STERILIZATION

Definition:
Killing or removing all forms of microbial life
(including endospores) in a material or an

object.

Terminologies
Disinfection: Killing of most microorganisms on a
substance (Inanimate Objects)
Antisepsis: Reduction or Inhibition of microbes found on
LIVING TISSUE
Bacteriostatic Agent: Agent that inhibits the growth of
bacteria, but does not necessarily kill them.
Suffix stasis: To stop or steady.
Germicide: Agent that kills certain microorganisms.
Bactericide: Agent that kills bacteria. Most do not kill
endospores.
Viricide: Agent that inactivates viruses.
Fungicide: Agent that kills fungi.
Sporicide: Agent that kills bacterial endospores or fungal
spores.

Microbial Control Methods

Five sterilization processes are described in the U.S.P:
1. By Steam
2. By Dry heat
3. By Filtration
4. By Gas
5. By Ionizing Radiation
All are commonly used for parenteral products,
except gas and ionizing radiation, which are
widely used for devices and surgical materials.

---sterilization-For sterilization purposes, microorganisms

can be categorized into three general
categories:
a) Easy to kill with either dry or moist heat
b) Susceptible to moist heat, but resistant to dry
heat (e.g. Bacillus subtilis)
c) Resistant to moist heat, but susceptible to
dry heat (e.g. Clostridium sporogenes)

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

A) Sterility test
It is done by detecting the presence of viable
forms of bacteria, fungi and yeast in
parenteral products. The test for Sterility
must be carried out under strict aseptic
conditions in order to avoid accidental
contamination of the product during test.
All glass-wares required for the test must be
Sterile.

--- Sterility test--The test for Sterility may be carried out either by:
a) membrane filtration method
b) Direct inoculation method
Sterility testing attempts to reveal the presence
or absence of viable micro-organisms in a
sample number of containers taken from batch
of product. Based on results obtained from
testing the sample a decision is made as to the
sterility of the batch.

Major factors of importance in sterility

testing:
o
o
o
o
o

The environment in which the test is conducted

The quality of the culture conditions provided
The test method
The sample size
The sampling procedure

Environmental conditions:
a. Environmental conditions avoid accidental
contamination of the product during the test.
b. The test is carried out under aseptic conditions
regular microbiological monitoring should be
carried out.
Culture conditions: Appropriate conditions for
the growth of any surviving organism
should be provided by the culture media
selection.

Factors affecting growth of bacteria:

Nutrition
Moisture
Air
Temperature
pH
Light
Osmotic pressure
Growth inhibitors

Culture media for sterility testing:

Culture media for sterility testing capable of
initiating and maintaining the vigorous growth
of a small number of organisms
Sterile Types of media:
1. Fluid thioglycollate medium,
2. Soya-bean casein digest medium

1. Fluid thioglycollate medium:

specific role of some ingredients primarily
intended for the culture of anaerobic
bacteria.
incubation of the media: 14 days at 30 -35C

2. Soya-bean casein digest medium :

Soya-bean casein digest medium primarily
intended for the culture of both fungi and
aerobic bacteria specific role of some
ingredients.
Incubation of the media: 14 days at 20 -25C

Membrane filtration method

In this method, the test sample of parenteral
preparation are transferred (aseptically) into
sterile nutrient media, suitable pH and
incubated for suitable period of time at
optimum temperature.
Evaluated in the presence of turbidity.

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) solutions to be examined must be
introduced and filtered under aseptic conditions.
All steps of this procedure are performed
aseptically in a Class 100 Laminar Flow Hood

Selection of filters for membrane

filtration:
pore

size

of

0.45

mm

effectiveness

established in the retention of microorganisms

appropriate composition the size of filter
discs is about 50 mm in diameter

The procedure of membrane filtration:

Sterilization of filtration system and
membrane filtration of examined solution
under aseptic conditions (suitable volume,
dissolution of solid particles with suitable
solvents, dilution if necessary).
The membrane is removed, aseptically
transferred to container of appropriate
culture medium passing the culture media
through closed system to the membrane,
incubation in situ in the filtration apparatus
(sartorius, millipore).

Direct inoculation of the culture

medium:
suitable quantity of the preparation to be
examined is transferred directly into the
appropriate culture medium volume of the
product is not more than 10% of the volume
of the medium suitable method for aqueous
solutions, oily liquids, ointments an creams

CLARITY TEST
It is performed to ensure that the parenterals are
free from visible foreign particles. Each parenteral
preparation in its final container is subjected
individually to a visual inspection to exclude the
possibility of foreign particles.
The un -labeled containers are held by the neck
against strongly illuminated black (for dark
particles) & white screen (for light color particles).
The contents of the container are slowly inverted &
rotated, then examined.

--Clarity Test- It may be dangerous when the particle size

is larger than R.B.C. & may block the blood

vessel. This type of products is immediately

rejected from the batch.

The limit test for particulate matter is

prescribed in I.P. 1996 (A- 125)

---Clarity test-- Applicable for: 100 ml or more volume

containers of single dose LV given by IV
infusion.
Not applicable for: Multi-dose injections
Single dose SVP injectable solutions
constituted from sterile solids.