Bioburden Analysis technique

Bioburden refers to the number of microorganisms, such as bacteria, fungi, and viruses, present on a surface or object. Bioburden is measured in terms of colony-forming units (CFUs) per unit of surface area or volume.

Bioburden is an important consideration in many industries, including healthcare, pharmaceuticals, and food production. In healthcare, bioburden can be a potential source of infection for patients and healthcare workers. In pharmaceuticals, bioburden can affect the safety and efficacy of drugs. In food production, bioburden can lead to spoilage and contamination of food products.

To control bioburden, industries use various techniques, including sterilization, disinfection, and cleaning. Sterilization is the process of eliminating all microorganisms from a surface or object. Disinfection is the process of reducing the number of microorganisms to a safe level. Cleaning involves removing dirt and debris from surfaces to reduce the number of microorganisms present.

Bioburden testing is a common practice in many industries to ensure that surfaces and objects meet acceptable levels of cleanliness and safety. Testing can be performed using various methods, such as swab sampling, environmental monitoring, and air sampling. Results of bioburden testing can be used to identify potential sources of contamination and to implement appropriate control measures.

OBJECTIVE

• To lay down the procedure for Bioburden Analysis.

 SCOPE

• This Procedure shall be applicable for the Bioburden Analysis in Microbiology Department.

RESPONSIBILITY

• Microbiology Personnel shall be responsible for the receipt of Bioburden samples and making entries in sample receipt record.
• Officer microbiology or above shall be responsible for carrying out the established procedure of Bioburden analysis.

 ACCOUNTABILITY

• Head Microbiology/ designee shall be accountable for the proper implementation of the SOP.

 DEFINITIONS

• Bioburden: Total numbers of microorganisms present in or on an object/ sample prior to sterilization.

  PROCEDURE

• General Requirement:

• Safety Considerations:

  • The Person entering the area should follow correct gowning and entry procedure for clean room and should strictly adhere to these practices.
  • The test must be carried out under LAF.
  • Before handling the samples, disinfect the hands with disinfectant and air dry.

• Equipment:

  • Laminar Air Flow
  • Incubator set at 22.5°C ± 2.5°C
  • Incubator set at 32.5°C ± 2.5°C
  • Cooling Incubator set at 2-8°C
  • Colony Counter
  • Vacuum pump

• Material:

  • Sterile Petriplates
  • Sterile Filtration Assembly
  • Sterile Membranes Filter (0.45µm- 47mm Diameter)
  • Manifold
  • Filtration Cups
  • Flask
  • Forceps
  • 70 % IPA

• Media:

  • Soyabean Casein Digest Agar
  • 1 % Peptone Water or Buffer sodium chloride peptone water

• Sample Requirement of Pre-filtered and In-process Product Sample:

  • 100 mL sample of product solution (pre-filtered or in-process product sample) shall be collected for bioburden analysis in pre-sterilized and sterilized container from the manufacturing tank.

• Test Procedure for Enumeration of Bacterial and Yeast Count:

  • Transfer the in-process samples and all the required material for testing to the micro lab-1 through dynamic pass box.
  • Place all the materials required for the testing inside the Laminar Air Flow.
  • Make the attachments of manifold unit to the suction pump and waste water reservoir.
  • Fix the sterilized filtration cup/ funnel on the manifold unit and place the sterilize membrane filters on the sterile filtration cup/ funnel with the help of sterile forcep taking care that the forcep does not damage the filter and touch anywhere.
  • Prewet the membrane filter with approx 20-50 mL of sterilized (0.1 % peptone water/BSCP). Open the knob of the manifold unit so that the rinsing fluid filters through the membrane with the help of vacuum pump.
  • After completion of filtration, transfer 100 mL of sample in the filtration cup and open the knob of manifold unit so that the sample filters through the membrane.
  • Transfer aseptically 100 mL of rinsing fluid (0.1 % peptone water/ BSCP) in the filtration cup. Filter the rinsing fluid with the help of vacuum pump.
  • Remove the filtration cup and aseptically transfer the membrane filter on the surface of pre incubated SCDA media plates with the help of sterile forcep.
  • Place the plates with the membrane filter in inverted position for incubation at 22.5°C ± 2.5°C for 72 hrs and 32.5°C ± 2.5°C 48 hrs.
  • Prepare one diluent negative control by filtering 100 mL of sterile 0.1 % peptone water/ BSCP through the membrane and put the membrane on the surface of pre-incubated SCDA plate and incubate along with test sample in inverted position.
•  
  

  Observation and Interpretation of the Results:

  • After completion of incubation period withdrawn the plates from incubator and observe the plates under Colony Counter.
  • Report the results in Annexure-I.

  • Report the results as CFU/ 100 mL as per Annexure-I. Compare the results obtained with the specification of product for compliance of sample.

ABBREVIATIONS

• • SOP :           Standard Operating Procedure
  • LAF :           Laminar Air Flow
  • ºC :           Degree Celsius
  • µm :           Micrometer
  • mm :           Millimeter
  • % :           Percentage
  • IPA :           Iso Propyl Alcohol
  • mL :           Milliliter
  • BSCP :           Buffer Sodium Chloride Peptone
  • SCDA :           Soyabean Casein Digest Agar
  • Hrs :           Hours
  • CFU :           Colony Forming Unit

REFERENCES

• • Nil

ANNEXURES

• • Nil

DISTRIBUTION DETAILS

• • QA & MB

 REVISION HISTORY

• • New

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