MircoBio Lab Exam 2

Where most of cell’s functions/reactions occur

Cytoplasm

Water movement across a semipermeable membrane

osmosis

Concentration of solutes outside the cell is higher than inside the cell

hypertonic

Excessive water loss causing the cell membrane to shrink/pull away from the cell wall

plasmolysis

Hypertonic = water moves ____ of the cell; Hypotonic = water moves _____ of the cell

out; into

Concentration of solutes outside of the cell is lower than inside

Hypotonic

Created by influx of water into a cell

Turgor Pressure

Internal salinity of most bacterial cells?

0.9% NaCl

Organisms that grow at higher salt concentrations due to their ability to maintain high cytoplasmic concentrations of solutes

Halophiles

Bacteria that can grow in both low-salt and high-salt enivironments

Facultative Halophile

Bacteria that can grow only in the presence of high salt

Obligate Halophile

Higher absorbance number = ?

Higher bacterial numbers (better growth)

Besides a spectrophotometer, how else can you observe growth of bacteria in different salt concentrations?

Turbidity (sediment)

Is radiation of physical or chemical control for microbial growth?

Physical

2 forms of radiation that are used to kill microorganisms

gamma and ultraviolet

How does gamma radiation kill microorganisms?

Collides with molecules in the cell and creates free radicals, which are toxic to the cell

How does ultraviolet radiation kill microorganisms?

Damages DNA and RNA by affecting bonding relationships within the molecule

Formation between two adjacent pyrimidines (usually thymine and cytosine) in a DNA strand as a result of UV light

Dimers

What wavelength of UV light is the most lethal and why?

200-290 nanometers because they are most absorbed by DNA

UV-C light

100-280 nm; cause more damage than cell system can repair

What is a limiting factor of using UV light?

Poor penetrating ability because it does not go through plastic, glass, and other materials

What is UV mainly used for?

Killing microbes on surfaces, air purifiers, and water treatment

Medicine that kills or inhibits microorganisms

Antimicrobial drugs

3 antibiotics with broad spectrum activity

Ciprofloxacin, tetracylcine, and trimethoprim

“Sensitivity” part of laboratory test

Testing the bacteria patient is infected with against a variety of different antibiotics

Kirby-Bauer Method

1. Cover surface of plate with bacteria

2. Add paper discs containing different antimicrobials

3. Observe and measure zones of inhibition

A clear circular area appearing around an antimicrobial disc, indicating inhibition

Zone of Inhibition

What agar is required for the Kirby-Bauer Test?

Mueller-Hinton agar

How should you measure zones of inhibition?

Measure in millimeters and measure the radius

Generates offspring genetically identical to the parent cell

Binary Fission

How does bacteria generate genetic diversity?

Horizontal gene transfer

Explain conjugation

• Bacteria in direct contact using a sex pilus

• DNA transferred from donor cell to the recipient

Explain transduction

• Bacteriophages transfer DNA from one bacterium to another

Explain transformation

bacterial cells take up DNA from the surrounding environment (requires cells to be competent)

How does Calcium Chloride make cells competent?

It opens the pores in the cytoplasmic membrane allowing greater intake of plasmid DNA

Small circular self replicating DNA molecules

Plasmids

Describe the process of lab transformation

1. Add competent cells to DNA+ tube and DNA- tube

2. Flick the tube to mix and ice for 20 min

3. Place tubes in 37 degree celcius water bath for 5 min

4. Add nutrient broth the both tubes

5. 20 more mins in water bath

6. Swab both bacteria onto ampicillin plate

On ampicillin plates, bacteria that are resistant will …?

Grow

An organisms survival depends on its ability to find and metabolize suitable forms of _______.

Nitrogen

Most predominant form of nitrogen?

Nitrogen Gas (N2) ; about 80% of earth’s atmosphere

Bacteria that convert nitrogen gas into a combined form of nitrogen or into an organic form of nitrogen

Nitrogen-Fixing Bacteria

Most common genus of bacteria that fixes nitrogen?

Rhizobium

Nitrfying bacteria convert _____ to _____ and then to _____.

Ammonium; nitrite

_____ is immediately usable by plants

Nitrate

Denitrifying bacteria reduce ____ to _____ or to one of the _____

nitrate; nitrogen gas; nitrous oxides (N2O)

What enzyme reduces nitrate to nitrite?

Nitrate Reductase

What enzyme reduces nitrite to nitrogen gas or other nitrous oxides?

Nitrite reductase

What does nitrate reagent A and B react with?

Nitrite

Nitrate reduction test: If the broth turns red with addition of reagents A and B, then ...?

Nitrite is present and nitrate reduction occurred (positive)

Nitrate Reduction Test: If the broth remains colorless after reagents A and B are added, then…?

That means nitrate is still in the tube or it was converted to nitrogen gas; further test is needed.

In a nitrate reduction test, the tube was colorless after A and B were added. Zinc and HCl were added. The tube turned red. What does this indicate?

This is a negative result. Zinc under acidic conditions catalzyes conversion of nitrate to nitrite. Since A and B are already in the tube it will turn red. This is a negative result because it indicated that nitrate was still in the tube.

In a nitrate reduction test, the tube was colorless after A and B were added. Zinc and HCl were added. The tube still remained colorless. What does this indicate?

This indicates a positive result because it indicates there is no nitrate present for zinc to convert to nitrite. This indicates nitrate reduction occurred and the final product was either nitrogen gas or a nitrous oxide.

An inorganic form of nitrogen that can be used as a nutrient or as a terminal electron acceptor in the respiratory chain

Nitrate

Extracellular enzyme that breaks down macromolecular DNA to the label of the mono or polynucleotide

DNase

Building blocks of nucleic acids

Nucleotides

_____ has been linked to an organism’s ability to degrade biofilms

Production and secretion of DNase

DNase Agar

Mixing powdered DNA into nutrient agar

What is added to a DNase plate to indicate positive or negative results?

HCl because it denatures DNA molecules in the medium

Describe a DNase positive result

The medium surrounding the colony is clear because it is free of DNA.

Describe a DNase negative result

The medium is oquae/white because the DNA in the medium surrounding the colony is denatured by HCl

A high molecular weight, water-soluble protein derived from collagen

Gelatin

When gelatin is chilled on ice, what does it do?

It crosslinks to itself to create a semisolid gel

Enzyme that hydrolyzes bonds in gelatin molecules to produce smaller peptides and amino acids so it can diffuse through the medium and be transported in the cell

Gelatinase

Nutrient gelatin medium (broth and gelatin) is used to detect…?

Production of an extracellular gelatinase

Describe a gelatinase-negative test

After being refrigerated, if the medium gels, this is a negative result. Gelatin molecules are still present and crosslinked on ice.

Describe a gelatinase positive result.

After being refrigerated and the medium remains liquid, this is gelatinase positive. Gelatinase is present and broke down gelatin into amio acids, so it did not cross link.

An abundant naturally occurring plant polysaccharide

Starch

Starch is a mixture of _____ and _____

Amylose and Amylopectin

An extracellular enzyme that catalyzes hydrolysis of amylose and amylopectin (limited tho); And how?

Amylase; Breaks glycosidic bonds to produce maltose, glucose and short chain polysaccharides

Amylose and Amylopectin are both composed of repeating units of _____ joined by _____ bonds.

glucose; glycosidic

Starch Agar

Soluble starch + nutrient agar

What reagent is used for the starch hydrolysis test?

Iodine

Positive Starch Hydrolysis Test

If an organism produces amylase, then agar surrounding the colony will be colorless (have a halo) because the amylase broke down the starch

Negative Starch Hydrolysis Test

If an organism does not produce extracellular amylase, the agar surrounding the colony will be blue-black because starch is still present.

Large, globular protein that gives milk its white, opaque color

Casein

An exoenzyme that hydrolyzes casein into amino acids and short pepetides; what is the formula?

Casease; Casein + Water —→ Amino Acids

Casein/Milk Agar Plate

Skim Mik + general growth agar

Positive Casein test

If casease is produced, the medium surrounding the colony will be clear and colorless because casease broke down casein in the milk

Negative Casein Test

If casease is not produced, the medium surrounding the colony will be white/opaque just like the agar because no casease broke down the casein.

Dentrification

Reduces nitrate to nitrogen gas in anaerobic conditions

Catabolic pathway whose end products include two molecules of pyruvate and ATP

Glycolysis

What does phenol red broth contain?

pH indicator (phenol red), sugar of interest (glucose and lactose), salts, and peptones and a Durham tube

Positive sugar fermentation result (phenol red sugar broth)

If the carbohydrates were metabolized (organism contains the transmembrane sugar transporter) (went through fermentation), acid will be released. The acid causes phenol red to change from red to yellow. A yellow tube always indicates a positive result. Sometimes gas will be released from fermentation and collected in the durham tube but sometimes not

Negative Phenol red sugar broth test

Carbohydrates were not metabolized (does not contain a transmembrane sugar transporter and did not go through fermentation). Organisms that are negative may metabolize the peptones in the medium instead and release ammonia which increases pH turning phenol red a bright fuscia color

Reversion

Once bacteria metabolize all carbohydrates, they will metabolize the peptones which release ammonia turning the yellow tube to fuscia.

What does Triple Sugar Iron Agar (TSIA) differential for and what does it test for? (one you STAB to the butt)

Gram Negative Enteric Bacteria and ability to ferment selected carbohydrates and produce hydrogen sulfide

What does TSIA plate contain?

Glucose, sucrose and lactose, pH indicator (phenol red), two sources of sulfur, iron salt, and nutrient base

TSIA: organism metabolizes glucose and another sugar

Slant + butt = Acid reaction (A/A); yellow

TSIA: organism can only use glucose

Butt = Acid yellow ; Slant = Red (may metabolize peptones)

TSIA: if the organism produces hydrogen sulfide what happens?

Iron sulfide is visible as a black precipitate in the medium and classified as H2S positive

Litmus Milk Broth

Contains pH indicator (litmus), lactose sugar and casein protein

Litmus Milk: bacteria fermented lactose?

Acid is produced turning the litmus pink

What can acid production in the litmus milk cause?

Can cause milk proteins to coagulate and form an acid clot, causing them to appear pink and white on the bottom

Litmus Milk: organism uses protein as energy source

Releases ammonia and increases pH, causing an alkaline reaction turning the litmus blue

Litmus Milk: If the milk remains purple and turns white on the bottom, this is an indication of ______, which means _____ has been removed.

reduction; oxygen

Litmus Milk: digestion reaction

Protein is completely broken down and and becomes clear with a brownish tinge

If gas is produced in litmus milk test, what happens?

Cracks/air bubbles form (product of fermenation)

SIM stands for?

Sulfide, indole and motility

A nitrogen containing compound that is a by-product of amino acid catabolism and nitrogenous waste in urine

Urea (NH2)2CO

Extracellular enzyme that hydrolyzes urea to release ammonia and carbon dioxide (show equation)

Urease ; (NH2)2CO + H20 —→ CO2 + 2NH3