Lab 6: Metabolic Activities of Bacteria

different bacteria species

produce different types of enzymes that allow them to carry out a characteristic set of biochemical reactions

each bacterial species has

a characteristic metabolism that can be used to distinguish them from other species; unknown bacteria are often identified based on both appearance under the microscope & metabolic properties

pH indicators

chemicals that change color depending on pH (phenol red & bromothymol blue); has a range of pH values over which it changes color

phenol red

pH indicator that turns yellow in acidic media (<pH 6.8), red in neutral media (pH 6.8-7.4), & pink/magenta in basic media (pH >7.4)

bromothymol blue

pH indicator that turns yellow in acidic media (<pH 6.0), green in neutral media (pH 6.1-7.5), & blue in basic media (pH >7.5)

fermentation

metabolic process that some bacteria use to break down glucose when O₂ is not available; includes the reactions of glycolysis (1 glucose molecule is broken down into 2 pyruvate molecules)

durham tube

a small inverted tube that is place within the larger glass tube containing the fermentation medium; detects gases produced when some bacteria ferment a carbohydrate

medium used to test carbohydrate fermentation

a nutrient broth that contains a fermentable carbohydrate (usually a monosaccharide or disaccharide), peptone (amino acids), as well as a pH indicator

carbohydrate fermentation

a metabolic process that some microorganisms use additional chemical reactions to convert other monosaccharides as well as disaccharides into glucose; therefore, bacteria can be differentiated both based on their ability to ferment various carbohydrates, as well as the end products that result from the fermentation process

urea agar slants (urea hydrolysis)

used to test for the presence of a urease enzyme that is produced by some bacteria

bacteria that produce ureases

can break urea down into ammonia and carbon dioxide

urease reaction

CH₄N₂O + H₂O —urease→ 2NH₄OH + CO₂

ammonia that results from the breakdown of urea causes

the medium of the slant to become more alkaline (basic); this pH change is detected by the phenol red indicator and change the medium to a hot pink color

organisms that do not break down urea (urea hydrolysis)

may grow on the slant, but the slant will remain its original color, or will become more yellow (due to the production of acids)

citrate permease

an enzyme that organisms that can survive using citrate as the sole source of carbon have; can transport citrate molecules into the cell, where citrate is converted into pyruvate, which can be converted into a variety of different products

Simmon’s citrate (citrate utilization)

a chemically define medium that contains sodium citrate as the sole carbon source as well as the pH indicator bromothymol blue; bacteria that grow on this medium produce alkaline byproducts that will change the medium color from green (neutral pH) to blue (basic pH)

by products of aerobic respiration (catalase activity)

2 toxic compounds: superoxide free radicals (2O₂^-) and hydrogen peroxide (H₂O₂)

superoxidase dismutase (SOD)

enzyme used by cells to convert superoxide free radicals to hydrogen peroxide

catalase

breaks down hydrogen peroxide into water and oxygen

catalase reaction equation

2H⁺ + O₂^- —superoxide dismutase→ H₂O₂ + O₂ (gas) —catalase→ 2H₂O + O₂

if catalase is present (simple test)

the hydrogen peroxide will be broken down into water and oxygen gas (results in bubbles)

test of catalase production should never

be performed on organisms that have been grown on blood agar bc there is catalase activity in blood that would produce a false positive result

most aerobic and facultative anaerobic organisms

produce SOD and catalase

obligate anaerobes (catalase activity)

lack SOD and catalase enzymes, which is why they cannot survive in an atmosphere containing oxygen

SIM test includes

H₂S test, indole test, & motility

SIM media contains (sulfide test)

ferrous ammonium sulfate and sodium thiosulfate which can be converted by some bacteria into hydrogen sulfide (H₂S), a black precipitate in the otherwise colorless medium

presence of H₂S (sulfide test)

occurs anaerobically and indicates that an organism is capable of producing energy through anaerobic respiration; no reagents are added to this test to detect H₂S

tryptophanase (indole test)

an enzyme that breaks down tryptophan down into indole, ammonia, and pyruvate; the ammonia and pyruvate are converted into other molecules, but the indole accumulate, and can then be detected in the media

indole reaction equation

tryptophan —tryptophanase→ indole + NH₃ + pyruvate

presence of indole

indicates that an organism produces the enzyme tryptophanase

Kovac’s reagent

a chemical that detects indole

after the tryptophan broth as been inoculated, incubated, and had Kovac’s added

the presence of indole will cause a pink or cherry red ring to develop at the top of the broth

motility

movement (flagella) in the deep; any agar deep can be used, though deeps with lower concentrations (0.2-0.5% agarose) are used as they allow motile organisms to move more freely with the deep

cytochrome c oxidase

detects an organism’s ability to carry out aerobic respiration; what the oxidase test is testing

sugars used for fermentation test

glucose, sucrose, lactose