Bioburden Testing

🏾 Content :

• Definition
• The Bioburden Control Trial
• Procedure and steps
• Sample Preparation
• Methods

🏾 Bioburden Control Trial

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 (Pharmacopoeia recommends 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 (the pharmacopoeia recommends Saboraud dextrose agar).
• Search for more specific germs

🏾 Procedure and steps

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).
• Fluids or solids in aerosol form: Aseptically transfer the product to a sterile container for later sampling. Although not specified in the USP, the container can be cooled in a mixture of alcohol and dry ice for about 1 hour. Then, at room temperature and using aseptic techniques, cut the container open and allow the propellant to escape before removing the item.
• 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 .

🏾 Methods

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.

Membrane filtration

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 pour plate and spread plate. The pour plate method is preferred due to its greater theoretical accuracy

1. Pour Plate method

The sterilized culture medium is added to a Petri dish containing the sample to be tested and allowed to solidify.

• Take 1 mL of the test liquid or its dilution from each Petri dish aseptically, add to each dish 15 to 20 mL of sterilized agar medium, previously melted and kept below 45°C, and mix (45 °C is just above freezing point to minimize heat-induced cell death).
• For the detection of bacteria, use soybean casein digested agar medium and for the detection of fungi, use Sabouraud glucose agar medium, to which an antibiotic has been previously added.
• Once the agar has solidified, incubate for at least 5 days at 30–35°C for the detection of bacteria and at 20–25°C for the detection of fungi.

2. 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.

Most probable number method

The most probable number (MPN) method is a quantitative method used to determine the approximate bacterial concentration in a sample.

The method involves taking the original solution or sample and subdividing it by orders of magnitude (often 10× or 2×) in broth culture and to assess the presence/absence of microorganisms in several subdivisions.

The main weakness of MPN methods is the need for a large number of replicates at the appropriate dilution to reduce the confidence intervals. MPN is only effective for examining bacteria, as it does not provide reliable results for enumerating fungi.

To determine the accuracy and sensitivity of the test methods used for microbial limit testing, 10 g or 10 ml samples of the test material are examined. Care must be taken to ensure that the test method does not introduce bacteria into the sample or kill bacteria in the test sample.

Note: The most probable number method is less used for precision reasons, it is reserved for bacterial counts when no other method is available. However, its use is recommended for raw materials with low microbial contamination.

🏾 Limits set for acceptance criteria

The limits set for the acceptance criteria are established by considering the following factors: the nature of the product used, its use, the route of administration and the type of patients targeted (children, pregnant women, immunocompromised patients).

The limits have been set taking into account these various factors and are as follows

• DGAT < 102 GFU/g or CFU/mL
• DMLT < 101 CFU/g or CFU/mL
• Manipulation controls must be clear and contain 0 CFU/mL