Biochemical Identification of an Isolate

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 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

   • • Culture plate
   • • Control organisms
   • • Glass slides
   • • Plastic loops
   • • Hydrogen peroxide
   • • Oxidase strips
   • • Plasma and saline
   • • Disinfectant spray

   What you decide by

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

   Run the bench tests10 steps · 1 by hand

   Run the catalase, oxidase and coagulase tests in turn, each with its controls.

   The method for this part

   1. 1

      Check the culture is fresh and pure

      Look at the culture plate: overnight growth, and only one kind of colony.

      Old cultures lose enzyme activity and mixed ones give mixed answers.

   2. 2

      Smear the catalase controls

      From the control organisms, smear Staphylococcus aureus (positive) and Streptococcus pyogenes (negative) onto a labelled slide.

      A positive and a negative control prove the peroxide works and that you are reading it correctly.

   3. 3

      Touch the top of a colony onto the slide

      With a plastic loop, touch only the top of one colony and smear it on the slide beside the controls. Take no agar.

      Red blood cells in the agar contain catalase, so agar carried over bubbles by itself.

   4. 4

      Add a drop of peroxide to each smear

      Put one drop of 3% hydrogen peroxide on each smear and watch at once for bubbles.

      Catalase splits hydrogen peroxide into water and oxygen, which shows as bubbles straight away.

   5. 5

      Test the oxidase controls

      From the control organisms, rub Pseudomonas aeruginosa (positive) and Escherichia coli (negative) onto an oxidase strip and read them at 10 seconds.

      The reagent goes off in air and light, and only the controls show whether it still works.

   6. 6

      Rub a colony on a strip and read itWatching the strip: 5–10 s

      With a plastic loop, rub a colony onto a fresh oxidase strip. Read it between 5 and 10 seconds, and no later.

      Purple within 10 seconds is positive; colour that comes later is the reagent oxidising in the air.

   7. 7

      Test the coagulase controls

      From the control organisms, mix Staphylococcus aureus (positive) and Staphylococcus epidermidis (negative) each into a drop of plasma on a slide.

      Plasma that has lost its activity clumps nothing, and only the positive control shows it.

   8. 8

      Emulsify in saline then add plasma

      Put two drops of saline on a slide and emulsify a colony in each until smooth. Add plasma to one drop, rock the slide and read within 10 seconds.

      The plain saline drop shows whether the strain clumps by itself, so clumping in plasma can be trusted.

   9. 9

      Discard the used loops and slides

      Put the used loops, slides and strips straight into the discard pot.

      They carry live organisms until they are disinfected or autoclaved.

   10. 10

       Disinfect the benchDone by hand, to the whole bench

       Spray the whole work area with disinfectant and wipe it.

       Bubbling peroxide throws fine droplets of culture onto the bench around the slide.

   What goes wrong here

   • Dig the loop in for plenty of growth. Digging in carries blood agar over with the colony. Red cells contain catalase, so the agar bubbles by itself: a false positive.
   • Use the nichrome loop on the strip. Nichrome wire can turn the oxidase reagent purple by itself: a false positive.
   • Mix a colony straight into plasma. With no saline drop beside it, you can't tell real clumping from a strain that clumps by itself.
4. 4

   Read the reactions3 tests to judge

   Look at both controls and the isolate for each test, then call it.

   The method for this part

   What you check

   • • Positive control
   • • Negative control
   • • Your isolate

   What you decide by

   • • Read the controls first. If either is wrong, or wasn't run, the test is not valid.
   • • Catalase: bubbles at once are positive.
   • • Oxidase: purple within 10 seconds is positive. Colour that comes later doesn't count.
   • • Slide coagulase: clumping in plasma within 10 seconds, with a smooth saline drop, is positive.
   • • For this exercise all three tests are run on the isolate, and oxidase is read at 10 seconds. In routine work the Gram stain decides which tests are needed, and the reading time is the one in your method.

   What you can decide

   Positive · Negative · Not valid: repeat

5. 5

   Name the isolate3 questions

   Put the Gram stain and the reactions together.

6. 6

   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

7. 7

   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

• Test a fresh, pure overnight culture, and run a known positive and a known negative control with every test.
• Catalase: bubbles in 3% hydrogen peroxide are positive. Never carry blood agar over: red cells contain catalase too.
• Oxidase: purple within the reading time is positive, and later colour doesn't count. Use plastic, platinum or wood, never nichrome.
• Slide coagulase: clumping in plasma but not in saline is positive, as for Staphylococcus aureus. Confirm doubtful results in a tube.
• A control that fails, or results that don't fit the Gram stain, mean repeat: never explain them away.

Equipment unlocked

• Hydrogen peroxide
• Oxidase reagent
• Coagulase plasma