Antimicrobial Susceptibility Testing

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 kit10 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

   • • 0.5 McFarland standard
   • • Densitometer
   • • Plastic loops
   • • Culture plate
   • • Saline tubes
   • • Vortex mixer
   • • Cotton swabs
   • • Mueller-Hinton plates
   • • E. coli ATCC 25922
   • • Antibiotic discs

   What you decide by

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

   Pre-use checks2 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

   • • Incubator
   • • Mueller-Hinton plates
   • • 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. 4

   Standardise the inoculum5 steps · 1 by hand

   Make a suspension of the isolate at 0.5 McFarland.

   The method for this part

   1. 1

      Check the densitometer

      Put the 0.5 McFarland standard in the densitometer: it should read 0.5.

      A meter that misreads the standard sets every suspension wrongly.

   2. 2

      Touch 3 to 5 similar colonies

      With a plastic loop, touch the tops of 3 to 5 well-separated colonies that look alike on the culture plate.

      Several colonies stand for the whole culture, where one alone might be an unusual variant.

   3. 3

      Rub the growth off into saline

      Rub the loop on the inside of a saline tube, just below the surface, until the growth comes off.

      Saline carries the cells without feeding them, so their number stays as you set it.

   4. 4

      Vortex the suspension

      Hold the saline tube on the vortex mixer for a few seconds, until no clumps are left.

      Clumps give a false turbidity reading and a blotchy lawn.

   5. 5

      Adjust the density on the meterDone by hand, to 0.45–0.55 McFarland

      Stand the tube in the densitometer and add growth a little at a time until it reads 0.5 McFarland. For this exercise 0.45 to 0.55 is accepted.

      The breakpoints only hold for a lawn grown from a 0.5 McFarland suspension.

   What goes wrong here

   • Pick one large colony. One colony can be an unusual variant, so its result may not hold for the rest of the culture.
   • Judge the turbidity by eye alone. With no standard beside it and no meter, the density is a guess, and zone sizes follow the density.
5. 5

   Set up the plates7 steps · 1 by hand

   Swab the plates, apply the discs and incubate, keeping each stage within 15 minutes.

   The method for this part

   1. 1

      Dip a swab and press out the excess

      Within 15 minutes of adjusting the suspension, dip a cotton swab in the tube, then press and turn it against the inside.

      A dripping swab floods the agar and makes the lawn too heavy.

   2. 2

      Swab the plate for an even lawnDone by hand, to the whole square, in both directions

      For this exercise swab the whole surface two ways: side to side, then up and down. At the bench it is three ways, turning the plate 60° each time, then once round the rim.

      Strokes that cross leave an even lawn, so the zones come out round with sharp edges.

   3. 3

      Swab a plate with the control strain

      From its own 0.5 McFarland suspension, swab a second plate with the control strain, Escherichia coli ATCC 25922.

      If the control strain's zones are in range, the agar, the discs and your technique are all performing.

   4. 4

      Let the surface dry with the lid ajarDrying: 180–900 s

      Leave the lids of the plates ajar for 3 to 5 minutes so the moisture soaks in, and never longer than 15.

      On a wet surface the discs slide, and after 15 minutes the bacteria have a head start that shrinks the zones.

   5. 5

      Apply the six discs well apart

      Place the six antibiotic discs on each plate, well apart and clear of the edge, and press each one flat. Six is the most a 90 mm plate takes.

      Discs too close give zones that overlap, and a disc in poor contact gives a lopsided zone or falls off when the plate is turned over.

   6. 6

      Invert the plates and incubate

      Within 15 minutes of applying the discs, turn the plates agar side up and put them in the incubator: 35 °C, in air, for 16 to 20 hours.

      Left on the bench, the antibiotics spread before the bacteria grow and the zones come out too large.

   7. 7

      Discard the swabs and suspensions

      Put the used swabs and suspension tubes into the discard pot.

      They carry live culture until they are autoclaved.

   What goes wrong here

   • Fit two extra discs on the same plate. More than six discs on a 90 mm plate sit too close, so their zones overlap and can't be measured.
   • Slide a disc that landed off its spot. Antibiotic starts to diffuse the moment a disc touches the agar, so a moved disc leaves a distorted zone.
   • Wait to incubate with the next batch. At room temperature the antibiotics diffuse before the bacteria grow, so the zones come out falsely large.
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 zones6 discs to judge

   Check the control strain, then measure and interpret each zone.

   The method for this part

   What you check

   • • Control strain
   • • Measure the zone

   What you decide by

   • • The breakpoints and control ranges here are for this exercise only. In real work use the current EUCAST or CLSI table for the organism and the disc.
   • • Check the control strain first: if its zone for a disc is outside the range, the isolate's result for that disc is not valid.
   • • Measure the whole diameter to the nearest millimetre. At or above the S figure is susceptible, below the R figure is resistant, and anything between is intermediate.
   • • Intermediate is the CLSI name for the middle band. EUCAST calls it susceptible, increased exposure.

   What you can decide

   Susceptible · Intermediate · Resistant · Not valid: repeat

8. 8

   Report the results3 questions

   Decide what can go on the report.

9. 9

   Clear down3 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

• Touch 3 to 5 similar colonies and adjust the suspension to 0.5 McFarland: a heavier lawn gives smaller zones, a thinner one larger.
• Keep to 15-15-15: use the suspension within 15 minutes, apply discs within 15 minutes of swabbing, incubate within 15 minutes of applying discs.
• Swab the whole plate in three directions for an even lawn. No more than six discs on a 90 mm plate, pressed flat and never moved.
• Incubate inverted at 35 °C for 16 to 20 hours, then measure each zone to the nearest millimetre.
• Check the control strain against its range before reporting anything, and read S, I or R from the current breakpoint table.

Equipment unlocked

• Antibiotic discs
• Zone ruler
• Mueller-Hinton agar