1. Sterilize velvet squares with widths at least 50% longer than the diameter of a Petrii plate. - Streak strains to be printed for single colonies. - Prepare a printing master plate. Place a grid sheet under the base of a sterile plate. Typically, the grids contain 24 or 50 squres, but grid between 9 and 121 squares have been used. Use a grid system of the appropriate size for the number of strains to be printed. - Pick a single colony of each strain and hatch an 'X' shape in the middle of a grid square. Note which grid contains what strain. Make 'X' shapes rather small since they tend to get larger on subsequently printed plates. Include control strains on the last few grid-squares. - Grow the master plate overnight. - Prepare media for printing. Each plate should be dried so that the velvet is not excessively wet after printing. - Place a sterile velvet face up on a print column and fix the sides with a collar. Print the master plate onto the velvet surface pressing lightly but evenly to transfer all patches to the velvet. - Remove the master plate and immediately cover the velvet with the first replica plate. Note the orientations of all plate so that the layout of the strains is known. Transfer cells to the replica plate by pressing lightly and evenly against the velvet. - Repeat for all replicas. Two to six replicas can easily be made from a single master. Up to fifteen replicas have been successfully made without too much patch-spreading. - To reduce cell-debris transfer, print to a minimal-media replica first, then discard it. To vastly reduce cell-debris transfer, make a minimal media replicate, then immediately use that replicate as a master. Print the new master to a fresh velvet and print as usual. - Always print last to a plate on which all strains will grow, like LB. This ensures that all strains were, in fact, transfered to the velvet. - To avoid nutrient carry-over, print to nutrient-deficient media first, then print to nutrient-rich media. This will prevent contamination of the velvet with trace nutrients. [](http://dx.doi.org/10.5281/zenodo.13551)