General microbiology

The sterility of all sterile products produced in accordance with GMP must be tested regularly. However, not all sterility testing methods are equivalent. Quality, convenience and application may vary significantly, affecting the reliability of results. False negative results may be related to the release of contaminated products and can have serious consequences for both patients and manufacturers. On the other hand, cross-contamination and false-positive results would mean long, costly tests, as well as the product delays or rejection. Merck offers a solution for sterility testing: closed membrane filtration system and a wide range of culture media and rinse buffers suitable for all sterility tests.

Bioburden testing is used for detection of viable aerobic microorganisms that are found in raw materials or final product, which has not been sterilized. The bioburden test is carried out in two stages. The total count of aerobic microorganisms, as well as total yeast and mold count are determined during the first quantitative stage (microorganism count). The second, qualitative stage (detection of microorganisms) includes the detection of presence or absence of specific microorganisms. Merck offers equipment and tools, such as: membrane filtration modules, nozzles, vacuum pumps, membrane filters, filter dispensers, culture media, reagents etc.

For many years Merck has been producing high-quality culture media for the pharmaceutical, cosmetic, food and beverage industries. Culture media for these industries are: dehydrated pellets, ready for use in a liquid form, ready to use in solid form (agars). All products meet highest quality requirements, have certificates of quality and are safe for consumers.

PyroDetect is the latest pyrogen detection system based on the monocyte activation assay (MAT) described in the European Pharmacopoeia (EP Chapter 2.6.30) in 2010. Using the body's innate immune defence reaction, PyroDetect system simulates the human fever reaction better than any animal pyrogen test. Pyrogens contained in the sample activate monocytes in the human blood, forming cytokines which are detected by ELISA method using specific antibodies and enzymatic colourimetric reactions. Monocyte activation test is based on the reaction of the frozen blood and interleukin-1β. Three major setting steps of the test with PyroDetect system: frozen (or fresh) blood incubation, IL-1β ELISA assay and data analysis.

ACC is one of the largest manufacturers in the world supplying products to the market for the detection and count of Gram-negative bacterial endotoxins and (1→3)-ß-D-glucan. These products are used by the world's leading pharmaceutical and medical device companies seeking to ensure the safety of their parenteral drugs, biological products and medical devices. Application: testing of pharmaceutical injectables, material and process control, control of biological agents, dialysis water testing etc.

Limulus Amebocyte Lysate (LAL) tests can used to identify and quantify the endotoxins secreted by outer membrane of gram-negative bacteria. The main component of the LAL agents used in these tests is a protein involved in endotoxin-initiated blood coagulation mechanism and derived from the amebocyte lysate extract of the Atlantic horseshoe (Limulus polyphemus). LAL tests have been described in the US Pharmacopoeia bacterial endotoxin detection section, the European Pharmacopoeia (chapter 2.6.14) and Japanese Pharmacopoeia (General tests, no. 4.01).

Chromogenic (colour) method. LAL agent has an appropriate synthetic substrate, producing yellow colour after the reaction with endotoxin-activated protein. The test is read in a microplate reader at 405 nm wavelength. It can be performed using the final result or kinetic method. Maximum sensitivity – 0.001 EV/ml. 

Turbidimetric (turbidity) method. Kinetic method, based on the appearance of turbidity, by treating lysate with endotoxin. Turbidity optical density is measured with incubating microplate reader or incubating tube reader. Test sensitivity – 0.001 EV/ml.

Gel-clot (gel lump) method. LAL agent affected by endotoxin forms gels. This is the simplest method. Results are evaluated by inverting the tube. Maximum sensitivity – 0.03 EV/ml. It is the cheapest method as it does not require an optical scanner. It is suitable for opaque and coloured samples of suspensions.