The document provides comprehensive guidelines on sterility testing, highlighting its importance in ensuring medical products are free from viable microorganisms. It details specific procedures, including required culture media, incubation conditions, and testing methods such as membrane filtration and direct inoculation. Additionally, it emphasizes the significance of method validation, personnel training, and environmental controls to maintain sterile conditions during testing.

1. USP <71> Sterility Testing
http://www.gibraltarlabsinc.com/sterility-testing.html
By: Minal Patel
Proprietary and Confidential © Gibraltar Laboratories, Inc.

2. • Definition
• Culture Media and Incubation Temperature
• Growth Promotion and Method Validation /
Method Suitability Test
• Number of Articles and volume to be tested
• Test Method
• Observation and Interpretation of Results
• Essential thing to know regarding Sterility
testing

3. What is sterility Testing?
• A test conducted to ensure the batch of products is sterile or has been
sterilized, meaning free of viable micro organisms ( Bacteria, yeast or
mold).
• Sterility testing is qualitative test which reveals quality of samples
tested.
• Sterility testing of medical devices is required during the sterilization
validation process as well as for routine quality control.
• The test is applied to substance, preparations, or articles which
according to pharmacopeia are required to be sterile. However, a
satisfactory results only indicated that no contaminating microorganism
has been found in the sample examined under the conditions of the test.
• Sterility testing is a very tedious and artful process that must be
performed by trained and qualified laboratory personnel.

4. Culture Media and Incubation Conditions
→ Fluid Thioglycollate Medium (FTM) and incubation temperature is
30⁰ to 35⁰C for 14 days.
→ Soybean-Casein Digest Medium – SCDB (previously called as
TSB) and incubation temperature is 20⁰ to 25⁰C for 14 days.

5. Fluid Thioglycollate Medium (FTM)
Ingredients Amount Significance
L-Cystine 0.5 g
Reducing Agent, prevent accumulation of
Peroxides which are lethal to some
organism.
Sodium Chloride 2.5 g Osmotic Balance
Dextrose
Monohydrate/Anhydrous 5.5/5.0 g
Energy Source
Agar 0.75 g
Prevent rapid uptake of Oxygen and helps
to maintain anaerobic environment
Yeast Extract (water-soluble) 5.0 g
Growth Factor
Pancreatic Digest of Casein 15.0 g
Growth Factor
Sodium Thioglycollate 0.5 g Reducing agent, remove Oxygen
or Thioglycolic Acid 0.3 mL
Resazurin Sodium Solution (1
in 1000), freshly prepared 1.0 mL
Redox Indicator, red in oxidized state and
colorless under anaerobic conditions
Purified Water 1000 mL pH – 6.9 to 7.3

6. Soybean-Casein Digest Broth (SCDB / TSB)
Ingredients Amount Significance
Pancreatic Digest of Casein 17.0 gram Provide Nitrogen, Vitamins
& Minerals.
Papaic Digest of Soybean Meal 3.0 gram Provide Nitrogen, Vitamins
& Minerals.
Sodium Chloride 5.0 gram For Osmotic Balance
Dibasic Potassium Phosphate 2.5 gram Buffering Agent to control
pH
Dextrose Monohydrate / Anhydrous 2.5 / 2.3 gram Energy source to promote
organisms growth
Purified Water 1000 mL pH - 7.1 to 7.5

7. Diluting and Rinsing Fluids
• Fluid A – 0.1% Peptone water; pH 6.9 to 7.3
• Fluid D – 0.1% Peptone Water with 0.1% Tween 80; pH
6.9 to 7.3
• Fluid K ; pH 6.7 to 7.1

8. Aerobic bacteria
Staphylococcus aureus ATCC 6538, CIP 4.83, NCTC 10788, NCIMB 9518,
NBRC 13276
Bacillus subtilis ATCC 6633, CIP 52.62, NCIMB 8054, NBRC 3134
Pseudomonas aeruginosa 1 ATCC 9027, NCIMB 8626, CIP 82.118, NBRC 13275
Anaerobic bacterium
Clostridium sporogenes 2 ATCC 19404, CIP 79.3, NCTC 532 or ATCC 11437,
NBRC 14293
Fungi
Candida albicans ATCC 10231, IP 48.72, NCPF 3179, NBRC 1594
Aspergillus brasiliensis
(Aspergillus Niger)
ATCC 16404, IP 1431.83, IMI 149007, NBRC 9455
1 An alternative microorganism is Kocuria rhizophila (Micrococcus luteus) ATCC 9341.
2 An alternative to Clostridium sporogenes, when a nonspore-forming microorganism is
desired, is Bacteroides vulgatus (ATCC 8482).
Table: Strains of the Test Microorganisms Suitable for Use in the
Growth Promotion Test and the Method Suitability Test

9. Growth Promotion Test and Method Validation
USP <71> Sterility Test contains two qualifying Assays which must be
performed prior to sterility testing. They are “ Growth Promotion Test” and
“Validation Test” ( Bacteriostasis and Fungistasis Test)
Growth Promotion Test:
Test each lot of medium prior to use for sterility testing to ensure that the
medium used support the growth of <100 viable micro-organisms.
Inoculate portions of the media with less than 100 cfu of appropriate organisms.
Inoculate FTM with less than 100 cfu of Pseudomonas aeruginosa,
Staphylococcus aureus and Clostridium sporogenes . Inoculate SCDB / TSB
with less than 100 cfu of Bacillus subtilis, Candida albicans and less than 100
spores of Aspergillus brasiliensis. Incubate for not more than 3 days in case of
bacteria and not more than 5 days in the case of fungi.
The media are suitable if a clearly visible growth of the microorganisms occurs.

10. Method Validation / Suitability Test /
Bacteriostasis and Fungistasis Test
• The Validation Test is used to determine if the test sample will
inhibit the growth of microorganisms in the test media.
• In terms of microbiology, is defined as the inability of a
microorganism to grow and proliferate in microbiological
media.
• The Validation Test must be performed on each product prior
and / or during sterility testing.
• Microbiological growth in the presence of the test samples is
compared to controls without test samples. If microbial growth
is present in the sample and control, then the test is valid.
• The microorganisms used for suitability test are same as
Growth Promotion Test.

11. Test Methods
1. Membrane Filtration
2. Direct Inoculation
Membrane Filtration
After transferring the content of the container or containers to be tested to the
membrane, add an inoculum of a small number of viable microorganisms (not more
than 100 cfu) to the final portion of sterile diluent used to rinse the filter.
Direct Inoculation
After transferring the contents of the container or containers to be tested (for catgut and
other surgical sutures for veterinary use: strands) to the culture medium, add an
inoculum of a small number of viable microorganisms (not more than 100 cfu) to the
medium.
In both cases use the same microorganisms as those described above under Growth
Promotion Test of Aerobes, Anaerobes, and Fungi. Perform a growth promotion test as
a positive control. Incubate all the containers containing medium for not more than 5
days.

12. Quantity per Container
Minimum Quantity to be Used
(unless otherwise justified and authorized)
Liquids
Less than 1 mL The whole contents of each container
1–40 mL Half the contents of each container, but not less than 1 mL
Greater than 40 mL, and not greater than 100 mL 20 mL
Greater than 100 mL 10% of the contents of the container, but not less than 20 mL
Antibiotic liquids 1 mL
Insoluble preparations, creams, and ointments to be suspended
or emulsified
Use the contents of each container to provide not less than 200 mg
Solids
Less than 50 mg The whole contents of each container
50 mg or more, but less than 300 mg Half the contents of each container, but not less than 50 mg
300 mg–5 g 150 mg
Greater than 5 g 500 mg
Catgut and other surgical sutures for veterinary use 3 sections of a strand (each 30-cm long)
Surgical dressing/cotton/gauze (in packages) 100 mg per package
Sutures and other individually packaged single-use material The whole device
Other medical devices The whole device, cut into pieces or disassembled
Table : Minimum Quantity to be Used for Each Medium

13. Number of Items in the Batch*
Minimum Number of Items to be Tested for Each
Medium (unless otherwise justified and authorized)**
Parenteral preparations
Not more than 100 containers 10% or 4 containers, whichever is the greater
More than 100 but not more than 500 containers 10 containers
More than 500 containers 2% or 20 containers, whichever is less
For large-volume parenterals 2% or 10 containers, whichever is less
Antibiotic solids
Pharmacy bulk packages (<5 g) 20 containers
Pharmacy bulk packages ( 5 g) 6 containers
Bulks and blends See Bulk solid products
Ophthalmic and other noninjectable preparations
Not more than 200 containers 5% or 2 containers, whichever is the greater
More than 200 containers 10 containers
If the product is presented in the form of single-dose containers,
apply the scheme shown above for preparations for parenteral
use.
Catgut and other surgical sutures for veterinary use 2% or 5 packages, whichever is the greater,
up to a maximum total of 20 packages
Not more than 100 articles 10% or 4 articles, whichever is greater
More than 100, but not more than 500 articles 10 articles
More than 500 articles 2% or 20 articles, whichever is less
Bulk solid products
Up to 4 containers Each container
More than 4 containers, but not more than 50 containers 20% or 4 containers, whichever is greater
More than 50 containers 2% or 10 containers, whichever is greater
* If the batch size is unknown, use the maximum number of items prescribed.
** If the contents of one container are enough to inoculate the two media, this column gives the number of containers
needed for both the media together.
Table : Minimum Number of Articles to be Tested in Relation to the Number of Articles in the Batch

14. Things to know Regarding Membrane Filtration
►This test is method of choice for pharmaceutical products.
►Use membrane filters having a nominal pore size not greater than 0.45 µm, in
which the effectiveness to retain microorganisms has been established.
►Cellulose nitrate filters are used for aqueous, oily, and weakly alcoholic
solutions.
►Cellulose acetate filters are used for strongly alcoholic solutions.
Specially adapted filters may be needed for certain products (e.g., for antibiotics).
►Do not exceed a washing cycle of five times 100 mL per filter.
Use the same volume of each medium as in the Method Suitability Test.
►If the product has antimicrobial properties, wash the membrane not less than
three times by filtering through it each time the volume of the chosen sterile
diluent used in the Method Suitability Test.
►Transfer the whole membrane to the culture medium or cut it aseptically into
two equal parts, and transfer one half to each of two suitable media.

15. Things to know Regarding Direct Inoculation
►This test is method of choice for medical devices because the device is in direct
contact with media through out the incubation period.
►Transfer the quantity of the preparation to be examined directly into the culture
medium so that the volume of the product is not more than 10% of the volume of the
medium, unless otherwise prescribed.
►If the product to be examined has antimicrobial activity, carry out the test after
neutralizing with a suitable neutralizing agent or by dilution in a sufficient quantity of
culture medium.
►For extremely large devices, immerse those portions of the device that come in
contact with the patient in a volume of medium sufficient to achieve complete
immersion of those portions.
►Articles can be immersed intact or disassembled.

16. Product Membrane Filtration
Method
Direct Inoculation Method
Oil & Oily
Solution
Low Viscosity – Can be
filtered directly without
Dilution
Viscous – Dilute in Diluent
such as IPM and wash with
Fluid K
Add Emulsifying agent to Medium
such as Tween 80
Ointment and
Creams
IPM – can be warm up to
40C but not more than 44C
Filter rapidly
Dilute Product in Emulsifying
agent containing Medium. Add to
medium, shake every day but
minimum for FTM to keep
Anaerobic Condition
Antibiotics for
Solution -<5g
20 containers and 300 mg
from each into 200 mL of
Diluent or constitute as label
Antibiotics for
Solution -≥5g
6 container and 1 gram from
each into 200mL Diluent

17. Product Membrane Filtration
Method
Direct Inoculation
Method
Antibiotics Solids, Bulk &
Blends
Equivalent to 6 grams of
solids in 200 mL of Diluent
Aerosol Freeze in Alcohol – dry ice
mixture at -20C for 1 hour
and then remove the
content and add 100 mL
of Fluid D
Syringes Remove the content,
pooled in vessel
Immerse the whole
product
Devices pathway labeled
sterile
Flush with Fluid D, not
less than 10 pathway
volume
Fill the lumen.

18. OBSERVATION AND INTERPRETATION OF RESULTS
• Technician must be trained as to how to detect growth during the incubation
period.
• At intervals during the incubation period and at its conclusion, examine the
media for macroscopic evidence of microbial growth.
• After 14 incubation if the presence or absence of microbial growth can not
be determined because of the product nature which cause turbidity in the
media transfer portions (each not less than 1 mL) of the medium to fresh
vessels of the same medium, and then incubate the original and transfer
vessels for not less than 4 days.
• If no evidence of microbial growth is found, the product to be examined
complies with the test for sterility.
• If evidence of microbial growth is found, OOS should be conducted. This
investigation should include: Clean Room environmental test data, media
sterilization records, technician training record, the relative difficulty of the
test procedure, media controls data, personal monitoring data, bioburden
data.

19. OBSERVATION AND INTERPRETATION OF RESULTS – CON’T
The test may be considered invalid only if one or more of the following conditions are
fulfilled:
• Error in test procedure used for the test.
• Microbial growth is found in the negative controls.
• After determination of the identity of the microorganisms isolated from the test,
the growth of this species (or these species) may be misunderstanding to faults
with respect to the material and or the technique used in conducting the sterility
test procedure.
• If the test is declared to be invalid, it is repeated with the same number of units as
in the original test.
• If no evidence of microbial growth is found in the repeat test, the product
examined complies with the test for sterility.
• If microbial growth is found in the repeat test, the product examined does not
comply with the test for sterility.

20. The four pillars of Sterility testing
•Personnel training & monitoring
•Environmental monitoring
•Facilities design
•Aseptic Technique

21. If people are a major source
of contamination how do we
avoid contaminating the
product while testing?

22. Gowning

23. Personnel: Gloves
Aseptic technique of wearing gloves
How often should gloves be examined and sanitized?

24. Gowning Qualification
•Written ( photographic) procedures describing
methods used to do each gown component in an
aseptic manner and the creation of barriers by
overlapping gown components.
•Initial training and periodic assessment.
•Annual requalification.

25. Qualifying Personnel
• Assess after gowning/gloving
• Microbiological surface sampling of several locations
• Glove fingers
• Facemask
• Zipper and Head cover interface
• Boot tops
Periodic requalification is necessary

26. Personnel: Behavior
Minimize movement:
Work slowly and purposefully

27. Active Air Monitoring
RCS Plus
Passive Air Monitoring
Settling Plates
Environmental Monitoring:
→Viable Microbial Air Monitoring
→Non Viable Air Monitoring

28. Environmental
Monitoring: Surface Monitoring
Contact plates
- RODAC Plates
Swabs

29. Horizontal airflow
Unidirectional airflow
The operator should
never come between the
air source and the product.
Minimize movement: Work
slowly and purposefully

30. Facilities: General Cleanroom Design
•HEPA filters on ceiling
•Airlocks and interlocking doors to control air balance
•Seamless and rounded floor to wall junctions
•Readily accessible corners
•Floors, walls, and ceilings constructed of smooth hard
surfaces that can be easily cleaned
•Limited equipment, fixtures and personnel
•Layout of equipment to optimize comfort and movement of
operators

31. Facilities
Establishing and Maintaining an aseptic
environment
•Use clean-rooms of various classes to establish an aseptic area
•Clean rooms use combinations of filtration, air exchange, and positive
pressure to maintain “clean” environment
•Lower quality clean areas should not be placed next to high quality
areas

32. Facilities: Cleanroom Classification
FS209
Cleanroom
classification
ISO 14644-1
Cleanroom
classification
≥0.5um
particles/m3
Viable
Microbes
(cfu/m3)
Ave Airflow
Velocity
(fpm)
Air
changes/hr
100,000 8 3,520,000 100 5-10 5-48
10,000 7 352,000 10 10-15 60-90
1000 6 35,200 7 25-40 150-240
100 5 3,520 1 40-80 240-480

33. Facilities: HEPA Filters
http://people.deas.harvard.edu/~jones/lab_arch/nano_facilities/hepa.gif
High Efficiency Particulate Air
Minimum particle collection efficiency:
99.97% for 0.3µm diameter particles.
Disposable
Filter made of pleated borosilicate glass

34. Facilities: Pressure Differentials
•Used to maintain airflow in the direction of higher
cleanliness to adjacent less clean areas
•Differential pressure between rooms should be more
than 0.020 inches of water and 0.050 inches of water
between the cleanroom and external environment

35. http://news.thomasnet.com/images/large/451/451402.jpg
Facilities: Air Lock
• Permits the passage of objects and people
into a cleanroom.
• Consists of two airtight doors in series
which do not open simultaneously.
• Spray down materials with using a
validated disinfectant before placing in the
airlock

36. •Fiber-shedding materials such as cardboard and paper
•Cardboard packaging must be removed and items placed into
non-cardboard containers.
•Wood (i.e. wooden pallets)
•Undesignated charts
Facilities: Material NOT permitted in a Cleanroom

37. Aseptic Technique
Contact sterile materials only with sterile
instruments:
•Sterile instruments should be held under Class 100
conditions between uses and placed in sterile
containers
•Operators should not contact sterile products,
containers, closures, or critical surfaces with any part
of their gown or gloves

38. Which is correct aseptic technique?
Wrong Correct

39. Aseptic Technique
•Keep the entire body out of the path of unidirectional airflow
•Unidirectional airflow design is used to protect sterile equipment
surfaces, container-closures, and product. Disruption of the path of
unidirectional flow in the critical area can pose a risk to product
sterility.

40. Aseptic Technique
•Approach a necessary manipulation in a manner that does
not compromise sterility of the product
•Proper aseptic manipulations should be approached from the side
and not above the product (in vertical unidirectional flow
operations).
•Operators should refrain from speaking when in direct proximity
to the critical area.

41. Which is correct aseptic technique?
Wrong Correct

42. Which is correct aseptic technique?
Correct

43. 434343
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