A refresher class on streaking for single colonies

I think we might benefit from an explanation of how to streak bacterial cells on an agar plate to get single colonies.

The problem:  You want to begin your experiment with cells from a single colony so you know they're a genetically pure clone.  You have a source of bacterial cells whose density you don't know (an overnight culture, a colony or patch of cells on an agar surface, a frozen stock), and you want to spread them out on a fresh agar plate so some of them will grow up to be well-separated individual colonies.

The solution:  You do what's really a solid-phase, non-quantitative serial dilution.  You start with an agar plate and a quantity of the cells on a sterile inoculating loop or the tip of a glass pipette.  It really doesn't matter how many cells you start with - anything between a whole colony and a drop of dilute culture will work fine.  You spread these cells onto a small area of the plate; the objective is not to spread out all the cells; just some.   You then pick up some of the spread cells by passing the resterilized loop or fresh pipette through this area, and then wipe some of these cells onto a fresh area of the plate by streaking the loop in a zigzag.  Again the objective is to transfer a small fraction of the cells (less than 1%) onto the new area.  You repeat this once or twice more.  

The grey lines in the drawing show the path of each pass, and the blue is the cells after overnight incubation.  It doesn't really matter how dense the cells originally were; if the transferred fractions are small enough, the third pass will spread out only a few cells, and these will grow into well-separated colonies overnight.  If the cells were not very dense originally, the third pass may not give any colonies, but the second pass will give well-separated colonies.

A common error:  Beginners often forget that the point is to drastically decrease the number of cells being spread in each pass.  In the photo above we see that the overly meticulous researcher has done seven (!) sequential passes and still wound up with many crowded colonies in the last streak area.  The problem is that each pass picked up a large fraction of the cells spread by the previous pass - you can see that there's very little decrease in colony density from one pass to the next.  The reason each pass picked up so many cells is that each 'zig' of the zig-zag went back into the previous pass, picking up more cells each time.

Later:  here's an example of a good streak.  Note that this and the above are both done on small (60 mm) Petri dishes rather than on the standard 90 mm ones.

A norte on what tool to use for streaking:  I use sterile 0.1 ml glass pipettes, rotating 180° after the first pass and then using the still-sterile other side.  (We got lots of these free because of a supplier error.) As The Lorax points out in the Comments below, you can do the same thing with skinny wooden dowels (like long cylindrical toothpicks), but these and toothpicks have rough surfaces and don't slide as smoothly over the agar surface.  Disposable plastic loops work fine, but you can go through a lot if you're doing serious genetics.  The best non-disposable loops are made of platinum - these are quite expensive ($50?) but retain their smooth surfaces through many thousand cycles of flame-sterilization.  The cheap steel loops become very rough after even a few flamings.  As a grad student I developed a custom handle for my platinum loop to get exactly the right degree of flexibility and balance of weight (yes, I did a lot of streaking then).