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These days I shoot macro handheld, using the focus and recompose technique. I used AF-one shot. I used a Canon 100mm 2.8 on my Canon 550D. My shutter speed was 1/100s, but it was quite frustrating until I get a sharp image.
What is maybe the reason? The Canon 100m has IS, so I could avoid camera shake. According to Canon, having IS on your lens, you can slow down 2-3 stops. So what is possibly the reason? Is it a possible slight movement caused by the focus and recompose technique?
At macro distances wide apertures are difficult to work with. Even with a steady tripod 2.8 may not be enough depth of field. I tried doing this to get an eyeball shot and discovered that there wasn't enough depth of field for anything to appear in sharp focus.
Doing the focus and recompose, with the very shallow depth of field, will adjust your focus. Focus and recompose works in other situations because there should be enough depth of field that your minor movements keeps the subject in focus, but with the depth of field at macro distances and wide apertures that's not the case.
Also, the focus plane is flatter with that lens than others. Most lenses have a curved plane which, albeit slightly, helps with the focus and recompose technique, but with a flatter plane of focus it's slightly more likely to take your subject out of focus. I don't think it's a significant factor, but it's hurting, not helping.
When determining what an adequate shutter speed is, remember the 1/focal length rule (taking into account the 1.6 crop factor of your camera) and that the effectiveness of IS is advertised and may not be what you experience. If you have shaky hands you may need to adjust the rule to be a little faster. Also, I believe this only works for horizontal or vertical movements. If your DoF is so shallow your forward/backward sway may cause issues.
So, use a DoF calculator to determine the aperture you need which then determines the shutter speed you need, which then tells you if you need a tripod or not (I think most people find they need tripods)
From my experience working with macro at about 1:1 enlargement, it's practically impossible to shoot handheld reliably - what I have to do is to try more than one shot each time, possibly powered by off-camera flash to help freeze movement of the subject, usually in manual focus.
Many rules fall apart when shooting macro: even f/22 is not enough depth of field, 1/focal_length is not enough to hand-hold, autofocus is not reliable, etc.
Focus and recompose is not feasible too, as a movement of, say, a few millimeters in your position moves the focal plane where it isn't intended.
You should realize that when shooting at high subject magnification, the effect of camera shake on image blur is also magnified. Although the lens provides some image stabilization, its effectiveness decreases when shooting near 1:1, to the point where a shutter speed of 1/100s may not be sufficiently fast to avoid blur entirely.
That said, many photographers who shoot at high magnification can successfully do so handheld, provided there is sufficient subject illumination. There are a number of useful tricks that they typically employ: for example, instead of a tripod, they use a monopod or even just a stick against which to lean the camera. This reduces the degrees of freedom of movement and provides just enough stability, while not sacrificing positional flexibility and responsiveness that using a tripod would.
For many types of popular macro subjects, the subject itself is not perfectly motionless: as a result, a tripod and image stabilization are not very useful. Rather, the solution is to use a strobe (i.e., flash). Use the flash to freeze subject and camera movement: this has the benefit of permitting slower f-numbers, increasing depth of field and making critical focus less difficult. Then the real trick is to get the flash source properly positioned for your subject and lens, and to use modifiers to achieve the desired lighting effects. The resourcefulness and variety exhibited in the numerous contraptions that have been devised are almost as diverse as the subjects these photographers shoot.
