Lab. No.2 Sterility Testing. applied to products intended to be sterile. (Ophthalmic and Parenteral preparations) applied to products intended to be sterile

Lab. No.2Lab. No.2

Sterility TestingSterility Testing


applied to products applied to products intended to be intended to be sterile. (Ophthalmic sterile. (Ophthalmic and Parenteral and Parenteral preparations)preparations)

Performed on Performed on random samples random samples from the batch.from the batch.


Must be carried out under aseptic Must be carried out under aseptic condition. condition.

Medium used in Medium used in sterility testingsterility testing

Trypticase Soya Broth Trypticase Soya Broth (TSB):(TSB):

It is used to detect the growth of It is used to detect the growth of aerobic bacteria, but when aerobic bacteria, but when incubated at 25incubated at 25ooC it will be C it will be suitable for the growth of fungi.suitable for the growth of fungi.


Fluid thioglycolate:Fluid thioglycolate: It is used to detect the growth of It is used to detect the growth of

anaerobic bacteria, It also could anaerobic bacteria, It also could be used to detect the growth of be used to detect the growth of aerobic bacteria.aerobic bacteria.


It is anaerobic medium due to presence of:It is anaerobic medium due to presence of: Sodium thioglycolate and cystaine which act Sodium thioglycolate and cystaine which act

as reducing agents.as reducing agents. Small amount of agar to increase viscosity of Small amount of agar to increase viscosity of

the medium thus decreasing convection the medium thus decreasing convection current.current.

Redox indicator (Methylene Blue) which Redox indicator (Methylene Blue) which change colour of the medium when 30% of change colour of the medium when 30% of the medium become oxidized. the medium become oxidized.

Fluid thioglycolate:Fluid thioglycolate:


Fluid thioglycolate:Fluid thioglycolate: It should be prepared in long tubeIt should be prepared in long tube

O2

aerobic bacteria

anaerobic bacteria


Sabaroud’s dextrose Sabaroud’s dextrose medium:medium:

It is suitable medium used to detect It is suitable medium used to detect the growth of fungi because:the growth of fungi because:

it has acidic pH.it has acidic pH. contains dextrose which is readily contains dextrose which is readily

fermentable sugar.fermentable sugar.

Incubation ConditionIncubation Condition• IncubationTemperature:

• Incubation Period:

It should be not less than It should be not less than 7 7 days.days.

Incubate the media intended for Incubate the media intended for detection of bacteria at detection of bacteria at 35oC. Incubate the media intended for Incubate the media intended for detection of fungi at detection of fungi at 25oC.

Performed exactly under the Performed exactly under the same condition as the test.same condition as the test.

Used to test the media used in Used to test the media used in the sterility test.the sterility test.

Control testsControl tests

Control testsControl tests

Negative control:Negative control: It is done by incubation of It is done by incubation of

uninoculated tubes of each medium uninoculated tubes of each medium used in the test.used in the test.

It is done to test sterility of the It is done to test sterility of the medium.medium.

Control testsControl tests Positive control:Positive control:

It is done to test incubation It is done to test incubation condition to show if the condition to show if the microorganism will actually grow microorganism will actually grow under the condition of the test or under the condition of the test or not.not.

Clostridium sporogenes Staph.aureus Candida albicans

35oC 35oC 25oC

ThioTSB TSB

Methods of Sterility Methods of Sterility testtest

Direct Inoculation:Direct Inoculation: Suitable for samples with small volumes.Suitable for samples with small volumes.

ThioTSB TSB

Methods of Sterility Methods of Sterility testtest

Membrane Filtration:Membrane Filtration: Suitable for samples with large volumes.Suitable for samples with large volumes. Applied by:Applied by:

1. Filtering the sample through a membrane filter.1. Filtering the sample through a membrane filter.

2. Aseptically cut the membrane into three equal pieces.2. Aseptically cut the membrane into three equal pieces.

3. inoculate them on appropriate 3. inoculate them on appropriate

culture media. culture media.

Application of Sterility Application of Sterility test to Different Dosage test to Different Dosage FormsForms Aqueous Solutions:Aqueous Solutions: Tested directly by direct Tested directly by direct

inoculation method if it is of small inoculation method if it is of small volume or by membrane filtration volume or by membrane filtration method if it is of large volume.method if it is of large volume.

Application of Sterility Application of Sterility test to Different Dosage test to Different Dosage FormsForms

Soluble Solids:Soluble Solids: Dissolve in a Dissolve in a suitablesuitable solvent solvent

* Sterile.* Sterile.

* Has no antimicrobial * Has no antimicrobial activity.activity.

Such as: meat peptone or caseine Such as: meat peptone or caseine peptonepeptone


Oily Preparations:Oily Preparations: Add a Add a suitablesuitable emulsifying agent emulsifying agent


* Has no antimicrobial activity.* Has no antimicrobial activity.

Such as: Polysorbate or Polyethoxyethanol.Such as: Polysorbate or Polyethoxyethanol. During incubation, oily preparation During incubation, oily preparation

should be shaken gently every day.should be shaken gently every day.


Ointments & creams:Ointments & creams: Dilute with a Dilute with a suitable suitable diluentdiluent


* Has no antimicrobial * Has no antimicrobial activity.activity.

Such as: isopropyl myristate.Such as: isopropyl myristate. add a add a suitablesuitable emulsifying agent. emulsifying agent.

Antimicrobial agents (antibiotics Antimicrobial agents (antibiotics & Preservatives):& Preservatives):

Inactivate the inhibitory agent by either:Inactivate the inhibitory agent by either:1. Dilution the preparation to less than MIC 1. Dilution the preparation to less than MIC

(Minimum Inhibitory Concentration) of (Minimum Inhibitory Concentration) of the antimicrobial agent.the antimicrobial agent.

2. Addition of inactivating agent.2. Addition of inactivating agent. For For examples:examples:

addition of penicillinase solution to inactivate addition of penicillinase solution to inactivate Sodium benzyl pencillin.Sodium benzyl pencillin.

Addition of Para amino benzoic acid (PABA) to Addition of Para amino benzoic acid (PABA) to inactivate sulphathiazole.inactivate sulphathiazole.

Application of Sterility Application of Sterility test to Different Dosage test to Different Dosage

FormsForms

Sterility Test for Sterility Test for Water for InjectionWater for Injection

Procedure:Procedure: Using the Direct inoculation

method.

ThioTSB TSB

1 ml 1ml 1ml

Carry out the control tests under the same condition of the experiment.

بخير وأنتم عام بخير كل وأنتم عام كل

With my Best Wishes,,,Manal Abu El-Khair

Documents

Lab. No.2 Sterility Testing. applied to products intended to be sterile. (Ophthalmic and Parenteral preparations) applied to products intended to be sterile