Plating a suspension

The purpose of this procedure is to obtain well separated yeast colonies on a surface of solid media, starting from a yeast suspension in a test tube, for later selection.
The suspension could be a master storage in distilled water, slurry from a previous batch, dregs from a commercial bottle, etc.

Since each colony on the solid media in most cases originate from a single yeast cell, to get well separated colonies we need to spread and dilute the suspension as much as possible. Our goal is to inoculate the solid media with less that 50 yeast cells.
To obtain such a low number, we can dilute the original suspension (possibly 1:100) or we can streak 2 plates in a row. Results are similar, but I prefer the 2 plates method.

As an alternative, you can perform a single streak with a sterile cotton swab. With the loop you deposit a drop of suspension on the media, then you spread it with the cotton swab.

The usual precautions apply: scrub the table surface, the test tube and your hands with alcohol. Wear a mask, keep your work area as dust-free as you can, work in flame zone.
Since you need to work as quickly as possible, you should practice with empty test tubes and Petri dishes ahead of time, to gain the necessary dexterity.

Place on the table 2 Petri dishes upside down (media on top, cover on bottom), test tube with suspension (standing in its rack), inoculation loop.
When ready, proceed as follows:

  • Hold inoculation loop with right hand
  • Flame loop from tip to handle until it's red hot, then keep it in the flame zone
  • While the loop is cooling near the flame, take test tube with left hand
  • Grab tube cap with right hand little finger, unscrew by turning tube with left hand. Hold cap between the little finger and the palm of your right hand
  • Flame tube mouth
  • Working in the flame zone, dip loop in the solution trying not to touch the glass walls
  • Remove the loop and keep in flame zone. Be extra careful not to heat the yeast on the tip
  • Flame the tube mouth again and place cap back on, again using left hand to screw it
  • Put away tube on rack
  • With your left hand, lift the media half of the first Petri dish and hold it vertical ( to minimize the chance of dust falling on the agar), leaving the cover on the table
  • Streak the loop on 1/2 of the media surface, in a zig-zag pattern, being careful not to scar the surface
  • Turn the plate 60° clockwise and repeat streaking. Do not flame the loop between streaks
  • Turn the plate 60° clockwise once more for final streaking
  • Put plate back on its cover. From this moment on, Petri dishes must be kept upside down (media on top, cover on bottom) at all times
  • Without reinoculating, repeat the above steps with the second Petri dish
  • Flame inoculation loop and put it away
  • Incubate plates in a warm (>27 °C) clean and dry place

Depending on the concentration of yeast cells in the original suspension, yeast colonies on the first dish will probably grow too close to each other. However on the second dish the number of cells carried over on the loop should be minimal, and colonies should grow well separated and healthy.

petri1 petri2
First plate Second plate

The rule is that if you can see yeast on the inoculation loop, then there is too much of it: after all, a yeast cell size is less than 10 µm.
It may seem that nothing remains on the loop after streaking the first plate, but there will be a few cells anyway, and this is exactly what we want.

After a few days (depending on the viability of original cells) you will have large, well separated colonies on the second plate, suitable for further selection or storage.

Should mold or bacteria colonies be present, I suggest you discard the plate and repeat the procedure.