Microbial Metabolic Tests                                         Lab Handout/Sprenkle    

 

Why are we doing this?  Why do I care?  Because it will help you figure out what your unknown organism is.  Not every microbe carries out the same set of biochemical reactions.  The varied metabolic activities of different microbes helps the lab investigator to identify what type of bacterium is in an unknown sample. 

 

How bacteria ‘EAT’:

 

Bacteria produce exoenzymes that work outside the cell to break down large molecules that can’t be transported into the cell.  These enzymes are usually hydrolytic, in that they break the molecule by enzymatic addition of water.

Carbohydrates: molecules with C, O, H in the ratio (CH2O)n

Carbohydrates can also be classified by size: 

monosaccharide (ex: glucose)< di- (ex: sucrose)< poly-<oligo- (ex: starch)

Proteins:  polymers of amino acids (a.a) Examples of amino acids: glycine, arginine, cysteine…almost anything with the suffix “ine” at the end.)

 

The exoenzymes we looked at in lab were:

          Amylase:  breaks down starch; an oligosaccharide, a carbohydrate.  (What are the monomers that result?)

          Gelatinase:  breaks down gelatin, a protein.  (What are the monomers that result?)

          BIO 406 only: DNAse, Lipase

Once big polymers are broken down with exoenzymes the sugars or amino acids that are transported into the cell can be metabolized, used for nutrient or energy sources.

 

Proteins can be metabolized but usually only when carbohydrates are not available.  Steps in the breakdown of individual amino acids can be deamination or decarboxylation.

 

Utilization of Carbohydrates:

          Oxidation/Fermentation Tubes (O/F tubes: set of two green tubes +/- oil on top).  There is a sole carbon source added; in our case, glucose, though other sugars can be tested.  The culture is inoculated into two tubes and one is covered with oil.  The ‘open’ tube is the O tube.  The oil covered tube is the F tube.  If the media turns yellow , the organism is said to be an O, an F, O/F, or neither.

 

Fermentation of Carbohydrates:

The ability to ferment various carbohydrates to acidic products +/- gas can be tested with fermentation tubes.  A pH indicator reveals acidic products (color change to YELLOW), and an inverted tube (Durham tube) traps gas.  Results are reported as +/- acid and +/- gas.

Large groups of related organisms can be differentiated based on which of two different fermentation pathways they carry out.  The MIXED ACID PATHWAY or the BUTYLENE GLYCOL pathway.  Products of these paths are detected in the MR-VP test.  If an organism uses the mixed acid pathway, the products are very low pH (~4), and are detected with the Methyl Red pH indicator (MR).  If the butylene glycol pathway is used, a product of the path, acetoin, is detected in the VP test (Voges-Proskauer).

 

Aerobic or facultative organisms have different subsets of oxygen detoxifying enzymes, and can be differentiated in the detection of catalase.  Catalase is an enzyme that converts hydrogen peroxide to oxygen and water.  To test for the enzyme, cells can be placed directly in hydrogen peroxide.  If bubbles are observed, they are the oxygen gas produced by catalase. 

 

Cytochrome C Oxidase is the terminal electron transport protein present in some organisms that carry out oxidative respiration.  It is detected by the Oxidase test on a “dry slide”, a filter paper that has been saturated with a chemical that turns dark blue in the presence of cytochrome C oxidase.

 

The tests we use in lab are:

 

Use of Oxygen

Carbohydrate catabolism

Protein/amino acid  catabolism

 

Fermentation Tubes

Urea slant

Catalase Test

MR-VP

MIO deep

Oxidase Test

Starch Hydrolysis

Phenylalanine slant

 

OF Glucose

Peptone Iron deep

 

 

Nutrient gelatin

 

One of these tests is a ‘multiple test medium’, meaning you get answers to multiple aspects of bacterial metabolism all in one tube.  Which one is it?

 

For BIO 406 only: additional multiple test media: TSI slants, SIM deeps

 

Make sure you know what each test tells you if you are given a description of the appearance of the result.

 

What is ORNITHINE?

What does the INDOLE test look for?

 

Also make sure you understand how to read and use a “dichotomous key.”.