Media Preparation

Laboratory technician performing sterile filtration of red cell culture media in a flask during media preparation.

Cell culture media preparation significantly impacts mammalian cell growth and experimental outcomes. Similarly, there are specific media formulated to support the cultivation of microorganisms in vitro. Culture media provide a source of energy for cell growth and compounds that regulate cellular processes. Both prokaryotic and eukaryotic cultures may have specific growth requirements. Media selection depends on the culture type, purpose of cultivation, and cell density requirements.   

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Media Preparation

Commercial culture media are provided in dehydrated or powdered form, in liquid concentrate, or as working solutions. Dehydrated media for broth cultures need to be prepared by dissolving in distilled water and adjusting the final pH prior to sterilization. Powdered media for agar cultures must be dissolved in distilled water, stirred, then boiled or autoclaved prior to pouring into sterile petri dishes using aseptic techniques. Culture media provided as liquid concentrates must be aseptically diluted in distilled water, adjusted for pH, and dispensed into sterile containers for storage. Culture media provided as working solutions are ready to use after the addition of any required serum or other supplements under sterile filtration and/or aseptic conditions, and warming to the appropriate temperature for cell growth.

Culture Media Sterilization

Culture media prepared from powders must be sterilized by filtration, heating, or autoclaving prior to use. Although liquid media are often supplied sterile and at ready-to-use concentration, sterile filtration is recommended, particularly if sera or other supplements are to be added before use. Following sterilization, aseptic techniques should be used when handling culture media to prevent cross-contamination or the growth of unwanted microorganisms.

Culture Media Supplements

Certain supplements required for cell cultivation or regulation are unstable or heat-labile; therefore, they must be added by the user to heat-sterilized basal culture medium prior to use. Serum typically derived from bovine sources (FBS) is a common supplement used in animal cell culture media. Hormones, growth factors, and specific signaling molecules may be required for the growth of certain cell types. Additives such as IPTG are used to induce protein expression. Antibiotics may also be added to culture media to control the growth of bacterial and fungal contaminants, though contamination can be well-controlled with appropriate handling and storage of media.

Culture Media Storage and Usage

Commercial and prepared culture media in liquid form, poured agar form, or working solutions are typically stored at 4 °C in the dark to suppress the growth of opportunistic microorganisms and to prevent light-induced degradation of culture media components. Prior to inoculation with cultures, media should be warmed to an appropriate temperature for cell growth.

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