M465:Antibiotic Resistance: Difference between revisions

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==Antibiotic Production==
==Antibiotic Resistance==


Many microbes secrete antimicrobial compounds to help them compete with other microorganisms for habitat. Some of the bacteria that are common antibiotic producers are the Actinomycetes (including ''Streptomycetes'' species), many of the ''Bacillus'' species, and the fruiting myxobacteria, to name just a few among many, many antibiotic producing bacteria.  You can also test for the opposite: the sensitivity of your organisms (or known stock bacteria) to manufactured or secreted antibiotics.<BR> <BR>
Antibiotics are molecules that kill bacteria or inhibit their growth. Common antibiotics target important cellular functions in bacteria (such as cell wall remodeling or protein synthesis).  Believe it or not, many microbes are naturally resistant to antibiotics, either because they produce them or because that resistance is advantageous in the habitat they occupy.  One place in which such selection is currently taking place, is in the human microbiome.  Unfortunately, due to the overuse of antibiotics (both in medicine and agriculture), widespread antibiotic resistance has become the norm.  The figure below captures the trend and shows the increasing prevalence of antibiotic resistance in human pathogens. <br><br>


(This testing will take 3 weeks.) <BR>
[[Image:Antibiotic_resistance_image.gif]] <br> <br>
''' Week 1:''' <BR>
Identify how many potential antibiotic producers you might have. It's possible that you might discover the next great antimicrobial drug and get very rich by selling the patent for your discovery to a drug company. Remember that the discovery of penicillin was completely accidental. <BR><BR>


Using aseptic technique, transfer an isolate that's likely to be an antibiotic '''producer''' to a microfuge tube containing 500μL of sterile water. Vortex to mix. Using a sterile swab, dip the swab in the diluted bacteria and make an inoculation (as shown below) down the middle of a plate of appropriate agar media. Make a second plate exactly like the first for each isolate to be tested. Label them carefully and incubate the plates for ~1 week at RT.<BR><BR>
Today you will be testing whether or not your Drosophila isolates are resistant to antibiotics.  


[[Image:strep1a.jpg]]<BR>
You will be using a soft agar overlay technique to test the degree of resistance in your bacterial isolates. First, you will take cellulose disks, impregnated with antibiotics, and put it on your agar plate. You will then take 2 mL of molten agar (45C water bath) and add 100 uL of an overnight culture of your isolate to that 2mL *very quickly* .  Pour your soft agar over the surface of your agar plate, nutating the plate in order to spread the soft agar evenly.  Don't take too long or the agar will solidify and you will not have time to  pour it!  <br> <br>
 
'''Week 2'''<BR>
 
We will provide you with 3 fresh cultures of ''Escherichia coli'' (Gram negative), ''Staphylococcus aureus'' (Gram positive) and grown in nutrient broth. <br>


'''PROTOCOL'''<BR><BR>
'''PROTOCOL'''<BR><BR>
'''Use the plate(s) from week 1<UL><LI>  
Set up an overnight culture the day before! For each isolate, and each antibiotic, perform the steps below. .<br>
Use a sterile swab and aseptically apply a line of inoculation of each of the provided broth cultures of : ''E. coli'' and ''S. aureus''.
1. Grab tube with cellulose disks impregnated with antibiotic. You will need one for each antibiotic we are testing. <br>
as shown below. Draw a line perpendicular to the antibiotic producer's (''Streptomyces'') inoculation. Be careful not to touch the antibiotic producer's growth. Using the same swabs, inoculate a new NA plate (one plate for all three cultures) by making a line across the plate for ''E. coli and S. aureus''. Incubate this plate along with your test plate. It will serve as a control to make sure that lack of growth is due to antibiotic sensitivity and not to no living cells in the inoculum.<LI>
2. Label an agar plate of the appropriate medium with your isolate. <br>
 
3. Using sterile tweezers, grab a single disk from the tube and place it on agar plate. Gently tap disk into agar with sterile tweezers. Repeat procedure for other antibiotic disks making sure to spread the disks evenly around the agar surface. <br>
[[Image:strep2.jpg]]
4. Grab a tube of melted agar from the 45C bath. <br>
<LI>
5. Add 100 ul of your overnight culture of your isolate to the melted agar. <br>
 
6. Vortex briefly to mix the bacteria into the soft agar and immediately pour onto the appropriate agar plate. <br>
<LI>
7. Tilt your agar plate back and forth to distribute the agar evenly over the surface. <br>
Incubate for another week.</LI></UL>
8. After the agar hardens (about 10 minutes), invert the plate and incubate overnight at 25C under appropriate atmosphere. <br>
 
9. Results should be visible in the next few days.  Come in to check on your experiment and if you see growth in the agar lawn, take your plate out of the incubator and measure the zone of inhibition around the antibiotic disk. <br>
'''Week 3 <BR>'''
<UL><LI>
Examine the plate and look for evidence of inhibition of growth of "test" organisms near the antibiotic producer's midline streak. <LI>
Draw the results and evaluate whether or not there was evidence that an antibiotic was produced by the organism and, if so, which of the bacteria tested were sensitive to it and to what degree. If you found no inhibition of growth, does that mean that your potential antibiotic producer does not secrete an any antimicrobial compounds? Why or why not? </LI></UL>
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Revision as of 14:04, 29 January 2015

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Antibiotic Resistance

Antibiotics are molecules that kill bacteria or inhibit their growth. Common antibiotics target important cellular functions in bacteria (such as cell wall remodeling or protein synthesis). Believe it or not, many microbes are naturally resistant to antibiotics, either because they produce them or because that resistance is advantageous in the habitat they occupy. One place in which such selection is currently taking place, is in the human microbiome. Unfortunately, due to the overuse of antibiotics (both in medicine and agriculture), widespread antibiotic resistance has become the norm. The figure below captures the trend and shows the increasing prevalence of antibiotic resistance in human pathogens.



Today you will be testing whether or not your Drosophila isolates are resistant to antibiotics.

You will be using a soft agar overlay technique to test the degree of resistance in your bacterial isolates. First, you will take cellulose disks, impregnated with antibiotics, and put it on your agar plate. You will then take 2 mL of molten agar (45C water bath) and add 100 uL of an overnight culture of your isolate to that 2mL *very quickly* . Pour your soft agar over the surface of your agar plate, nutating the plate in order to spread the soft agar evenly. Don't take too long or the agar will solidify and you will not have time to pour it!

PROTOCOL

Set up an overnight culture the day before! For each isolate, and each antibiotic, perform the steps below. .
1. Grab tube with cellulose disks impregnated with antibiotic. You will need one for each antibiotic we are testing.
2. Label an agar plate of the appropriate medium with your isolate.
3. Using sterile tweezers, grab a single disk from the tube and place it on agar plate. Gently tap disk into agar with sterile tweezers. Repeat procedure for other antibiotic disks making sure to spread the disks evenly around the agar surface.
4. Grab a tube of melted agar from the 45C bath.
5. Add 100 ul of your overnight culture of your isolate to the melted agar.
6. Vortex briefly to mix the bacteria into the soft agar and immediately pour onto the appropriate agar plate.
7. Tilt your agar plate back and forth to distribute the agar evenly over the surface.
8. After the agar hardens (about 10 minutes), invert the plate and incubate overnight at 25C under appropriate atmosphere.
9. Results should be visible in the next few days. Come in to check on your experiment and if you see growth in the agar lawn, take your plate out of the incubator and measure the zone of inhibition around the antibiotic disk.

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