Salmonella Detection in Food

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

   • • Food sample
   • • Sterile blender bag
   • • Buffered peptone water
   • • Salmonella control strain
   • • Pipette
   • • RVS and MKTTn broths
   • • Inoculating loop
   • • XLD and BGA agar plates
   • • Nutrient agar plates
   • • TSI, urea and lysine tubes
   • • Salmonella antisera and saline
   • • Salmonella worksheet

   What you decide by

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

   Receive samples5 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 checks5 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

   • • Pipette
   • • Incubator, 37 °C
   • • Water bath, 41.5 °C
   • • Buffered peptone water
   • • XLD and BGA agar 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

5. 5

   Enrich and plate10 steps · 1 by hand

   Take the samples from weighing to incubated selective plates.

   The method for this part

   1. 1

      Weigh 25 g of sample into the bagDone by hand, to 24.5–25.5 g

      Weigh 25 g of each food sample aseptically into its own labelled blender bag.

      The result is reported per 25 g, so 25 g is what has to be tested.

   2. 2

      Add 225 mL of buffered peptone water

      Pour 225 mL of buffered peptone water onto the sample in the bag: nine parts to one.

      A non-selective broth gives damaged cells the chance to repair and multiply.

   3. 3

      Homogenise in the paddle blender

      Run the blender bag in the paddle blender for a minute or two.

      Salmonella inside the food has to be shaken out into the broth.

   4. 4

      Set up a positive and a negative control

      Add the Salmonella control strain to one portion of broth and leave another with nothing added. Handle the control strain last.

      The positive control shows the run can find Salmonella; the negative shows the laboratory added none.

   5. 5

      Pre-enrich at 37 °C for 18 h (1 s = 1 h)Pre-enriching, 1 s for each hour: 16–20 s

      Put the blender bags in the 37 °C incubator. On the clock one second stands for an hour: stop it between 16 and 20.

      Stressed cells need this time to recover before they can survive a selective broth.

   6. 6

      Transfer 0.1 mL to RVS and 1 mL to MKTTn

      Pipette 0.1 mL of each pre-enriched culture into 10 mL of RVS broth and 1 mL into 10 mL of MKTTn broth, with a fresh tip each time.

      RVS broth is made for one part of culture in a hundred. Some Salmonella grow better in one broth than the other, so both are used.

   7. 7

      Incubate the RVS broth at 41.5 °C

      Put the RVS tubes in the 41.5 °C water bath and the MKTTn tubes in the 37 °C incubator, each for 24 hours.

      RVS broth needs 41.5 °C to hold back the background flora.

   8. 8

      Incubate the MKTTn broth at 37 °C

      Put the RVS tubes in the 41.5 °C water bath and the MKTTn tubes in the 37 °C incubator, each for 24 hours.

      MKTTn broth is selective at 37 °C: the two broths are incubated at different temperatures.

   9. 9

      Streak both broths onto XLD and BGA

      With the loop, streak each broth onto XLD agar and onto brilliant green agar (BGA), for single colonies.

      Two agars that work differently catch strains that one alone would miss.

   10. 10

       Incubate the plates inverted at 37 °C

       Turn the plates upside down and put them in the 37 °C incubator for 24 hours.

       Inverted plates keep condensation off the agar, so colonies stay separate.

   What goes wrong here

   • Go straight to the selective broths. Stressed cells die in a selective broth unless they have recovered in a non-selective one first.
   • Put both broths in the 37 °C incubator. At 37 °C RVS broth lets the background flora outgrow Salmonella.
   • Use one pipette tip for every transfer. A used tip carries Salmonella from one culture into the next, and into the negative control.
   • Leave the controls out to save media. Without controls nothing shows that the run could find Salmonella, or that it added none.
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 plates5 samples to judge

   After incubation, say what each set of plates shows.

   The method for this part

   What you check

   • • Plates from RVS
   • • Plates from MKTTn

   What you decide by

   • • On XLD, typical Salmonella colonies are red with a black centre. Yellow colonies are not typical.
   • • For this exercise the second agar is brilliant green agar (BGA): typical colonies are pink, and turn the agar around them red.
   • • Typical colonies on any one plate make the sample presumptive.
   • • A plate too crowded to pick a single colony from can't be read.
   • • The positive control should show typical colonies, and the negative control none.

   What you can decide

   No typical colonies · Typical colonies · Can't be read

8. 8

   Confirm the colonies6 steps

   Typical colonies are only presumptive. Take them through confirmation before anything is reported.

   The method for this part

   1. 1

      Pick a single typical colony from each plate

      Touch the loop to one well-separated typical colony on each XLD or BGA plate that has them.

      One separate colony is one organism, which is what the tests need.

   2. 2

      Streak each pick onto nutrient agar

      Streak each colony onto a nutrient agar plate and incubate it at 37 °C for 24 hours.

      Confirmation tests only mean something on a pure culture.

   3. 3

      Inoculate TSI, urea and lysine tubes

      With the loop, take growth from the pure culture into the TSI, urea and lysine tubes, and incubate them at 37 °C for 24 hours.

      Salmonella has a pattern of its own: a yellow (acid) butt and blackening on TSI, urease negative, lysine positive.

   4. 4

      Mix a little growth in a drop of saline

      Before any antiserum, mix growth from the pure culture into a drop of the saline and watch for clumping.

      A strain that clumps in saline alone would clump in any antiserum, so its serology means nothing.

   5. 5

      Test with O and H antisera

      Mix growth into a drop of each antiserum, O and then H, and look for clumping within a minute.

      Agglutination with Salmonella antisera is the serological half of the confirmation.

   6. 6

      Record every reaction on the worksheet

      Write each result against its sample on the worksheet before anything is reported.

      The report is only as good as the record it is drawn from.

   What goes wrong here

   • Report 'detected' from the XLD plates. Other organisms look the same on XLD, so typical colonies are presumptive until they are confirmed.
   • Take a good loopful of the typical growth. A loopful of growth is a mixture, and tests on a mixture give reactions that belong to no one organism.
9. 9

   Report each sample3 samples to judge

   Weigh up the plates, the confirmation and the run itself, then decide what can be reported.

   The method for this part

   What you check

   • • Plate reading
   • • Confirmation results
   • • Controls and run record

   What you decide by

   • • Detected in 25 g: typical colonies confirmed by biochemistry and by serology.
   • • Not detected in 25 g: nothing confirmed, in a run done to the method with both controls as they should be.
   • • Salmonella on confirmation: TSI with a yellow (acid) butt and blackening, urease negative, lysine positive, agglutinates (clumps) with O and H antisera but not in saline.
   • • For this exercise every Salmonella isolate shows that full pattern. Real strains vary, and doubtful ones go to a reference laboratory.
   • • A missing or failed control means nothing from the run can be relied on.

   What you can decide

   Not detected in 25 g · Detected in 25 g · No result yet: confirm or repeat

10. 10

    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

11. 11

    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 buffered peptone water, 18 hours at 37 °C: pre-enrichment lets stressed cells recover before they meet a selective broth.
• 0.1 mL into RVS broth at 41.5 °C and 1 mL into MKTTn broth at 37 °C: two broths, two temperatures.
• On XLD, typical Salmonella colonies are red with a black centre. They are presumptive, not a result.
• Confirm suspect colonies by biochemistry and by serology before reporting 'detected in 25 g'.
• A positive and a negative control go through every step: without them, no result can be relied on.

Equipment unlocked

• Paddle blender
• Selective broths
• XLD agar