How can I culture Propionibacterium acnes ( P. acnes )?

[sciencenewbie]

I need P. acne bacteria culture for my science project since I can't buy it i have to culture it so now i need to know is how. This is for my procedure. I need specific steps so I can follow. My school is letting me use their incubator, agar plates, beakers, and stuff. HELP ME!! I NEVER CULTIVATED ANYTHING BEFORE!

[donnahardy2]

Hi,

This is a challenging but very interesting product. The science buddies website contains excellent information for basic microbiological techniques:

https://www.sciencebuddies.org/science- ... ndex.shtml

Propionibacterium acnes is normally present as normal flora on human skin and can be an opportunistic pathogen. It is available for purchase from the American Type Culture Collection, but you would probably need to obtain it through your school or a local university:

http://www.atcc.org/

If you tried to isolate it from its native environment, you would have to identify it using classical serological techniques or newer DNA techniques:

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC274142/

http://www.ncbi.nlm.nih.gov/pubmed/18201989

Here is a paper that describes optimum growth conditions for Propionibacterium acnes. Please note that this organism is anaerobic and very slow growing. It would be very challenging to grow this for a first time project.

http://www.springerlink.com/content/kt6262h336782290/

Also, you would need approval to do this project. Here is information on the scientific review committee from the science buddies website:

https://www.sciencebuddies.org/science- ... _src.shtml

What are you trying to do for your project? Do you have access to an anaerobic incubator? I recommend continuing to do background reading to make sure you will be able to do your project. Let me know if you can't open any of the links.

Donna Hardy

[sciencenewbie]

Thank you Donna.
Which acne medication can effectively inhibit the growth of bacteria that causes acne?
That is my project but i need p acnes culture to do it.
If i do and hopefully able to culture this do i need to streak it?

[deleted-71615]

Here is a study that tested in vitro susceptibility of 96 isolates of Propionibacterium acnes to 17 antibiotics. With the exception of the aminoglycosides, these organisms are susceptible to virtually all of the commonly used antimicrobial agents. http://www.ncbi.nlm.nih.gov/pmc/articles/PMC351938/

You can purchase P.acnes from ATCC, (ATCC#6919) here is the link. http://www.atcc.org/ATCCAdvancedCatalog ... e=bacteria Keep in mind that speical permit might be required for purchase.

Michael

[donnahardy2]

Hi,

Michael has given you very good information on the susceptibility of P. acnes. Here is information for the rest of your question.

If you can obtain the culture and find a laboratory to work in that has the anaerobic incubators that you would need to grow this organism, you would make a “lawn” of bacteria on an agar plate and place discs with antibiotic on the surface and then measure the zone of inhibition of growth. Here is a website with a detailed procedure for measuring antibiotic sensitivity:

http://www.rapidmicrobiology.com/test-m ... esting.php

If you are not able to obtain a culture of P. acnes, then I recommend that you consider doing this project with an aerobic bacterium. You would gain valuable experience and it would still be a very worthwhile project. Frequently, one of the challenges in doing a science project is to adapt it to the resources that you have available and I think you have a really good idea here, but it will be really difficult to do as a first time microbiology project.


Donna Hardy

[sciencenewbie]

how do i make this "lawn"?
Okay so i plan on culturing it myself
I found this and it seems helpful
http://ph.answers.yahoo.com/question/in ... 944AAmzdAP
and i am doing this for a science project
http://all-science-fair-projects.com/pr ... 132_2.html

so after i culture it what do i do ???
on the procedure it asks for 10ml of P. acne bacterium
but it's on an agar plate if i culure it? isn't it?
and what is this "streaking" thing do i need to do this to get my 10 ml of p acnes?

[donnahardy2]

Hi,

You have found some good information. Here is information on streaking an agar plate to isolate colonies:

http://www.umsl.edu/~microbes/pdf/streakplates.pdf

Once the bacteria are grown, you will suspend a colony in water and use a sterile swab to inoculate the surface of a plate. Or, you can grow a broth culture and transfer a small amount to the surface of the agar plate. After you have spread the pure culture on the plate, you would place discs containing the antimicrobial agents on the surface before incubating the culture. You will measure the zones of inhibition around each disc.


Donna Hardy

[sciencenewbie]

Thanks donna! Can you explain more thoroughly and be a bit more specific? What is streaking though? What's the purpose? Why do I streak it? What do I do after I streak it? How does it work?? Omg I am nervous. Wait. Do I do the streaking before suspending a colony in water and using a sterile swab to inoculate the surface? How do I suspend a colony in water??? How much water? What does streaking have to do with suspending a colony in water? Wait do I cut the agar where the streaks show single colonies? Is that how it is? Any specific directions? D: help me, my project depends on you!

Wait when you say inoculate does that mean #4 in the procedure link I showed you? It says:
"Mix the fourth test tube containing 10ml of P. acne bacterium with 40ml of water. The swab is washed with disinfected water and dipped into the test tube containing the bacteria before gently swabbed all over the agar surface of the 3 Petri dishes. Ensure that you swab in at least 3 directions to cover the entire surface of the dishes."

Oh and how does the broth culture work? Which one is easier to do?

Oh and does growing it on nutrient agar from agar powder work ???
Sorry for bombarding you with questions but without you I would be clueless!
THANK YOU!

[donnahardy2]

Hi,

Here's a video that shows the basic technique for streaking an agar plate. The results of the plate after incubation are not shown, but the idea is to grow single colonies to ensure that you have isolated a pure culture.

http://www.wonderhowto.com/how-to-strea ... te-221130/

Here's a photograph of a plate after incubation showing what looks like a pure culture (single type of colony) and individual colonies. You would pick a single colony to prepare the suspension of bacteria to make the lawn of bacteria for the sensitivity testing:

http://media.nowpublic.net/images//a1/1 ... 58a41d.jpg

Here's a photograph of a lawn of bacteria with antibiotic discs. The clear zone of inhibition is what you will measure to obtain quantitative results.

You will need to grow the Propionibacterium on an agar plate first to verify it is a pure culture, and then you can either make

Now, I have some questions for you to help you plan your project. What type of culture medium are you going to use to grow the Propionibacterium acnes? If you isolate the bacterium from a human source, how will you identify the organism? How are you going to keep oxygen from your culture (Propionibacterium won't grow in an oxygen environment)? What is the biological safety level of the lab facilities you have available to use and what safety precautions will you need to follow to work with Propionibacterium?

It sounds like you are reading all of the links and that is good, but it would be helpful if someone could show you the basic techniques for making agar and growing bacteria. Do you know anyone who works in a microbiology lab? Could your teacher show you how to do these experiments? It would be really helpful if you could find someone just to show you the basics. If this is not possible, then let me know if you do have any other questions.


Donna Hardy

[sciencenewbie]

Thanks for the link and video I checked it out (:

but did something get cut off?

"Here's a photograph of a lawn of bacteria with antibiotic discs. The clear zone of inhibition is what you will measure to obtain quantitative results. You will need to grow the Propionibacterium on an agar plate first to verify it is a pure culture, and then you can either make "

I want to grow it on Nutrient Agar but can I? I think that is the agar I am using...it's the one where you mix agar powder and then water and heat it up or something...
BUT can i grow it on nutrient agar?
Uh is there a way to identify them? can i look them under a microscope?
My high school has an incubator, but i don't know if it is an anerobic one. I think I will ask tomorrow.

If it isn't then I would do what the yahoo answers person said to blow CO2.

To grow your samples anaerobically, keep them in a sealed container like a jar with a screw-on top or a plastic Tupperware container. Blow N2 or CO2 over the samples to displace the O2 ("sparge" the container), then put the lid on. (You'll want to do this each time you open the container.) A CO2 cartridge, like those used by cyclists to inflate tires, is probably the most accessible source of gas. The pressure in the cartridge is very high, so try to borrow an inflater and a bicycle tube from a cyclist friend. Inflate the tube, then release the CO2 at lower pressure from the tube.

I don't know about the biosafety level or precautions...lol need more research and ask the teacher about this....I think I will ask the teacher for help and ask around to see if anyone knows how to streak or show me. Unfortunately I don't know anyone that works in a microbiology lab.

I'm going to ask the head of the science department tomorrow and hopefully get some answers! Will keep you posted (:
THANK YOU!

[donnahardy2]

Hi,

Sorry, I forgot to include the photo of the sensitivity plate. A good photograph is actually included on the science buddies website for this project:

https://www.sciencebuddies.org/science- ... p019.shtml

I don't know if P. acnes will grow on nutrient agar. Most of the references I found used a medium that contained yeast extract. Here's a reference that used a thioglycolate medium, which contains a reducing agent that helps maintain anaerobic conditions. Please search for more information on this topic, as you have to make sure the bacterium will grow on the medium you have available.

http://aac.asm.org/cgi/reprint/33/3/326.pdf

What types of bacterial medium do you have available in your lab?

Donna Hardy

[sciencenewbie]

I talked to my teacher and the incubator is not anerobic =/

Okay I did some searching and I found this:
http://books.google.com/books?id=HcgGLf ... 20&f=false

That means it can grow on it right?

Oh! and I talked to the head and I think she knows how to streak bacteria i think lol i asked her and she was like "I know a lot of things.." so i guess that is a yes? I will be seeing her after class tomorrow. (=

Uhm. The single colonies is like a dot right? How do I do the suspension of bacteria? Do I cut out the dot and then put water? Oh. and was there something after the word "make" on your previous post?

first to verify it is a pure culture, and then you can either make

-Thanks!

[donnahardy2]

Hi,

Great! You are much better at web searches than I am. You have confirmed that P. acnes will grow on regular culture media like nutrient agar. This is good news because many anaerobes require blood or other special nutritional supplements to grow.

Yes, a colony is a single dot, or colony forming unit, presumably originating from a single organism. To transfer a colony, you can use a sterile inoculating loop or swab and scoop it up from the surface of the agar and transfer it to another plate, or to a tube of sterile water to make the suspension.

Now, I’ll try to finish my thought from yesterday. When you make a streak plate, you observe the morphology of the colonies to make sure they are all the same, and presumably growing as a pure culture. Since P acnes grows slowly, it may be overgrown with contaminants so it would be essential to verify the identity of the organism. Two basic techniques are to verify the colony morphology and the Gram stain results with a microscope. Here is a link that describes the colony and microscopic morphology for this organism:

http://books.google.com/books?id=vqVN-B ... es&f=false

Do you have access to Gram stain reagents or to a microscope? Can you find any other techniques that can be used to verify the identity of this organism?

Donna Hardy