Below is a list of our most frequently asked questions to help answer any uncertainties you may have. We hope this will enhance your knowledge and understanding of our products. If you have any other questions, please don’t hesitate to contact us. We are here to help.
The serum producing process includes allowing the blood to clot, then removing the serum by centrifugation. Fibrinogen is a large fibrous glycoprotein found in blood which functions in hemostasis and injury healing, and is essential to forming clots. Fibrinogen is insoluble; the conversion to fibrin occurs during clotting. Not all of the fibrinogen is converted during clotting and some may remain in the serum during filtration. Thermodynamic changes will cause fibrinogen to convert to fibrin, such as incubation at 37°C or more for prolonged periods or the freeze/thaw processes. As the fibrinogen is converted to fibrin during the thermodynamic changes, flocculent material or turbidity will be observed.
If you believe that your serum is contaminated, try plating it out or putting some in media and incubating it. DO NOT put a bottle of thawed serum directly into an incubator; always add it to cell culture media first.
Mycoplasma species are often found in research laboratories as contaminants in cell culture. Mycoplasmal cell culture contamination occurs from individuals or contaminated ingredients in the cell culture medium. The name was chosen because Mycoplasmas were observed to have a fungi-like structure (“Myco”) and because it also had a flowing plasma-like design without a cell wall (“plasma.”)
Mycoplasma cells are physically small and difficult to detect with a conventional microscope. Detection techniques include Polymerase Chain Reaction (PCR), plating on sensitive agar, and staining with a DNA stain including DAPI or Hoechst. Mycoplasmas may induce cellular changes, including chromosome aberrations (changes in metabolism and cell growth). Mycoplasmas can grow in tissue fluids and grow in live tissue cells without killing the cells, unlike viruses. Severe Mycoplasma infections may destroy a cell line.
Atlas Biologicals proprietary filtration scheme uses three separate 0.1-micron membranes to effectively remove mycoplasma from our sera. Every lot is tested for mycoplasma using the method from “FDA Points to Consider for the Mycoplasma testing of Cell Cultures and Biologicals” derived from cell substrates. This procedure is recommended for master cell banks, working cell stocks, and cell substrates used to manufacture these products and consists of the Direct Culture method combined with the Indicator Cell Culture procedure.
Endotoxin is a complex lipopolysaccharide (LPS) found in gram-negative bacteria’s outer cell membrane and is typically waterborne. Bacteria shed endotoxin in large amounts upon cell death and when they are actively growing and dividing. Endotoxin is measured in Endotoxin Units per milliliter (E.U./mL). One EU/mL equals approximately 0.1 to 0.2 ng/mL. Endotoxin is directly related to the quality of collection and processing of serum; the more endotoxin, the more exposure to gram-negative bacteria.
The industry standard for endotoxin in fetal bovine serum is less than 10 E.U./mL; even at that level, it is suitable for most cell culture applications. Some exceptions are the production of vaccines and injectables, in which case the lowest levels are desirable. Some manufacturers will tell you that the lowest levels are better across the board and charge a premium for products that have levels typically below 1 E.U./mL. If your application does not require these low levels, you can probably save some money using standard grade material. There is a wealth of online information available regarding this subject.
Hemoglobin in finished fetal bovine serum results from the lyses of red blood cells during the transportation and processing of blood into serum. When collection techniques were not as good, hemoglobin concentration was used as an indicator of the quality.
There are no reported effects on cell cultures from hemoglobin concentrations of less than 100 mg/dL. Normal levels of total hemoglobin in finished fetal bovine serum range from 10–30mg/dL. If you are using a Certificate of Analysis to compare the quality of fetal bovine serum and are considering hemoglobin as a factor, call the manufacturer; ask about the assay they are using and their opinion regarding hemoglobin’s effect on cell cultures.
“U.S. Origin” serum is collected in facilities located within the contiguous United States of America undergoing continuous inspection by the United States Department of Agriculture (USDA). “USDA Origin” serum is collected from facilities located outside the U.S. which are approved for the import of ruminant meat products and byproducts. Current eligible countries that may export bovine serum into the U.S. can be found on the USDA website. https://www.aphis.usda.gov/aphis/ourfocus/animalhealth/animal-and-animal-product-import-information/animal-and-animal-products-imports.
Why may U.S. and USDA Origin serum behave differently? The characteristics of U.S. origin serum and USDA origin serum may vary with particular applications. In the U.S. and other large-volume producers, most of the cattle are raised on feedlots. Feedlots are the final stage of production before slaughter, focusing on the growth of the animals. In many Latin American countries, animals are left out in pastures or open range. The serum’s chemistry profile can vary in these situations, which is why end users may see different results with a specific source.
The price of different sources varies for several reasons. There is a perception that countries like the U.S., Australia, and New Zealand have stricter controls on meat processing than second and third world countries, creating a greater demand and commanding higher prices. An additional misconception is that Australia and New Zealand are safer sources because of their geographic isolation. Australian origin serum must be tested for different viruses, like Akabane, which is not endemic in many other FBS sources.
Historically many large commercial manufacturers have required Australia and N.Z. origin serum and recently have allowed U.S. origin serum, creating an additional demand on the available serum supply. The extra market has created a more significant burden on the serum supply, pushing up pricing.
The limitation of the available supply from USDA-approved sources to the United States has increased U.S. pricing. Brazilian FBS, which the USDA does not currently approve, is accepted throughout Europe and many Far East countries but is not allowed in the United States because of Foot and Mouth Disease (FMD) prevalence. Countries with cases of FMD are the most inexpensive sources of FBS.
In almost every instance, the serum is NOT contaminated. Occasionally thawed serum will appear cloudy. It is usually the result of normal serum components. Fibrin (a non-globular protein involved in the clotting of blood) is most common. Fibrin appears in small quantities as a fine flake or may occur as a lump of a semi-solid substance. The presence of this material does not alter the serum’s performance and may be mistaken for microbial contamination. If the turbidity is a concern, it can be removed by filtration through a 0.45µm filter. However, the sediment may clog many lab-scale filters. If necessary, allow the serum to stand. The sediments will eventually fall to the bottom of the bottle and the serum can be decanted.