inoculum size is not always a reliable indictaor of likely spoilage

➢ Low levels of aggressive pseudomonads in a weakly preserved solution may

➢ Formulations contain: crude vegetable, animal and microbial products

➢ Eg: additives like sugar, amino acids, alcohols – microbial nutrients

➢ Demineralized water produce by ion-exchange resins may contain nutrients

➢ Primary contaminants may produce metabolites which could be used by

3) Moisture content (Water activity):

Organism Water activity

3) Moisture content (Water activity):

➢ Reduce Aw: adding sugars or polyethylene glycols - resist microbial attack

➢ Reduce Aw: drying (tablets, capsules, powders)

- ability of microbes to grow in an environment is thus influenced by its

- Presence or absence of oxygen

- Presence of dissolved oxygen increases redox potential of the product

➢ Reconstituted syrups and multi-dose eye-drop packs: domestic fridge (8°–

➢ Pseudomonads and Gram negative spoilage: mouthwashes, antacids,

➢ Above pH 8 Eg: soap-based emulsions – spoilage is rare

➢ Below pH 6 Eg: fruit juice-flavoured syrups with a pH 3–4 - mould or yeast

➢ Low pH is used to preserve food stuffs - Eg: Pickles, yogurts

➢ Multi-dose containers: contamination by user itself

➢ Change in designs of these containers may minimize contamination and

8) Protection of microorganisms within pharmaceutical products:

• Polymers such as starch, acacia, gelatin – makes microbes resistant to heat

• Adsorption to particulate matter – aid in survival

• suspended particles such as kaolin, magnesium trisilicate or aluminium

Microbial Limit Test

Total Aerobic Viable Test for specified

Total Aerobic Microbial Total Combined Yeast

• Soyabean-Casein Digest Agar • Sabourauds Dextrose Agar

- If the sample contains antimicrobial activity it may be neutralized by adding

Type of antimicrobial Inactivator

Iodine, Quaternary ammonium Lecithin, Sodium Lauryl

Alcohols, aldehydes, sorbates Dilution

Mercurials, halogens Sodium Thiosulphate

Pour Plate Technique

3) Incubate at 30-35℃ for 3-5 3) Incubate at 20-25℃ for 5-7

4) Count the number of colonies, 4) Count the number of colonies,

Spread Plate Technique

2) Spread the sample on the 2) Spread the sample on the

3) Incubate at 30-35℃ for 3-5 3) Incubate at 20-25℃ for 5-7

4) Count the number of colonies, 4) Count the number of colonies,

b) For Staphylococcus aureus :

- Transfer loopful of sample in 5ml

- Incubate at 30℃ for 3 hrs

- Production of coagulase enzyme

- Coagulation is positive for S. aureus

- Cytochrome oxidase enzyme transfers

- Aerobic organisms use oxygen as e- acceptor

- The test reagent, N, N, N’, N’-tetramethyl-p-

- The dye is reduced to deep purple color

• Identification: Triple sugar ion slant

- Salmonella ferments glucose and