Microbiology Resources

Types of microbes for research

Working in a microbiology laboratory

When working in a microbiology laboratory, aseptic technique is fundamental to the success and safety of an experiment. Generally, aseptic techniques are procedures or processes performed by scientists under sterile conditions to ensure that microbial contaminants do not harm colleagues and are not introduced into sterile solutions, supplies, or experimental cultures. To minimize the possibility of microbial contamination, the below rules and recommendations are encouraged. Additional information on risk assessment and precautions can be found in the Center for Disease Control (CDC) publication “Biosafety in Microbiological and Biomedical Laboratories, (BMBL)” at http://www.cdc.gov.

Maintain a sterile work area

• Before and after use, disinfect all work surfaces with 70% ethanol or an appropriate disinfectant. This is especially important after any spills.
• Maintain an uncluttered work space; all work surfaces should only contain equipment that is required for your experiment.
• Ensure that you have all necessary supplies before beginning an experiment. Being prepared will reduce the likelihood of careless contamination.
• Work may be performed in a thoroughly sterilized biosafety cabinet. Biosafety cabinets can be sterilized via ultraviolet light in conjunction with 70% ethanol or an appropriate disinfectant.
• Do not open windows or use fans that circulate outside air. If possible, work in laboratory settings that have air vents covered with filters. This will prevent the contamination of cultures by airborne particles.
• Frequently clean water baths used for thawing or warming media or solutions.
• Routinely sterilize incubators used for microbial propagation.

Handling media

• Before and after use, sterilize the outside container of all media and reagents with 70% ethanol. Also, do not leave containers of media open longer than necessary.
• Aliquot sterile solutions into smaller volumes whenever possible. If you are unsure of the sterility of your media, it is best to discard it immediately.
• Avoid pouring sterile liquids from one container to another, this increases the likelihood of media contamination. Rather, use sterile pipettes for the aseptic transfer of media.
• Never mouth pipette. This poses a health risk for personnel as well as increases the risk of contamination.
• Always use sterile glass or disposable plastic pipettes to work with liquid media. Use each pipette only once to avoid cross contamination.
• Do not open sterile media, petri dishes, or pipette containers until you are ready to use them

Handling microbial cultures

• Before working with media and microbial cultures, wipe your work area with 70% ethanol or an appropriate disinfectant.
• Ensure you are wearing appropriate protective clothing. This will protect you from the culture as well as reduce accidental culture contamination.
• Only use sterile glassware, equipment, media, and reagents. Check media for contamination by observing for turbidity.
• Handle only one microbial culture at a time. The risk of cross contamination or misidentification increases when more than one strain is handled at a time.
• When working with test tube cultures, hold cultures at an angle after you remove the lid to avoid airborne particles from falling into the culture. Sterilize the outside of the culture tube using a Bunsen burner flame.
• When working with plated cultures, hold the petri dish lid at an angle after you remove the lid to avoid airborne particles falling into the culture dish.
• When handling a microbial culture, work quickly and carefully in an environment that has minimal distractions. Do not leave the lid off your culture for extended periods of time. ƒ Never take cultures outside of the laboratory.
• Notify the laboratory supervisor immediately of any spills.

Culturing techniques

A culture medium is a solution of nutrients that is required for microbial growth. Depending on their composition or use, culture media can be categorized into several groups; these include defined, complex, selective, and enrichment medium. In a defined medium, the exact chemical composition is known. These types of media are usually composed of pure biochemicals and are often used to study the minimal nutrient requirement of a microorganism. In contrast, the exact chemical composition of a complex medium is not known. This latter medium type often contains reagents of a biological origin, such as yeast extract and peptone, where the exact chemical composition is unknown. Complex media usually provide a large range of growth factors that assist in the cultivation of unknown and fastidious bacterial species.

Medium may also be formulated as selective or to enrich. A selective medium is formulated to inhibit the growth of certain bacterial species and/or promote the growth of a specific species. These media can consist of additional selective reagents, such as high salt concentration to select for halophiles, or can be used under selective growth conditions. An enrichment medium also allows for the growth of specific bacterial species; however, enrichment media are supplemented with a reagent that permits, rather than inhibits, the growth of a species.

Generally, bacterial culture media are mixtures of proteins, salts, trace elements, amino acids, and carbohydrates. The presence and volume of these components can vary significantly among bacterial species depending on the macro- and micro-nutrient requirements of each strain. The manner of which bacterial strains are cultured also varies widely. Liquid media are often used for the growth and propagation of pure batch cultures, while solid agar-based media are used for the isolation of pure cultures. All culture media must be sterilized prior to its use. This is often accomplished by heating the media at high temperatures within an autoclave. This instrument provides 15 pounds per square inch of steam pressure, allowing for temperatures to reach and be maintained at 121°C. All media must be loosely capped prior to sterilization to equalize the pressure of the container and to prevent contamination upon removal from the autoclave. Below, we will describe how to inoculate various broth and agar cultures using aseptic technique.

Counting microbes

Bacterial cell counts are necessary in order to establish or monitor bacterial growth rates, determine population doubling time, or to set up new cultures with known cell counts. Bacterial cultures can be titered by determining the viable cell count, which is the number of live colony forming units per milliliter (CFU/mL), or by analyzing total cell count by measuring the optical density at a wavelength of 600 nm (OD₆₀₀) using spectrophotometry. It must be noted that the growth rate and culturing requirements of bacteria can vary drastically between species, thus making it difficult to quantify a bacterial titer. For more information on how to culture a strain, refer to Bergey’s Manual of Systematic Bacteriology 2nd Edition. Automation of this step is encouraged to minimize errors.

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