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NEED HELP WITH MY UNKNOWN GRAM POSITIVE ORGANISM PLEASE??

About microscopic forms of life, including Bacteria, Archea, protozoans, algae and fungi. Topics relating to viruses, viroids and prions also belong here.

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NEED HELP WITH MY UNKNOWN GRAM POSITIVE ORGANISM PLEASE??

Postby SweetSarah » Mon Feb 27, 2012 3:47 am

My unknown organism I am trying to figure out is Gram positive cocci (it was in "tiny" purple clusters when I observed it under microscope). So far I've done the following tests below. Which bacteria or microorganism would match the following:
- Gram positive cocci, in clusters
- Catalase positive (+) (meaning that it forms bubbles/gas on catalase test)
- Mannitol negative (-) (on MSA-mannitol salt agar- it doesn't grow or ferment salts)
and -Fructose negative (-) (So, that means it doesn't ferment fructose)
-- I also want to mention that from my agar plates I used all turned to a black/brownish coloration. I am unsure if this would indicate it's due to H2S (hydrogen sulfide) and that's the main thing that throwing me off. However I've done the streaking sevral times, and seem to have a pure culture according to my gram stains so I don't know if that shoudl be happening. THe gram stains came out fine, mainy in 1 color. Also, the colonies for my gram positive are mostly little, grayish/clear dots. I used blood agar (which had really dark black color around the tiny colonies), chocolate agar and Macconkey agar. Only my PEA agar didn't come out with the black color but all the other ones did. I find it strange my Mac agar came with the color since I looked it up and have been told that too.

:arrow: Does this sound right? Could my organism still be a micrococccus or staph (since my tests point to those) even with the black color on my plates? And if so what type would produce the H2S or black pigmentation??

Also, from these tests I know that the organism is a type of Staph (or it may be a Micrococcus?), but it isn't Staph aureus since it doesn't ferment mannitol, but am not sure what else it could be or narrowed down to. There was absolutely NO growth at all on my MSA plate so that makes me unsure if it could be Staph epidermidis since I read that grows on MSA but doesn't ferment the manitol so wouldn't be yellow like S. aureus would be. But what type of Staph wouldn't grow at all on MSA?? IS it taht my organism actually isn't a Staph? (all my tests seem to point that it is though so I don't know.) What about Micrococcus species-do those not grow at all on MSA?

That confuses me since isn't staph supposed to be halophiles, that is, salt loving organisms? that's what I always thought anyways :/

Please explain your answer or if you used a link if you could put it in your answer. Sorry for all these questions. am just confused about my results especially the black color. This is for my unknown lab project by the way. I've been looking this up but am having hard time finding what it is. Thanks for all answers!!
SweetSarah
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Re: NEED HELP WITH MY UNKNOWN GRAM POSITIVE ORGANISM PLEASE??

Postby mariah000 » Sun Mar 18, 2012 7:50 pm

if your unknown organism is staphylococcus aureus (s.aureus) then
1) under the microscope you would generally observe:
gram positive (blue/purple )
cocci
clusters (because staphylococci divide in 2 planes)
2) on nutrient agar = golden/yellow looking medium-large sized colonies
3) on blood agar = beta hemolysis
4) on mannitol salt agar = growth (yellow looking colonies) because s.aureus is able to grow in high salt concentrations and the medium turns from pink to yellow because s.aureus ferments mannitol.
5) catalase positive because it produces o2
6) coagulase positive because it has the protein coagulase that converts fibrinogen to fibrin (the clump looking clot that forms).

if your unknown organism is staphylococcus epidermidis (s.epidermidis) then:
1) under the microscope you would generally observe:
gram positive (blue/purple )
cocci
clusters (because staphylococci divide in 2 planes)
2) on nutrient agar = white looking small-medium sized colonies.
3) on blood agar = gamma hemolysis (no hemolysis).
4) on mannitol salt agar = growth (pink looking colonies) but the medium stays pink because s.epidermidis does not ferment mannitol.
5) catalase positive because it produces o2.
6) coagulase negative.

if your unknown organism is from the genus streptococcus (strep) then:
1) under the microscope you would generally observe:
gram positive (blue/purple )
cocci
chains (because strep divide in a single plane)
2) on blood agar = alpha, beta or even gamma hemolysis (no hemolysis). it all depends on the species of streptococci you have isolated.
3) on mannitol salt agar = no colony growth in media whatsoever and the medium stays pink.
4) catalase negative.
5) coagulase negative.

I suggest that you re-streak a new mannitol salt agar plate because i think that based on your gram stain and catalase result then you might be dealing with a staphylococcus organism, so you should have some growth on mannitol.
Or it could be that you did not have a pure culture, so in that case you should do a subculture from your "pure culture" and now try using those colonies.

oh and by the way, i noticed that you said you used a MaConkey agar? i dont know why you did that because that medium selective for gram negative organisms and you should have eliminated that from your choices when you did the gram staining and found that you're dealing with a gram positive organism.
You also didn't need to use PEA agar and chocolate agar.
when dealing with a gram positive organism it is best to use nutrient agar, blood agar(with 5% sheep or horse blood) and mannitol salt agar.

Anyway I hope that this answer has been somewhat helpful to you. :-)
Just a few days ago I was in your position and I was that confused too so I decided to answer your question because I know how it feels to get results that don't match and you feel so lost!!

Good luck xxx
mariah000
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