Transcript Sterilization and disinfection

STERILIZATION AND
DISINFECTION
1
Prepared by
Anfal Mohammed
M.Sc.
INTRODUCTION
Sterilization
A physical or chemical process that completely destroys
or removes all microbial life, including spores.
 Disinfection
It is killing or removing of harmful microorganisms
 Disinfectant
Products used to kill microorganisms on inanimate
objects or surfaces. Disinfectants are not necessarily
sporicidal, but may be sporostatic, inhibiting
germination or outgrowth
 Antiseptic
A product that destroys or inhibits the growth of
microorganisms in or on living tissue.
2
 Aseptic
Characterized by the absence of pathogenic microbes.

METHODS OF STERILIZATION
1. Physical methods
 Heat (thermal steri.)
Dry
 Moist


Radiation (cold sterilization)
U.V. light
 Ionizing radiation


Filtration
2. Chemical Methods
3
METHODS OF STERILIZATION
Radiation:

U.V. light- Has limited sterilizing power because
of poor penetration into most materials. Generally
used in irradiation of air in certain areas eg.
Operating Rooms and T.B. laboratories.

Ionizing radiation- e.g. Gamma radiation: Source
Cobalt60 has greater energy than U.V. light,
therefore more effective. Used mainly in industrial
facilities e.g. sterilization of disposable plastic
syringes, gloves, specimens containers and Petri
4
Dishes.
METHODS OF STERILIZATION

Filtration (for sol. And gases)
 May be done under either negative or
positive pressure. Best known example
is the membrane filter made from
cellulose acetate. Generally removes
most bacteria, but viruses and some
small bacteria e.g. Chlamydias &
Mycoplasmas may pass through. Thus
filtration does not technically sterilize
items but it is adequate for
circumstances under which it is used.

Main use: for heat labile substances
e.g. sera, antibiotics.
5
The recommended size filter that will exclude the
smallest bacterial cells is 0.22 micron
METHODS OF STERILIZATION
Sterilization by Heat: Most common
method
 Dry Heat


Simplest method is exposing the item to
be sterilized to the naked flame e.g.
Bunsen
burnerfor
sterilizing
bacteriological loops, knives, blades.

Hot air oven expose items to 160°C for 1
hour. It has electric element in the
chamber as source of heat plus a fan to
circulate air for even distribution of heat
in chamber. Oven without fan is
dangerous.

Used for Metals, Glassware, Ointment,
Oils, Waxes, Powders i.e. items that are
lacking water
6
METHODS OF STERILIZATION
Moist Heat: Uses hot water. Moist heat kills
microorganisms by denaturing proteins.
1. Boiling – quite common especially in domestic
circumstances.
2.Tyndallization named after John Tyndall
 Lengthy process designed to reduce the level of
activity of sporulating bacteria that are left by a
simple boiling water method.
7
METHODS OF STERILIZATION
Moist heat:
Tyndallization

The process involves boiling for a period (typically 20
minutes) at atmospheric pressure, cooling, incubating
for a day, boiling, cooling, incubating for a day, boiling,
cooling, incubating for a day, and finally boiling
again(4).
The three incubation periods are to allow heatresistant spores surviving the previous boiling period to
germinate to form the heat-sensitive vegetative
(growing) stage, which can be killed by the next boiling
step.
8
 The procedure only works for media that can support
bacterial growth - it will not sterilize plain water.

METHODS OF STERILIZATION
Moist heat:
3. Pasteurization
 It aims to reduce the number of viable pathogens
in liquids so they are unlikely to cause disease
 It uses heat at temperatures sufficient to
inactivate harmful organism in milk. Does not
achieve sterilization (does not destroy pyrogens).
 Temperature may be 138°C for a fraction of a
second (flash method), 71.7°C for 15-20 seconds
or 62°C for 30 minutes.
9
METHODS OF STERILIZATION
Moist heat:
4. Autoclaving – Standard sterilization
method in hospitals.


The Autoclave works under the same principle
as the pressure cooker where water boils at
increased atmospheric pressure i.e. because of
increased pressure the boiling point of water is
>100°C.
The autoclave is a tough double walled chamber
in which air is replaced by pure saturated
steam under pressure.
10



The air in the chamber is evacuated and
filled with saturated steam. The chamber is
closed tightly, the steam keeps on filling into
it and the pressure gradually increases.
The items to be sterilized get completely
surrounded by saturated steam (moist heat)
which on contact with the surface of
material to be sterilized, condenses to
release its latent heat of condensation which
adds to already raised temperature of steam
so that eventually all the microorganisms in
what ever form –are killed.
The usual temperature achieved is 121 °C at
a pressure of 15 pps.i. at exposure time of
only 15-20 mins. By increasing the
temperature, the time for sterilizing is
further reduced.
11
Advantages of Autoclave:
 Temperature is > 100°C therefore spores are killed.



Condensation of steam generates extra heat (latent
heat of condensation).
The condensation also allows the steam to penetrate
rapidly into porous materials.
Note: that autoclavable items must be steam
permeable. Can not be used for items that are lacking
water.
12
Monitoring of autoclaves
 Physical- use of thermocouple to
measure accurately the
temperature.

Chemical- it consists of heat
sensitive chemical that changes
color at the right temperature and
exposure time.



Autoclave tape
Browne’s tube.
Biological – where a sporebearing organism is added during
the sterilization process and then
cultured later to ensure that it has
been killed.
13
Sterilization by Chemical Methods
Useful for heat sensitive materials e.g. plastics and
lensed instruments endoscopes.
 Ethylene Oxide (EO) Chamber:

Ethylene oxide alkylates DNA molecules and thereby
inactivates microorganisms.
 Ethylene oxide may cause explosion if used pure so it is
mixed with an inert gas e.g. Neon, Freon at a ratio of 10:90
 It requires high humidity and is used at relative humidity
50-60%, Temperature : 55-60°C and exposure period 4-6
hours (to reduce the sterilization time).


Activated alkaline Glutaraldehyde 2%:

Immerse item in solution for about 20 minutes if organism
is TB. In case of spores, the immersion period is extended to
2-3 hours.
14
DISINFECTANTS

Factors influencing activity of Disinfectants
 Directly proportional to temperature.
 Directly proportional to concentration up to a point –
optimum concentration. After this level no advantage in
further increases in concentration.
 Time: Disinfectants need time to work.
 Range of Action : Disinfectants are not equally effective
against the whole spectrum of microbes.
e.g.
Chlorhexidine is less active against GNB than Gram
Positive Cocci.
 May be inactivated by
Dirt, organic matter
 Proteins, Pus, Blood, Mucus, Faeces
 Cork and some plastics.


Hypochlorites and Glutaraldehyde are more
active against hepatitis viruses than most other
15
disinfectants.
DISINFECTANTS
Types of Disinfectants
Phenol and phenolics
 Phenol (carbolic acid) is seldom used today. Derivatives of the
phenol molecule, however, are widely used.
 Phenolics injure plasma membrane, inactivate enzymes, or
denature proteins. They are stable, persistent, and are not
sensitive to organic matter.
O-Phenylphenol
 It is the main ingredient in most formulations of Lysol.
Hexachlorophene
 It is main ingredient of a prescription lotion, pHisoHex, used
in nurseries and for surgical and hospital microbial control
procedures to control gram positive skin bacteria such as
staphylococci and streptococci.
 Excessive use can cause neurological damage.
Triclosan
 It is a widely used found in many household products. It has
broad spectrum of activity, especially against gram positive16
bacteria. It is also effective against gram negative bacteria and
fungi.
DISINFECTANTS
Biguanides
 Chlorhexidine, a member of the biguanide group, is not a phenol, but
its structure and applications resemble hexachlorophene. It is
frequently used for surgical skin preparation and surgical hand
scrubs.
Halogens
 Iodine: is effective against all kinds of bacteria, many endospores,
fungi, and some viruses. Its mechanism of activity may be its
combination with the amino acid tyrosine in enzyme and cellular
proteins.
 An iodophore is a combination of iodine and an organic molecule.
Iodophores do not stain and are less irritating than iodine. Examples
are Isodine and Betadine.
 Chlorine: is used as a gas or in combination with other chemicals.
Chlorine gas is used for disinfecting local water supplies, swimming
pools, and sewage. Sodium hypochlorite – ordinary household bleachis good disinfectant.
 Chloramines consist of chlorine and ammonia. They are more stable
than most chlorine. The U.S. military uses tablets for field
disinfection of water.
17
 Chlorine dioxide in gaseous form is used for area disinfection, most
commonly to kill endospores of anthrax bacteria.
DISINFECTANTS
Alcohols
 Both ethanol and isopropanol (rubbing alcohol) are
widely used, normally at a concentration of about
70%.



Concentrations of 60% to 95% are effective.
They are bactericidal and fungicidal but are not
effective against endospores or non-enveloped
viruses.
Alcohols enhance the effectiveness of other
chemical agents.
18
Heavy metals and their compounds
 Tiny amount of heavy metals (e.g. silver and copper) are effective
antimicrobials. A silver coin on an inoculated nutrient medium
will inhibit growth for some distance.




1% silver nitrate solution has been used to prevent gonorrheal
ophthalmia neonatorum, which the infants might have contracted
as they passed through the birth canal (recently been replaced by
antibiotics).
Silver-sulfadiazine is used in wound dressings. Available as
topical cream for use on burns.
Mercuric chloride is highly bactericidal, but is toxic and corrosive
and is inactivated by organic matter. Organic mercury compounds
such as Mercurochrome are less irritating and less toxic than
inorganic mercury.
Copper sulfate is often used to destroy green algae in reservoirs or
other water.
19

Zinc chloride is used in mouthwashes, and zinc oxide is used in
paints as antifungal.
20
 Filtration: The separation of solid from a fluid by means of a
porous medium that retains the solid but allows the fluid to
pass.
 Clarification: This term is applied when solid do not exceed
1.0% and filtrate is the primary product.
 Ultra-Filtration: Separation of intermicellar liquid from solid by
the use of pressure on a semi permeable membrane.
 Cake Filtration: If recovery of solid is desired, the process is
called cake filtration.
21
 Feed or Slurry: The suspension of solid and liquid
to be filtered is known as the slurry or feed.
 Filter Medium: The porous medium used to retain
the solids is described as the filter medium.

Filter Cake: The accumulation of solids on the
filter is referred to as the filter cake.
 Filtrate: The clear liquid passing through the filter
is the filtrate.
22
 The flow of solid is resisted by the filter medium while
the liquid is allow to pass.
 As the filtration proceeds, the retention of the solid on
the filter media goes on increasing which acts as a
secondary and some times more efficient filtering media.
23
1. Area of filter surface.
2.
3.
4.
5.
6.
7.
24
Particle size of cake solids.
Pore size of filter media.
The resistant of the filter cake and filter media.
Viscosity of liquid to be filtered.
Temperature.
Pressure difference across the filter
Where:
V= Volume of filtrate
T= Time
A= Filter area
P= Total pressure drop through cake and filter media.
µ= Filtrate viscosity
α= Average specific Cake resistant
W= Weight of dry cake solids.
R=Resistant of filter medium.
“The surface upon which solids are deposited in a filter
is called the Filter medium”.
PROPERTIES OF IDEAL FILTER MEDIA:
1. Chemically inert.
2. High retention power.
3. Sufficient mechanical strength.
4. Absorbs negligible amount of material.
5. Resistant to the corrosive action of liquid.
Selection of filter media depends on followings:
1. Size of particle to be filtered.
2. Amount of liquid to be filtered.
3. Nature of product to be filtered.
4. Purpose of filter.
26
 Filter paper:
Filter paper is a common filter
medium since it offers controlled
porosity,
limited
absorption
characteristic, and low cost
• It has different grades and
qualities, different pore size such
as coarse medium and fine.
Disadvantages:
• They shed very fine particle to the
filtrate.
• Absorb small quantity of liquid.
27
Woven Material:
Cotton, silk, wool, nylon & glass etc.
Nylon cloth:
• Superior to the cotton cloth.
• Not affected by molds, fungus and bacteria.
• Has negligible absorption properties.
• It is extremely strong as compared to cotton cloth.
Woven wire cloth:
• Made from stainless steel.
• Easily cleaned.
• Long lasting.
• Resistant to the chemicals.
28
Cotton Wool:
• Commonly used.
• Small tough of cotton wool
placed in the neck of
funnel.
 Glass wool:
• Use for filtering highly
corrosive chemicals.
• May contaminate the
filtrate with glass fibers
29
Asbestos:
• Also used for filtering the
corrosive liquid.
• They impart alkalinity to the
filtrate.
• Alkaloids may get absorbed.
• May contaminate the filtrate.
30
 Membrane Filter:
• These are very common
among the ultra filtration
methods.
• Made up of cellulose,
Polyvinylchloride,
Nylon
and
other
cellulose
derivatives.
• They are very fine having a
very wide range of pore size
from 8µ down to 0.22µ.
31
Pore size (in µ)
Particles removed
0.2
0.45
0.8
1.2
All bacteria
All coliform group bacteria
All air born particles
All Non living particles considered dangerous in I.V. Fluid.
5
All Significant cell from body fluid
32

•
•
•
•
•

•
•
•
Advantages:
Bacteria are removed by sieving
Absorption of medicament is negligible
In every new operation, a new disc is used
Filtration is quite rapid
Don’t liberate particles to the filtrate.
Disadvantages:
Fine pores may get clogged easily
Soluble in certain organic solutions e.g. ketones and esters
Very brittle when dry.
 Craft Paper:
•
•
•
•
Mainly used in plate and frame filters.
They offer controlled porosity
Limited absorption.
Quite cheap.
 Sintered Glass:
•
•
•
•
•
•
33
Consist of Pyrex glass powder.
Used for filtering parenteral preparations.
Useful for filtering the corrosive liquid and oxidizing agent.
Don’t shed particles.
Don’t absorbs any liquid.
Can be easily washed.
“The substances which when added to the liquid to be
filtered, reduce the resistance of the filter cake and
increase the filtration”.
 Properties of Filter Aids:
• Chemically inert
• Low specific gravity
• Insoluble in liquids
• Form a porous cake
• Free from impurities
• Suitable particle size with irregular shape
• Able to remain suspended in liquids
• Free from moisture
34
Materials
Chemical
composition
Advantages
Disadvantages
Diatomaceous earth
Silica
Wide size range
Slightly soluble in
acid and alkalies
Perlite
Silica
+Aluminosilicate
Wide size range
More soluble
Asbestose
Aluminosilicate
Very good retention
on coarse screen
More soluble
Cellulose
Cellulose
Chemically inert
Expensive
Carbon
Carbon
Non reactive with
strong alkalies
Expensive
35
DIATOMACEOUS EARTH
36
ASBESTOSE
CELLULOSE
PERLITE
CARBON
Precoating: It requires suspending the filter aid in a
liquid and circulating the feed until the filter aid is
uniformly deposited on the filter septum.
Quantity required:
• 5-15 pounds / 1002 feet of filter area.
• Or filter cake thickness=1/16 to 1/8 inch
Body Mix:
• Direct addition of filter aid to the filter feed.
• Ratio of filter aid=0.1 to 0.5 % of total liquid.
• OR 1 kg filter cake : 1-2kg of filter aid.
37
Selection of method and equipment
required for filtration of a liquid depends
on nature of the material and quantity to
be filtered as well as the object of the
operation.
38
FILTER FUNNEL:
• Funnels are conical shaped
devices
• Made up of Glass, Aluminum,
Polythene, Stainless steel OR
any other suitable material.
• Neutral Glass made funnel
are most commonly used.
• White filter paper of suitable
pore size is folded in such a
way that it fits in the funnel.
39
BUCHNER FUNNEL:
• Made up of porcelain.
• It has a perforated plate.
• Used for filtration under reduced
pressure.
HOT WATER FUNNEL:
• These are doubled wall funnels made
up of metals.
• Viscous substances such as liquid
paraffin, Glycerin, Castor oil and fatty
substances like wool fat, bees wax,
ointments and cream etc are filtered
easily from this filter.
• Boiling water or steam is circulated in
jacketed funnel.
40
FILTER PRESS:
•
•
•
•
41
It consists of hollow frames and solid plates.
Plates have grooved surface to support the filter cloth.
Each plate has an outlet for filtrate.
Frames are opened with an inlet for the liquid to be
filtered.
Advantages:
• Construction is very simple.
• Used for coarse to fine filtration.
• Operation and maintenance is easy.
• Filter cloth can be easily replaced.
Disadvantages:
• Not economical for filtration of small quantities
• Leakage between the plates may take place
• Suitable when the slurry contain less than 5%
solids.
42
ROTARY FILTERS:
• They are used when the proportion of solid content in slurry is 15
to 30%.
• It consists of perforated matter drum wrapped with filter cloth.
• Drum is partially immersed in the tank containing the materials to
be filtered.
• Drum rotates at the slow speed and creates vacuum due to which
filtrate enters into the drum.
• Filter cake deposits on outer surface of the filter media.
• Cake is removed by scrapping with a knife.
43
Advantages:
• Labor costs are very low.
• Area of filtration is very high.
• Thickness of the filtration cake is controlled by
altering with the speed of rotation of drum.
Disadvantages:
• Very expensive.
• Can’t be used for slurries containing low
percentage of solids.
44
FILTER LEAF:
•
•
•
•
45
It consists of frame in which drainage screen is enclosed.
Whole unit is covered with a filter media.
Outlet is connected to the vacuum pump.
Frames may be shaped in round, square or rectangular.
Advantages:
• Liquid can be filtered form any vessel.
• Filter cake can be removed simply by washing or
blowing air.
• It is very economical.
Disadvantage:
• It is not effective when solid content in the liquid is
more than 5%.
46
 VACUUM FILTRATION
:
• Vacuum filtration is used primarily to collect a
desired solid (cake filteration).
• Vacuum filtration uses a Buchner funnel and a sidearm flask.
• Vacuum filtration is faster than gravity filtration,
because the solvent or solution and air is forced
through the filter paper by the application of
reduced pressure.
47
To perform a vacuum
filtration:
• Clamp the flask securely to a
ring stand.
• Add a Buchner funnel with a
rubber funnel adaptor.
• Obtain a piece of filter paper in
the funnel that is small enough
to remain flat but large enough
to cover all of the holes in the
filter. If necessary, you can cut a
larger piece of filter paper
down to size.
48
• Place the paper in the
funnel.
• Connect the side arm flask
to a vacuum source. Always
use thick-walled tubes.
• Wet the paper with a small
amount of the solvent to be
used in the filtration. This
causes the paper to adhere
to the plate and keeps
materials from passing
under the paper during
filtration.
49
• Turn on the vacuum source.
• Pour the mixture to be
filtered onto the filter
paper. The vacuum should
rapidly pull the liquid
through the funnel.
• Rinse the cake with a small
amount of cold fresh
solvent to help remove
impurities
that
were
dissolved in the filtrate.
50
• Carefully disconnect the
rubber tube.
• Remove the filter paper
and the collected solid
that is on it.
51
STERILE PRODUCTS
52
PARENTERAL
Parenteral refers injectable
route of administration.
It derived from Greek words Para (Outside) and
enteron (Intestine).
So it is a route of administration other than the
oral route. This route of administration bypasses
the alimentary canal
53
PRIMARY PARENTERAL ROUTES
Routes
Usual volume
(mL)
Needle
commonly
used
Formulation
constraints
Types of
medication
administered
Sub cutaneous
0.5-2
5/8 in. ,
23 gauge
Need to be isotonic
Insulin, vaccines
Intra muscular
0.5-2
1.5 in. ,
23 gauge
Can be solutions,
emulsions, oils or
suspensions
Isotonic preferably
Nearly all drug
classes
Intra venous
1-100
Vein puncture
1.5 in. ,
20-22 gauge
Solutions, emulsions
and liposomes
Nearly all drug
classes
LVP (LARGE VOL.
PAR.)
101 and larger
(infusion unit)
Venoclysis
1.5 in. ,
18-19 gauge
Solutions and some
emulsions
Nearly all drug
classes
SVP
54
S. No.
ADVANTAGES
DISVANTAGES
1.
Quick onset
Wrong dose or over dose can
be fatal
2.
Vomiting and
Pain at site
unconscious patients can
take
3.
Prolonged action by
modified formulation
( Depot)
4.
Nutritive fluids (glucose, Expensive
electrolytes) can be given
5.
Drugs with poor
absorption or instability
from GIT
Trained person required
NECESSITY OF ASEPTIC
CONDITIONS IN
PRODUCTION,
COMPOUNDING AND
ADMINISTRATION
55
A. CONTAINERS:
1. Glass:
Highly Resistant Borosilicate Glass
 Treated Soda lime Glass
 Regular Soda Lime Glass
 N.P (Non-parenteral) Glass
Type 4 is not used for parenteral packaging,
others all are used for parenteral packaging.

56
2. Plastic:
Plastic containers are used but they face following problems
 Permeation
 Sorption
 Leaching
 Softening
3. Rubber:
To provide closure for multiple dose vials, IV fluid bottles,
plugs for disposable syringes and bulbs for ophthalmic
pipettes, rubber is the material of choice.
Problems associated with rubber closures are
 Incompatibility
 Chemical instability
 Physical instability
57
B. CLOSURE:

•
•
•
•

Characteristics of Good Pharmaceutical rubbers :Good ageing qualities
Satisfactory hardness and elasticity
Resistance to sterilization conditions
Impermeable to moisture and air
Examples:

Butyl Rubbers

Natural Rubbers

Neoprene Rubbers

Polyisoprene rubbers

Silicone Rubbers
58
INTRAVENOUS ADMIXTURE SYSTEM




“Admixture system” refers to sterile IV
solutions that are prepared by using one or
more medications or electrolytes and will be
administered via the parenteral route.
It requires the measured addition of a
medication to a 50 ml or larger bag or bottle of
IV fluid.
It can be provided to the patient in his/her
home.
Many hospitals involved in compounding IV
solutions and medications to outpatient
settings.
59
METHODS FOR SAFE & EFFECTIVE USE OF
IV ADMIXTURE
Proper training to nurses & pharmacist
 Instruction regarding labeling Information for
stability & compatibility to the hospital
pharmacy dept.
 Information for the formulation skills to the
pharmacist.

60
PROCESSING OF PARENTERALS
S.No.
STEPS
1.
Cleaning of containers, closures and equipments
2.
Collection of materials
3.
Preparation of parenteral products
4.
Filtration
5.
Filling the preparation in final containers
6.
Sealing the containers
7.
Sterilization
8.
Evaluation of parenteral preparation
9.
Labeling and packaging
61
FORMULATION OF PARENTERAL PRODUCTS

In the preparation of parenteral products, the following substances are
added to make a stable preparation:
The active drug
 Vehicles

Aqueous vehicle (e.g. water for injection, water for injection free
from CO2 )
 Non-aqueous vehicle (e.g. Ethyl alcohol, propylene glycol, almond
oil)


Adjuvants
Solubilizing agents (e.g. Tweens & polysorbates)
 Stabilizers & antioxidants (e.g. thiourea, ascorbic acid, tocopherol)
 Buffering agents (e.g. citric acid, sodium citrate)
 Antibacterial agents (e.g. benzyl alcohol, metacresol, phenol)
 Chelating agents (e.g. EDTA)
 Suspending, emulsifying & wetting agents (e.g. MC, CMC)
 Tonicity factor (e.g. sodium chloride, dextrose)
62

PRODUCTION FACILITIES OF PARENTERALS

The
production
area
where
the
parenteral
preparation are manufactured can be divided into
five sections:

Clean-up area

Preparation area

Aseptic area

Quarantine area

Finishing & packaging area
63

Clean-up area:





It is not aseptic area.
All the parenteral products must be free from foreign
particles & microorganism.
Clean-up area should be withstand moisture, dust &
detergent.
This area should be kept clean so that contaminants
may not be carried out into aseptic area.
Preparation area:


In this area the ingredients of the parenteral
preparation are mixed & preparation is made for filling
operation.
It is not essentially aseptic area but strict precautions
are required to prevent any contamination from
outside.
64

Aseptic area:

The parenteral preparations are filtered, filled into final
container & sealed should be in aseptic area.

The entry of personnel into aseptic area should be
limited & through an air lock.

Ceiling, wall & floor of that area should be sealed &
painted.

The air in the aseptic area should be free from fibers,
dust and microorganism.

The High efficiency particulate air filters (HEPA) is
used for air.

UV lamps are fitted in order to maintain sterility.
65

Quarantine area:




After filling, sealing & sterilization, the parenteral
product are held up in quarantine area.
Randomly samples were kept for evaluation.
The batch or product pass the evaluation tests are
transfer in to finishing or packaging area.
Finishing & packaging area:




Parenteral products are properly labelled and
packed.
Properly packing is essential to provide protection
against physical damage.
The labelled container should be packed in
cardboard or plastic container.
Ampoules should be packed in partitioned boxes 66
EVALUATION OF PARENTERAL
PREPARATIONS
 The
finished parenteral products are
subjected to the following tests, in
order to maintain quality control:
 A) Sterility test
 B)Clarity test
 C)Leakage test
 D)Pyrogen test
 E)Assay
67
A) STERILITY TEST

It is a procedure carried out to detect and confirm
absence of any viable form of microbes in or on
pharmacopeia preparation or product.
1)
Method of sterility testing
i ) METHOD 1 Membrane filtration method
ii) METHOD 2 Direct inoculation method
68
MEMBRANE FILTRATION METHOD
(METHOD 1):

Membrane filtration Appropriate for : (advantage)

Filterable aqueous preparations

Alcoholic preparations

Oily preparations

Preparations miscible with or soluble in aqueous
or oily (solvents with no antimicrobial effect)

All steps of this procedure are performed aseptically
in a Class 100 Laminar Flow Hood
69
Membrane filter 0.45μ porosity
Filter the test solution
After filtration remove the filter
Cut the filter in to two halves
First halves (For Bacteria)
Transfer in 100 ml culture media
(Fluid Thioglycollate medium)
Incubate at 30-350 C for not less then 7
days
Observe the growth in the media
Second halves (For Fungi)
Transfer in 100 ml culture media
(Soyabeans-Casein Digest medium)
Incubate at 20-250 C for not less then 7
days
70
Observe the growth in the media
DIRECT INOCULATION METHOD (METHOD
2):
Suitable for samples with small volumes
 volume of the product is not more than 10% of the
volume of the medium
 suitable method for aqueous solutions, oily liquids,
ointments and creams
 Direct inoculation of the culture medium: suitable
quantity of the preparation to be examined is
transferred directly into the appropriate culture
medium & incubate for not less than 14 days.

71
OBSERVATION AND RESULTS
Culture media is examined during and after at the end of
incubation. The following observations are possible:
1)
No evidence of growth
2)
There is evidence of growth
Pass the test for sterility.
Re-testing is performed
same no. of sample, volume & media as in original test
No evidence of growth
3)
There is evidence of growth
Pass the test for sterility.
isolate & identify the
organism.
Re-testing is performed with twice no. of sample if:
No evidence of growth
Pass the test for sterility.
72
B)CLARITY TEST
Particulate matter is defined as unwanted mobile
insoluble matter other than gas bubble present in
the product.
 If the particle size of foreign matter is larger than
the size of R.B.C.. It can block the blood vessel.
 The permit limits of particulate matter.are
follows:

73
METHODS FOR MONITORING PARTICULATE
MATTER CONTAMINATION:
1)
2)
3)
4)
Visual method
Coulter counter method
Filtration method
Light blockage method
74
C)LEAKAGE TEST

The sealed ampoules are subjected to small cracks which
occur due to rapid temperature changes or due to
mechanical shocks.
Filled & sealed ampoules
Dipped in 1% Methylene blue solution
Under negative pressure in vacuum chamber
Vacuum released colored solution enter into the ampoule
Defective sealing
Vials & bottles are not suitable for this test because the
sealing material used is not rigid
75
D)PYROGEN TEST
Pyrogen = “Pyro” (Greek = Fire) + “gen” (Greek =
beginning).
 Fever
producing, metabolic by-products of
microbial growth and death.
 Bacterial pyrogens are called “Endotoxins”. Gram
negative
bacteria
produce
more
potent
endotoxins than gram + bacteria and fungi.
 Endotoxins are heat stable lipopolysaccharides
(LPS) present in bacterial cell walls, not present
in cell-free bacterial filtrates

76
1. RABBIT TEST: METHOD:









Dissolve the subs being examined in, or dilute it with a
pyrogen free saline solution
Warm the liquid being examined to approx. 38.5o C temp
before injection into rabbit
The volume of injection is NLT 0.5ml/kg & NMT 10ml/kg of
body weight
Withhold water during test
Clinical thermometer is inserted into the rectum of rabbit to
record body temp.
2 normal reading of rectal temp. are should be taken prior to
the test injection at an interval of half an hr & its mean is
calculated- initial temp
The solution under test is injected through an ear vein
Record the temp of each rabbit in an interval of 30 min for 3
hrs
The difference between initial temp & maximum temp is
recorded- taken as response
77
INTERPRETATION OF RESULTS
78
2. LIMULUS AMEBOCYTE LYSATE [LAL]
TEST
Limulus amebocyte lysate [LAL] test another
method for the determination of pyrogenic
endotoxins
 In this method the test solution is combined with
a cell lysate from the ameabocyte [blood cells] of
horse shoe crab
 Any endotoxin that might be present will be
coagulated with protien
fraction of the
ameabocytes and results in the formation of a gel
 This consider to be simple,rapid and of greater
sensitivity that the rabbit test

79
E)ASSAY
Assay is performed according to method given In
the monograph of that parental preperation in
the pharmacopoeia
 Assay
is done to check the quantity of
medicament
present
in
the
parenteral
preperation

80
THANK YOU……..
81