Expert: John Locke - 6/24/2011

Question
Hi,

I'm looking for a wizard for guidance.
My lab report is based on identifying a specific Gram Positive Bacillus from the following tests.
I'm presently searching in EBSCO, JSTOR, and other sites for information.
If I can ask for your help in guiding me through the tunnel?

I've identified a Gram Positive Bacillus, but the following tests are difficult to understand.

1. Blood Agar Plate– Tested Pos., Very little color change = Gamma Hemolysis.   Sample Color: Yellowish/ White
2. Bile Esculin: - Tested Positive – Bacteria Type???- PH Indicator Changed to Black.
   Possible monocytogenes                 http://www.cmpt.ca/pdf_id_tables/gram_positive_bacilli_id_table_10.pdf      http://www.textbookofbacteriology.net/Listeria.html

3. Coagulase Test- Neg Result- No color change
4. Citrate Test: Tested Pos. Bacteria Type?? Difficult to see a strong blue color, Placed Petri dish on a white napkin for contrast to compare to a new unused Citrate Plate – got a blue color
5. Catalase: neg no bubbling
6. Spirit Blue: blue medium gone, huge clear area around bacteria, shd be a halo.
Possible: Bacillus subtilis  http://www.uwyo.edu/molb2210_lab/info/biochemical_tests.htm#spirit_blue

Thank you so much,

Barry

Answer
Thanks for using AllExperts, Barry. You should find a good microbiology reference text, as from a college library, that includes flowcharts of the various microbes according to their microbiologic characteristics. They do exist: Bergey’s Manual of Determinative Bacteriology, for instance, is probably the standard.

Let's consider your unknown. You do know that it's a Gram-positive rod, so consider first the most common possible organisms:

Bacillus spp
Clostridium
Lactobacillus
Listeria
Corynebacterium

The bile esculin test is important, as it rules out a number of these species: Listeria, Corynebacterium, and Lactobacillus are bile esculin positive or variably positive. Now consider the catalase test: yours was negative, so we could rule out Listeria and Corynebacterium (we can also rule out Bacillus on this alone, as Bacillus spp. are catalase-positive). The gamma hemolysis is consistent with Lactobacillus, so far as I am aware. It is coagulase negative also. The results seem to be consistent with Lactobacillus as the unknown organism. However, you will want to get hold of a copy of Bergey's Manual, if possible, to confirm. Without a clear structure for investigating the unknown bacterium, you wind up performing multiple tests that may not be necessary. Those tests include the potential for contamination and inaccurate results that complicate your overall identification. Good luck.