Media Preparation and Transfer Techniques

The availability of a variety of preformulated dehydrated media has, in most cases, dramatically shortened the time required for preparation in the modern media-preparation laboratory. In most cases, all one has to do is add distilled water and sterilize. Further, most suppliers handle a variety of specialized media and, depending on demand, can prepare others as needed. Even when fully formulated specialized media are not available, preformulated components are readily acquired in most cases. 

M gor suppliers of laboratory equipment handle microbiological media as well. Brands such as Difco, and BBL (Baltimore Biological Laboratories) are routinely available. Others brands include Oxoid (now marketed in North America by Unipath, a division of UL Canada, Inc. Ogdensburg, NY). For price-conscious individuals, house-brand formulations of routine media are also available from most major suppliers at very competitive prices. 

Dehydrated media is very hygroscopic and has a limited shelf life when opened. As reported in Standard Methods for Examination of water and waste water, unopened containers should be used within 1 year of receipt and, once opened, the contents should be used within 6 months. In any case, media should be stored under cool ( 30°C/86°F) dry conditions, preferably out of direct sunlight, and should be discarded if clumping/ caking or off-colors and odors develop. Because media in open bottles deteriorates relatively rapidly, and cost considerations not withstanding, it is better to order smaller volumes or duplicated smaller volumes than a single larger container. Such a practice minimizes the potential deterioration of an opened container over time. 

Several criteria must be met in order to successfully cultivate microbes. The medium must contain a utilizable source of organic carbon (sugar) for energy and nitrogen for amino acid, protein, and nucleic acid synthesis as 

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Appendix B outlines the basics of laboratory media preparation and skills involved in handling microorganisms without contamination. For those who need to brush up on technique, the fundamentals of routine aseptic transfer are covered in detail. 

The introduction and implementation of heteronuclear-based multidimensional techniques have revolutionized the protein NMR field. Large proteins (> 100 residues) are now amenable to detailed NMR studies and structure determination. These techniques, however, necessarily require a scheme by which and isotopes can be incorporated into the protein to yield a uniformly labeled sample. Additional complications, such as extensive covalent post-translational modifications, can seriously limit the ability to efficiently and cost effectively express a protein in isotope enriched media - the c-type cytochromes are an example of such a limitation. In the absence of an effective labeling protocol, one must therefore rely on more traditional proton homonuclear NMR methods. These include two-dimensional (1) and, more recently, three-dimensional H experiments (2,3). Cytochrome c has become a paradigm for protein folding and electron transfer studies because of its stability, solubility and ease of preparation. As a result, several high-resolution X-ray crystal structure models for c-type cytochromes, in both redox states, have emerged. Although only subtle structural differences between redox states have been observed in these... 

The technique of membrane filtration is used whenever the nature of the product permits. The membrane is transferred to the growth medium, or the medium is transferred onto the membrane. Alternatively, the prepared sample is inoculated directly into the appropriate media. This method is only used when the product (e.g. some vaccines) cannot be dissolved or diluted in a nontoxic diluent. The media used are fluid thioglycolate medium (FTM) for aerobic, micro-aerophilic and anaerobic bacteria, and Soybean casein digest broth (SCDB) for aerobic bacteria and fungi. FTM and SCDB are incubated at 30-35 °C and 20-25 °C respectively, both for a period of not less than 14 days. This relatively long incubation period seems to be justified, because an unacceptable proportion of contaminants would be missed by limiting incubation to 7 days [60]. 

Table 5.1 illustrates the differences in scale of operations between normal analytical TLC, preparative layer chromatography and preparative column chromatography. Separations which have been successfully achieved by analytical TLC may be transferred directly to the preparative layer, because the same sorption media with the same grain size are used in each technique. However, it is not always possible to transfer directly, separations which have been successfully achieved by analytical TLC to preparative column chromatography with equal success because appreciably different adsorbent grain sizes are used in the two techniques. 

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