Unknown Identification

[zoidia]

So i have a very big delema. What i'm basically doing is taking an unknown culture (says theres only just 2 types of bacteria) and growing it on specific and differencial mediums to conclude which two types i have. So i may have one gram-postive and one gram-negative bacteria in the culture tube. First, i inculate PEA (phenylethanol agar) which is particularly for isolation of gram-postive bacteria. Then i inoculate EMB (Eosin Methylene Blue agar) is a selective medium for gram-negative bacteria.

So after incubation i have postive growth on both mediums. Now i take both plates and inoculate MSA (Mannitol salt agar) from the PEA (if the MSA is fermented the pH of the medium is lowered and the medium turns yellow, which is typical of Staphylococcus aureus and under heavy inoculation Enterococcua faecalis but not Staph epidermidis. Also i inoculated Enterococcosal agar, which is a plated medium to identify enteric cocci. This differential medium in that enteric cocci turn this agar black which signifies the hydrolyzing of esculin. Other organisms may grow on this plate but will not turn the color of it.

There was positive growth on my MSA plate So it turned yellow meaning its acidic. Now i look at my enterococcosel plate AND IT WAS MOSTLY YELLOW WITH A LITTLE TINY SPOT OF BLACK!!! Not the kind of thing i wanted to deal with since this project is very crucial to my future. My professor is not allowed to help any student out with this assignment but he was nice enough to give us a clue.. and so i told him the 2 types of bacteria i think it is and he said one of them is wrong. Well for sure gram-negative bacteria is right which is Proteus Vulgaris. And the other answer is gave him was the bacteria that made the enterococcosel agar mostly not black which would be Staphylococcus aureus, and its not right!!! So now i'm thinking the bacteria that made the little spot of black is right which is Enterococcus faecalis. I dont know why or how or what im so lost. I would be very appreiciated if someone could help me out..

after i conducted this experiment i came to conclusion that i have the correct gram-negative bacteria right but not the gram-postive.

 

 

 

Here is a flow chart of how i think i got to the gram-postive

 

 

 

PEA- MSA - Growth or no growth (was growth)- Yellow or not yellow (yellow)- Enterococcosel plate - Black or not black- IDK!!?? Black??

[Genecks]

Buy an enterotube and manual, throw the sucker in, and get through the game.

Other than that, your flow chart isn't showing.

 

What most people don't realize early in the "unknown" game is that if you throw the thing into an enterotube and decipher the result, you can be done within a week: That's for generic bacteria.

 

If I remember correctly, black means that it uses iron sulfide? Or something like that..

[CharonY]

For starters enterotubes were developed for the identification of Enterobacteriaceae.

Thus if you get thrown at with random bacteria it is pretty much useless. Second, the reason for doing these plate assays is train yourself on the technique and the logic behind it and not to stay on the level of a kit user. That being said it would be helpful if you could provide a list of from which you have to select the bacterial species in question, as the combinations in question leave room for quite a number of potential species.

Edited December 7, 2009 by CharonY

[Genecks]

You're right, CharonY. They don't cover all spectrum of bacteria.

Still, I'm sure there is some quick kit to take care of this business.

And if a person can understand how to make a kit and the logic behind the kit, well, then that kind of takes care of making a bunch of dishes.

 

@ OP

Is it even possible to determine the second bacteria with this much data?

I'm not sure.

 

Enterococcus faecalis is gram-pos.

Staph. aur. is gram-pos.

Notice you had two selective plates.

 

You say one is Proteus vulgaris.

 

If you're sure it's not S. aureus, then there should be some other test you did to differentiate between the Enterococcus faecalis idea and S. aureus. That's what will help point you. Perhaps you had contamination on that last test you mentioned, which is why it had yellow and black.

Edited December 7, 2009 by Genecks

[CharonY]

Dishes serve a very different purpose: sterile working and actually seeing the colonies. Also you will notice that many will not behave the way they are supposed to do (which is also part of the training).

Moreover, to my limited knowledge there are only kits for a couple of well-defined groups and even so the identification rate is not stellar. Even well-established one like the enterotube hover at around 90%- again, for well characterized groups.

A good microbiological training should cover all the basics and then move on to using kits.

 

In this particular case for instance, it is important to note, how the black dots look like. Are there in the middle of the colonies or (as it supposed to be) diffusing out of the colony (essentially a zone)? Generally the reaction is quite strong with Enterococci and type D streptococci, and if actually have active growth it should be easily distinguishable.

 

What about colony size? What is the color of the colony? Staphylococci tend to be largish white whereas e.g. corynebacteria tend to be smallish yellowish and micrococci whitish grey, each without a brown or black zone.

 

Also regarding the MSA, where the colonies yellow (with a yellow zone) or more orange?

 

Note that I am not primarily a microbiologist and am just saying something from memory. Apparently the training I received a decade or so back was not that bad as I can still remember some stuff.

[zoidia]

I only need help with my gram-postive bacteria.

 

Anyway, my enterococcosel agar plate is mostly yellow, but where i have streaked the plate looks like its white but there are no specific colonies growing just where i have streaked the plate. The spot that is black is still white streaks but around the streaks is black.

 

So all the possible gram-positive bacteria to chose from are: Bacillus megaterium (no), Staphylococcus aureus (maybe), Enterococcus faecalis (maybe) and Staphylococcus epidermidis (nope, i dont think at least). The only bacterium that turns the palte black is Enteroccus faecalis.

 

-thx zoidia

[Genecks]

Well, seeing as getting that entero would be unusual, since most people use gloves when playing with germs, I'm going to throw out the idea it was the entero. It'd be kind of unusual for it to show up. S. aureus is around a lot, but for it to give a black dot? Probably not.

[CharonY]

It is a bit unfortunate that you have not diluted it more to obtain colonies. However, if they form streaks it is extremely unusual to see only black dots instead of a large brown-black zone exuding from the streaks. What do you define as spot?