1. Lab. No.2 Sterility Testing

2. applied to products intended to be sterile. (Ophthalmic and Parenteral preparations) applied to products intended to be sterile. (Ophthalmic and Parenteral preparations) Performed on random samples from the batch. Performed on random samples from the batch.

3. Sterility Testing Must be carried out under aseptic condition. Must be carried out under aseptic condition.

4. Medium used in sterility testing Trypticase Soya Broth (TSB): Trypticase Soya Broth (TSB): It is used to detect the growth of aerobic bacteria, but when incubated at 25 o C it will be suitable for the growth of fungi. It is used to detect the growth of aerobic bacteria, but when incubated at 25 o C it will be suitable for the growth of fungi.

5. Medium used in sterility testing Fluid thioglycolate: Fluid thioglycolate: It is used to detect the growth of anaerobic bacteria, It also could be used to detect the growth of aerobic bacteria. It is used to detect the growth of anaerobic bacteria, It also could be used to detect the growth of aerobic bacteria.

6. Medium used in sterility testing It is anaerobic medium due to presence of: Sodium thioglycolate and cystaine which act as reducing agents. Sodium thioglycolate and cystaine which act as reducing agents. Small amount of agar to increase viscosity of the medium thus decreasing convection current. Small amount of agar to increase viscosity of the medium thus decreasing convection current. Redox indicator (Methylene Blue) which change colour of the medium when 30% of the medium become oxidized. Redox indicator (Methylene Blue) which change colour of the medium when 30% of the medium become oxidized. Fluid thioglycolate: Fluid thioglycolate:

7. Medium used in sterility testing Fluid thioglycolate: Fluid thioglycolate: It should be prepared in long tube It should be prepared in long tube O2O2 aerobic bacteria anaerobic bacteria

8. Medium used in sterility testing Sabarouds dextrose medium: Sabarouds dextrose medium: It is suitable medium used to detect the growth of fungi because: It is suitable medium used to detect the growth of fungi because: it has acidic pH. it has acidic pH. contains dextrose which is readily fermentable sugar. contains dextrose which is readily fermentable sugar.

9. Incubation Condition IncubationTemperature: Incubation Period: It should be not less than 7 days. It should be not less than 7 days. Incubate the media intended for detection of bacteria at Incubate the media intended for detection of bacteria at 35oC. Incubate the media intended for detection of fungi at Incubate the media intended for detection of fungi at 25oC.

10. Performed exactly under the same condition as the test. Performed exactly under the same condition as the test. Used to test the media used in the sterility test. Used to test the media used in the sterility test. Control tests

11. Negative control: Negative control: It is done by incubation of uninoculated tubes of each medium used in the test. It is done by incubation of uninoculated tubes of each medium used in the test. It is done to test sterility of the medium. It is done to test sterility of the medium.

12. Control tests Positive control: Positive control: It is done to test incubation condition to show if the microorganism will actually grow under the condition of the test or not. It is done to test incubation condition to show if the microorganism will actually grow under the condition of the test or not. Clostridium sporogenesStaph.aureusCandida albicans 35 o C 25 o C Thio TSB

13. Methods of Sterility test Direct Inoculation: Direct Inoculation: Suitable for samples with small volumes. Suitable for samples with small volumes. Thio TSB

14. Methods of Sterility test 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. 2. Aseptically cut the membrane into three equal pieces. 3. inoculate them on appropriate culture media. culture media.

15. Application of Sterility test to Different Dosage Forms Aqueous Solutions: Aqueous Solutions: Tested directly by direct inoculation method if it is of small volume or by membrane filtration method if it is of large volume. Tested directly by direct inoculation method if it is of small volume or by membrane filtration method if it is of large volume.

16. Application of Sterility test to Different Dosage Forms Soluble Solids: Soluble Solids: Dissolve in a suitable solvent Dissolve in a suitable solvent * Sterile. * Sterile. * Has no antimicrobial activity. * Has no antimicrobial activity. Such as: meat peptone or caseine peptone

17. Application of Sterility test to Different Dosage Forms Oily Preparations: Oily Preparations: Add a suitable emulsifying agent Add a suitable emulsifying agent * Sterile. * Sterile. * Has no antimicrobial activity. * Has no antimicrobial activity. Such as: Polysorbate or Polyethoxyethanol. During incubation, oily preparation should be shaken gently every day. During incubation, oily preparation should be shaken gently every day.

18. Application of Sterility test to Different Dosage Forms Ointments & creams: Ointments & creams: Dilute with a suitable diluent Dilute with a suitable diluent * Sterile. * Sterile. * Has no antimicrobial activity. * Has no antimicrobial activity. Such as: isopropyl myristate. add a suitable emulsifying agent. add a suitable emulsifying agent.

19. Antimicrobial agents (antibiotics & Preservatives): Antimicrobial agents (antibiotics & Preservatives): Inactivate the inhibitory agent by either: Inactivate the inhibitory agent by either: 1. Dilution the preparation to less than MIC (Minimum Inhibitory Concentration) of the antimicrobial agent. 2. Addition of inactivating agent. For examples: addition of penicillinase solution to inactivate Sodium benzyl pencillin. addition of penicillinase solution to inactivate Sodium benzyl pencillin. Addition of Para amino benzoic acid (PABA) to inactivate sulphathiazole. Addition of Para amino benzoic acid (PABA) to inactivate sulphathiazole. Application of Sterility test to Different Dosage Forms

20. Sterility Test for Water for Injection Procedure: Procedure: Using the Direct inoculation method. Thio TSB 1 ml Carry out the control tests under the same condition of the experiment.

21. كل عام وأنتم بخير With my Best Wishes,,, Manal Abu El-Khair