Gram (+) and Gram (-) Bacterial Biochemical Tests

• test microbes' ability to utilize citrate as it's carbon source and ammonium as it's nitrogen source

• sodium citrate, ammonium dihydrogen phosphate, pH indicator bromothymol blue

• positive: medium turns blue as a result of bacteria metabolizing citrate and breaking it down into alkaline byproducts which raise the pH

• no color change occurred (remains green) as a result of bacteria not being able to use citrate

• Gelatinase

• Gelatinase hydrolyzes gelatin into amino acids

• positive: medium remains liquid, enzyme broke down gelatin in medium

• negative: medium solidifies, no gelatinase enzyme present

• Sulfur reduction

• Indole production

• Motility

differential, identifies for enteric bacteria

Tests for production of hydrogen sulfide

• positive: a black precipitate forms in the liquid

• negative: no changes occur in the medium

Tests for the enzyme tryptophanase that breaks down tryptophan

• Kovac's Reagent

• positive: red ring forms at the top of medium, bacteria contain enzyme

• negative: no change, bacteria do not contain enzyme

Tests for bacteria with flagella

• positive: growth radiates outward from stab, bacteria are flagellated

• negative: no growth radiating from stab, bacteria are not flagellated

Tests for the urease enzyme that breaks down urea into ammonia and carbon dioxide

• positive: medium turns hot pink as a result of ammonia production raising the pH, activating the pH indicator of phenol red

• negative: no color change occurred, enzyme urease is not present

Methyl Red, Vogues-Proskauer

• Two biochemical tests used to identify bacterial species by determining their glucose fermentation pathway.

• broth contains glucose, peptone, phosphate buffer

Detects acid fermentation, drops pH below 4.4

After adding methyl red,

• positive: medium turns red

• negative: medium does not change color

Detects butanediol fermentation pathway, neutral pH acetoin

After adding a-naphthol and potassium hydroxide,

• positive: pink/red color change, contains acetoin

• negative: no color change, no acetoin

Used to determine if bacteria can use the enzyme nitrate reductase to convert nitrate into nitrite, or further into nitrogenous gases

1) Check the Durham Tube for a gas bubble inside

• positive: bacteria reduced nitrate all the way to nitrogenous gas

• negative: move on to step 2.

2) Add Reagent A (sulfanilic acid) and B (a-naphlylamine)

• positive: broth turns red, bacteria reduced nitrate to nitrite

• inconclusive: no color change, move on to step 3

3) Add Zinc dust

• positive: red color develops, negative for nitrate reduction because zinc forced reduction

• negative: no color develops, positive for nitrate reduction all the way to nitrogenous gas

Determines which antibiotics will effectively treat a bacterial infection. The zone of inhibition's (zone where bacteria cannot grow) diameter determines how effective antibiotic is against bacteria

This medium grows gram (-) bacteria and separates them into lactose fermenters and non-fermenters.

• selective agents: crystal violet and bile salts inhibit gram (+) bacterial cell growth

• differential agents: lactose allows for expression of fermenters and non-fermenters

• lactose fermenters turn a bright pink or red

• non-fermenters stay a colorless yellow

Triple Sugar-Iron Agar

• differential medium used to identify gram (-) bacteria based on carbohydrate fermentation & hydrogen sulfide production

• triple sugar's glucose, lactose, and sucrose along with phenol red and ferrous sulfate

• slant/butt

• phenol red turns yellow (A) in the presence of acid and remains red (K) under alkaline (basic) conditions

1) Red/Yellow (K/A) - glucose fermentation only

2) Yellow/Yellow (A/A) - glucose fermentation & lactose or sucrose fermentation

3) Red/Red (K/K) - no sugars fermented

• gas production - bubbles in the agar, cracking or lifting up in agar

• hydrogen sulfide production - black precipitate forming in tube

Identifies if a bacterium can breakdown glucose to produce acid and gas.

made out of:

• basal broth (nutrients, peptone, 0.5% glucose)

• pH indicator (phenol red)

• Durham Tube (traps gas)

1) Yellow broth + gas bubble in tube (A/G) - glucose fermented, and gas produced

2) Yellow broth + no gas (A) - glucose fermented, no gas production

3) Red broth + no gas (-) - no glucose fermented, bacteria might use peptone instead

Determines if bacteria produce cytochrome c oxidase, an enzyme used in the aerobic electron transport chain

• positive: dark blue/purple color change in the first 10-60 seconds, organism is aerobic

• negative: no color change, organism is anaerobic