Preparation of Materials for Making a Plate Count
The following is for making ONE plate count. ..Your experiment will probably require making many plate counts. ..Thus merely multiply this "recipe" by the number of plate counts you need to make (and maybe add a few more in case something is spilled or otherwise "messed up").
- Put a tip onto a micropipetter and make sure that this can reach to the bottom of the small test-tubes you are about to use.
- Make up 100 ml of Normal Saline ("NS" = 0.15M or 0.9% NaCl). ..(Percent solutions are a handy notation: 0.9% means "0.9 per hundred". ..So add 0.9 grams of NaCl solid to a container and add sufficient water to make 100 ml. ..(Do NOT put the salt into a graduated cylinder and then water. ..Often the solid cements itself to the bottom of cylinders and is very hard to get out or up into solution. ..Add 0.9 grams of salt to a beaker, add about 50 ml of water, then mix to dissolve. ..THEN add to a graduated cylinder and add water up to 100 ml.) It has been found that for counting E.coli and Bacillus spp, NS is not needed. Use just plain water.
- Make sure that you have some sterile petri plates.
- Put 900 microliters (900 μL) of saline into each of 7 small test-tubes, which are set in a metal rack.
- Cover the tubes with aluminum foil.
- Put 10 pipetter tips into a "tip box".
- Make up 50 ml of the appropriate agar in a flask that is covered by a small inverted beaker.
- Autoclave the covered tubes, the tip box and the flask for 15 minutes.
- Take out and allow the tubes to cool, while the "tip box" is set in a warm oven (60c) with its top slightly ascew so that extra moisture can evaporate. ..Pour the agar into two empty but sterile petri plates. ..If the agar surface has any bubbles, waft a flame from a burner over the surface. ..You will see that the bubbles burst! ..Allow the agar to solidify.
- NO condensation may be on the surface of the agar. NEAT TRICK:
- Find a flat place that is free from drafts.
- Unroll a couple of feet of fresh paper towel (these are usually sterile inside of the roll), and lay the paper towels on the flat surface.
- Lay a pencil near one end of the paper.
- Open a plate and lay the agar-fill bottom such that the agar surface is facing downwards. ..The lip of the plate rests on the pencil, and lay the top down so that its lip rests on the bottom of the plate.
- After about an hour, the agar surfaces should be free of condensation droplets.
- Put the plates back together again.
- Appropriately label the BOTTOMS of the plates. ..(Do NOT write in the middle of the bottoms. ..Write small around the edge. ..This will not obscure your later "reading" the plates.
- Do not remove the aluminum foil, but look under it to check the tubes, and disgard any that are not filled or are overfilled from what you started with. ..You are now ready to do the serial dilution.
- Put a tip onto your 100 μL pipetter. ..(Do NOT touch the pointed end with your non-sterile fingers unless you have thoroughly flamed your fingers!)
- Immediately, remove 100 μL from the source container. (To use a micropipetter: ..Many pipetters have two "stops" when pressing down the button on top. ..The distance between the up position and the first stop is the 100 μL, and the second stop is used to blow out the contents (from top to second stop). ..To obtain your 100 μL, first push the button to the first stop, and THEN put the tip into the liquid. ..Slowly release the button, and the tip will fill to the correct amount. ..Failure to first push the button down before going into the liquid will sometimes result in a bubble's being expelled from the tip and the bubble's remaining on the point to be sucked right back in giving a smaller than wanted volume's being transferred.)
- Now squirt the contents in the tip into the first of the several serial dilution tubes you made.
- DISGARD the tip! ..If you don't do this, go home! You have wasted the day
- Put on a new tip and transfer from tube #1 to tube #2.
- DISGARD the tip!
- Continue transferring until you have put 100 Ál into the last tube. ..You would then be ready to put these dilutions onto a petri plate.
- Put a tip onto the 10 μL pipetter.
- Now imagine that you are going to put FIVE droplets onto the plate in this fashion:
- Here is a trick that will help you in subsequent days. You are wasting your time transferring from tubes will give you unproductive TMTC's. Which will then be the productive tubes? See if you can figure this out: IF your original is visibly turbid, you know it must have more than a million cfu/ml. Will tube #1 yield TMTC? What about #2? And so on.
- Transfer liquid from the most dilute tube onto to place where the fifth droplet should go. ..Be careful to not press the tip against the agar. ..If a dent is formed, you will not be able later to spread the droplet out eveningly as some will remain in the dent's micro-puddle.
- Keep the plate horizontal until further notice!
- Next transfer 10 μL from the next more concentrated tube to where the 4th droplet is to be placed. ..Note, so long as you are moving UP the concentrations you do not need to change pipetter tips!
- Keep doing this until you have moved UP a total of five tubes.
- Take a flame sterilized inoculation loop, and
- jab the hot loop into the agar at the edge of the plate to cool it.
- spread the most dilute drop into a narrow wet line on the plate.
- Do the same spreading of the remaining droplets. Always move from low to higher concentrations. ..NEVER go back to a lower one (or return to GO and do not collect $200). ..If you go from low to high, again you do not have to resterilize your loop between spreadings.
- The horizontal plate is covered and occasionally checked for the disappearance of the moisture of the droplets.
- Once all moisture is gone, turn the plates upside down a place them somewhere to incubate (on your desk, in a cabinet, or, if you are fortunate, in a heated incubator).
Now return to the main serial dilution page.