Appendix 1.1 Problems and Solutions

The matter given below lists some potential problems and offers possible solutions that may help you troubleshoot your cell culture experiments. Note that the list given below includes only the most commonly encountered problems in cell culture and provides guidelines to solutions only.

Appendix 1.2 Equipment for Cell Culture

• • Laboratory refrigerator/freezer combination. Separation of refrigerator from the freezer unit for tissue culture media and ingredients is recommended to minimize contamination.

• • Two-stage vacuum trap for aspiration. Construct an aspirator for the culture hood using two Buchner or Erlenmeyer flasks, two 1-hole or 2-hole rubber stoppers, glass Pasteur pipettes or glass tubes, and Tygon® tubing. Use a 1000 ml or larger flask for the first stage trap. Connect the second stage to a vacuum supply or pump through a vacuum filter to protect the pump from residual water vapor.

• • Stainless steel pipette sterilization boxes. Obtain at least two sterilization boxes and keep one autoclaved box of pipettes in the culture hood at all times (suggested model: Fisher scientific 03-475-5 rectangular box for 9 inch Pasteur pipettes).

• • Set of pipettes for flow hood. Keep a separate set of autoclavable 2–20 μl, 20–200 μl, and 100–1000 μl pipettes to be used only for cell culture. Autoclave the pipettes according to the manufacturer's instructions. Autoclave repeatedly every one to two months or in case of contamination.

• • Tissue culture microscope. An inverted, phase contrast microscope with 10x, 20x, and 40x phase-contrast objectives and a long working distance condenser should be used for observing the extent of cell extraction, for counting cells, and for cell maintenance.

• • Clinical centrifuge. A fixed-angle or swinging bucket centrifuge for 15 ml conical tubes with a maximum speed of 1,300 g can be used for pelleting nematodes and floating eggs on sucrose (suggested model: Fisher scientific 228 benchtop centrifuge).