Measuring Bacterial Colony Height Using Confocal Microscopy

Bacterial colonies were observed under a Zeiss Axioskop phase contrast microscope at a magnification of 400x. Bacterial movement was recorded using a Sony NEX-5N camera. To measure the height of colonies, S. aureus HG001(pRPO-gfp), S. aureus CGL005(pRPO-gfp) and S. aureus CGL007(pRPO-gfp) were inoculated on 25 ml TSA-0.4 plates that contained 1/100th of its volume of 0.5 μm carboxylate-modified FluoSphere beads (Invitrogen, Ex580/Em605) and cultured at 37 °C for indicated time. At each time point, one plate was moved out from the incubator, cover removed, and placed on the stage of an upright microscope for observation. Different plates were used for the indicated time point. A Z stack in which the planes were separated by 10 μm was acquired using an upright laser-scanning confocal microscope (Leica, TCS-SP2). Green fluorescence indicated the distribution of bacteria in the spreading colony. The last plane showing red fluorescence was used to indicate the plate surface. The height of the colony was determined by green fluorescence using the method described elsewhere16 (link). 3-D images of colonies were generated from the Z stacks using Imaris software.