6.2: Principles of Aseptic Technique

Biotech Focus

In 1912, Alexis Carrel established the first cell line derived from fragments of explanted chicken embryo heart. This cell line could be sub-cultured maintained in vitro and even sub-cultured multiple times but his cultures eventually become contaminated with bacteria. In response, Carrel developed a series of procedures that minimized this contamination and, in doing so, introduced the concept of aseptic technique to cell culture. Through aseptic technique, cultures can now be maintained in the absence of microbe contamination.

Introduction

Successful in vitro cell culture depends on keeping the cultured cells free from contamination by micro-organisms, thus ensuring the safety and integrity of the process, products, the environment, and the user. Sources of contamination in the culture lab include: non-sterile supplies, media, and reagents, airborne micro-organisms, unclean incubators, and dirty work surfaces. The easiest way to eliminate a contamination is to prevent one in the first place. Prevention can be achieved through a combination of aseptic and sterile techniques. Sterile technique eliminates contamination through sterilization. Aseptic technique maintains the sterility of something that has been sterilized.

Learning Objectives

Preventing microbe contamination of cell cultures through proper aseptic technique is essential. At the end of this section, you will be able to:

• List and describe the types of microbe contamination
• Explain autoclaving and filtration
• Describe the fundamentals of aseptic technique

Types of Contamination

Contamination of cell cultures is easily the most common problem encountered in cell culture laboratories. Cell culture contaminants can be divided into two main categories: 1) chemical contaminants, such as impurities in media, sera, and water, endotoxins, plasticizers, and detergents, and 2) biological contaminants, such as bacteria, molds, yeasts, viruses, mycoplasma, as well as cross contamination by other cell lines. Chemical contaminants can significantly alter the chemical properties of the culture and quickly kill cells. Unfortunately, chemical contaminants can be difficult to detect as they are often invisible. Biological contaminants are more common in the culture lab and are easier to detect using simple methods, such as microscopy. The biological contaminant competes with host cells for nutrients. They also secrete acidic or alkaline by-products which can inhibit growth of the culture cells or kill them. Microbes can degrade essential culture components, like arginine and purine, inhibiting synthesis of histones and nucleic acids by the cultured cell. Finally, microbes produce significant amounts of hydrogen peroxide, which is directly toxic to cells.

The major types of biological contamination are:

1. bacterial
2. fungal: mold
3. mycoplasma
4. eukaryotic: yeast & cell lines

Bacterial Contamination

Bacteria are a large and ubiquitous group of unicellular microorganisms. They are typically a few micrometers in diameters, and can have a variety of shapes, ranging from spheres to rods and spirals. Because of their ubiquity, size, and fast growth rates, bacteria are one of the most commonly encountered biological contaminants in cell culture. Fortunately, bacterial growth can be prevented with good aseptic technique and the with addition of antibiotics, such as penicillin and streptomycin, to the growth medium.

Bacterial contamination is easily detected by visual inspection of the culture within a few days of it becoming infected. Infected cultures usually appear cloudy (i.e., turbid), sometimes with a thin film on the surface. Sudden drops in the pH of the culture medium is also frequently encountered. Under a low-power microscope, the bacteria appear as tiny, moving granules between the cells, and observation under a high-power microscope can resolve the shapes of individual bacteria.

Mycoplasma Contamination

Mycoplasma are simple bacteria that lack a cell wall and they are considered the smallest self-replicating organism. Because of their extremely small size (typically less than 1 µm), mycoplasma are very difficult to detect until they achieve extremely high densities and cause the cell culture to deteriorate; until then, there are often no visible signs of infection. Some slow growing mycoplasma may persist in culture without causing cell death, but they can alter the behavior and metabolism of the host cells in the culture. Chronic mycoplasma infections might manifest themselves as decreased rate of cell proliferation, reduced saturation density, and agglutination in suspension cultures. The only assured way of detecting mycoplasma contamination is by the testing of cultures periodically using assays such as fluorescent staining, ELISA, PCR, or microbiological assays.

Yeast Contamination

Yeasts are unicellular eukaryotic microorganisms found in kingdom Fungi that range in size from a few micrometers (typically) up to 40 µm (rarely). Like bacterial contamination, cultures contaminated with yeast become turbid, especially if the contamination is in an advanced stage. There is very little change in the pH of the contaminated culture until the contamination becomes heavy, at which stage the pH usually increases. Under microscopy, yeast appear as individual ovoid or spherical particles. In cultures with significant numbers of yeast, the cultures can actually smell like bread.

Mold Contamination

Molds are eukaryotic microorganisms in found kingdom Fungi that grow as multicellular filaments called hyphae. A connected network of these multicellular filaments are referred to as a colony or a mycelium. Similar to yeast contamination, the culture medium becomes turbid upon advanced contamination and the pH of the culture remains stable in the initial stages, then rapidly increases as the culture become more heavily infected. Under microscopy, the mycelia usually appear as thin, wisp-like filaments, and sometimes as denser clumps of spores. Spores of many mold species can survive extremely harsh and inhospitable environments in their dormant stage, only to become activated when they encounter suitable growth conditions such as those encountered during cell culture.

Cross-Contamination

While not as common as microbial contamination, extensive cross-contamination of cell cultures with HeLa and other fast growing cell lines is a clearly-established problem with serious consequences. Obtaining cell lines from reputable cell banks, periodically checking the characteristics of the cell lines, and practicing good aseptic technique are practices that will help you avoid cross-contamination. The main elements of aseptic technique are a sterile work area, good personal hygiene, sterile reagents and media, and sterile handling.

Sterilization: Autoclaving and Filtration

There are two main methods for sterilization in a culture lab: autoclaving and filtration. Autoclaving is used to sterilize solid materials, such as culture glassware. Both autoclaving and filtration can be used to sterilize most culture liquids. An autoclave (Figure \(\PageIndex{1}\)) uses pressurized steam to destroy microorganisms, and is the most dependable system for the sterilization of laboratory glassware, media, and reagents, in addition to the decontamination of laboratory waste.

Figure \(\PageIndex{1}\): The autoclave. (Sterilisator by KOchstudiO, CC BY-SA 3.0)

Not everything used in a cell culture lab can be autoclaved. For example, culture media, serum and antibiotics cannot be autoclaved because the high heat will destroy their component parts. For these heat-sensitive solutions, sterilization is typically done through filtration using a vacuum-driven filtration system (Figure \(\PageIndex{2}\)). When filtering a liquid, there are two important considerations: pore size and filter composition. A filter with a pore size of 0.2 µm will effectively remove the most common microorganisms, including fungus, yeast and bacteria. Viruses can be removed using filters with smaller pore sizes (20 to 50 nm). However, the smaller the pore size, the lower the flow rate through the filter. The composition of the filter is also important because it may bind certain constituents of the liquid being filtered (e.g. proteins) or may not be very chemically resistant. Typical cell culture filters are made of polyethersulfone (PES) owing to their low protein-binding capacity.

Figure \(\PageIndex{2}\): A vacuum-driven filtration unit. The top of the filtration unit is a reservoir for the unfiltered liquid. A filter is found at the bottom of the reservoir. The bottom of the unit is a sterile reservoir for the filtered liquid. An attachment for a vacuum line is found attached to the upper reservoir chamber. The vacuum pulls the liquid from the upper reservoir, down through the filter, and into the bottom reservoir (filtration direction shown with an arrow). The upper reservoir and filter can be unscrewed and discarded and a sterile lid attached for storage. Filtration units of various sizes are available depending on the amount of liquid needing to be filtered. Bottle-top filters (not shown in figure), comprised of the just the upper reservoir, filter, and vacuum attachment (i.e., the area of figure above dotted line), can be used as an alternative. These units simply screw on top of sterile glass or plastic bottles. (Filtration unit by Patricia Zuk, CC BY 4.0; adapted from Bottle top disposable filter by Lilly_M, CC BY-SA 3.0)

Concept in Action

Video: Aseptic Technique

Aseptic Technique Fundamentals

Aseptic technique is a set of procedures that is designed to create a barrier between micro-organisms in the environment and the sterile cell culture. Good aseptic technique reduces the chance of contamination and maintains the sterility of the culture environment and reagents.

Aseptic technique requires that the user do the following:

1. Wash their hands prior to beginning culture work
2. Wear the appropriate personal protective equipment (PPE) when working in the lab - e.g. gloves, lab coat
3. Use sterile culture reagents (e.g. culture medium, saline, serum, antibiotics) or properly sterilize them prior to use
4. Decontaminate the biosafety cabinet before and after use
5. Maintain all cell culture equipment properly (e.g. incubators, biosafety cabinet) and confirm their sterility on a regular basis

Good Personal Hygiene & PPE

In cell culture, the chief source of contaminants, such as bacteria and yeast, is hands. Therefore, washing hands before cell culturing is essential. The use of PPE, such as gloves and lab coats, will also reduce the probability of contamination from shed skin, as well as dirt and dust from clothes. The use of PPE will also provide protection from potentially hazardous materials.

The Sterile Work Area

The biosafety cabinet (BSC) is a sterile work area for cell culture. However, it must be used properly in order to maintain this sterile environment. To learn more about the BSC, go to Chapter 6.3 Cell Culture Equipment.

The following list should be followed when using a BSC:

• The BSC should be located in an area that is exclusively for cell culture
• The BSC area should be free from drafts from doors, windows, and other equipment, and with no through traffic
• The work surface inside the BSC should be uncluttered and contain only the items required for a particular procedure; place those items needed inside the BSC prior to the procedure and remove them when complete
• The stainless steel work surfaces inside the BSC should be disinfected thoroughly or autoclaved on a regular basis
• Raise the glass sash and turn on the BSC blower at least 30 minutes before use to allow for adequate recirculation of air through the BSC
• Prior to use, wipe the BSC's work surface with 70% ethanol; continue to wipe the surface during use and especially after any spillage
• Between use, the ultraviolet light may be used to sterilize the air and exposed work surfaces in the BSC; do not use the UV light when personnel are near the BSC
• Do not use a Bunsen burner inside a BSC

Aseptic Handling Principles

• Using 70% ethanol, wipe off the outside of materials that have been removed from the water bath, refrigerator or other non-sterile areas before placing them in the BSC
• Do not spray flasks and dishes that have been removed from an incubator with 70% ethanol before transferring them to the BSC unless contamination of the outer surface is suspected
• Use only sterile glassware (i.e., autoclaved) or disposable plastic-ware in sealed packages
• Avoid pouring media and reagents directly from bottles or flasks; use sterile glass or disposable plastic serological pipettes and a pipette-aid
• To ensure sterility, open sterile culture plastic-ware packages and culture reagents in the BSC immediately before use and close immediately after
• Seal open plastic-ware packages in their plastic packaging with tape or place them in re-sealable bags to prevent contamination
• To prevent contamination, open culture dishes immediately before use and close immediately after
• Upon removing a culture cap or culture dish lid, place it on the BSC work surface, with the opening facing up
• Perform all experiments as rapidly and aseptically as possible to minimize contamination
• If the sterility of a pipette or culture dish is suspected, discard and obtain an aseptic replacement
• If the sterility of a reagent such as medium is suspected, sterilize it by filtration or autoclaving
• If the sterility of a culture is suspected, properly dispose of it

Key Concepts

Aseptic technique refers to the practices and procedures used in cell culture that are designed to maintain a sterile environment and prevent contamination of cultures by microorganisms

Some important concepts to remember are:

• cell culture contaminants can be chemical and biological (i.e. micro-organisms)
• common micro-organisms that contaminate cell cultures are bacteria, yeast, molds, and mycoplasma
• the most common source of biological contamination is the user
• biological contaminants compete with cultured cells for nutrients
• contamination can be minimized through good aseptic technique
• sterility can be achieved through autoclaving and filtration, the use of good personal hygiene and proper PPE

Glossary

Aseptic technique - a series of procedures designed to ensure the sterility of the culture environment and reagents

Autoclave - laboratory equipment that uses high pressure stem to sterilize materials

Biological Safety Cabinet (BSC) - laboratory equipment used to aseptically work with biological materials; designed to protect users from exposure to potential biohazards through its filtering and circulation of air inside of the cabinet

Contamination - the presence of unwanted microorganisms (bacteria, yeast, mold) in a cell culture or cross-contamination from other cell lines

Decontamination - the process of removing or neutralizing contaminants to make an object or surface safe for handling

Disinfection - the process of reducing or eliminating microbes on surfaces

Filter - a device or material used to separate, remove, or block unwanted particles, substances, or impurities from a fluid or other materials

Filtration - a physical separation process that separates solid matter from a fluid using a filter with a complex structure through which only the fluid can pass

Personal Protective Equiment (PPE) - protective clothing, gloves, masks, and eyewear that is used to prevent contamination and exposure to biological or chemical hazardous materials

Sterilization - a process that kills or removes all microorganisms and other biological agents from a surface, fluid, or object; common methods include autoclaving and filtration