Bacteria Identification Help

[Lisa McEm]

Hi, everyone! I'm currently enrolled in an Introductory Microbiology course and our final project is to separate and identify two unknown bacteria. I successfully identified one, but the other is proving difficult. Here is what I have so far:

 

Culture morphology - the bacteria grows very fast, with large colonies (about 5mm or so). Colonies are opaque, cream-to-brown in color, irregular, with undulate margins. The texture is like rippled glass.

 

 

Cell morphology - they are Gram positive rods that form oval spores. They look fairly large and form chians. Some of the chains can be pretty long.

 

Gram Stain:

 

Spore Stain:

 

Biochemical Tests:

Blood Agar - there was no hemolysis on the culture I grew, which ended up filling about a third of the plate.

Extracellular amylase on starch agar - postive

Glucose fermentation - positive for acid, no gas produced

 

According to my flow chart, I have narrowed the possibilities down to three: B. cereus, B. subtilis, and B. megaterium. The flow chart says only B. megaterium would be gamma hemolytic, but that it also would be negative for amylase on the starch agar. Mine was definitely positive. Also, the colony morphology just does not look like what I've seen for B. megaterium. The cultures, to me, look like B. cereus, but the flow chart says B. cereus would not ferment glucose. I've read conflicting information about this on several sites. It could be B. subtilis, but then why was it not beta hemolytic?

 

Are there any other tests I can do to figure this out?

[CharonY]

Depending on strain hemolysis is sometimes not terribly strong. Are the cells motile (B. cereus)? Another test is the Voges-Proskauer test (B. megaterium, would be negative, the others positive). But that being said, I am pretty sure that B. cereus does ferment glucose anaerobically. However, they still grow very poorly if the medium is not very rich (which may lead to false negatives).

[Lisa McEm]

Thank you so much for your reply, CharonY! Thanks for the tip on the V-P test, I will try that out. I've read from several sources that all three of those Bacillus species ferment glucose. I'm not sure why my flow chart for this project says that B. cereus does not. I'm also going to test the bacteria on MSA. I've read that B. cereus cannot ferment mannitol, but B. subtilis can. Is that true?

[CharonY]

I am pretty sure about the inability of B. cereus, but I would have to look it up for B subtilis. Yes, you are correct.

However, colony and cell morphology are somewhat distinguishable between those two as an additional factor. But that really only helps if you got a reference or experience with them.

Edited July 26, 2013 by CharonY

[Lisa McEm]

Thank you again for your help. Everything, except the hemolysis factor, points to B. cereus - it was positive for the V-P test and negative for mannitol fermentation. It looks like B. cereus to me, too, so that's what I'm going with. Hope I'm right!