Micro lab practical

Glucose, Lactose, and Mannitol fermentation tests

Glucose

• ferment glucose to produce acidic and gaseous end products

Lactose

• ferment the sugar lactose to produce acidic and gaseous end products

Mannitol

• ferment the sugar alcohol mannitol, producing acidic and gaseous end products

(+) = yellow acid and maybe gas

G, L, or M —→ fermentation —→ acid/gas

Oxidase Test

determines if a bacterium produces the enzyme cytochrome c oxidase, which is part of its electron transport chain

(+) = blue

Catalase test

determines if a bacterium produces the enzyme catalase, which breaks down toxic hydrogen peroxide into water and oxygen,

(+) = bubbles

H2O2 —→ via catalase —→ H2O + Oxygen

Nitrate reduction test

determine if bacteria can utilize nitrate as an electron acceptor during anaerobic respiration, specifically by producing the enzyme nitrate reductase to convert nitrate into nitrite

(+) = red

Nitrate —→ via nitrate reductase —→ nitrite

Indole test

determines the ability of bacteria to produce the enzyme tryptophanase, which breaks down the amino acid tryptophan into indole

(+) = red

Tryptophn —→ via tryptophanase —→ indole

Methyl red test

determines if bacteria perform mixed-acid fermentation, producing stable, high-concentration acid end-products from glucose

(+) = red

Glucose —→ via mixed-acid ferm. —→ acid

Vogues-Proskauer test

detects acetoin (acetylmethylcarbinol), a neutral byproduct produced during the fermentation of glucose via the 2,3-butanediol pathway

(+) = red

Glucose —→ via fermentation —→ acetoin

citrate test

determines if a bacterium can use citrate as its sole carbon source and ammonium salts as its sole nitrogen source

It specifically tests for the enzyme citrate permease,

(+) = blue

Starch agar test

determines if a microorganism produces extracellular enzymes, specifically amylase and oligo-1,6-glucosidase

(+) = halo

Skim milk agar test

to detect if microorganisms produce extracellular enzymes, specifically caseinase, which break down the milk protein casein

(+) = halo

Spirit blue agar test

to detect and identify microorganisms that produce the enzyme lipase, which breaks down lipids (fats) into fatty acids and glycerol.

(+) = halo

Lipids —→ via lipase —→ glycerol + fatty acids

Urea agar test

to differentiate microorganisms based on their ability to produce the enzyme urease, which hydrolyzes urea into ammonia and carbon dioxide

(+) = pink

urea —→ via urease —→ ammonia + CO2

Kligers iron agar test

Kligler’s Iron Agar (KIA) is used to differentiate Gram-negative enteric bacilli by detecting their ability to ferment glucose (dextrose) and lactose, produce hydrogen sulfide, and produce gas

Yellow (A)→ Acidic (fermentation)

Red (K)→ Alkaline (no fermentation, peptone use)

Black→ H₂S hydrogen sulfide

Motility test

to determine if bacteria can move independently using flagella

Colony morphology

the visual characteristics of bacterial or fungal colonies grown on agar plates

Agar slant growth

describes the visual appearance of bacterial growth along the inoculated line, used for identification

Protease production (gelatin slab)

identifies microorganisms that produce extracellular gelatinases, which hydrolyze gelatin into amino acids, causing liquefaction

(+) = liquid

Oxygen requirements

Obligate aerobes - Growth exclusively at the top of the tube, where oxygen concentration is high

Obligate anaerobes - Growth only at the very bottom, where oxygen is absent

Facultative anaerobes - Growth throughout the tube, but usually with a higher concentration near the top.

Aerotolerant anaerobes - uniform growth throughout tube as they do not use oxygen but are not harmed by it

Microaerophiles - Growth in a thin band just below the surface, requiring low levels of oxygen

Gram stain

Distinguishes between Gram-positive (thick peptidoglycan layer) and Gram-negative (thin layer) bacteria.

(+) = purple

acid fast stain

It works by staining wax-like, mycolic acid-rich cell walls red with carbolfuchsin, which resists decolorization by acid-alcohol, unlike non-acid-fast bacteria.

(+) = pink

Bacteria used in capsule stain

K. pneumoniae

Function of capsule

protection and attatchment

Step excluded in the capsule stain

heat fixation (it will destroy the capsules)

Bacteria used in UV experiment

S. aureus and B. cereus

Grows unexposed to UV light

S. aureus

Growth exposed and unexposed to UV light

B. cereus (endospores)

Bacteria used in salt concentration experiments

E. coli, S. aureus. H. salinarium

Will not thrive in a salty environment (non-halophile)

E. coli

Will grow in low or high salt concentration (Halotolerant bacteria)

S. aureus

Favorable growth in hight salt environment (halophile)

H. salinarium

Why will an increase in temperature decrease the pigment (prodigiosin) that ________ produces?

1. S. marcescens

2. proteins denature as temp increases (42 C)

Blood agar test

looking for beta-hemolysis from alpha toxin in staph

Antiseptic

• slow down bacteria growth, will not kill them

• resistance is rare

Antibiotic

• kills bacteria

• Resistance is common

Kirby-Bauer method

• uses Mueller-Hinton agar

• pH 7.2-7.4 (same as human)

• incubated at 37 C (same as human)

• observe zones of inhibition

Gram negative bacteria in the antimicrobial experiments (Kirby-Bauer method)

• E. coli

• P. aeruginosa

• P. vulgaris

Gram positive bacteria in the antimicrobial experiments

S. aureus (penicillin can’t penetrate peptidoglycan)

Implications of small zone of inhibition

bacteria = resistant, antibacterial not effective

implications of large zone of inhibiiton

bacteria = sensitive, antibacterial was effective

Yogurt fermentation

homolactic fermentation: convert sugars (like glucose) almost exclusively into lactic acid

First step in yogurt fermentation

S. thermophilus ferments lactose to lactic acid (thermophile)

Second step in yogurt fermentation

L. bulgaricus ferments in fridge, provides flavor and aroma (mesophile)

% of staph infections from MRSA

2%

what most staph infections are caused by

S. aureus