Identifying a mixture of unknown bacteria

I’m a pre-vet student and I took microbiology this year. For our lab practical we were given a vial of two bacteria mixed together and told to find out what they were and we had to do it with no aid from our professor.

The first thing I did was make a streak plate on nutrient agar to separate the unknowns. Each dot is an individual colony started off by a single bacteria separated from the others. The white colony is different than the clearish one. One of these colonies was very stinky! I could already tell what it was by the smell!

The next step was to make a gram stain of each of the colonies. The first being the white colony. Gram stains will make gram + bacteria purple and gram - bacteria red under the microscope. As you may be able to tell, the white colony consisted of many tiny, purple and round bacteria. Purple indicates gram + and round indicates a cocci. That bunching pattern is also indicative of staph aureus but I couldn’t just go by that!

My other colony was a gram - rod. Meaning it was red and, well, shaped like a rod. I didn’t get a quality picture through the microscope though.

Aftermath of the gram staining had me looking like a gram positive bacteria for a week!

After that gram stain I knew one of the colonies was gram + so I put them both on mannitol salt agar for a day and observed the results.

Mannitol salt agar is an agar used to grow gram positive bacteria and turns yellow when acids are being produced. I mislabeled this and switched the bacteria around. On the left you can see little growth occurred but it is yellow, this is because I’m not perfect and got a mixed sample from my nutrient plate. This is the gram - mixed with the staph aureus or staph epidermis.

At this point I knew I either had staph aureus or staph epidermis. Both grow on mannitol salt agar, but only staph aureus makes it yellow. Hmmmmm! But I couldn’t prove it yet!

The final step to proving the first bacteria was staph aureus was by performing a coagulase test. In this you expose the bacteria to fibrinogen and if a coagulant forms (which it most definitely did!) you have staph aureus! Yay my first bacteria is now known!

Here is a picture of my growth plate after letting it grow some more. You can see certain spots had spread out because I collected bacteria from them and they continued to replicate afterwards in the incubator.
But now I need to focus on the stinky clear colony...

I knew the clear colony was gram negative but put both colonies on MacConkey’s agar. This agar selects for gram - bacteria and lactose fermenters will grow pink on it. As you can see, our clear colony LOVED it and completely took over! And it was pink! This ruled out pseudomonas (another stinky bacteria), shigella, and salmonella, but, left E. coli. I knew my next step.

I put the clearish colony on Eosin Methylene Blue agar, EMB for short. This agar tests for lactose fermentation which can by signified by a metallic green sheen on top of the colonies. Lactose fermentation is something that E. Coli is known for, that and stinking. I knew it was E. Coli all along because of the unique smell but now I had proven I was correct!

I had so much fun in this class and I hope some of you have learned something from this process I did ^^ if you have any questions I’d love to chat. I am seriously considering going into microbiology now after this!

Completely unamused cat tax