Proper culture conditions are critical for optimal CFU growth in MethoCult™ media. Culture dishes should be incubated at 37°C, in 5% CO2, with ≥ 95% humidity. Use of water-jacketed incubators with water pan in chamber and routine monitoring of temperature and CO2 levels is recommended.

Maintaining the proper level of humidity in the cultures is critical for supporting optimal colony growth. Without the appropriate levels of humidity, CFUs may not develop properly and counts may be underestimated.  This is especially important for erythroid colony development.  

Dehydrated cultures may appear thinner than usual, and can appear speckled or cracked when viewed under the microscope.

Optimal humidity is particularly important when using the STEMvision™ instrument for imaging and scoring of colonies. In addition to sub-optimal colony growth, if cultures are dehydrated, the images taken by STEMvision™ will show a gradient or darkening on one side of the well, which can interfere with colony enumeration and identification.

In order to prevent dehydration, please follow the recommendations below, for CFU assays plated in 35 mm Culture Dishes (Catalog #27100/27150) or SmartDish 6-well Plates (Catalog #27370/27371).

35 mm Culture Dishes:

• After plating 1.1 mL of the MethoCult™ medium and cell mixture per 35 mm Culture Dish, place two of these 35 mm culture dishes inside a larger 100 mm Culture Dish (Catalog #100-0082) with a lid.
• Add approximately 3-4 mL of sterile water to an additional uncovered 35 mm dish and place this inside the 100 mm dish as an open water dish to maintain humidity.
• Alternatively, 35 mm culture dishes can be placed inside a 245mm x 245mm Square Treated Tissue Culture Dish (Catalog #38039/100-0084), or other cultureware of an appropriate size with a loose-fitting lid.  Ensure that a sufficient number of open water dishes are placed inside the outer dish (i.e. 4 water dishes inside the 245 x 245mm square dish) and that the 35mm culture dishes are equidistant from the water dishes.

SmartDish™ 6-well Plates:

• After plating  1.1 mL of the MethoCult™ medium and cell mixture per well, and distributing evenly across the surface, add 4 mL of sterile water to the inter-well space of the SmartDish™ (2 mL per space, as shown in blue below).  

• Place SmartDish™ plates inside a larger tray, such as a 245mm x 245mm Square Treated Tissue Culture Dish (Catalog #38039/100-0084).
• It is recommended that at least four additional uncovered 35 mm Culture Dishes, each containing 3 ­ 4 mL of sterile water, are placed around the SmartDish™ 6-well plates.