STERILITY

TESTING

By
PresenterMedia.com
 In pharmaceutical manufacture, the sterility of a parenteral product lot is checked by

a statistically valid sampling procedure.

After years of experience, most manufacturers of parenteral products will sterility

test 10 to 20 units of product per lot. The number of units tested may be doubled

when the deliverable volume is 1 ml or less

 SAMPLING REQUIREMENTS
The number of units sampled depends on :

1. the number of units in the batch,

2. the volume of liquid per container,

3. the method of sterilization,

4. the use of a biological indicator system, and

5. the good manufacturing practice requirements of the regulatory agency for the

particular product.
 SAMPLING REQUIREMENTS
For example,

Jika ukuran batch lebih besar dari 500 artikel, minimum 20 unit artikel yang di

sampling

Jika ukuran batch akhir antara 100 dan 500 artikel, maka tidak kurang dari 10 artikel

yang diuji sterilitas

Untuk produk LVP (volume 100 mL/ wadah), paling sedikit 2% dari batch atau 10

wadah yang disampling.

Persyaratan sampling untuk Pengujian Sterilitas yang di spesifikasikan dlm USP dan

European Pharmacopoeia (Tabel 1.2)

 CULTUR MEDIA
1. FLUID THIOGLYCOLATE MEDIUM
(FTM)

The USP and EP describe two primary types of culture media to be

used in the sterility testing of parenteral products.

One type is called fluid thioglycollate medium (FTM), which was

introduced by Brewer (13) in 1949.

The formulation ingredients of FTM and their basic purpose in the

medium are listed in Table 1.3.

 CULTUR MEDIA

FTM provides both aerobic and anaerobic environments within

the same medium.
Thioglycollate and L-cysteine are antioxidants or reducing
agents that maintain anaerobiasis in the lower levels of the
culture tube.
FTM solution has a two-color appearance.
1. The pinkish color of the top part of the solution is indicative of
the presence of resazurin sodium, an oxygen-sensitive
indicator.
2. The pink color should consume no more than one-third of the
medium volume.
 CULTUR MEDIA

Because of the need for two environments in the same test

tube or container, the ratio of the surface to the depth of the

medium is very important

To provide adequate depth for oxygen penetration, a 15-ml volume of

FTM must be contained in a test tube with dimensions of 20 -150

mm.

A 40-ml volume of FTM is to be contained in 25 -200 mm test tubes,

and 75100 ml FTM in 38 -200 mm test tubes.

CULTUR MEDIA
 CULTUR MEDIA
ADVANTAGES & DISADVANTAGES OF FTM

FTM is an excellent medium for the detection of bacterial

contamination.
Thioglycollate also has the advantage of neutralizing the
bacteriostatic properties of the mercurial preservatives.
One disadvantage of FTM is that it will not support the growth of
Bacillus subtilis spores entrapped in solids or material that
locates itself in the anaerobic lower portion of the medium.
Bacillus subtilis spores require an environment of high surface
tension for normal growth.
 CULTUR MEDIA
2. SOYBEAN CASEIN DIGEST (SCB) ATAU
TRYPTICASE SOY BROTH (TSB)
medium

TSB has a slightly higher pH (7.3 0.2) than FTM (7.1 0.2).

TSB replaced Sabouraud medium in the 19th edition of the USP (1970) because TSB was

found from experience tobe a better medium.

It possesses a higher pH and thus was considered a better nutrient for fungal contaminants.

TSB promotes growth of fungi and bacteria and is also considered a better medium for than

FTM slow-growing aerobic microorganisms.

 CULTUR MEDIA
2. SOYBEAN CASEIN DIGEST (SCB) ATAU
TRYPTICASE SOY BROTH (TSB)
medium
CULTUR MEDIA
CULTUR MEDIA
CULTUR MEDIA
 CULTUR MEDIA

Culture media may be purchased in either the dehydrated state or

the ready-to-use fluid state.
Dehydrated media are less expensive and have a longer shelf life.
media tubes must be obeyed, provided that the proper storage
conditions (usually refrigeration) have been met
Strict adherence to the expiration date on the label of premixed culture
 CULTUR MEDIA
When membrane filtration is used for the sterility
test, a diluting fluid must be used to rinse the
filtration assembly to ensure that no microbial
cells remain anywhere but on the filter surface.
The diluting fluid may also be used to dissolve a
sterile solid prior to filtration.
Diluting fluids are intended to minimize the
destruction of small populations of vegetative
cells during the pooling, solubilizing, and filtering
of sterile pharmaceutical products
 TIME AND TEMPERATURE OF INCUBATION

No ideal incubation time and temperature condition exists for the

harvesting of all microorganisms.
Most organisms grow more rapidly at 37C than at lower temperatures.
However, a temperature of about 23C may reveal the presence of some
organisms that might remain undetected if incubations were done at higher
temperatures.
Pittman and Feeley (19) demonstrated that temperatures of 22C and 30C
were more favorable for the recovery of yeasts and fungi in FTM than a
temperature of 35C.
 TIME AND TEMPERATURE OF INCUBATION

The Division of Biologics Standards of the National Institutes of Health

discovered that a pseudomonad contaminant in plasma grew in FTM at

25C, but was killed at 35C.

As a result of this finding, the incubation temperature range of FTM was

lowered from 3235C to 30 35C as required by the USP/NF (20th edition).

The time of incubation for sterility testing by membrane filtration (MF) is 7 or 14

days.
TIME AND TEMPERATURE OF INCUBATION
TIME AND TEMPERATURE OF INCUBATION
TIME AND TEMPERATURE OF INCUBATION
 STERILITY TESTING

The USP and EP sterility tests specify two basic methods for performing
sterility tests :
1. the direct transfer (DT) or direct inoculation method and,
2. the MF (Membrane Filtration) method, with a statement that the latter,
when feasible, is the method of choice.
. In fact, in some cases, membrane filtration may be the only possible choice.
 STERILITY TESTING

The DT method is the more traditional sterility test method.

Basically, the DT method involves three steps:

1) Aseptically opening each sample container from a recently

1.
sterilized batch of product
DIRECT
TRANSFE 2) Using a sterile syringe and needle to withdraw the required
R volume of sample for both media from the container

3) Injecting one-half of the required volume sample into a test

tube containing the required volume of FTM and the other half

volume of sample into a second test tube containing the

required volume of TSB

 STERILITY TESTING

The USP and EP tests require a minimum volume of sample per

container volume to be transferred to a minimum volume of each

culture medium.
1.
DIRECT The sample volume must be a sufficient representation of the entire

TRANSFE container volume and the volume, of medium must be sufficient to

R
promote and expedite microbial growth, if present.

Adequate mixing between the sample inoculum and the culture

medium must take place to maximize interaction and facilitate

microbial growth.
 STERILITY TESTING

1.
DIRECT
TRANSFE
R
 STERILITY TESTING
The MF sterility test became official in the 18th edition of the

USP in 1970.

It has since become the more popular and widely used

method over the DT method and, when feasible for

pharmacopeial articles, should be preferred.

2.
Specific application of the MF sterility test method has been
MEMBRANE
FILTER concerned with the sterility testing of antibiotics , insulin, and

LVPs.

The successful employment of this technique requires more

skill and knowledge than that required for the DT method.

 STERILITY TESTING

Five basic steps are involved in the use of the MF sterility test method:

1. The filter unit must be properly assembled and sterilized prior to use.

2. The contents of the prescribed number of units are transferred to the

filter assembly under strict aseptic conditions.

3. The contents are filtered with the aid of a vacuum or pressure

2.
differential system.
MEMBRANE
FILTER 4. The membrane is removed aseptically and cut in half.*

5. One-half of the membrane is placed in a suitable volume (usually 100

ml) of FTM, and the other membrane half is placed in an equal volume

of TSB
 STERILITY TESTING

2.
MEMBRANE
FILTER
STERILITY TESTING

A membrane suitable for sterility testing has a rating of 0.45

m and a diameter of approximately 47 mm.

2.
MEMBRANE
FILTER
 STERILITY TESTING

ADVANTAGES OF MF

1. Greater sensitivity

2. The antimicrobial agent and other antimicrobial solutes in the

2. product sample can be eliminated by rinsing prior to transferring
MEMBRANE the filter into test tubes of media, thereby minimizing the incidence
FILTER of false-negative test results.
3. The entire contents of containers can be tested, providing a real
advantage in the sterility testing of large-volume parenterals and
increasing the ability to detect contamination of product lots
containing very few contaminated units.
 STERILITY TESTING

ADVANTAGES OF MF

4. Low-level contamination can be concentrated on the membrane by

2. filtering large volumes of product. This results in faster reporting of

MEMBRANE test results since MF requires only 7 days incubation (for most terminally
FILTER
sterilized products).

5. Organisms present in an oleaginous product can be separated from the

product during filtration and cultured in a more desirable aqueous medium

 STERILITY TESTING

DISADVANTAGES OF MF

1) There exists a higher probability of inadvertent contamination in

manual operations because of the need for greater operator skill
and better environmental control in disassembling the filtration
2.
unit and removing, cutting, and transferring the membrane.
MEMBRANE
(Newer systems such as the Steritest have eliminated this
FILTER
disadvantage.)
2) The method is unable in differentiate the extent of contamination
between units, if present, because all product contents are
combined, filtered through a single filter, and cultured in single
test tubes. Also, if accidental contamination has occurred, rather
than this being detected in one or more vessels of the DT method,
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