By Derek Kinchington, Raymond F. Schinazi

ISBN-10: 0896035611

ISBN-13: 9780896035614

Royal London college of drugs, united kingdom. moment writer, Raymond F. Schinazi, is at Emory Univ., Decatur, GA. bargains simple and scientific researchers versions to guage compounds potent opposed to acute and protracted infections. comprises entire assays, caliber checking out, and unpublished equipment. DNLM: Hepatitis, Viral, Human - drug treatment.

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Pool aliquots as necessary (typically 1–2 L) and centrifuge at 7000g, 10 min to remove cellular debris. Add the supernatant to an Amicon “stirred cell” (8400 series) fitted with a YM100 membrane (cat. no. 13642) 400 mL at a time. 54 Schmidt and Korba 54. 55. 56. 57. 58. 59. 60. 61. 62. 63. 64. 65. 66. 67. 68. 69. 70. 71. 72. 73. 74. 75. 76. 77. Filter at approx 20 psi nitrogen with constant stirring, at 4°C (approx 3–5 h), ensuring that the membrane is not allowed to dry. This will result in approx a 40fold concentration by volume.

Pipet the stain to a waste bottle. 11. Add one drop of mountant to a labeled slide and place the coverslip cell side down onto the appropriate slide. 12. Examine the slide at ×100 magnification with oil immersion under UV epifluorescence. Cell nuclei will fluoresce. In mycoplasma-negative cultures, the nuclei will be seen against a dark background. In mycoplasma-positive cultures, the cell nuclei will be seen among fluorescing thread-like or coccal structures (Fig. 1). In an alternative system, cells of the test culture can be inoculated onto coverslips preinoculated with an indicator cell line, such as the Vero African Green Monkey cell line.

However, in the case of irreplaceable stocks this may not be practical. 1. Elimination of Contamination 1. Culture cells in the presence of the chosen antibiotic(s) for a period of 10–14 d, during which time most cultures will be passaged approx 4 times. Each passage should be performed at the highest dilution of antibiotic that the cell will tolerate following the manufacturer’s guidelines. 2. Test the culture for the presence of mycoplasma by a Hoechst stain. If mycoplasma is still detectable, it is unlikely that this antibiotic will be successful and an alternative should be tried on a fresh batch of cells.

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