MIC-02Foundation

Media Preparation & Sterilisation

Scenario brief

A batch of plate count agar is needed for tomorrow's testing. Weigh, dissolve, sterilise and pour it, then check the batch before it is released for use.

About 9 min
8 parts

What you'll do

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
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
Collect your kit8 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
- • Plate count agar powder
- • Balance
- • Purified water
- • Media bottle
- • pH meter
- • Petri dishes
- • Marker pen
- • Reference strain
What you decide by
- • Take only what the test uses: anything extra on the bench is clutter to reach over.
3
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
- • pH meter
- • Autoclave
- • Incubator
- • 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
4
Make up and sterilise9 steps · 1 by hand
Turn the powder into sterile, molten agar.
The method for this part
1. 1
  Check the powder's label and expiry
  Read the label on the agar powder: plate count agar, in date, dry and free-flowing. Note the grams per litre.
  Each maker's recipe is different, and old or damp powder makes a medium that won't perform.
2. 2
  Weigh the powder for the batchDone by hand, to 6.8–7.2 g
  The label says 17.5 g per litre, so 400 mL needs 7.0 g. Weigh the powder onto the balance: for this exercise, within 0.2 g is accepted.
  Too little powder and the gel is soft; too much and it is not the recipe on the label.
3. 3
  Suspend the powder in the water
  Measure 400 mL of purified water into the media bottle, add the weighed powder and swirl until none is left dry.
  Purified water is the same every time; tap water's salts and chlorine vary. A bottle with room to spare leaves space for the froth.
4. 4
  Heat and stir until it boils clear
  Stand the media bottle on the stirrer hotplate and heat it until it just boils and the liquid is clear, with no specks.
  Agar only dissolves at the boil: any left undissolved gives a weak, lumpy gel.
5. 5
  Check the pH of a cooled sample
  Pour off a small sample, cool it to 25 °C and read it on the pH meter. For this exercise it should already be within the final limit of 7.0 ± 0.2.
  A pH far from its limit points to the wrong powder, the wrong water or a weighing mistake, while there is still time to start again.
6. 6
  Load the bottle into the autoclave
  Loosen the media bottle's cap half a turn, stand the bottle in a tray inside the autoclave and close the door.
  A sealed bottle can't vent and may burst. The tray catches a boil-over before it reaches the chamber drain.
7. 7
  Autoclave at 121 °C for 15 minutesHolding at 121 °C: 900–1200 s
  Start the autoclave's liquid cycle. Stop the clock once the load has held 121 °C for 15 minutes and before 20: that is 900 to 1200 s.
  Fifteen minutes at 121 °C is the hold proved to kill bacterial spores. Much longer overheats the medium and weakens the gel.
8. 8
  Open once the pressure reads zero
  Open the autoclave door only when its pressure gauge reads zero and the load has cooled well below 100 °C.
  Liquid under pressure is hotter than its boiling point: opened early, it boils over violently. The load is still hot, so it is lifted out in heat-proof gloves.
9. 9
  Cool the bottle in the water bath
  Move the hot media bottle to the 47 °C water bath and leave it to come down to 45–50 °C.
  At 45–50 °C the agar is still liquid, but cool enough to pour without flooding the lids with condensation.
What goes wrong here
- Fill the bottle to the top to make more. Agar froths as it boils: with no headspace it boils over on the hotplate and in the autoclave.
- Screw the cap tight to keep it sterile. A sealed bottle can't vent, and can burst: caps are always left loose in an autoclave.
- Open the door when the timer ends. The agar is still above its boiling point: let the pressure go and it erupts, and the steam scalds.
5
Pour the plates8 steps · 1 by hand
Pour the plates cleanly, then set up the checks that prove the batch.
The method for this part
1. 1
  Check the bath reads 45–50 °C
  Read the water bath's display: it should show 45–50 °C. Give the bottle time to come down to it.
  The bottle only cools as far as the bath: if the bath is wrong, so is the agar.
2. 2
  Swirl the bottle gently to mix
  Swirl the bottle of molten agar slowly, without lifting the agar into froth.
  Agar settles in the autoclave: unmixed, some plates set soft and others hard.
3. 3
  Pour 15–20 mL into each dishDone by hand, to 15–20 mL
  Lift the lid of each Petri dish just enough, pour in 15–20 mL of molten agar so that the base is covered, and close it.
  A plate poured too thin dries out in the incubator; one poured too thick wastes the batch.
4. 4
  Leave them level to set with lids on
  Leave the Petri dishes flat and closed until the agar is firm.
  Moved early, the agar sets with a slope; left open, it catches whatever falls from the air.
5. 5
  Label with batch and expiry
  With the marker pen, write the medium, the batch number, the date made and the expiry date on the base of each plate.
  A plate that can't be traced to its batch record can't be shown to have passed its checks.
6. 6
  Incubate a few plates uninoculated
  Put a few of the poured dishes into the 30 °C incubator with nothing on them: this is the sterility check.
  Plates with nothing put on them should grow nothing: if they do, the batch is not sterile.
7. 7
  Inoculate plates for a growth check
  Spread a known, low number of the reference strain on plates from this batch and from the last released batch.
  Sterile is not enough: the batch must also grow what it should, as well as the last one did.
8. 8
  Bag the rest and hold at 2–8 °C
  Bag the rest of the dishes base-up, mark them as on hold and put them in the media fridge until the checks are read.
  Cold, sealed plates don't dry out, and a batch on hold can't be used before it is proved.
What goes wrong here
- Pour hot before the agar can set. Agar stays liquid down to about 40 °C, so there is time. Poured near boiling, it throws heavy condensation onto the lids, which runs back over the surface.
- Shake the bottle to mix it well. Shaking fills the agar with bubbles, which set in the surface and can be mistaken for colonies.
- Leave the lids off so they set faster. Open plates collect bacteria and mould spores from the air while they set.
6
Release or reject3 batches to judge
The checks have been incubated. Decide what happens to each batch.
The method for this part
What you check
- • Look at the plates
- • Read the sterility check
- • Read the growth check
- • Read the batch record
What you decide by
- • Sterility check: nothing may grow on the uninoculated plates.
- • Growth check: the count must be at least 70% of that on the last released batch.
- • Final pH for plate count agar: 7.0 ± 0.2 at 25 °C.
- • The record must show the label's recipe, and 121 °C held for 15 minutes.
- • Water on the agar is put right by drying the plates. A fault in the gel is not.
- • For this exercise, a batch more than a fifth out on its powder, or poured thinner than 15 mL a plate, is rejected.
What you can decide
Release · Dry, then release · Reject
7
Check your reasoning3 questions
Three questions on the reasoning.
8
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

Read the label first: the grams per litre differ between makers, and caked or out-of-date powder is not used.
Dissolve the agar completely at the boil, and check the pH: 7.0 ± 0.2 for plate count agar.
Autoclave at 121 °C for 15 minutes with the cap loose, and never open the door until the pressure has fallen.
Cool to 45–50 °C before pouring 15–20 mL into each 90 mm dish: hotter agar floods the lids with condensation.
Label every plate with its batch and expiry, and hold the batch until its sterility and growth checks have passed.

Equipment unlocked

Autoclave
Water bath
Top-pan balance