Subculturing

Mammalian cell cultures have been used as the basis of several systemsin detect ng the potential carcinogenic activity of chemicals. Basically, two general approaches have been utilized continuous cell lines and primary cell cultures. Cell lines have the advantage of ease of use, in that cultures do not have to be obtained fresh from animals prior to each test, but may be maintained for months to years by proper subculturing techniques. They have the disadvantage of possessing one or more transformed characteristics (e.g., immortality). In some cases cell lines may also lack certain enzyme systems required for metabolic activation of chemicals. Some of the cell lines used for transformation assays include the murine (BALB/3T3) A31 system ( ), and the baby kidney-21 (BHK-21) systems (43). 

Primary cell cultures have advantages over cell lines in that the cells are not initially immortal, and usually have none of the transformed characteristics which may be seen in some cell lines Additionally, the embryonic cells which are most 

A useful in vitro transformation assay which may be particularly applicable for metals, is the the enhancement of hamster embryo cell transformation by simian adenovirus. Casto, et al (46) and DiPaolo et al ( ) reported that all metal salts (of a series of 38 tested) with known carcinogenic activity, increased the frequency of simian adenovirus SA-7 induced transformations. 

ACS Symposium Series American Chemical Society Washington, DC, 1980. 

Metals were divided into three groups high (positiveat 0.05 mM), moderate (positive at 0.05 to 0.6 mM), and low (positive only at concentration greater than 0.9 mM) activity. 

Cells in tissues and cells growing as monolayers on glass or plastic surfaces are held together and to the substratum by mucoproteins, and sometimes by collagen (see 2.4). In addition, many cell monolayers and tissues, especially epithelial tissues, require divalent cations (Ca2+, Mg2+) for their integrity. 

Thus for tissue dissociation or for releasing cells from monolayers various protease solutions are used, sometimes in association with chelating agents. 

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L. mesenteroides strain NRRL B-512F produces a water-soluble dextran with 95% a(l 6) main-chain linkages and 5% a(l 3) branch linkages (78). This strain was subcultured from NRRL strain B-512, isolated in 1943. Strain NRRL B-512F is the strain used for commercial dextran production in the United States and most other countries. Nearly all of the studies done on the industrial production and utilisation of dextran have used this strain. 

The discovery and biological properties of lincomycin (1, R = OH, R = H) were described ia 1962 (1). This antibiotic is active in vitro and in vivo against most of the common gram-positive pathogens. Resistance by Staphylococci is developed slowly ia a stepwise manner, based on in vitro serial subculture experiments, and its activity is not iafluenced by body fluids up to concentrations of 50% ia the assay medium (2). 

Available Chlorine Test. The chlorine germicidal equivalent concentration test is a practical-type test. It is called a capacity test. Under practical conditions of use, a container of disinfectant might receive many soiled, contaminated instniments or other items to be disinfected. Eventually, the capacity of the disinfectant to serve its function would be overloaded due to reaction with the accumulated organic matter and organisms. The chlorine germicidal equivalent concentration test compares the load of a culture of bacteria that a concentration of a disinfectant will absorb and still kill bacteria, as compared to standard concentrations of sodium hypochlorite tested similarly. In the test, 10 successive additions of the test culture are added to each of 3 concentrations of the hypochlorite. One min after each addition a sample is transferred to the subculture medium and the next addition is made 1.5 min after the previous one. The disinfectant is then evaluated in a manner similar to the phenol coefficient test. For equivalence, the disinfectant must yield the same number of negative tubes as one of the chlorine standards. 

Microbial strains must be maintained in such a way that they do not lose their desirable characteristics. Some strains are maintained by regular subculturing, whereas others are lyophilised (freeze-dried), or frozen under nitrogen, or held at -80°C in a freezer. To ensure that a standard inoculum can be obtained on demand, great care is taken to ensure that foe stored cultures are pure and foe viability is known. 

Secondary cell cultures, which can be prepared by taking cells from some types of primary culture, usually those derived from embryonic tissue, dispersing them by treatment with trypsin and inoculating some into a fresh batch of medium. A limited number of subcultures can be performed with these sorts of cells, up to a maximum of about 50 before the cells degenerate. 

There are now available a number of lines of cells, mainly originating from malignant tissue, which can be serially subcultured apparently indefinitely. These established cell lines are particularly convenient as they eliminate the requirement for fresh animal tissue for such sets or series of cultures. An example of these continuous cell lines are the famous HeLa cells, which were originally isolated from a cervical carcinoma of a woman called Henrietta Lacks, long since dead but whose cells have been used in laboratories all over the world to grow viruses. 

The activity of diamidines is reduced by acid pH and in the presence of blood and serum. Microorganisms may acquire resistance by serial subculture in the presence of increasing doses of the compounds. Propamidine and dibromopropamidine, as the isethionate salts, are the major diamidine derivatives employed as antimicrobial agents propamidine in the form of eye-drops (0.1%) for amoebic infection and dibromopropamidine for topical treatment of minor infections. 

The disinfectant is assessed on its overall performance, namely its ability to kill microorganisms, as judged by subculture recovery or lack of it and not by comparison with phenol, i.e. a disinfectant would pass or fail according to its performance. A use-dilution concentration of a disinfectant must pass the test at three replications. 

Fungal spores or mycelium may be added to the solution under test. At selected time intervals, samples can be subcultured into suitable media and the presence or absence of growth noted after incubation. A quantitative assessment similar to that described for bactericidal activity (section 3.2, Table 11.3) can also be undertaken. 

Thus, the culture may be mixed and transferred to a hypodermic syringe surrounded by a constant-temperature jacket at desired intervals, the mixture is subcultured by ejecting small volumes from the syringe nozzle into subculture medium. 

Solid disinfectants may be evaluated in vitro by applying them to suitable test organisms growing on solid medium. Discs may be cut from the agar and subcultured, observing the usual precautions. 

Bacteria grown under different conditions may show wide response to biocides. For example, fattened cells of Staph, aureus obtained by repeated subculturing in glycerol-containing media are more resistant to benzylpenicillin and higher phenols. 

Ideally, when considering the level of treatment necessary to achieve sterility a knowledge of the type and total number of miciooiganisms present in a product, together with their likely response to the proposed treatment, is necessary. Without this information, however, it is usually assumed that organisms within the load are no more resistant than the reference spores or than specific resistant product isolates. In the latter case, it must be remembered that resistance may be altered or lost entirely by laboratory subculture and the resistance characteristics of the maintained strain must be regularly checked. 

The individual may then withdraw from society into a drug subculture. 

Changes in wall architecture occur during isodiametric cell expansion, cell elongation, and the thickening of a growing wall to its mature thickness, and so it is important to define whether the synthesis and secretion of particular cell-wall molecules correlates with differentiation events or with general cell expansion. Ceils are stimulated into producing wall polysaccharides upon subculture, so we used a non-inductive medium, in which the cells only expand, for comparison with inductive medium at all times. 

Results of pyrolysis mass spectrometric analyses can be influenced by both phenotypic drift and instrument drift. Phenotypic drift can result from variations in culture growth immediately prior to analysis, and from variations during serial subculturing before analysis.125,126 However, this type of drift is not perceived as an obstacle in microbiological work because it can be largely overcome by standardizing culture conditions and by analyzing more than one sample from each culture. 

When isolates are believed to be S. schenckii, they should be subcultured on an enriched medium such as blood agar to determine whether they are dimorphic that is, whether they grow vegetatively as hyphae at 25°C and as yeast cells at 35°C. The conversion from the mold form to the yeast form is enhanced by incubation of the inoculated medium in a candle jar. 

Phaeococcomyces exophialae forms slimy, mucoid, slow-growing, smooth colonies that are grayish black. Budding yeast cells which are at first subhyaline are abundant. With age, some of the cells become darker, with thickened cell walls. Some pseudohyphae usually are present. Hyphal development may become dominant in some isolates of this species with subsequent subculture. 

These changes in morphology induced by chemical substances are usually temporary, since reversion to normal form occurs promptly when the filamentous bacteria are subcultured in the absence of the inhibitory agents. Irradiation, on the other band, may give rise to a temporary or permanent induction of filamentous cells. 

When a fraction of the cells from an existing culture is placed in a new flask these transferred cells are said to have advanced in passage number. This action is called passaging, splitting cells, or subculture, and each cell line has... 

In order to reduce the time required to confirm the accumulation of a given recombinant protein, we have developed a cell culture system in which transgenic alfalfa callus material produced at the proliferation step of Agrobacterium-based transformation is used to initiate cell cultures. These cell suspensions can be subcultured to sustain batch production of modest protein amounts. The protein blot shown in Fig. 1.2 demonstrates our ability to detect a recombinant protein in total... 

Indeed, Galison explains that In the trading zone both sides impose constraints on the nature of the exchange (806), and the collaboration itself can even reinforce the various subcultures involved (829). 

The bottles are subcultured as necessary throughout the expression period to maintain subconfluency. This involves retrypsinization and determining the cell titre for each treatment. For each culture a fresh roller bottle is reseeded with a minimum of 106 cells. 

Induction ofmRNA for pS2 and secretion of cell-type-specific proteins. pS2 was measured in the culture medium of MCF7 cells with the ELSA-pS2 immunoradiometric assay (CIS Bio International, Gif-sur-Yvette, France). Cells were subcultured in 24-well plates for 144 h in 10% CDHuS. The culture medium was centrifuged at 1200g for 10 min to eliminate floating and detached cells. Results are expressed as ng of secreted protein per million cells. The relative-induced protein potency (RIPP) was calculated as 100 X the ratio between the dose of E2 and that of the chemical needed to produce maximal expression of cell-type-specific proteins (pS2). 

Similarly, Charles Rosenberg has written that knowledge is "a central element in shaping the structure of disciplinary cultures and subcultures."9 In this interpretation, ideas are the engine, not the accoutrements, of discipline building. 

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