1. Sterility test and Rapid
microbiological methods
BY/ MOHAMMED FAWZY
MICROBIOLOGIST AT NODCAR

2. Agenda
Sterility test

Introduction

Definition

Media in sterility test

Methods

Preparation of test products
Incubation period


Growth promotion tests

Interpretation of results
Rapid microbiological methods
Introduction


ATP-bioluminescence

Colorimetric growth detection

Autofluorescence detection

Cytometry systems

3. Sterility test
introduction

The test is applied to substances, preparations, or articles
which, according to the Pharmacopeia, are required to be
sterile. However, a satisfactory result only indicates that no
contaminating microorganism has been found in the
sample examined under the conditions of the test

Sterility test is applied only for sterile, non-pyrogenic
products

Example as parenteral, ophthalmic products, Pulmonary
drug delivery system, and sterile medical devices as ocular
inserts.

4. Introduction

All products labeled sterile must be pass sterility test

This test is suitable to reveal the presence of viable form of
bacteria, fungi and yeasts in pharmaceutical and medical
devices

Sterile product must be free from
 Microorganisms
 Spores

Pyrogens
 Pathogens

5. Definition

Sterility is define as freedom from the presence of viable
microorganisms, is a strict, uncompromising requirement of
an injectable dosage form.

Sterility test is define as:
 Microbiological
test applied to sterile product to
show are products manufactured and processed
under specification guided by cGMP.
 Or

to confirm the products either sterile or non.
sterility test is a destructive test thus, it is impossible to test
every item for sterility.

6. Media in sterility test

USP and EP describe two primary types of culture
media to be used in the sterility testing of
parenteral products.
Fluid
thioglycollate media (FTM)
Soybean
casein digest broth

FTM used for detection of aerobic and
anaerobic bacteria.

SCDB used for detection of molds and molds.

7. Media in sterility test
FTM
1.
Detect aerobic and anaerobic
bacteria.
2.
Contain O2 sensitive substance
(resazurin sodium)
3.
The top part should not
consume more than 1/3 of
tube.
4.
Neutralize the bacteriostatic
properties of mercuric
compounds.
SCDB
1-Detect molds and yeasts
2-possesses a higher pH
3- a better nutrient for
fungal contaminants
4-Suitable for aerobic
bacteria

8. FTM
CONPONANTS
FUNCTION
L-Cysteine 0.5 g
Antioxidant
Agar, granulated (moisture 0.75 g Nutrient and viscosity content
_15%)
Sodium chloride 2.5 g
inducer
Isotonic agent
Dextrose 5.5 g
Nutrient
Yeast extract 5.0 g
Pancreatic digest of casein 15.0 g
Nutrient
Nutrient
Sodium thioglycollate or 0.5 g
thioglycollic acid 0.3 ml
Resazurin sodium solution 1.0 ml
(1:1000), freshly prepared
Purified water QS 1000 ml
Antioxidant
Nutrient
Oxidation indicator
pH after sterilization 7.1 _ 0.2.

9. SCDB
Components
Function
Pancreatic digest of casein 17.0 g
Nutrient
Papaic digest of soybean meal 3.0 g
Nutrient
Sodium chloride 5.0 g
Isotonic agent
Dibasic potassium phosphate 2.5 g
Buffer
Dextrose 2.5 g
Nutrient
Purified watera QS 1000 ml
Solvent
pH after sterilization 7.3 _ 0.2.
---------------------

10. Minimum Quantity to be used for Each
Medium
Minimum Quantity to be Used
(unless otherwise justified and authorized)
Quantity per Container
Liquids (other than antibiotics)
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
Other preparations soluble in water or in isopropyl myristate
The whole contents of each container to provide not less than 200 mg
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
Devices
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

11. Methods
I.
Direct transfer method
It is most usable method and carried out by transfer the test
product after preparation to the medium and incubate for not
less than 14 days.
II.
Membrane filtration method
It is usually used in sterility test of antimicrobial agents as
antibiotic and other. It is carried out by using a membrane filter
having a pore size about (0.45) micrometer that retain most of
bacteria, after filtration of the test product, the membrane is
aseptically transfer and washed with a diluting fluid such as
fluid A,D and K. then aseptically transfer to the medium and
incubate for not less than 14 days.

12. Preparation of test products
a- Oily liquid
Added the tween 80 as 10gm.to 1000 ml of media for emulsification
and to be easily dispersion to media.
b- Ointment and creams:
Preparing by taking about 1 gm. of test product and aseptically
transfer to 10 ml of diluting fluid as fluid A, then transfer to a medium not
contain an emulsifying agent. And incubate for not less than 14 day.
Observe the cultures several times during the incubation period. Shake
cultures containing oily products gently each day. However, when
thioglycollate medium or other similar medium is used for the detection
of anaerobic microorganisms, keep shaking or mixing to a minimum in
order to maintain anaerobic conditions.

13. Preparation of test products
c- devices with pathways labeled sterile
Aseptically pass not less than 10 pathway volumes of Fluid
D through each device tested. Collect the fluids in an
appropriate sterile vessel, and proceed as directed for
Aqueous Solutions or Oils and Oily Solutions, whichever
applies.
d- solids:
Transfer the quantity as described before in the previous
table either directly in a dray form or after making a
suspension by adding sterile diluent to the immediate
container.

14. Preparation of test products
e-Sterile devices:
Articles can be immersed intact or disassembled. To
ensure that device pathways are also in contact with the
media, immerse the appropriate number of units per medium
in a volume of medium sufficient to immerse the device
completely, and proceed as directed above. For extremely
large devices, immerse those portions of the device that are
to come into contact with the patient in a volume of
medium sufficient to achieve complete immersion of those
portions.

15. Preparation of test products
f-For catheters:
where the inside lumen and outside are required to be sterile, either cut them into
pieces such that the medium is in contact with the entire lumen or fill the lumen
with medium, and then immerse the intact unit.
g-Surgical dressing:
Three sample each of about 1 gm. or 10 cm are taken from each
dressing after opening the wrappers. They are chosen from different places
including regions where the contamination is more likely (center and outside). If
dressing is less than 1 gm. Or 10 cm2, it is used entire or divided in to equal pieces
for inoculation in the bacterial and fungal medium. OR each sample is shaken for
10 minutes in 50 ml. of sterile broth and then the liquid is pass through a membrane
filter, the membrane is washed with diluting fluid and then incubated in to
medium.

16. Incubation period
 For
bacteria:
 Temp
 Time
 For
30-35°C.
NLT 14 days.
molds and yeasts:
 Temp
 Time
20-25°C
NLT 14 days.

17. Growth promotion tests

Aerobic bacteria


Bacillus subtilis


Staphylococcus aureus
Pseudomonas aeruginosa
Anaerobic bacterium


Clostridium sporogenes
Fungi

Candida albicans

Aspergillus brasiliensis
(Aspergillus Niger)

18. Growth promotion

Inoculate media


Not more than 100 CFU
Incubate

Bacteria


Fungi


Not more than 3 days
Not more than 5 days
Clear growth of microorganism:

Suitable for use

19. Interpretation of result

examine the media for macroscopic evidence of
microbial growth.

If the material being tested renders the medium turbid so
that the presence or absence of microbial growth cannot
be readily determined by visual examination

14 days after the beginning of incubation 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.

20. Positive and negative control
Positive control


For validation of nutrient
medium.
For testing the growth ability of
media.
Negative control

For validation of nutrient medium.

For testing the sterilization of
media.

Done by exposure to the same
environmental condition as test
exposure.

Done by inoculation of NMT
100 CFU.

Incubate at 30-35 for 14 days.

Incubate at 30-35 for 14 days.

If growth where obtain →used
in sterility test.

If growth where obtain
→sterilization failed or improper
storage of media

21. Interpretation of result
 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, the
product to be examined does not comply with
the test for sterility, unless it can be clearly
demonstrated that the test was invalid for causes
unrelated to the product to be examined.

22. Interpretation of result

The test may be considered invalid only if one or more of the following
conditions are fulfilled:
a.
The data of the microbiological monitoring of the sterility testing facility
show a fault.
b.
A review of the testing procedure used during the test in question reveals
a fault.
c.
Microbial growth is found in the negative controls.

23. Interpretation of result
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

24. RMM
Introduction

Current harmonized compendial sterility test methods using either
membrane filtration or direct inoculation require at least 14days of
incubation.

In cases where drug products either possess an intrinsic turbidity, or
because of their formulation become opaque or cloudy during the
incubation period, identification of microbial contamination based on
visual confirmation of turbidity of growth media becomes difficult. In
such instances, at the end of the 14-day incubation, a portion of the
sample is sub-cultured into fresh medium for an additional 4–5 days to
allow detection, further extending the incubation period.

25. Introduction

The replacement or the supplement of the conventional sterility
test by a rapid microbiology test will have significant benefits.


a test based on current technologies can increase throughput and allow better
data handling.

a greater assurance of product safety.


A rapid method has the potential to produce test results much faster at enhanced
sensitivity.
Time saving.
Characters of ideal method:



simple
economic
relatively easy to implement.

26. ATP Bioluminescence
Adenosine triphosphate (ATP) bioluminescence is a well
established rapid method utilizing a specific substrate and enzyme
combination, luciferin/luciferase, to break down microbial ATP from
growing cells and produce visible light, which can be measured
using a luminometer. Several commercial systems have been
developed for a range of pharmaceutical test applications, including
sterility testing, especially for filterable samples where non-microbial
ATP in the sample is less of a concern. The test time can be
reduced considerably because detection of microbial growth in
culture media is accomplished by ATP-bioluminescence, rather than
by visible turbidity. Typically, results equivalent to those of
compendial tests are available within 7 days or less.

27. Celsis Rapid Detection Advance luminometer
system, AkuScreen™ reagents
which use the enzyme adenylate kinase to increase the
quantity of microbial ATP produced and reduce detection
times by 25-50%.

28. ATP Bioluminescence

ATP (adenosine triphosphate) act as energy source for all a live
organisms That includes microorganisms, and it is for the
detection of bacteria, yeasts, and molds that ATP
bioluminescence has been applied most successfully.

ATP bioluminescence is well-established technique in food
industry

Used for hygienic monitoring and finished product testing

ATP bioluminescence today used in cosmetic industry and
pharmaceutical industry

29. ATP Bioluminescence
 Bioluminescence
is an enzymatically catalyzed
reaction that generates light.
 based
on a reaction that occurs naturally in the
North American firefly, Photinus pyralis.
 The
reaction was famously first described by McElroy
1947.

30. ATP Bioluminescence
 ATP
bioluminescence reaction depend on
production of light that can be detected by an
instrument after magnification .
 The
amount of emitted light is proportionally to
amount of ATP.
 SO.
that there is a linear relationship between the
amount of ATP in a sample and the amount of light
generated.

31. Working mechanism

luciferin-based bioluminescence reaction makes use of the
firefly enzyme luciferase (luciferin 4-monooxygenase, EC
1.13.12.7),which catalyzes the oxidation of D-luciferin in the
presence of ATP, magnesium ions and molecular oxygen.

The reaction yields a quantum of yellow light at 564 nm for
each molecule of the luciferin substrate oxidized.
 ATP
+ Luciferin + O2 →AMP + Oxyluciferin + CO2 + PPi + Light
(Bioluminescence). In presence of luciferase

Light produced from the luciferin–luciferase reaction is
proportional to the amount of ATP used and can be
measured in Relative Light Units (RLU) by a luminometer.

32. ATP Bioluminescence-based
Pallchek™

33. Milliflex Rapid Microbiology
Detection and Enumeration System
The Milliflex® Rapid Microbiology Detection and Enumeration
system from Millipore also uses ATP-bioluminescence to detect
microbial cells and is designed specifically for monitoring
microbial contamination in filterable samples. It is automated,
employing image analysis technology to detect microcolonies
growing directly on the surface of a membrane filter after the
addition of bioluminescence reagents. The system is designed to
be quantitative, but a method has been developed and validated
to use it for a rapid sterility test with an incubation time of just five
days

34. Milliflex Rapid Microbiology
Detection and Enumeration System
prons
cons
 Equivalent
to
compendial tests
 Results in 7 days
 Non-microbial
detection
ATP

35. Colorimetric Growth Detection
Colorimetric growth detection methods rely
on a colour change being produced in a
growth medium as a result of microbial
metabolism during growth, often as a result
of CO2 production.

36. Colorimetric Growth Detection
mechanism
1. Media (two different media are used) as SCDB supplemented with
complex amino acids and carbohydrates and are designed both to
support growth and to ensure optimal C02 production.
2. CO2 sensor :a C02 sensor is bonded to the bottom of each bottle
and is separated from the broth medium by a semipermeable
membrane.
3. The semipermeable membrane is permeable to CO2 and
impermeable to H2 ion and most ion are exist in the media

37. Colorimetric Growth Detection
mechanism
4. Carbon dioxide produced by growing organisms diffuses across the
membrane into the sensor and dissolves in the water, thereby generating
hydrogen ions according to the following equation:
C02 + HO2 +H2CO3 -> H+ + HCO35. Free hydrogen ions can interact with the sensor, which is blue to dark
green in the alkaline state. As C02 is produced and dissolves in the water,
the concentration of hydrogen ions increases and the pH decreases. This
causes the sensor to become lighter green and eventually yellow, which
results in an increase of red light reflected by the sensor.

38. BacT/ALERT® 3D Dual-T Microbial
Detection System
The system is automated and employs sensitive colour
detection and analysis technology to produce a result in
as little as three days. It can detect both aerobic and
anaerobic bacteria, as well as yeasts and molds.
1-Results 3 days.
2-Aerobic/anaerobic bacteria.
3-Yeasts and molds.

39. Autofluorescence detection

All living cells produce a small amount of fluorescence (autofluorescence) and this
can be used to detect microbial colonies growing on a solid surface long before
they are visible to the naked eye.

particularly useful for filterable samples.

where a membrane filter can be incubated on a conventional nutrient medium
and scanned using highly sensitive imaging systems to detect microcolonies.

Automatic detection. uses a large area CCD imaging system without
magnification to detect developing microcolonies.

detect both aerobic and anaerobic bacteria, as well as yeasts and molds.


produce a result in as little as three days.
BacT/ALERT® 3D Dual-T Microbial Detection System from bioMerieux.

40. Cytometry systems






Cytometry does not rely on microbial growth to detect contamination.
but instead uses cell labelling techniques to detect viable microorganisms.
detect a wide range of organisms, including yeasts and molds, within
minutes.
the cells are labelled using a fluorescent dye or a non-fluorescent
substrate, which is converted to a fluorochrome in viable cells.
Detection of the labelled cells occurs by laser scanning in either a flow cell
(flow cytometry), or on a solid phase platform such as a membrane filter
(solid phase cytometry).
AES Chemunex has developed solid phase cytometry detection systems.
The company’s Scan®RDI (also known as ChemScan RDI) system is
capable of detecting 1 CFU per sample and has been evaluated as a
possible RMM for sterility testing. The technology has been developed for
the Stereal®-T sterility testing system.

41. Rapid Sterility Methods
 AES
 BD
Chemunex Scan®RDI (ChemScan RDI) system
Diagnostic Systems BD FACSMicroCount™
 bioMérieux
(industrial applications) BacT/ALERT® 3D
Dual-T Microbial Detection System
 Celsis
Rapid Detection Advance luminometer
system, AkuScreen™ reagents

42. Rapid Sterility Methods
 Merck
KGaA Milliflex® Rapid Microbiology
Detection and Enumeration System
 Pall
Life Sciences Pallchek™ Rapid Microbiology
System
 Rapid
Micro Biosystems Growth Direct™ System