Maintenance medium

Nutritional and cultural techniques for growing Ochromonas danica in light and darkness have been described (A2, H18). It was grown under constant illumination from five 40-watt warm-white fluorescent lights, ca. 1.0 m from the cultures. Transfers were made weekly in 10 ml of maintenance medium 1ml was used for transfer (Table 6). 

The organism is L. casei ATCC No. 7469. The basal medium (J6) has been modified it contains twice the concentration listed in Table 10a, and is stable when stored in an amber bottle in the cold. It is kept free from contamination with a volatile preservative. L. casei was maintained in medium given in Table 10b 10 ml of this medium is dispensed into screw-capped tubes and autoclaved for 30 minutes at 118°, 16 psi. It should be transferred every 2 weeks one drop of an 18-hour culture into 10 ml of maintenance medium suffices with subsequent 18-hour incubation at 37°. Cultures should be stored at 4°. 

Blood of normal subjects was obtained from an antecubital vein, diluted 1 5 with pH 4.5 buffer,2 and autoclaved 30 minutes to convert bound cobalamin into its microbiologically active form serum was treated like blood. This procedure allowed estimation of total vitamin Bi2. For the subsequent inoculation of specimens (a) E. coli as a loopful from nutrient agar suspended in 25 ml of medium, (b) L. leichmannii, an 18-hour culture diluted 1 10 in basal medium, (c) E. gracilis, strain Z, and (d) O. malhamensis are inoculated directly from a 5-day culture grown in liquid maintenance medium. One drop into a culture flask served as inoculum. The bacteria required 18-hours for full growth protozoa, 4-5 days. 

Vero maintenance medium 500 mL 199, supplemented with 5 mL glutamine, 10 mL antibiotics, and 10 mL fetal bovine serum. 

Thaw stock HS V rapidly and prepare correct dilution in Vero maintenance medium. Add 100 mL of virus to all wells to be infected (do not tip off culture medium). 

Dilute drug to twice the test concentrations to be assayed in Mewo maintenance medium. 

Add virus in 100 pL of maintenance medium to the growth medium in each well and allow to adsorb for 1 h at 37°C. 

Add 0.75 mL Mewo maintenance medium containing twice test concentrations of drugs, and 0.75 mL Mewo maintanance medium supplmeneted with 0.3% agarose (1/10 dilution of stock agarose). Controls should include a set of wells with dilutions of a known effective drug (typically acyclovir, assayed at 0.2-200 xM). 

Maintenance medium, Minimal Eagles Medium and Liebovitz L15, 1 1, without serum, but with glutamine, penicillin, streptomycin, and fungizone. For virus culture add 0.1% TPCK-treated trypsin (Worthington) at 2 pi./ml.. 

After adsorption, add 4 mL of maintenance medium containing drug at the 50% inhibitory concentration (IC50) to four wells. 

Pick any plaques present on plates one and two using a sterile pipet tip. Place agarose/plaque plug into maintenance medium. 

After adsorption, remove inoculum and add 1 mL of maintenance medium containing drug at approx 2 pg/mL to four wells only. 

Add maintenance medium to the fifth well for the comparative growth of the diluted virus in the absence of drug. 

After overnight incubation, remove growth medium and add 100 pL of maintenance medium to each well. 

To duplicate wells, add log10 dilutions of drug in 1 mL of maintenance medium from 30 to 0.003 pg/mL. Leave one pair of wells without drug for the control. 

Add maintenance medium containing 0.5% fetal calf serum, this maintains the cell integrity better than without the serum. 

Comparable results were reported by Morris (595). It was shown that the standard Gamborg B5 maintenance medium resulted in only low yields of serpentine, whereas no ajmalicine was produced. The cultures were maintained in continuous diffuse light. When the sucrose concentration of the medium was increased to 60 g/liter, a 3-fold increase in serpentine yield was observed. A further increase of the sucrose concentration did not enhance serpentine production. At sucrose concentrations of 100 g/liter, biomass yield decreased. On transfer of the cells from the maintenance medium to the production medium devised by Zenk et al. 20) ajmalicine was accumulated in excess of serpentine, but toward the end of the culture period (22-48 days) ajmalicine levels declined to zero while serpentine continued to accumulate during the entire culture period. 

Growth of cells in microplates secretion of immunoglobulin into maintenance medium... 

Maintenance medium A medium that will support cell survival and limited biological function but not sustained proliferation. 

Neuronal maintenance medium Neurobasal medium (GIBCO, Grand Island, NY), lx B27 (GIBCO, Grand Island, NY), 1 mM glutamine (GIBCO, Grand Island, NY). 

Plate the cells onto a laminin- and poly-D-lysine-coated culture dish at a density of 2 x 10 cells in 2 ml of OptiMEM I/glucose solution. Allow the cells to adhere to the dish for 1-2 h in CO2 incubator (37°, 5% CO2). After 1-2 h, exchange the neuronal plating medium with 3 ml of the neuronal maintenance medium and incubate the plates in CO2 incubator. In case the culture is maintained for more than one week, half of the medium is exchanged with the fresh culture medium every five days after plating. 

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