Good laboratory practice in culture media preparation

Good laboratory practice in culture media preparation

Good laboratory practice in culture media preparation in pharmaceutical industry

Media preparation

Culture media may originate from one of three sources: laboratory-prepared from component raw materials, commercially manufactured and presented in either a dehydrated, or ready-to-use form. The first of these is now rarely encountered in the pharmaceutical industry, and most microbiology quality assurance laboratories will employ dehydrated or ready-to-use products. However, in some instances where unusual formulations are required, there may be no alternative to the use of laboratory-prepared media. Pharmaceutical microbiologists use a narrow range of media in the main, all of which are available from a commercial source. The decision of whether to use a dehydrated medium concentrate or a short-life, ready-poured culture medium will be influenced by considerations of economy, space and available staff time for both production and quality control. Whatever the source, the microbiologist’s expectations of his or her culture media remain the same: a freshly prepared quality product in all instances.


Good laboratory practice in culture media preparation


Storage of incoming raw materials

All incoming raw materials should be purchased from an approved supplier and dated on receipt. Particular care should be given to the sourcing of media used in the production of pharmaceutical materials with respect to the bovine spongiform encephalopathy status of herds providing beef extracts (Garland, 1999). The suitability and security of packaging should be checked. Raw materials should be stored as advised by the manufacturer; humid environments (in the vicinity of steam pipes) and those with fluctuating temperatures are clearly unsuitable.

Water

Water quality can markedly influence the performance of culture media. Fresh, good quality water must be readily available and provided by distillation, deionization or reverse osmosis. Tap water is unsuitable owing to its inherent impurities (traces of magnesium, calcium and other metals, as well as chlorine and fluorine), all of which may affect the characteristics of selective culture media. Water quality should be regularly monitored. Conductivity measurements should be made daily and should be within the manufacturer’s specified low range. Higher readings suggest that equipment maintenance may have been neglected. The pH of water is not usually checked, unless there is a problem with the pH of prepared media. As with pharmaceutical grade water, water quality can only deteriorate on storage, and therefore ideally supplies should be produced and used as required. If water must be stored, clear glass containers with lids are preferable to polythene containers as the pH may become acid on storage.

Preparation of culture media

Good laboratory practice requires that culture media are made to pharmaceutical and not ‘cook book’ standards. Media should be made according to master formulae and written procedures. Documentation should be completed for each batch of media made; a batch number and expiry date should also be allocated. Manufacturer’s instructions should be closely followed.

When weighing out powders, a suitable face mask should be worn by the operator. Excessive dust can be avoided by weighing out each ingredient into a suitable weighing boat. Depending on the quantity involved, a top pan balance (accuracy ±0.01 g) or analytical balance for smaller weights will be required. Where very small quantities are involved, it may be necessary to prepare a concentrated stock solution and then dilute this to the required strength. When dissolving powders in water, the aqueous phase (1/3 volume) should be added first, followed slowly by the powder, dispersed by gentle swirling, and finally the remaining liquid can be used to wash down any adhering powder. Balances should be cleaned thoroughly after use; they also require weekly calibration and regular maintenance. Appropriate records should be kept, as discussed below. Equipment used for dispensing culture media should be thoroughly cleaned after use.


Measurement of pH

The properties of a given culture medium are pH-dependent. Incorrect pH may result not only in physical changes such as precipitation of components or soft gelling of agar, but also significant chemical changes, such as a loss or change in the indicator or selective system. Incorrect pH may also affect the recovery of stressed cells and influence cell growth. Since pH is temperature-dependent, measurements are best taken at a standardized temperature, e.g. 25°C using a suitable instrument (i.e. measuring to 0.1 pH units). pH meters should be calibrated before use using standardized buffer solutions (e.g. pH 4, 7 and 10) and these should be renewed at least weekly. As before, planned preventative maintenance is required, and records should be maintained. The pH of laboratory prepared media should be checked and adjusted if necessary before dispensing for sterilization. Changes in pH may occur during sterilization; allowance for this can be made on the basis of experience. In contrast, commercially available dehydrated media usually require no pH adjustment if properly prepared. The pH of the final sterilized product can be measured on a single unit (plate or bottle), but this must be discarded after use. pH measurements should be recorded in the batch records.

Sterilization

The sterilization of culture media is a critical control point in assuring their quality: the media must be sufficiently processed to ensure sterility, but any over-processing may affect their nutritive properties and result in the accumulation of toxic residues. Thus, as in pharmaceutical production, individual sterilization cycles should be properly validated with thermocouples to ensure that all containers in the load achieve the required temperature. Furthermore, for each batch processed the accompanying temperature time chart recording should indicate that the correct conditions in the load have been achieved. These charts should be retained with the accompanying batch documentation.
Autoclaves
Loading patterns in autoclaves should be considered to allow free circulation of steam and adequate steam penetration. Similar volumes should also be processed at the same time whenever possible. Correct segregation of sterilized and unsterilized media should be ensured through a streamlined work-flow process. Autoclave tape may also be used as a supplementary aid, but should not be regarded as hard evidence that a satisfactory sterilization cycle has been achieved. Guidance on the design, installation, operation, maintenance and process monitoring of autoclaves is available in BS 2646 (1988, 1990), and the importance of following these recommendations cannot be overemphasized. As before, all records should be retained.
Agar preparators
The sterilization of large volumes (i.e. in excess of 1 litre) of culture media in an autoclave can result in a nutritionally inferior product if an extended heat transfer occurs during heating up and cooling down. In contrast, agar preparators provide greater control of the heating process with rapid heating and cooling cycles, resulting in a good quality product. Here, liquid medium is introduced directly into the jacketed heating chamber, continuously mixed throughout the sterilization cycle, cooled rapidly and then pumped directly into containers. After use, the bowl must be thoroughly cleaned.
Membrane filtration
Membrane filtration is used to sterilize heat-sensitive media and components. Filters with a pore size of 0.45 or 0.22 μm may be used, supplied in a ready-to-use disposable filtration unit or as a re-usable and sterilizable filter unit with a separate filter.

Plate pouring

Depending on the chosen method of isolating and counting microbial colonies, the product sample will either be spread as a dilution onto the surface of a pre-poured set and dried agar plate—a technique known as surface inoculation—or alternatively the sample can be mixed with molten agar, then poured into a Petri dish and allowed to set—known as the pour plate technique. In both instances the agar should be carefully tempered to 46±1°C. Above this temperature, there may be damage to microbial isolates or heat-labile supplements, or excessive condensation may occur; below this temperature, the gel may begin to set before plates have been poured. Care should be taken to avoid the introduction of air bubbles by gently swirling the contents to ensure thorough mixing of ingredients and possible supplements. When pouring plates by hand, an aseptic technique should be used and ideally within a laminar flow cabinet. A 15–20 ml volume is required in each 90-mm Petri dish, in order to minimize batch-to-batch variations in microbial performance. Smaller volumes may result in the medium drying out. Automated plate pouring systems may be used where there is a high demand for plates of a limited range of media types. However, before embarking on such a system, the microbiologist should consider the plate throughput per hour, the destacking and stacking mechanisms for loading plates, the likelihood of blockage and ease of clearing, the acceptability of Petri dishes from alternative suppliers, and finally the proposed savings in staff time offset against the capital cost of the system.

Good laboratory practice in culture media preparation in pharmaceutical industry


2 comments

  1. Buenas noches

    que medios de cultivo recomienda para incubar bacterias en temperaturas por encima de los 75 °C, ya que como ustedes dicen por encima de 46°C el medio se tiende a perder. Muchas gracias

  2. How can I will enroll myself in “good laboratory practice in culture media preparation in pharmaceutical industry

Comments are closed.