Biochemical Tests for Unknown

Phenol Red Broth –

Phenol red broth serves three purposes.

1.  A single carbon sources is added to test whether the organism can utilize that carbon source.  In our experiment we utilized lactose, mannitol, sucrose, and glucose.

2.  Phenol red turns yellow in acidic conditions, since acid production is a sign of fermentation we can determine whether a carbon source is fermented by the test organism.

3.  The small tube inside the larger tube is called a Durham Tube; it is used to capture gases produced during growth.  Fermentation produces a large amount of gas and the Durham Tube is utilized to determine this metabolic activity.

So in this experiment we will determine the following:  growth/no growth, acid/no acid, gas/no gas.

Nutrient Gelatin –

Gelatin is a solidifying agent that some microorganisms can digest.  This will be a simple yes/no answer as to whether gelatin can be digested.

Motility Media –

Motile organisms will move out into the soft agar from the point where the organism is inoculated.

Thioglycollate Media –

Thioglycollate media determines whether an organism is an obligate aerobe or a facultative anaerobe.  Obligate aerobes will grow in the upper third of the tube, while facultative anaerobes will grow through out the tube.

Casein Media –

Casein media is to test whether the organism can digest the majority protein of milk, casein.  Digestion will result in a clear zone around the bacteria.

Starch Plate –

Starch digestion is determined by a zone of clearance around the bacterial colony when the plate is exposed to iodine.

Catalase –

Catalase is an enzyme that breaks down hydrogen peroxide.  A catalse positive organism will produce bubbles when exposed to hydrogen peroxide.  A catalase negative organism will produce no bubbles.

ENTEROTUBE                                             POSITIVE                  NEGATIVE

Glucose:  Acid from Glucose                            YELLOW                    RED/ORANGE

Gas:  Gas from Glucose                                    GAS BUBBLE             NO GAS

LYS:  Lysine Dehydrogenase                            PURPLE                      YELLOW

ORN:  Ornithine Dehydrogenase                       PURPLE                      YELLOW

H2S:  Hydrogen Sulfide Production                   BLACK                       TAN

ADO:  Acid from Adonitol                                YELLOW                    RED/ORANGE

LAC:  Acid from Lactose                                 YELLOW                    RED/ORANGE

ARA:  Acid from Arabinose                              YELLOW                    RED/ORANGE

SOR:  Acid from Sucrose                                 YELLOW                    RED/ORANGE

DUL:  Acid from Dulcitol                                  YELLOW                    GREEN

PAD:  Phenylalanine Dehydrogenase                 BROWN                     GREEN

URE:  Urea Hydrolysis                                     PINK/FUSCHIA         TAN

CIT:  Citrate Hydrolysis                                   PURPLE/BLUE            GREEN

API STAPH                                                               

GLU:  Acid from Glucose                                 YELLOW                    RED/ORANGE

FRU:  Acid from Fructose                                YELLOW                    RED/ORANGE

MNE:  Acid from Mannose                              YELLOW                    RED/ORANGE

MAL:  Acid from Maltose                                YELLOW                    RED/ORANGE

LAC:  Acid from Lactose                                 YELLOW                    RED/ORANGE

TRE:  Acid from Trehalose                               YELLOW                    RED/ORANGE

MAN:  Acid from Mannitol                               YELLOW                    RED/ORANGE

XLT:  Acid from Xylitol                                    YELLOW                    RED/ORANGE

MEL:  Acid from Melbiose                               YELLOW                    RED/ORANGE

RAF:  Acid from Raffinose                                YELLOW                    RED/ORANGE

XYL:  Acid from Xylose                                   YELLOW                    RED/ORANGE

SAC:  Acid from Sucrose                                 YELLOW                    RED/ORANGE

NAG:  Acid from NAG                                    YELLOW                    RED/ORANGE

ADH:  Arginine Dehydrogenase                        RED                             YELLOW/ORANGE

URE:  Urea Hydrolysis                                     RED                             YELLOW/ORANGE

All chambers not listed above can be ignored at this time.  The unused chambers require additional reagents that are slightly hazardous and do not add significantly to you ability to identify the unknowns.

*  Both DUL and PAD are in the same chamber and a positive result for the PAD test will mask the DUL result.

Return to BIO220 Homepage