Lecture 21:
Identification of Bacterial Unknowns IV

I. The MR/VP test is used to differentiate between ________________________ ________________________ organisms based on the way they ______________ ___________________.


Organisms can usually only be _______________________________________. However, organisms can be MR-, VP-.

Examples:
Escherichia coli is ___________________.
Enterobacter aerogenes is ________ and VP+.
Pseudomonas aeruginosa is MR- and __________.

II. Simmon’s Citrate Agar

A. This is a ____________________________________ used to determine if an organism can use citrate as its ________________________________. It is often used to _____________________________________________ _________________________________.
B. In citrate-positive organisms, the enzyme ______________________ hydrolyzes citrate into oxaloacetic acid and acetic acid. The oxaloacetic acid is then hydrolyzed into pyruvic acid and ____________.
D. If CO2 is produced, it reacts with components of the medium to produce an _______________________________________ (e.g. Na2CO3). The alkaline pH turns the ____________________________________________ from __________________________________. This is a _____________________ result.
E. Escherichia coli and Shigella dysenteriae are ___________________ whereas Klebsiella pneumoniae and Proteus mirabilis are ___________________.

See Summary of Biochemical Tests (Simmon’s Citrate Agar)

III. Spirit Blue agar

A. This agar is used to identify organisms that are capable of producing the enzyme ___________________. This enzyme is secreted and _______________________________________ to ___________________ and three ____________________________________________. These compounds are ___________________________________ to pass through the bacterial cell wall.


1.) Glycerol can be converted into a ______________________ __________________________.
2.) The fatty acids can be ____________________________ and their fragments can eventually enter the ___________________ ______________________.

B. Spirit blue agar contains an emulsion of _________________________ ___________________________. Bacteria that produce lipase will hydrolyze the olive oil and produce a ______________ around the bacterial growth.

See Summary of Biochemical Tests (Spirit Blue Agar)

C. Bacillus subtilis is lipase ____________________.

IV. Starch hydrolysis test

A. This test is used to identify bacteria that can ______________________ _____________________________________________ using the enzymes ________________________ and oligo-1,6-glucosidase.
B. Often used to differentiate species from the genera _________________ __________________________________.
C. Because of the _______________________________ of amylose and amylopectin molecules, these starches ______________________________ through the bacterial cell wall.

1.) In order to use these starches as a carbon source, bacteria must _____________________ a-amylase and oligo-1,6-glucosidase into the extracellular space.
2.) These enzymes ________________________________________ ______________________________________ subunits which can then be utilized directly as carbon sources.

D. After incubation of the starch plates, _____________________ is added to the top of the agar. The iodine reacts with the starch to form a ____________________________________. Hydrolysis of the starch will ___________________________________________________around the bacterial growth.

See Summary of Biochemical Tests (Starch Hydrolysis Test)

E. Bacillus subtilis is amylase ___________________.

ALSO TODAY:
*Perform ____________________________________ needed to help determine your unknown: oxidase, catalase, various stains.
*Determine your unknown and _______________________________________ __________________________________.
*Papers - Consult the lab notebook for a description of the report format. If you have any questions about the report be sure to ask today.
* Remember the late policy: _____________________________________ that a report is late. Late reports are not accepted after 1 week.
* Reports are due on ___________________!
*Make sure that once you have correctly identified your unknown, you discard all your plates and slants in the biohazard, tape in the trash, and wash all the slides.

Rachel Watson, M.S.
AG 5010
766-3524
Cell: 307-314-9636
rwatson@uwyo.edu