Preparation and Culture Techniques

Successful cultivation of microorganisms for growth and identification requires use of various types of media, either liquid (referred to as hroths) or solid by inclusion of agar. For winemakers, a growth medium may he as simple as diluted sterile grape juice for the activation and expansion of yeast starter cultures or as complicated as that necessary to grow lactic acid bacteria. 

The availability of a variety of preformulated dehydrated media has dramatically shortened the time required for preparation. To prepare these media, frequently only distilled water is added and then the medium is sterilized, usually by autoclaving. Most suppliers handle a variety of specialized media and, depending on demand, can prepare others as needed. Even when specialized media caimot be obtained, individual ingredients are normally available. Major suppliers of microbiological media include Difco, BBL (Baltimore Biological Laboratories), and Oxoid. For price conscious individuals, house-brand formulations of routine media are also available from most major suppliers at competitive prices. 

Dehydrated media are hygroscopic and, thus, have a limited shelf life once opened (Flowers et al., 1992). In general, unopened containers should be used within 1 year of receipt and, once opened, the contents should be used within 6 months. Media should be stored under cool ( 30 C/86 F), dry conditions, preferably out of direct sunhght, and should be discarded if clumping/caking or off-colors and odors develop. Since media in opened bottles wiU deteriorate relatively rapidly, it is better to purchase smaller quantities depending on expected use. 

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One of the most sensitive biological effects of ionizing radiation is to increase the frequency of normally observed chromosome aberrations (but not to induce qualitatively special abnormalities). Peripheral blood lymphocytes are the most feasible cells for chromosome investigations, as blood samples are easy to obtain and the techniques to stimulate the lymphocytes to proliferate within a culture medium and to prepare suitable chromosome slides for microscopic analyzation have their routine protocoil (e. g. Yunis, 1965 Lloyd et al, 1982). 

Aseptic techniques are used to avoid the possibility of infection of the animals or ceU cultures. These include the preparation of the vaccines and spleens under aseptic conditions in a class 100 clean room equipped with a laminar airfiow hood, sterilization of instruments, and treatment of work surfaces with disinfectant before and after use, washing of the investigator s hands with an antiseptic surgical scrub preparation, and wearing of sterile gloves, face mask, and eyeglasses. 

Growth of C. versicolor on Wood. Samples of beech heartwood (Fagus syl-vatica) were added to culture plates of C. versicolor after 7d. growth, using the Bravery miniature woodblock technique (12). Plate cultures were covered with a 1mm mesh 60mm diameter sterile nylon net. Sterile sections of beech (30 x 10 x 3mm) were placed aseptically onto the net and cultured at 20° C for 4 weeks. The external growth of mycelium was removed and the wood block cut into segments of approximately 3 x 1 x 1mm for preparation for electron microscopy. Uninfected wood samples were prepared in a similar manner. 

Ascites production, however, suffers from a number of drawbacks. It is costly, and the product is contaminated by significant levels of various mouse proteins, rendering subsequent downstream processing more complex. As a result, monoclonal antibody production by standard animal cell culture techniques has become the method of choice for the production of pharmaceutical-grade monoclonal antibody preparations. 

In this chapter more detailed information on the double-cell voltage-clamp setup and protocols for assessing gap junctional conductivity is given, as well as a description of the cell-isolation procedure for this purpose and cell culture models. Information on immunocytochemical localization of gap junctions and on the experimental procedure of preparing specimens and slides for immunohistology is given. A protocol for isolation of gap junction proteins is also outlined. Readers interested in more details of the cell-culture technique regarding incubators, sterile technique, etc., and different isolation and culture protocols are referred to more specialized literature [Lindl and Bauer, 1994 Piper, 1990]. 

Because a-L-arabinofuranosidase is an extracellular enzyme, a crude preparation may be made simply by fractionation of the culture filtrate with ammonium sulfate. The enzyme can be purified from the crude enzyme-preparation by some suitable combination of ion-exchange chromatography, gel filtration, and similar techniques. Three examples of purification procedures, two from micro-organisms and one from a plant, are given here. 

Thus, the surfaces prepared by radiation techniques such as irradiation, grafting, etching, and pore-forming polymerization at low temperatures provide useful materials for cell cultures with better wettability, micro-porous heterogeneous structure and increased positive charges on the polymeric surfaces. 

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