To help answer any questions you may have, below is a list of our most frequently asked questions. 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.
Occasionally, after thawing serum it will appear to be cloudy. This is commonly confused with bacterial contamination and is usually the result of precipitated components, the most common being fibrin, a non-globular protein involved in the clotting of blood. Precipitated fibrin in serum is a fine whitish powder or flake. During the processing of blood into serum, some fibrin can remain in solution.
When frozen serum is thawed small amounts can precipitate out. The more freeze thaw cycles, the more chance of precipitates forming. These are for the most part cosmetic and have no ill effects on cell culture. It is not recommended to try and filter precipitates out as they will clog most lab scale filter apparatuses. If necessary, allow the serum to stand and the precipitates will eventually fall to the bottom of the bottle and the serum can be decanted.
In most every instance, the serum IS NOT contaminated, it is fine or flakey precipitate. This is not an uncommon occurrence and most FBS will contain small amounts upon thawing. How quickly the blood is processed into raw serum usually dictates the amount of precipitate, the longer the amount of time allowed for the red cells to clot, the less precipitates. This is because the fibrin is not completely clotted out and even though the raw serum is filtered during final processing it remains in solution. When the serum is frozen and then thawed, the fibrin can come out of solution giving rise to fine or flakey precipitates. If you do believe that your serum is contaminated, try plating it out or putting some in media and incubating it. DO NOT put a thawed bottle of 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 due to contamination from individuals or contaminated cell culture medium ingredients. The accepted name was chosen because Mycoplasmas were observed to have a fungi-like structure – hence “Myco,” and it also had a flowing plasma-like structure without a cell wall – hence “plasma.”
Mycoplasma cells are physically small and they are therefore difficult to detect with a conventional microscope. Mycoplasmas may induce cellular changes, including chromosome aberrations, changes in metabolism and cell growth. Severe Mycoplasma infections may destroy a cell line. Detection techniques include PCR, plating on sensitive agar and staining with a DNA stain including DAPI or Hoechst.
Today, over 100 documented species of mycoplasmas have been recorded to cause various diseases in humans, animals, and plants. Mycoplasma pneumonia as well as at least 7 other mycoplasma species have now been linked as a direct cause or significant co-factor to many chronic diseases including rheumatoid arthritis, Alzheimer’s, multiple sclerosis, fibromyalgia, chronic fatigue, diabetes, Crohn’s Disease, ALS, nongonoccal urethritis, asthma, lupus, infertility, AIDS and certain cancers and leukemia, just to name a few.
Mycoplasmas, unlike viruses, can grow in tissue fluids (blood, joint, heart, chest and spinal fluids) and can grow inside any living tissue cell without killing the cells, as most normal bacteria and viruses will do. Mycoplasmas are frequently found in the oral and genito-urinary tracts of normal healthy people and are found to infect females four times more often than males, which just happens to be the same incidence rate in rheumatoid arthritis, fibromyalgia, chronic fatigue and other related disorders.
Atlas Biologicals proprietary filtration scheme utilizes three separate 0.1 micron membranes which effectively removes mycoplasma from our sera. In addition, 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 for the manufacturing of these products and consists of the Direct Culture method combined with the Indicator Cell Culture procedure.
Endotoxin is a complex lipopolysaccharide (LPS) found in the outer cell membrane of gram-negative bacteria, typically water borne. 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 (EU/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 EU/mL and even at that level it is suitable for most cell culture applications. Some exceptions are 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 they charge substantially more for that premium product with levels typically below 1 EU/mL. If your application does not require these low levels you can probably save some money by using standard grade material. There is a wealth of information available online regarding this subject.
Hemoglobin in finished fetal bovine serum is a result of cell lyses during the transportation and processing of fetal blood into raw fetal bovine serum. Many years ago when collection techniques were not as good, hemoglobin concentration was used as an indicator of the collection and handling quality. Many suppliers of fetal bovine serum will use assays that report a portion of the hemoglobin as opposed to the total hemoglobin. One of the most common assays used today reports oxy-hemoglobin, which is approximately 40 – 60% of the total.
The fact is that hemoglobin concentrations up to 100 mg/dL have virtually no effect on cell culture. Normal levels of total hemoglobin in finished fetal bovine serum available in today’s market range from 10 – 20 mg/dL. If you are using a Certificate of Analysis to compare quality of fetal bovine serum and are considering hemoglobin as a factor, call the manufacturer and ask the questions regarding the assay they are using and their opinion regarding the effect of hemoglobin on cell culture. You might be surprised.
US Origin serum is collected in facilities located within the contiguous United States of America that undergoes continuous inspection during operation by the United States Department of Agriculture (USDA). USDA Origin serum is collected from facilities outside of the U.S. that have been inspected by USDA and approved for the import of ruminant meat products and byproducts. Current eligible countries that may export bovine serum into the U.S. include: Australia, Canada, Chile, Costa Rica, El Salvador, Guatemala, Honduras, Mexico, New Zealand, Nicaragua, and Panama.
Why may US and USDA Origin serum behave differently? The characteristics of US and USDA origin serum may vary with particular applications. In the US and other large volume producers most of the cattle are raised on feedlots. Feedlots are the final stage of production prior to slaughter with a focus on growth of the animals. This is achieved by providing a high-energy diet which cuts the time needed to achieve optimal weight. In many Latin American countries the animals are left out in pastures or open range. The chemistry profile of the serum can vary in these situations which is why end users may see different results with a specific source.
Pricing of different sources varies for several reasons. There is a perception in the industry that countries like the US, Australia and New Zealand have stricter controls on meat processing compared to second and third world countries creating a greater demand and increase in price. There is a perception that Australia and New Zealand are safer sources because they are geographically isolated countries. Theses origin have been the highest priced. Australian origin serum must be tested for additional viruses like Akabane which is not endemic in most all other FBS sources.
Many large commercial manufacturers historically have required Aus and NZ origin serum and most recently allowed US origin serum, creating an additional demand on the available serum supply. The additional demand has created an increase burden on the serum supply pushing up pricing.
The limitation of available supply from USDA approved sourcing to the United States has resulted in an increase in US pricing. Brazilian FBS, which is not currently approved by USDA, is accepted throughout Europe and many Far East countries but not allowed in the United States due to the prevalence of Foot and Mouth Disease (FMD). Countries with cases of FMD are the least expensive sources of FBS.