Reproducibility is crucial in scientific research, and contamination creates one of the biggest challenges to meeting that standard. To keep reagents, cell cultures, and even tools sterile, scientists must work carefully and consistently. In some cases, sterile technique matters the most, but the right tools can also help. Pipettes, for example, play a key role in keeping samples and reagents sterile.
Prep the pipette
In many scientific procedures, only a pipette comes in contact with reagents and samples. Consequently, scientists must keep pipettes clean and sterile, which requires regular attention and maintenance. For a refresher, see the Cleaning and Decontamination Guide for Sartorius Pipettes.
“Pipettes are precision instruments whose performance can be significantly impacted if not adequately maintained,” the Sartorius document notes. “To maintain the level of purity that is critical in many laboratory applications, adequate cleaning and decontamination of pipettes is necessary.” That decontamination should kill or remove bacteria, fungi, and viruses. Plus, a decontamination process should remove chemicals and radioactive materials.
Sartorius recommends maintenance on three time scales:
- daily—clean the outer surface with “a disinfectant liquid or mild detergent, for example 70% ethanol, and a soft, lint-free cloth.”
- every three months—clean the lower part of the pipette and check for wear.
- annually—a pipette should be calibrated.
In some cases, the required service depends on use. For example, Sartorius notes that “cleaning and decontamination should always be done after over-aspiration or when the pipette has clearly been contaminated.”
Like all scientific instruments, though, refer to manufacturer guidelines regarding suggested maintenance and timing.
It’s cultural
Cell culture science lives in a contamination jungle. The list of potential contaminants is long, and they seem to hide in every crevice. Like many stories, the battle against contamination starts at the beginning. “Raw materials used to prepare cell culture media should be tested for adventitious agents,” says Gary Gilleskie, executive director of the Biomanufacturing Training and Education Center at North Carolina State University.
Image: This scanning electron microscope image shows SARS-CoV-2 (round blue objects) emerging from the surface of cells cultured in the lab. Image courtesy of NIAID-RML
In preparing cell culture reagents, Gilleskie says that “materials of animal origin should be avoided if possible, but this is not always possible.” Still, a scientist can always use high-purity water and run all reagents through a 0.2 or 0.1 micron sterile filter. As Gilleskie points out, some reagents “cannot be sterilized in an autoclave.”
Plus, it’s not just the outside world that creates contamination in cell culture. Any plasticware can be a culprit, because the resins and additives in the polymers produce extractables. A scientist should check with the manufacturer for a list of extractables and the likely concentrations, but this really depends on how the labware is used, such as subjecting it to a high temperature or solvent. Plus, specific extractables damage some cells lines more than others. So, a researcher should explore the literature and look for information about the plasticware being used safely—or not—with a particular cell line in a specific application.
Keep it in a cabinet
A biosafety cabinet (BSC) is a must in a cell culture lab. “Any transfers of sterile material—that is, passaging cells or adding medium to additional bottles—should be performed in a biosafety cabinet,” Gilleskie explains. “Open-room additions can be made using either tube welding or aseptic connectors.”
Just installing a BSC, though, won’t prevent contamination. Much of that comes from how researchers use a BSC. Gilleskie describes good cell culture techniques as:
- spraying the BSC with 70% isopropyl alcohol before and after use
- spraying everything that is brought into the BSC
- not walking behind someone who is in the BSC with open flasks or bottles
- working six inches back in the BSC
- holding all caps while pipetting
- slow movement in the BSC
- removing any drips of medium from the outside of the flask.
To further reduce the odds of contaminating cell cultures, keep a BSC orderly. If something doesn’t need to be in the BSC, get it out.
Contamination consequences in industry
Circumstances also impact the level of precaution that must be taken in labs. For example, with labs around the world working with the SARS-CoV-2 virus, the U.S. Center for Disease Control and Prevention developed the Interim Laboratory Biosafety Guidelines for Handling and Processing Specimens Associated with Coronavirus Disease 2019 (COVID-19).
With this virus, a BSC is not enough. Instead, the guidelines state: “CDC recommends virus isolation in cell culture, and initial characterization of viral agents recovered in cultures of novel SARS-CoV-2 should be conducted in a Biosafety Level 3 (BSL-3) laboratory using BSL-3 practices.” The CDC adds: “To determine appropriate biosafety mitigation measures, laboratories should perform an activity-specific biosafety risk assessment that evaluates laboratory facilities, personnel and training, practices and techniques, safety equipment, and risk mitigation measures.”
Despite a long history of fighting viral contamination, manufacturers must stay especially vigilant. For example, making therapeutics from recombinant proteins involves culturing cells, which can be contaminated with viruses. So, scientists from MIT and biotechnology companies around the world teamed up to see how often viral contamination occurs in bioprocessing. The team reported: “These events are rare; we are aware of only 26 virus contaminations over the past 36 years.” Even though such viral contaminations are rare, they could be dangerous, because some of the contaminations came from viruses that are pathogenic in humans.
When contamination did occur in these examples of bioprocessing, the raw materials were the most common source, but even testing the raw materials rarely found the viral contamination. So, testing one thing is not enough. As this team stated: “Although testing is a key component of viral safety in biotechnology products, the data presented here indicate that testing alone is not enough to ensure that a given product is free of a viral contaminant, and that a holistic, multifaceted approach must be taken.”
Overall, keeping reagents and processes sterile always requires a multifaceted approach, including tools and techniques. Plus, scientists must always stay on guard against potential contamination. Microorganisms keep evolving new ways that prove problematic to all sorts of biological and industrial processes.