Culture Club

Managing media preparation can be difficult and dangerous; here Steve Robertson evaluates your options

AS WITH any living organism, microbes need nutrients and specific environmental conditions in order to survive and multiply. A culture medium essentially constitutes an aqueous solution containing all the necessary nutrients, which can be used as a liquid or solidified by adding agar, a naturally occurring polysaccharide produced by marine algae.

Media must be sterilised before inoculating with the desired microorganisms to prevent growth of contaminant bacteria or fungi, as the quality of media directly affects cultural characteristics. While sterilisation can be achieved using a number of techniques, such as irradiation, filtration, or the use of chemicals, the most common method used in the microbiology laboratory is heat. Bacteria are more readily destroyed by moist heat and so the use of pressurised steam autoclaves is ideal. Treatment at 121ËšC for 15 minutes is sufficient to destroy even the most thermo-tolerant organisms.

Laboratories can meet their culture media requirements by manually preparing, purchasing pre-made media or by using an automated media preparator. Each of these methods has certain advantages.

Media preparation is traditionally carried out by following a step-by-step recipe. Each of the particular nutrients required are weighed into a flask before making up to volume with water. Media made manually can be prone to contamination due to the step-by-step nature of preparation and the associated exposure to air, glassware and human contact, so must be sterilised before use.

Steam sterilisation using an autoclave is the most widely-used method; however standard autoclaves may not guarantee complete sterility. They often only measure the temperature of the chamber and, as it takes longer for a liquid to reach the required sterilisation temperature, the autoclave may finish its cycle too early. The temperature subsequently displayed after cooling may be misleading due to heat retention by the media. Transferring extremely hot media directly to cool in a water bath presents a potential safety hazard and another opportunity for contamination, as water baths provide a perfect environment for microbial growth. It can also be difficult to ensure homogeneity through uniform blending of ingredients, when preparing large quantities of media.

Even with its limitations manual media preparation still has its place, as contract microbiology lab manager Stuart Moore explains: “Preparing culture media manually can be labour intensive and very time consuming. Each step from boiling up the media and water on a hotplate, to autoclaving, cooling and pouring, has the potential to introduce contamination. However, as long as the appropriate precautions are taken, manual preparation is still useful when small quantities of media are required. If we only need 25 – 50 plates making up then we would often prepare the appropriate amount of media manually, as any excess would be wasted.”

Providing an alternative to manual in-house preparation, an increasing number of pre-prepared media products are now available. The most popular formats include ready-to-pour bottled liquid media, pre-poured plates and liquid media pouches, which are available in sizes ranging from 1-100 litres. Bags containing sterile powdered media to which sterile water is added are also available. Although an element of preparation still exists with this approach, mixing sterile components removes the need for sterilisation and is useful when preparing thermally-sensitive media. Care must be taken, however, to ensure effective mixing of media while it is contained within a bag or pouch. 

Bought-in media brings with it a number of benefits. The ratio of staff-to-sample throughput is improved as a direct consequence of the manual-preparation time saved. Quality control and associated regulatory requirements become the responsibility of the culture media supplier rather than the testing laboratory, and can therefore be easier to defend during an audit. For media that is particularly difficult to prepare manually, purchased media provides a clear advantage.

Stuart comments: “Pre-prepared media is often used in microbiology laboratories. Some culture media require a supplement to be added post-sterilisation. These supplements can either be bought in liquid format, or as a dehydrated powder that we would then rehydrate using an appropriate solvent before adding to the culture media. It may also be more cost-effective to purchase pre-prepared media if it is to be used sporadically. This will avoid the purchase of components that may be used once or twice then eventually discarded when reaching expiration date.”

Although pre-prepared media offers a time-saving solution, it does not address

Figure 1: Microbial growth requires specific environmental conditions and nutrients

the need to accurately predict the quantities required for future testing. In sudden high-volume testing situations, microbiologists may be left without sufficient supplies of a particular culture media. Alternatively, if testing slows down or the workload has been miscalculated, it may become necessary to dispose of media that has passed its expiration date. As with any pre-made products, the cost of purchasing pre-prepared media can also prove prohibitive to some non-commercial laboratories.

Another solution is to prepare media in-house using an automated media preparator. This approach allows preparation and sterilisation to be performed using a single piece of equipment, where nutrients and water are added initially to the main vessel. The mixture is heated and stirred to help dissolve agar, if required for plated media. Once this stage is complete, the media is brought up to sterilisation temperature and held for the appropriate length of time. Using a preparator, rather than a standard autoclave, provides a clear advantage in this situation. Effective sterilisation is ensured, as the actual temperature of the media is measured, as opposed to the autoclave chamber. Media can be maintained at a specific temperature if required, or cooled to allow dispensing. Users can be confident that media is at the temperature displayed and therefore reduce the risk of burns or scalds. To eliminate hot spots and ensure thorough mixing, media is continuously stirred without introducing air bubbles.

Most automated media preparators have ports to allow sterile additives to be

Figure 2: Liquid media can be solidified by adding agar

added aseptically following sterilisation. Stuart explains: “We either prepare heat-sensitive supplements by aseptically rehydrating a sterile powdered ingredient, or we use vials of pre-prepared liquid supplements. These can be added through the ports on the automated media preparator, once the media has been cooled to the appropriate holding temperature.”

Processed sterile media is then dispensed directly from the automated preparator with the aid of a peristaltic pump. It is also possible to connect media preparators to automated plate pourers, allowing prepared agar plates to be stacked ready for use. Stuart comments: “Combining an automated media preparator with a pourer/stacker is ideal as prepared media can be quickly poured into plates with minimal risk of contamination. The media preparator is connected to the pourer/stacker with sterilised tubing via a pump, allowing us to dispense over 700 plates in an hour. The system we use has an option for an integrated refrigeration plate for rapid cooling so that you get uniform, level plates.” In addition to automated pourers, it is also possible to connect media preparators to gravimetric diluters for precise dilution of bulk samples.

Every aspect of automated media preparation is controlled electronically on the

Figure 3: Small quantities of media can be prepared manually

equipment directly or via a computer. All programme parameters are displayed and can be adjusted to create customised sterilisation cycles. To ensure traceability, operator names and batch numbers can be entered and it is possible to add a chart recorder and/or barcode reader. The addition of a Petri dish labelling machine would also reduce potential labelling errors and truly automate the media production process.

Stuart continues: “There is a perception that automated media preparators are expensive, however when you consider the amount of time they save and the reduced chance of contamination they are an excellent way for producing consistent, high quality culture media.”

In considering the most suitable methods for preparing microbiological culture media, it would appear that the volume required dictates the need for automation. For laboratories with predictable workloads, purchased media certainly provides a time-saving option with additional regulatory benefits. Those requiring high-quality, homogenous media on tap can benefit from using automated preparators that offer safe, reproducible and traceable culture media. When considering the most appropriate method, care must be taken to balance the demands of laboratory workloads, budgets and specific requirements to guarantee sterile media preparation of the highest quality.