Medical Device Lot Release Testing

Sterility tests: (ISO 11737-2) 

Sterility tests are conducted in a sterility class A Isolator (LA Calhène/Gethinge) or in class A laminar flow with a class C-background.

Direct Transfer:

Devices are transferred aseptically to Fluid Thioglycolate Medium (FTM) and/or to Trypticase Soy Broth.

Membrane filtration:

Liquids or extracts of solid materials are filtered over a membrane (pore size 0.22 µM). Filters can be washed to remove possible bacteriostatic or fungistatic products. Filters are transferred to FTM or TSB.


Bioburden: (ISO 11737-1)

Bioburden validation

Exhaustive Recovery (repetitive treatment)

The bioburden of non-sterile devices is determined repeatedly. The recovery of the first extraction is divided by the sum of the recoveries of each repeated bioburden determination to determine the recovery percentage.

Spore Inoculation

An artificial bioburden of 30-100 CFU is introduced on sterile test items. After the inoculum has been allowed to dry the bioburden of the inoculated devices is determined. The recovery percentage can be determined by dividing the inoculated amount of CFU by the recovered amount of CFU.

Bioburden estimation

Solid samples are extracted in an isotonic extraction fluid. Extracts and fluid test items are vacuum filtered through a membrane filter with a pore size of 0.22 µM. Filters of that size will retain micro-organisms present in a solution.

The filter is aseptically transferred to an agar plate and incubated at the appropriate conditions.


Bacterial Endotoxins (LAL): USP 85, USP <161>, EP 2.6.14

Pyrogens are a group of substances capable of eliciting a fever response upon injection or infection. Endotoxin is a subset of pyrogens that is strictly of gram-negative bacterial origin. Dozens of microbiological compounds have been found to induce fever but many do so only weakly or only at very high doses.

Most reactive (by far), most occurring and most resistant to extreme conditions of all possible pyrogens are gram-negative bacterial endotoxins. This is why the focus of pyrogens is mainly on endotoxins.

LAL-activation is considered analogous to the response that is considered to be pyrogenic but is more specific for bacterial endotoxin and at much lower levels of detection.

Endotoxins or Lipopolysaccharides (LPS) are a part of the outer cell wall of gram-negative bacteria.

LAL chromogenic kinetic assay


Endotoxins catalyse the activation of a proenzyme in the Limulus Amebocyte Lysate (LAL). The rate of activation is determined by the concentration of endotoxin present. The activated enzyme induces the formation of colour which is measured photometrically continuously throughout the incubation period. The concentration of endotoxin present is characterised by the time needed to increase the optical density by 0.2 units. A calibration curve is developed from an E. coli endotoxin standard from which reaction times of unknowns can be translated into Endotoxin concentration.

The kinetic approach for the LAL test allows for a cost-effective, automatable and quantifiable method to detect gram-negative bacterial pyrogens. It is also one of the few existing methods with a theoretical detection limit of 0.001 EU/mL.

Inhibition & Enhancement

The constitution of the test liquid (or extract) may influence the proper functioning of the enzyme and thus result in an over- or underestimation of the endotoxin concentration. To exclude this inhibition or enhancement of the enzyme, a positive product control (PPC) for each sample is included in the assay.

Limits USP

  • 20 EU/device: USP <161>
  • 2.15 EU/device when in contact with cerebrospinal fluids (USP<161>)
  • 0.25 EU/mL WFI: USP <85> 


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Nelson Labs Europe | Medical Device Lot Release