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Fall 2011 Lab 8 antiseptics+antibiotics+ handshake

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					Lab 8: antiseptics and antibiotics (Exercises 19, 20)
Key Concepts:
    minimum inhibitory concentration (MIC) vs minimum bactericidal concentration (MBC)
    not all antibiotics inhibit all bacteria – some better on certain bacteria than others
    not all chemical agents inhibit all bacteria – some better on certain bacteria than others
    disinfectantion, antisepsis, sanitization
    cidal vs static
    what does sterile really mean?



Final Project Discussions
      come up with project that deals with a MICROBIOLOGY question and that is of interest
       to BOTH OF YOU and which you think can be experimentally addressed by the end of
       the semester




Exercise 20: Antibiotic sensitivity = Kirby-Bauer disk method

When antibiotic use is indicated and the causative microorganism has been identified, it is extremely
important that the correct antibiotic be prescribed to kill that particular organism. The Kirby-Bauer
disk sensitivity method is the current standard with which to test the antibiotic sensitivity of a bacterial
species isolated from a patient. It is simple, cheap and reproducible.

      The procedure for K-B sensitivity testing is described on page 170.

      Make sure to uniformly cover the agar plate with the test bacteria using a cotton swab to
       create a bacterial lawn. You should try and cover every square millimeter with bacteria!


      Each lab pair will swab one plate for each of these 4 bacteria:
              Staphylococcus aureus
              Escherichia coli
              Enterococcus faecalis
              Pseudomonas aeruginosa


Choose either antibiotic dispenser 1 or 2. Each dispenser has 6 different antibiotics in it. Use
forceps to gently push the disks onto (not into!) the agar and then incubate the plates at 37C.

Make sure to record the antibiotics and the doses used for this experiment in your notebook!
Lab 8: antibiotics + antiseptics                                                        Micro 310 Fall 2011


Exercise 20: Antibiotic sensitivity = MIC method

The Kirby-Bauer method for determining antibiotic sensitivity is good when determining the sensitivity
(or resistance) of bacteria to a series of different antibiotics. However it cannot tell you quantitatively
how much of a particular antibiotic it will take to inhibit the growth of or kill a particular bacterial strain.
To quantitatively determine this using dilutions of an antibiotic in tubes, the MIC and/or MBC test are
used. A description of tube dilution testing, MIC, and MBC is also found in the course textbook on
pages 376-377.

Although we’re not going to do a hands-on MIC experiment, MAKE SURE you know the difference
between MIC and MBC and why you would choose to do an MIC versus a Kirby-Bauer test.

        MIC: the lowest concentration of an antibiotic (or other chemical) at which bacterial growth is
         completely inhibited. Remember that some antibiotics are static and not cidal, so an MIC
         doesn’t necessarily mean the lowest concentration that kills.

        MBC: the lowest concentration of an antibiotic (or other chemical) at which bacteria are
         completely killed.



Exercise Die You Nasty Bacteria: Evaluation of Antiseptics = filter disk method

Chemical agents are widely used to control or kill microorganisms. These agents can be grouped
into several broad categories:
        Steriliants or -cidal agents – kill all organisms including spores
        Disinfectants – control or kill microbes on non-living things like tables (harsh)
        Antiseptics – control or kill microbes on living tissues like your arm (less harsh)
        Sanitizers – lower the microbe count to a safe level (usually food service)


We are going to test the activity of a variety of agents against Staphylococcus aureus (Gram-positive)
and Pseudomonas aeruginosa (Gram-negative).

        The technique for doing antiseptic / disinfectant testing is similar to the Kirby-Bauer technique.
         However, in this case, you’re making your own disks. Wet the sterile blank paper disks with
         your chosen chemical agents. It is important that the disks are NOT TOO WET when you put
         them on the plate of bacteria.

        Each lab pair will do one plate for each of the two bacteria.

                 -- Limit your testing to 4 chemical agents (record which ones and concentration!)

                 -- If you’re testing something you brought in, make sure to record the active ingredient
                     and its concentration

                 -- Do the same 4 chemical agents for the two different bacterial species so you
                    can compare results for Gram-positive versus Gram-negative

        Use forceps to gently push the disks onto the agar and then incubate the plates at 37C.

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Lab 8: antibiotics + antiseptics                                                 Micro 310 Fall 2011


Exercise 19: Handwashing

As you should know by now, washing your hands regularly throughout each day is the easiest and
single best way of preventing the spread on infectious agents that cause disease. But what type of
washing is the best?

Does that alcohol wipe a phlebotomist uses prior to sticking that needle in your arm really do any
good for reducing the number of bacteria that might be able to get inside? How well does a non-
alcohol wipe work? How do the hand sanitizers work?

        We’re going to test the following methods of handwashing. Random numbers will determine
         which method you will use…..

            o    Soap & water: do in bathroom and use air dryer to dry hands
            o    Soap & water: do in lab and use paper towels to dry hands
            o    Water alone: do in bathroom and use air dryer to dry hands
            o    Water alone: do in bathroom and use paper towels to dry hands
            o    Alcohol-based hand sanitizer: let hands air dry
            o    Alcohol-based sanitizing wipes: let hands air dry
            o    Non-alcohol-based sanitizing wipes: let hands air dry


    o    Each person will divide TSA agar plate into quarters and label the sections A, B, C, and D.

    o    Press your unwashed left thumb on section A and then immediately press the same thumb on
         section B. This tests how many microbes are removed just by touching the agar.

    o    Press your unwashed right thumb on section C.

    o    Perform the handwashing technique you have selected for about 20 seconds (sing “Happy
         Birthday”).

    o    Press your washed right thumb on section D.

    o    Incubate plates at 37°C.




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Lab 8: antibiotics + antiseptics                                                    Micro 310 Fall 2011


Exercise today: The nasty handshake

What is your student number for this lab ______________?

We always talk about diseases spreading so we’re going to start with one infected person and see
how quickly the disease spreads and after the epidemic is over, whether we can track the cases back
to the original source (the “index case”). Each person is going to participate in this by shaking hands
and inoculating a plate.

        Divide a TSA plate into thirds. Label one part as “control”, one part as “first round” and the
         other part as “second round”.

        Get 3 sterile cotton swabs. Open them and place them into your tube of sterile water for each
         access.

        Place a glove on one hand. This is what is going to be the hand receiving bacteria (or maybe
         no bacteria). You’ll need the other hand free to swab the glove and to inoculate the plate.

        You have a Petri dish on your bench with a caramel inside of it. One of the caramels in one
         of the dishes has been soaked in Serratia marcescens for a couple of hours while the rest
         have been soaked in sterile water.

             -- see if you can find out why we’re using Serratia marcescens

        Before you start with the caramels, do the control for the experiment: take one of your
         cotton swabs, press it against the inside glass of the water tube to make sure it’s not dripping
         wet, and then swab the palm of your glove by making a single streak from your fingers across
         the middle of your palm towards your wrist. Roll the swab over the third of your TSA plate
         marked “control”.

        Now, open your Petri dish and pick up the caramel with you GLOVED HAND. Roll it around
         and squeeze it so that any bacteria on it are now on your glove. When you’re done, put the
         caramel back in the dish but don’t touch anything with your glove!

        Student number one shakes hands with any other student. Don’t be shy – really squish your
         gloves together so that any bacteria are transferred – just like sneezing in your hand and then
         shaking hands with someone. That person then shakes hands with someone else and we
         keep going until everyone has been the “shaker”. Each time, I will write down who shakes
         with who so we can try and find the index case. Once you’re done, don’t touch anything with
         your glove!

        When we’re all done shaking, take one of your cotton swabs, press it against the inside glass
         of the water tube to make sure it’s not dripping wet, and then swab the palm of your glove by
         making a single streak from your fingers across the middle of your palm towards your wrist.
         Roll the swab over the third of your TSA plate marked “first round”. Put the swab on a paper
         towel for later discard.




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Lab 8: antibiotics + antiseptics                                                 Micro 310 Fall 2011


The nasty handshake - continued

        After you do the swab and inoculate the plate, discard your glove and get another one. We’re
         going to keep going with the handshaking, following the same procedure as for the first round
         EXCEPT that now 2 people will have contaminated caramels. After we’re done shaking,
         swab your palm as before and inoculate the last third of your TSA plate marked “second
         round”.

        Place the used swab on the paper towel. Carefully remove your glove by pealing it inside out.
         Discard the towel, swabs and gloves in the green biohazard bin up front.

        Place the TSA plates in the back incubator set at 25C.

        WASH YOUR HANDS – NOW!




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Lab 8: antibiotics + antiseptics                                                   Micro 310 Fall 2011


Interpretation of Unknown Staph 16s rRNA DNA Sequence Data

Your sequencing data are going to come back in the form of a PDF file with the actual
chromatograms like this:




You will also get a text file like the one below, which is much more useful. The first line contains the
name of your DNA sample and the number at the right (868 in this case) is how many bases of
sequence they got. You see the expected A, C, G, and T but you also see some N’s. These are
DNA bases that the software couldn’t clearly interpret from the chromatogram.


>100316-03_O03_CKC-515F.ab1                                                  868
NNNNNNNNNNNNNCNNATATTGGGCGTAAGAGCTCGTAGGCGGTTTGTCGCGTCTGTCGTGAAAGTCCGGGGCTTAACCCCG
GATCTGCGGTGGGTACGGGCAGACTAGAGTGCAGTAGGGGAGACTGGAATTCCTGGTGTAGCGGTGGAATGCGCAGATATCA
GGAGGAACACCGATGGCGAAGGCAGGTCTCTGGGCTGTAACTGACGCTGAGGAGCGAAAGCATGGGGAGCGAACAGGATTAG
ATACCCTGGTAGTCCATGCCGTAAACGTTGGGCACTAGGTGTGGGGACCATTCCACGGTTTCCGCGCCGCAGCTAACGCATT
AAGTGCCCCGCCTGGGGAGTACGGCCGCAAGGCTAAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGCGGAGCATGCG
GATTAATTCGATGCAACGCGAAGAACCTTACCAAGGCTTGACATGTTCTCGATCGCCGTAGAGATACGGTTTCCCCTTTGGG
GCGGGTTCACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTCG
TTCCATGTTGCCAGCACGTCGTGGTGGGGACTCATGGGAGACTGCCGGGGTCAACTCGGAGGAAGGTGAGGACGACGTCAAA
TCATCATGCCCCTTATGTCTTGGGCTTCACGCATGCTACAATGGCCGGTACAATGGGTTGCGATACTGTGAGGTGGAGCTAA
TCCCAAAAAGCCGGTCTCAGTTCGGATTGGGGTCTGCAACTCGACCCCATGAAGTCGGAGTCGCTAGTAATCGCAGATCAGC
AACGCTGCGGTGAATACGTTCCCGGGCCTTGCNNNNNNCGCCCCGTCA



Interpretation of Unknown Staph 16s rRNA DNA Sequence Data (continued)

The simplest way to begin your analysis is to copy the DNA sequence starting after any “N”s at the
beginning (sequence too close to the primer) and going until you hit a number of “N”s towards the
end. If the sequencing gods are with us, this is usually 500-800 bases. If not, copy what you can.


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Lab 8: antibiotics + antiseptics                                                   Micro 310 Fall 2011


Get out there on the web – we’re going to use the BLAST program at the NCBI (National Center for
Biotechnology Information) to search the public databases with your sequence.

BLAST: http://blast.ncbi.nlm.nih.gov/Blast.cgi

        On this page, go under Basic BLAST and choose Nucleotide Blast

        Paste your sequence into the box at the top (Enter Query Sequence)

        Go down to the next box (Choose Search Set)
         -- choose the radio button “others”
         -- the default in the menu immediately below should say “nucleotide collection (nr/nt)”

        Scroll down to the bottom of the page and hit the big blue “BLAST” button

        the results should come back within 30 secs or so and will give you a GRAPHIC display
         (which doesn’t help us much) and then DESCRIPTIONS (with links) and then ALIGNMENTS




        In the case of the sequence in this lab, you can see from the DESCRIPTIONS that it is
         probably a Micrococcus species, although Actinobacterium FB-11 is second on the list. You
         can’t tell which Micrococcus for sure since M. luteus and M. yunnanensis are both there.

        When you go to ALIGNMENTS, you can see how well your sequence (QUERY) matches up
         to each hit (SBJCT) listed in the DESCRIPTIONS. A vertical hash mark shows identity at that
         base between your sequence and the database entry and the numbers at the beginning and
         end of each line are the nucleotide numbers.




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Lab 8: antibiotics + antiseptics                                                  Micro 310 Fall 2011


Questions to Ponder
1. Why use an MIC test in tubes vs doing the Kirby-Bauer test? What kinds of information do you
   best learn from each test?




2. Do any patterns emerge from your results in the K-B test as to which species is generally more
   sensitive or resistant to the antibiotics tested? Any patterns for sensitivity of Gram positive vs
   Gram negative bacteria to particular antibiotics?




3. What’s the difference between an antiseptic and a disinfectant? Give an example of each one.
   Can any chemical be used as both?




4. Do any general patterns emerge for sensitivity or resistance of Gram positive vs Gram negative
   bacteria to chemical agents or to particular chemical agents?




5. Why is alcohol a good antiseptic for the skin?




6. Sometimes you see a few small colonies growing within the zone of inhibition in a K-B test. What
   do you think these colonies might be (other than contaminants) and why might they be important
   to note when considering how best to treat the patient from which the bacteria being tested were
   isolated?




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