Microbio Lab Practicum 1

facultative aerobe

Both aerobic and anaerobic growth; more growth in the presence of oxygen.

-gradient down, mostly at the top

-SOD and Catalyse

aerotolerant anaerobe

Only aerobic growth, but grows in the presence of oxygen.

-even distribution

-SOD and Peroxidase

gas pack reaction

2H2 + O2 = 2H2O

What is the OF test?

determine whether a bacteria uses oxidative or

fermentative carbohydrate metabolism or is inert

-indicator = bromthymol blue

- yellow = positive, green = negative (no color change)

What is the sugar fermentation test?

microbes ferment glucose to produce gases,mainly carbon dioxide and hydrogen, can use the sugar provided for carbon, hydrogen, and energy

-indicator = phenol red

- yellow = positive, red = negative, gas = positve

What is the Methyl Red (MR) test?

dentifies enteric bacterial ability to produce stable acid end products bymeans of a mixed-acid fermentation of glucose

-indicator = methyl red

- positive = red, negative = yellow

What is the VP test?

detect the production of acetoin, a neutral end product of glucose fermentation

- indicators = a-napthol and postassium hydroxide (KOH)

-positive = red ring no change = negative

What is the catalase test?

This test detects the presence of catalase.

- indicator = hydrogen peroxide

- positive = bubbles, negative = no bubbles

What is the oxidase test?

identifies bacteria that have cytochrome oxidase

- indicator = dimethyl-p-phenylenediaminehydrochloride

What is the nitrate reduction test?

tests for anaerobic respiration, can nitrate be reduced?

-indicator = sulfanic acid, α-naphthylamine, zinc

- initial = gas (+) no gas (-)

- add sulfanic acid/a-napth = red (+), no change/yellow (-)

-add Zn = red (-), no change (+)

What is the citrate utilization test?

This test helps differentiate among gram-negative bacteria, and citrate-permease.

-indicator = bromothylmol blue

- blue (+), growth w/o color change (+), no growth/green slant (-)

What is the decarboxylation test?

can the microbe can decarboxylate amino acids using decarboxylase.

-indicator = bromcresol purple

-purple = (+)

yellow = -

What is the Phenylalanine Deamination Test?

Detects if the organism produces phenylalanine deaminase, which removes the amino group from phenylalanine.

-indicator = ferric chloride (FeCl3)

- after adding FeCl3 = green (+) no change (-)

what is the Tryptophan Hydrolysis / Indole Test?

Tests for the enzyme tryptophanase, which breaks down tryptophan to indole.

-indicator = kovacs reagent

- red/pink on top (+)

- no change/yellow (-)

What is the hydrolytic enzyme (Bile Esculin) tests?

Tests for growth in the presence of bile salts, and

Hydrolyze esculin into esculetin and glucose.

indicator = ferric citrate

- change to dark = +

- no change = -

What is the starch hydrolysis test?

tests for amylase starch breakdown

indicator = iodine

results = clear zone = positive blue/black = negative

What is the urea hydrolysis test?

tests for urease, which breaks down ammonia to co2

- indicator = phenol red

- pink = positve yellow = negative

What is the OPNG test ?

Tests for β-galactosidase activity

indicator = ortho-nitrophenol

yellow=+

no color = negative

What is the gelatin hydrolysis test?

tests for gelantinase - breaks down gelatin

indicator = state of gelatin

liquid = positive

solid = negative

What is the Kligler's Iron Agar (KIA) test?

Tests for glucose and lactose fermentation and hydrogen sulfide (H₂S) production.

indicators = glucose, lactose, and phenol red

yellow butt, red slant = glucose fermentation

all yellow = glucose and lactose

pink/orange = no fermentation

black precipitate, bubbles, cracks = H2S

Gram Stain

Purpose:To differentiate bacteria based on the structure of their cell wall — specifically, the thickness of peptidoglycan.

Indicator/Dye System:

Crystal violet (primary stain)

Iodine (mordant)

Alcohol/acetone (decolorizer)

Safranin (counterstain)

Acid-Fast Stain (Ziehl-Neelsen or Kinyoun method)

Purpose:To identify bacteria with waxy, mycolic acid-rich cell walls (e.g., Mycobacterium species).

Indicator/Dye System:

Carbol fuchsin (primary stain, requires heat for Ziehl-Neelsen)

Acid-alcohol (decolorizer)

Methylene blue (counterstain)

Result Interpretation:

Acid-fast: Bright pink/red (retains carbol fuchsin)

Non–acid-fast: Blue (decolorized, takes up counterstain)

Capsule Stain (Negative stain)

Purpose:To detect bacterial capsules, which are protective layers that resist staining.

Indicator/Dye System:

India ink or nigrosin (stains background)

Crystal violet (optional, can stain the cell)

Result Interpretation:

Capsule: Appears as a clear halo around the stained cell and against a dark background

Cell: Stained; background is dark

Endospore Stain (Schaeffer-Fulton method)

Purpose:To detect endospores, which are highly resistant, dormant structures produced by some bacteria (e.g., Bacillus, Clostridium).

Indicator/Dye System:

Malachite green (primary stain, applied with heat)

Water (decolorizer)

Safranin (counterstain)

Result Interpretation:

Endospores: Green

Vegetative cells: Red/pink

Microaerophiles

growth in low oxygen concentration

-growth in middle

-SOD

-Peroxidase