Exp. No. 13 Bioburden testing

 

Bioburden testing

Introduction

The Bioburden test, bioburden or microbial load, is a term used to describe the presence and measure the level of viable microorganisms on a surface or in a specific device before sterilization.

Bioburden is the sum of microbial contributions from a number of sources:

• Raw materials used in the manufacturing process.
• Introduced via labor or the assembly/manufacturing environment.
• During cleaning processes and packaging of the finished product, etc.

The reason for performing the Bioburden test, varies and depends on the field and the final application of the product tested. Qualitative/quantitative measurements help determine whether the types and numbers of microorganisms present are satisfactory or not against the predefined acceptance criteria.

The bioburden test is designed to count the number of microorganisms (as colony forming units, CFU) in a product or material first non-sterile. The bioburden control test is done using different culture media conducive to the proliferation of bacteria and fungi. The test is either one or both of the compendial methods TAMC or TYMC, or an alternative.

·         Total viable aerobic count: TAMC, TYMC

·         TAMC: total aerobic microbial count, this is an estimate of viable aerobic mesophilic microorganisms that can be derived from a general-purpose medium (soy casein digest).

·         TYMC: total mold and yeast count, this is an estimate of aerobic mesophilic (yeast-like and filamentous, and dimorphic) fungi. The test uses a fungal medium for general use (Saboraud dextrose agar).

Aim

To check the viable microbial load of the given sample (bacteria and fungi).

Procedure/ Program

Estimating the bioburden of a medical device generally consists of four distinct steps:

·         A sample of the product to be tested is prepared based on the physical characteristics of the product.

·         Isolation, typically by membrane filtration, of microorganisms from the sample and then culture.

·         Counting of the collection sample containing the recovered microorganisms.

·         Characterization of the microbial load. The results are recorded, and a report is prepared detailing the test results.

Sample Preparation

In general, the sample preparation step consists of dissolving or suspending 10 ml or 10 g of the sample to be tested in a mild diluent such as a buffered solution of sodium chloride and peptone at pH 7, 0 or phosphate buffer at pH 7.2.

Usually a 1:10 dilution is prepared, the pH of the sample preparation should be adjusted to a range of 6-8 using sterile acidic or alkaline solutions. When testing a solid product that does not completely dissolve in the chosen diluent, the material can be reduced to a fine powder using, for example, a sterile mortar and pestle, for better dispersion of the sample in the buffer solution.

• Water-insoluble non-fatty products: The use of an emulsifying agent such as polysorbate 80 at a concentration of 1 g/L of diluent is recommended to promote homogenization of the sample preparation.
• Fatty products: Dissolve the product in lterized isopropyl myristate or homogenize with sterile polysorbate 80 or any other suitable sterile surfactant. If necessary, heat the sample preparation to a maximum of 40°C (or use a preheated diluent).
• Transdermal patches: Remove the protective sheets and place the product units, adhesive side up, in a sterile container such as a large Petri dish. Cover the adhesive side of the product units with a sterile porous material to prevent the units from sticking together. Aseptically remove each product unit and add to test diluent containing appropriate inactivators such as lecithin and polysorbate 80. Shake the product preparation for at least 30 min, before withdrawing the sample aliquot for testing.

Membrane Filtration Method

As a general rule, the microbiological control of non-sterile products is carried out either by the membrane filtration method or by the plate count method. The most probable number method will be used exceptionally, when the microbial count cannot be carried out by one of the first two, due to the nature of the product (fat) or the presumed number of OM.

This is the method of choice and should be applied to samples containing antimicrobial substances. With the method, the sample is passed through a membrane filter with a pore size of 0.45 μm or less. The membrane functions as a barrier and captures microorganisms larger than the pore size of the membrane.

Usually the test measures two test fluids of 10ml each, passing each sample through a separate filter. It is important to dilute the pretreated test liquid if the bacterial concentration is high, so that 10 to 100 colonies can grow per filter

The membrane is transferred to a culture medium and placed in an incubator for at least 5 days at 30–35°C for the detection of bacteria and at 20–25°C for the detection of fungi. At the end of the incubation period, the number of colonies is counted.

Direct Plating Techniques (plate count method)

Direct plating methods for bioburden testing include spread plate technique.

Spread plate method

·         Place 0.05-0.2ml of the test liquid on the solidified and dried surface of the agar medium and spread it evenly.

The microbial content of a product or device is expressed in colony forming unit (CFU) and this number is used to determine the bioburden level. Pharmaceutical products typically have an acceptable range and type within which the bioburden content should fall.

Bioburden testing is useful to:

·         Validate and control sterilization process

·         Monitor the quality of raw materials, components and packaging

·         Highlight manufacturing process weaknesses

·         Evaluate the effectiveness of cleaning measures

Result

The total number of bacteria, yeasts, and molds would be calculated and expressed in number per ml.