Lab. No.2

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PHT351
Lab. No.2
Sterility Testing
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Sterility Testing

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

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Sterility Testing

• Must be carried out under aseptic condition.

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Medium used in sterility testing

• Trypticase Soya Broth (TSB)
• It is used to detect the growth of aerobic
  bacteria, but when incubated at 25oC it will be
  suitable for the growth of fungi.

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Medium used in sterility testing

• Fluid thioglycolate
• It is used to detect the growth of anaerobic
  bacteria, It also could be used to detect the
  growth of aerobic bacteria.

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Medium used in sterility testing

• Fluid thioglycolate

• It is anaerobic medium due to presence of
• Sodium thioglycolate and cystaine which act as
  reducing agents.
• 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.

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Medium used in sterility testing

• Fluid thioglycolate
• It should be prepared in long tube

aerobic bacteria
anaerobic bacteria
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Medium used in sterility testing

• Sabarouds dextrose medium
• It is suitable medium used to detect the
  growth of fungi because
• it has acidic pH.
• contains dextrose which is readily fermentable
  sugar.

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Incubation Condition

• IncubationTemperature

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

• Incubation Period

• It should be not less than 7 days.

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Control tests

• Performed exactly under the same condition as the
  test.
• Used to test the media used in the sterility test.

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Control tests

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

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Control tests

• Positive control

Clostridium sporogenes
Staph.aureus
Candida albicans
TSB
TSB
Thio
35oC
35oC
25oC

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

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Methods of Sterility test

• Direct Inoculation
• Suitable for samples with small volumes.

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Methods of Sterility test

• Membrane Filtration
• Suitable for samples with large volumes.
• 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.

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Application of Sterility test to Different Dosage
Forms

• 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.

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Application of Sterility test to Different Dosage
Forms

• Soluble Solids
• Dissolve in a suitable solvent
• Sterile.
• Has no antimicrobial activity.
• Such as meat peptone or caseine peptone

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Application of Sterility test to Different Dosage
Forms

• Oily Preparations
• Add a suitable emulsifying agent
• Sterile.
• Has no antimicrobial activity.
• Such as Polysorbate or Polyethoxyethanol.
• During incubation, oily preparation should be
  shaken gently every day.

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Application of Sterility test to Different Dosage
Forms

• Ointments creams
• Dilute with a suitable diluent
• Sterile.
• Has no antimicrobial activity.
• Such as isopropyl myristate.
• add a suitable emulsifying agent.

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Application of Sterility test to Different Dosage
Forms

• Antimicrobial agents (antibiotics
  Preservatives)
• 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 Para amino benzoic acid (PABA) to
  inactivate sulphathiazole.

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Sterility Test for Water for Injection

• Procedure
• Using the Direct inoculation method.

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

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