Microbio Lab Quiz #5

Free radicals

When oxygen gets converted into a dangerous compound

• Molecular species capable of independence existence that contains an unpaired electron

Obligate aerobe

Requires presence of O2 to grow

• Capable of neutralizing free radicals that form O2 & convert them into safer/non-damaging molecules

• Perform aerobic cellular respiration

Microaerophile

Require lower levels of atmospheric O2

• Too much will kill it but not enough will kill it

Facultative anaerobe

Able to survive in anaerobic conditions

• Grow best and fastest when O2 is present but will grow without it as well

Obligate anaerobe

Unable to survive in O2

• Doesn’t express enzymes to mitigate harmful byproducts of aerobic respiration

Aerotolerant anaerobes

NO oxygen used for any metabolic processes but doesn’t die when exposed to oxygen

Fluid thioglycollate

Fluid (semi-solid) growth medium that forms oxygen gradient

• Autoclaving drives free oxygen out of medium

• Oxic zone (pink layer): contains atmospheric oxygen

• Anoxic zone (yellow layer): as you move down the tube, the broth becomes anoxic (no atmospheric oxygen present)

Enriched media

Type of cultural media used to grow microorganisms

• Fastidious organisms: any organism that has complex/particular nutritional requirements (very high nutrient concentration)

• Only grow when specific nutrients are included in its medium — difficult to culture & can’t be cultivated on general growth medium

Sheep’s blood agar

Enriched media used for cultivation of pathogenic organisms capable of producing extracellular enzymes that cause hemolysis of blood

Hemolysis

Breaking down RBCs for nutritional means

• Types: gamma hemolytic, alpha hemolytic, and beta hemolytic

Gamma hemolytic

Organism has NO ability to break down RBCs for metabolism

• Lack the proper enzymes that can be made & secreted in order to break down RBCs

• Color: NO color change on agar

Alpha hemolytic

Organism can perform partial RBC breakdown (incomplete lysis of RBCs)

• Reduce hemoglobin to a compound called met-hemoglobin

• Color: greenish-brown “bruise-like” appearance

Beta hemolytic

Organisms capable of fully breaking down and utilizing RBCs

• Fully destroy and utilize RBCs for metabolism

• Color: zone of clearing surrounding bacterial colonies — opaque red agar becomes see through

Fermentation

Anaerobic pathway in which an organic substrate (glucose or carbs) serves as final electron acceptor & produce organic alcohol/acid as waste byproduct

• Doesn’t produce as much energy as respiration (2 ATP molecules vs. 32-36 ATP molecules)

Glycolysis

Only energy extraction pathway during fermentation

• Metabolic pathway that converts glucose into pyruvate, generate small amounts of ATP, while producing NADH

Sugar fermentation results

Result 1: organism is negative for sugar fermentation

• Tube broth stays red & didn’t change color

Result 2: organism is positive for sugar fermentation

• Tube broth changes from red to yellow (acid present)

• Negative for CO2 gas production (no air space in Durham tube)

Result 3: organism is positive for sugar fermentation

• Tube broth changes from red to yellow (acid present)

• Positive for CO2 gas production (air space in Durham tube)

IMViC

Indole, Methyl Red, Voges-Proskauer, and in Citrate — group of tests run to identify bacteria in Enterobacteriaceae family

• Can differentiate between Escherichia’s, Proteus’s, Klebsiella’s, Salmonella’s, etc

Indole

Determine whether or not your unknown organism can produce tryptophanase — breaks down tryptophan

Indole test - tryptophan metabolic reaction

Tryptophan + H2O → indole + pyruvate + ammonium

• If end product (indole) is present, the reaction moved forward & contains tryptophanase

Kovac’s reagent (indole test results)

Kovac’s reagent binds to indole & produces bright cherry red color change in reagent liquid layer when binding takes place

• Reagent added AFTER inoculation & incubation

SIM agar

Sulfide, indole, and motility agar — used to determine these 3 test results

• Test results: hydrogen Sulfide production, Indole test results, and Motility

Methyl red

Used to determine if unknown organism can ferment glucose to an acidic end product (end pH of around 4)

• Red color change: pH below 6 — positive for acidic end product

• Slightly yellow/no color change: pH above 6 — negative for acid end product (non-acidic)

Voges-Proskauer

Used to determine if organism can ferment glucose to a non-acidic/neutral end product (end pH of around 6.5-7)

• Use two reagents: Barritt’s A & Barritt’s B

Voges-Proskauer test results

Organism A: tube turned red after addition of reagents

• Positive for glucose fermentation to neutral/non-acidic end product

Organism B: tube turned slightly yellow/no color change

• Negative for glucose fermentation to neutral/non-acidic end product

in Citrate (citrate utilization test)

Uses citric agar plate to determine whether unknown organism possess enzymes citrase & citrate permease → necessary to produce citrate as carbon source

• Determines organism’s ability to utilize citrate as the ONLY carbon source via enzymes citrase & citrate permease

Citrate permease enzyme

Enzyme involved in actively transporting citrate molecule into cell from environment

• Bromothymol blue: pH indicator on the agar

Citrate test results

• Dark blue color: reaction occurs & pH rises

  • Organism is positive for ability to use citrate as only carbon source

• Green color: no reaction & pH stays the same

  • Organism is negative for ability to use citrate as only carbon source

Cellular respiration

Use highly reduced chemical compounds like NADH and FADH2 to establish a electrochemical gradient across membrane

• Consists of aerobic & anaerobic respiration

Aerobic respiration

Biooxidative process where molecular oxygen serves as final (terminal) electron acceptor

Anaerobic respiration

Biooxidative process where inorganic ions (NO3 and SO4) serve as final (terminal) electron acceptor

Nitrate reduction

Determine if organism uses nitrate (NO3) as terminal electron acceptor & perform anaerobic respiration for energy production

No nitrate reduction (negative test result)

Organism doesn’t perform nitrate reduction & cannot utilize nitrite afterward

Partial/incomplete nitrate reduction

Contains nitrate reductase to reduce nitrate to nitrite

• Able to reduce nitrate as terminal electron acceptor & into nutrient absorption

• NO3 + 2H + 2e → NO2 + H2O (NO3 → NO2)

Complete nitrate reduction (positive test result)

Organism is able to fully reduce nitrate during anaerobic respiration & nitrate reduces to final form of nitrogen gas (N2)

• NO3 → NO2 → NO → N2O → N2 gas (complete)

Nitrate reduction test

Stab inoculation test

1) Add 5 drops of nitrate A & B

• If red color change: organism performs partial nitrate reduction

2) If no color change, 5 add drops of zinc

• If no color change: organism performs complete nitrate reduction

• If red color change: organism performs no nitrate reduction

Nitrate reduction test results (partial NO3 reduction)

NO3 → NO2 (positive result)

• Nitrate A & B binds to nitrite & produce red color

Nitrate reduction test results (complete NO3 reduction)

NO3 → NO2 → N2 (positive result)

• Complete NO3 reduction of nitrate to nitrogen gas

• Reagents had nothing to interact with because organism completely utilized NO3

Nitrate reduction test results (no NO3 reduction)

NO3 remained in broth unused by bacteria

• Zinc reduced nitrate to nitrite

• Nitrite reacted with Nitrate A & B (still in broth after first test) binds to produce red color

Nitrate A & Nitrate B

Two compounds when added together will bind to nitrite (NO2) that is present & produce a red color change

Zinc

Can catalyze the reduction of nitrate to nitrite (NO3 → NO2) by itself

• Partial nitrate reduction reaction

• If tube turns red after zinc, zinc takes unused nitrate & converts it into nitrite

  • Nitrite binds to nitrate A & B

  • Bacteria did NOT use nitrate — ZINC did the reduction (no nitrate reduction)

Cytochrome

Iron-containing hemeproteins that are critical to electron transport chains (ETCs)

• Different microorganisms use different ETCs that contain different _________ (carry electrons)

• To be effective e carriers, cytochromes must be repeatedly reduced & oxidized

• Organisms that contain them as part of respiratory system are oxidase-positive (present in aerobic organisms)

Oxidase

Enzymes that help organisms that perform aerobic respiration

• All Enterobacteriaceae are oxidase negative (don’t contain oxidase)

Oxidase test

Look for immediate change

• Purple color change within 20 secs: positive result — organism produce oxidase

• No color change within 20 secs: negative result — organism cannot produce oxidase

Catalase

Protects cells from hydrogen peroxide (oxygen-based free radical)

• Breaks down hydrogen peroxide into oxygen & water

• 2H2O2 → 2H2O + O2 (gas bubbles)

Catalase test results

Look for immediate response

• Positive result: bubbles occur; organism has catalase

• Negative result: no bubbles occur; organism doesn’t have catalase