Serial Dilution & Total Plate Count

This is the procedure you'll follow at the bench. Open a part to read it step by step, with the reason for each step, before you start.

1. 1

   Gown up5 steps, in order

   Choose the right protection for the work and put it on in the right order.

   The method for this part

   1. 1

      Tie hair back, remove jewellery

      First, before your hands are washed: you won't touch your head again once they're clean.

   2. 2

      Lab coat, fastened

      The coat goes on before you wash, so clean hands don't have to handle it, and before gloves, so the gloves can cover its cuffs.

   3. 3

      Wash your hands

      Hands are washed once your hair and the coat are dealt with, and before you touch clean eye protection and gloves. With rings and watch off, the whole hand and wrist can be reached.

   4. 4

      Safety glasses

      Eye protection goes on before gloves, with freshly washed hands.

   5. 5

      Nitrile gloves

      Gloves go on last, over the cuffs and onto clean, dry hands, so they're clean when you start work.

   What goes wrong here

   • Respirator. Routine bench work doesn't need a respirator. Wearing more than the risk calls for just makes the work clumsier.
   • Plastic kitchen apron. A kitchen apron leaves your arms bare. In a lab you need a fastened coat with sleeves.
   • Your own jacket. Outdoor clothes stay in the locker: they carry contamination in and out.
2. 2

   Collect your kit12 things to fetch

   Fetch what the test needs from the stores around the lab, or have it brought to you.

   The method for this part

   What you check

   • • Marker pen
   • • 10⁻² tube, 9 mL
   • • 10⁻³ tube, 9 mL
   • • Dish 10⁻¹
   • • Dishes 10⁻² (A, B)
   • • Dish 10⁻³
   • • Food sample
   • • Balance
   • • Sample in diluent, 10⁻¹
   • • Pipette
   • • Vortex mixer
   • • Molten agar, 45 °C

   What you decide by

   • • Take only what the test uses: anything extra on the bench is clutter to reach over.
3. 3

   Receive samples4 samples to judge

   Check every delivery at the hatch before it's logged in. Accept what is fit to test; reject what isn't.

   The method for this part

   What you check

   • • Check the container and seal
   • • Take its temperature
   • • Match it to the request form
   • • Check when it was sampled

   What you decide by

   • • Container closed and intact, with its seal unbroken.
   • • Chilled microbiology samples: 0–8 °C on arrival.
   • • The label must match the request form: what it is, where from, and what it's in for.
   • • Microbiology must start within 24 hours of sampling.
   • • An accepted sample is given a laboratory number, which goes on everything made from it.
   • • A rejected sample is never tested 'just to see': its result could not be trusted.

   What you can decide

   Accept: log it in · Reject: record why, tell the sender

4. 4

   Pre-use checks4 instruments to check

   Before you rely on anything, read its label and its record. Today's date matters.

   The method for this part

   What you check

   • • Balance
   • • Pipette
   • • Incubator
   • • Molten agar, 45 °C
   • • Read its label, for each
   • • Read its record, for each

   What you decide by

   • • Equipment that needs calibrating carries a label showing when it was done and when it is next due. Check 'Next due' against today's date.
   • • Each record form prints its own acceptance limit. A reading outside it means the equipment is not fit for use.
   • • Culture media are released batch by batch, after a sterility check and a growth check with a control strain.
   • • Anything out of date or out of limits is taken out of service: labelled 'Do not use', reported, and not used until it is put right.
   • • The limits and intervals here are set for this exercise. In your own laboratory, use the ones in its procedures.

   What you can decide

   Fit for use · Do not use: label it and report

5. 5

   Dilute and plate9 steps · 3 by hand

   Make the dilution series and pour the plates.

   The method for this part

   1. 1

      Label tubes and plates

      Label every tube and dish with the sample and its dilution.

      Unlabelled plates can't be traced back to a dilution.

   2. 2

      Weigh 25 g into 225 mL diluentDone by hand, to 24.5–25.5 g

      Weigh 25 g of sample aseptically into 225 mL of diluent.

      25 g in 225 mL is a one-in-ten dilution: 10⁻¹.

   3. 3

      Homogenise the sampleBlending: 50–75 s

      Run the paddle blender, then stop it at about a minute.

      Bacteria inside the food must be shaken out into the diluent.

   4. 4

      1 mL into 9 mL: 10⁻²

      With a fresh tip, move 1 mL of the 10⁻¹ into a 9 mL tube.

      Each 1 mL into 9 mL is ten times weaker.

   5. 5

      Mix the 10⁻² tube

      Vortex the tube before taking from it.

      Unmixed, the cells sit where the pipette left them.

   6. 6

      Fresh tip, 1 mL into 9 mL: 10⁻³

      Change the tip, then move 1 mL of the 10⁻² into the next tube and mix.

      A fresh tip stops cells being carried down the series.

   7. 7

      Pipette 1 mL of each dilution into its dishDone by hand, to 0.95–1.05 mL

      Put 1 mL from each dilution into its labelled dish.

      Plating several dilutions makes sure one lands in the countable range.

   8. 8

      Pour agar cooled to 45 °C and swirlDone by hand, to 15–20 mL

      Pour 15–20 mL of molten agar from the 45 °C bath and swirl gently.

      At 45 °C the agar is still liquid but won't kill the cells.

   9. 9

      Let set, invert and incubate

      Once set, turn the plates upside down and incubate at 30 °C for 72 hours.

      Inverted plates keep condensation off the agar.

   What goes wrong here

   • Use the same tip all the way down. Cells carried over on the tip make every later dilution count too high.
   • Pour agar straight from boiling. Boiling agar kills the bacteria, so the count comes out falsely low.
   • Incubate the plates lid-up. Condensation drips onto the agar and smears colonies together.
6. 6

   Shared incubator4 stacks to judge

   The incubator is shared. Find the plates with your sample's number, and take only those.

   The method for this part

   What you check

   • • Read its label

   What you decide by

   • • Everything in an incubator is labelled with its sample's laboratory number, the test, the date it went in and who set it up.
   • • Read the whole number. Two samples from the same day can differ by one digit.
   • • Other people's plates stay exactly where they are, and the door is closed again quickly so the temperature holds.

   What you can decide

   Take: these are yours · Leave: not yours

7. 7

   Read the plates4 plates to judge

   After incubation, decide what to do with each plate.

   The method for this part

   What you check

   • • Count the colonies

   What you decide by

   • • Countable range for this method: 30 to 300 colonies.
   • • Reject plates with spreading colonies over more than half the agar.

   What you can decide

   Count it · Too few · Too many · Reject the plate

8. 8

   Report the count2 questions

   Work out the result.

9. 9

   Clear down5 items to judge

   Put everything you used where it belongs. Nothing live leaves the laboratory untreated.

   The method for this part

   What you decide by

   • • Anything that has held culture or sample is autoclaved before it leaves the laboratory.
   • • Anything that could pierce a bag goes into a rigid container.
   • • What is left of a sample is kept, chilled and numbered, until its result is released.
   • • Only what never touched the work is ordinary waste.

   What you can decide

   Biohazard bag: for the autoclave · Rigid sharps container · Retained samples fridge · Ordinary waste

10. 10

    De-gown4 steps, in order

    Take it off in the right order, most contaminated first, and wash your hands last.

    The method for this part

    1. 1

       Take off: nitrile gloves

       Gloves are the most contaminated thing you're wearing: they come off first, peeled inside out.

    2. 2

       Take off: safety glasses

       Glasses come off by the arms once the gloves are off, so you don't touch your face with dirty gloves.

    3. 3

       Take off: lab coat, fastened

       The coat comes off after the gloves and glasses, and stays in the lab on its hook.

    4. 4

       Wash your hands

       Hands are washed last, after everything that might have contaminated them is off, and before you touch the door.

What you'll take away

• 25 g of sample in 225 mL of diluent is the 10⁻¹ dilution; each 1 mL into 9 mL is ten times weaker.
• Use a fresh pipette tip for every transfer, and mix each tube before taking from it.
• Pour agar cooled to about 45 °C: hotter agar kills the cells you are trying to count.
• Only count plates in the countable range; too few is unreliable, too many is crowded.
• CFU/g = colonies ÷ volume plated × the dilution factor.

Equipment unlocked

• Micropipettes
• Diluent bottles
• Colony counter