Biochemical Unknown Help Please - (Apr/25/2011 )

Hi all, I had to choose two unknown microorganisms at lab. one, pertaining to a number, and the other pertaining to a letter. in this case, my organisms are "3" and letter "o". i did several tests. six on plates and six on test tubes (broth and slants). To make things easy, I have attached the picture files that i have taken which I have observed in the lab along with their descriptions of what I saw. please find the attached copy. Please save the copy and zoom in the description details.

After incubation, my test results on plates and testtubes are as follows.
plates:
1)PEA - 3 grew, o was clear.
2)EMB - 3 was clear, o grew.
3)MSA - 3 grew and yellowish, o clear but reddish on side.
4)TSA - 3 grew, o grew. after catalase test, both bubbled but 3 faster and stayed longer whereas o slower but faded soon.
5)MAC - 3 clear, o grew.
6)Starch - 3 grew, o grew. Iodine test proved negative since none had clear zone of hydrolysis.
testtubes:
7)TSA slant - 3 showed pigmentation, o was clear.
8)Gelatin - 3 showed no change except for liquification, o had thin pink layer on surface and butt.
9)Citrate slant - 3 remained green -ve, o turned blue +ve.
10)Urea - 3 was orange -ve, o was orange -ve.
11)SIM - 3 very blackish and motile, o normal or no change in color but motile.
12)Phenol red fermentation broths - 3 luctose, sucrose and glucose all turned yellow and showed no presence of gas on durham tube, o had only one tube red (same color), rest yellow and no presence of gas in durham tube.

One out of the two microorganims can be mine which is a gram negative and the other is a gram positive. Here are the list of microorganims that can be mine for "3" and "o":
1)E.coli and L.lactis
2)E.aerogenes and P.mirabilis
3)Pseudomonas and S.epi
4)Klebsiella and S.aureus
5)Serratia and S.pyogenes
6)Citrobacter freundii and B.subtilis
7)Alcaligenes faecalis and M.luteus
8)P.vulgaris and E.faecalis

Help needed desperately. Will rate lifesaver. Its worth a 100 points and I really need to get this done. If you are not able to see my attached copy. Please let me know then I can email it to you.

Thank You and a Happy Easter to you all.

Note: I have concluded that "o" is gram -ve and "3" is gram +ve with EMB and PEA plates. I cannot conlude the same with gram stain though. My gram stain for "o" shows -ve because it is purple dots in color but I also saw red/pinkish inside those blue dots. And as for shape and arrangement it shows cluster and cocci. In contrast, "3" did not show anything. I think iwill have to do it again.

You need to make sure you start off right with either gram + or gram -. So do the gramstrain again.
And do you have a book like Bergey's Manual of Systematic Bacteriology to help you? DO you have a basic flow chart to identify bacteria?

something like here:http://toolboxes.flexiblelearning.net.au/demosites/series4/412/laboratory/forms/fmChartBacterialIdent.htm

And we cant see your pictures. I think its because you have only one post.. you can not attach pictures before you have more posts I think.

A quick one: can you do a 16S PCR and sequencing?

@pito - Thank you for your help. I just realized that my picture is 6.1mb because of merging all the lab pictures together. and the max limit a user can upload here is 2.5mb or so. Is it possible if I could email you guys the picture. That would be really great.

@adrian - No. We weren't taught those methods in our school. Does it work?

ps. I am new on here and I haven't made a thread before. And I am getting so many replies and help. Thank you so much. I really appreciate it. You could send me an email at nelsongomes007@hotmail.com. That way I could email the lab picture to you.

nelson on Mon Apr 25 15:55:12 2011 said:

its best not to put your email adres on a website.

Anyway: do the gram stain. that is your start.

If you did that, you can go further.

You have pairs of bacteria.. if you do the gramstain you allready elimenate some of the pairs...

The picture , is not needed right away.

about the 16S PCR and sequencing, this the way how they identify bacteria normally (well not always, but often). But I am guessing you are doing some schoolproject thats is about the basics of bacteriadetermination... So never mind the PCR techniques.

Hey man, I google your email and saw you sings on youtube... Is that you singing the Ryan Cabrera song?

I don't know this is your school project.... sorry about the PCR suggestion.

adrian kohsf on Mon Apr 25 19:50:00 2011 said:

Well I think it is because he said this in his first post:

So I am guessing its a schoolproject...

Yep. Right. And it's counted towards my final grade ='(

Here's an online guide to micro biochemical tests.

Biochemical Tests for Microbiology with pictures

and anotherPractical Review

Based on your phenol tests, if you have no indication of gas with fermentation, you probably don't have E. coli. Lactose and glucose would show gas accumulation and red color in sucrose means no fermentation.

Also, E. coli gives a clear SIM, not black.

What lab rat tells you is valuable information.

Now: as I said before, start with the gram stain, make sure you are 100% sure about gram+ or gram -
This is your start!
(however: you used 3 media to see the difference between gram + and - so you should allread be able to state that 3 is + and o is gram -)

combination 2 are both gram -
so they can be elimated very fast if you have a gram +

And then use the PEA to differentiate anaerobic vs aerobic.
Those two steps can say a lot allready.

And use the flow chart I added in this message.

Just write down what each test means, make a table of it and write down the basic characteristics of each bacterium (you can elimante pair 2 I think, so only 7 pairs left , 14 bacteria and write down 4 characteristics per bacterium. Start with the anaerobic vs aerobic, this will allready make a big difference.)

And for example: MSA is highly specific, so if you get growth on it, its most likely a staph.. and especially if the medium turns yellow...

So you see how you can use the information?

In general: start with a very basic differentation: gram - vs +
then anaerobic vs aerobic
(generals are easy to make and usefull to make a clear distinctions between the samples)

and then you can use certain very very speficif media you have with results to immediately make a very very big distinction. Like the MSA, very specific, and with the information you allready have a clear view on 1 bacterium.
So with this information is allready clear what bacterium you have... To be sure: just check the characteristics of the bacterium you think it is based on the MSA and check it with the rest of the plates.. if they are all + (a match) you know what you have...


(esp knowing Pseudomonas does not ferment lactose, while Klebsiella does..)