MB Lab Quiz 4

6-5 Start

GOOOOD is real god is real god is actaully real im not kidding this time god is real i wouldnt joke about this

Virus vs Bacteria

• Virus =

  • Smaller

  • Acellular

  • CANNOT grow on normal media, require hot cell

    • Lackding celllular machinery

    • ONLY have DNA/RNA

  • Can infect prok or euk

Phage/ bacteriophage + name for ecoli phage

• Virus that infect bacteria

• Coliphage = ecoli bacteria

Lyytic vs lysogenic virus

• Lytic virus = VIRULENT

  • Enters ell, takes over, replicates, lyse for release

• Lysogenic = TEMPERATE means phage does not immediately kill host

  • Enter cell

  • Integrate into DNA

  • Cell replicate normally

  • Eventually virus replicates + lyse cell

VIU

• Virus infection unit

• Smallest unit causing EFFECT on host

Plaque/ PFU

• Zone of lysis/growth inhibiton in a LAWN of bacteria

• 1 plaque = 1 virus

Assay count efficiency vs electron microscope

NOT as efficient, Why?

• Efficiency of infection by virus is NOT 100% so electron microscope is better

Enrichment purpose + teps

• Increase number of phage in raw sewage

• Combine

  • Sewage

  • E.coli = host

  • DSPB (10x stronger than ordinary TSB, accommodate dilution with sewage water)

  • Enriches the HOST bacteria for growth, which then increases phage number

DSPB

• Is NOT a broth for growth of phage directly

• Phage CANNOT grow in the broth alone

  • Requires host

  • E.coli acts as HOST and SELECTS for type of virus, that being coliphage

• DSPB increases number of E.coli which indirectly increases phages in broth

Filtration purpose

• Seperate E.coli from phage

Filtration steps + pore size + type of filter apparatus

• Use 0.2 MCM filter, allows virus to pass but NOT bacteria

• Sample MUST be centrifuged prior to filtration

  • Removes cellular debris = no clogging

• Nalgene filtration apparatus

  • only need 2ml

Seeding purpose

• Plate dilution of filtrate WITH E.coli to see phage evidence and find concentration of phage in original filtrate

Seeding steps + type of agar

• Use SOFT NUTRIENT AGAR

  • Liquefied in water bath so that they can be spread evenly on plate

• Add diluted filtrate + E.coli to agar tube and ROLL BETWEEN HANDS

• Pour contents evenly in prewarmed TSA plate

  • Why prewarmed TSA?

    • Prevents solidification as soon as it hits plate, allows agar to flow over entire surface

How long for plaques to appear on plate

2-2.5 hours of incubation

• IF YOU WAIT LONGER, plaques will merge together = difficult to count them individually

know?

Oxygen requirments 2-6,2-7,2-8 START

Aerotolerance

• Organism ability or lack of ability to grow in presence of oxygen

Aerobes

• REQUIRE oxygen, use it as final electron acceptor

Facultative anaerobes

• Can grow in both BUT prefer oxygen

  • If oxygen, they can utilize it like normal aerobes

  • If no oxygen, can obtain from alternate source, like nitrate

Anaerobes

• CANNOT grow with oxygen

  • Oxygen = inhibitory or lethal to bacteria

  • Lack enzymes to deal with harmful products of oxygen metabolism

Microaerophiles

• Require free oxygen, ONLY in limited amounts

  • Think micro = small amount of oxygen cool!

Capnophiles

• Microaerophile that require ELEVATED levels of CO2 to survive

  • Simple enough

TSA Deep + oxygen status

• Solid agar

• Removed oxygen from atuoclaving diffuses back into media as solidifies

  • Creates oxygen gradient, MORE at top (aerobic) less or none at bottom (anaerobic)

TSA shake

• Same preparation as TSA deep but kept as liquid in warm water bath

  • After inoculation, tube is GENTLY rolled between palms to disperse bacteria

  • THEN allow to solidify

• As media solidifies, oxygen gradient is created with bacteria

FTM (stand for and what it contains)

• Fluid thioglycolate medium

• Basic medium that contains sodium thioglycolalte, Lcystinine, reazurin, agar, gelatin

• OXYGEn gradient formed during media cooling

Thioglycolate + L-cystine function

• Reducing agents

  • Reduce oxygen present in media to water

Resazurin

• Dye used as oxygen indicator

• With oxygen becomes = PINK

  • This is why the top is PINK, and fades out

• Absence of oxygen = YELLOWISH (natural color media)

PINK is hard to see following inoculation and bacterial growth

Agar + gelatin

• Small amounts

• Helps to localize the organisms as they grow

TSA plates + GasPak purpose

• GasPak- used to create an anaerobic enviornment

Gas generator sachet (3 things it contains and functions)

• BBP -peruvians

• Contains sodium borohydride + sodium bicarbonate

• Borohydride

  • Upon air exposure, sachet becomes activated

  • Hydrogen from borohydride COMBINES with oxygen with help of palladium to fomr water

• Bicarbonte

  • Produces CO2 gas

• Contains palladium - IMPORTANT

  • Catalyst between hydrogen and oxygen

Anaerobic indicator strip

• Paper strip with RESAZURIN

• Pink with oxygen, cololress/white without oxygen

3 bacteria used

• Clostridium sporogeneses - white/cream

  • Endospore former

  • Can contaminate imporperly canned food / stored

• Escherichia coli - white/cream

  • Found all over + in intestines

  • Can produce endotoxins = UTIS, food poisning, etc.

• Micrococcus luteus - yellow due to CAROTENOID PIGMENTS

  • Found in soil, dust, water, human skin

  • Sign of improper decomination in labs

Oxygen growth status of 3 bacteria

• Clostridium sporogeneses = OBLIGATE ANAEROBE (no oxygen)

  • Will grow in lower part

• E.coli = FACULATIVE ANAEROBE (better with oxygen)

  • Will grow all over, but HEAVIER at top

• M. luteus (Obligate AEROBE) = REQUITES OXYGEN

  • Only grow at top

TSA plate results

LOOK AND KNOW, it goes with other stuff

Temperature 2-9 start

Temperature =

• ONE OF THE MOST IMPORTANT FACTORS FOR BACTERIAL ENZYME ACTIVITY

Cardinal temperatures

• Range of temps that enzymes can function at

Minimum vs maximum vs optimum cardinal temp

• Minimum = lowest, lower will = INACTIVE

• Max = highest, higher = inactive + destructure

• Optimum = growth rate is highest, enzymes at max speed

Psychrophiles CHRO

• 0-20 (CANNOT grow above 20)

• Common in arctic + antartic akak PERMANENTLY COLD TEMPS

Mesophile

• 15-40

• Human bacterial pathogens fall in this category

  • WHY? because human body temp is around 37

• MESO = middle

Thermophile

• Above 40

• Typically in hot springs

Extreme thermophile

• 65-110

• Optimum of around 80

Bacillus Stearothermophilus KNOW SPELLING

• Cream color

  • Endospore forming

  • Thermophile

• Expeirment = 50 + 65

E. coli

• Cream colored

• Mesophile

• Often incubated at 37, lives inside human

• Experiment = 25-37

Serratia marescens

• at 25-30 = Red pigment prodigiosin

• 37 = growth but CREAM COLORED like ecoli

• Experiment = 25-37

2-11 OSMOTIC start

hurray? you said now its gone away, brought back we can try things over, as if there wasnt ever pain, SWEETER this time will be sweeter, i can be much sweeter, now back inside your cage

Water

• Principal component of cytoplasm + source of electrons + hydrogen ions

• ALSO, required for cellular turgor pressure

Turgor Pressure

• Counter pressure required to maintain integrity of cell shape and microorganism

  • ESSENTIALLY, the outward force pushing out that retains shape

2 components of turgor pressure

1. High potassium or sodium ions concentration- promotes inward diffusion of water

2. Compatible solutes - composed of amino acids to help maintain turgor pressure

Bacteria in saline environment + turgor rpessure

• Saline/ high salt environment = CONSTANT effort to maintain turgor pressure as cell is fighting against shrinking

Osmosis

• Movement of water from region of high concentration to low concentration ACROSS a semipermeable membrane

Hypotonic vs isotonic vs hypertonic

• Hypo

  • Environment has low solute = constant movement water inward

  • Swelling/ burst

• Isotonic

  • Equal solute concentration

  • Movement in and out = equal

• Hypertonic

  • Environment has HIGH solute = constant movement out

  • Shrinkage of membrane = plasmolysis

Halotolerant

• TOLERATE high salt, but do not REQUIRE

  • If it can grow in media with lower salt then it does not REQUIRE and thus is not a halophile

Halophile

• Salt loving = REQUIRE NaCl

  • NaCl must be 3% or higher

  • MUST HV

Extreme halophile

• 15-25% NaCl

• WIll NOT survive in low solute env

Osmophile

• Require high concentrations of ORGANIC SOLUTE

  • Like sugar

Xerophile

• Grow in LOW WATER ACTIVITY env

  • This means DRY enviornment, or enveiornments where there is littel available salts

• What dose this mean

E.coli

• NON-halophile

• Cream colored

• E. coli = requires STANDARD salt concetnariton, around 0.5%

Halobacterium salinarum

• EXTREME halophile

• Reddish colored colonies

• Found in great salt lake + dead sea = high saline

• Utilizes potassium chloride + compatible solutes to reduce

Staphylococcus aureus, RG 2

• Halotolerant

• RISK GROUP 2, must use biosafety cabinet

• Golden color

  • WHY? Carotenoid pigments

• Can grow in 0.5-10% but thats abou tit

  • Aka DOESNT need ti

Results know percentages and growth

porbably

Oxidation and Fermentation Tests, unkonwn day 3

WOAH

Catalase test

• Using slants

• Tests for:

  • CATALASE enzyme = breaks down hydrogen peroxide into water and oxygen

• Reagent

  • Hydrogen peroxide 3% to slant

• Positive = bubbles

• Negative = no bubbles

Sugar fermentation tubes/ Durham tubes

• Liquid TSB broths

• Tests for:

  • Fermentation of sugars (gluc, lac, mannitol)

• Reagent:

  • Phenol red = pH indicator

    • Red over 7, yellow under 7

  • Inverted/durham tube = detects gas w/ fermetnaiton

    • Bubbles will appear if gas produced

    • DONT VORTEX, will make bubbles

• Results:

  • Positive ferment = yellow

  • Positive gas production = bubble

  • Negative ferment = red

  • Negative gas production = no bubble

What is the nitratase test really asking (2 questions)

1. Does the organism have nitratase (nitrate reductase)?

This enzyme converts nitrate (NO₃⁻) → nitrite (NO₂⁻).

2. If nitrite is NOT present, did the organism…

• reduce nitrate even further (to ammonia, nitrogen gas, etc.)
  OR

• not reduce nitrate at all?

Why add zinc in nitratase test

• Its possible that the organism reduced nitrate PAST nitrite

• OR it didnt reduce nitrate at all (aka doesnt have nitratase)

• SO

  • Zinc chemically reduces nitrate to nitrite (acting like enzyme

• SO SO:

  • If it turns red after zinc = NO nitratase, because nitratase would have done it

Nitratase test

• Test for:

  • Nitrate presence as electron acceptor in anaerobic respiration

  • AND Nitratase presence, converts nitrate to nitrite

• Reagents - SA DAN

  • Sulfanilic acid

  • Dimethyl - a - naphthylamine

    • Both of these detect NITRITE

      • If present = turn red

      • Not present = no red

• Steps

  • Add 10-15 of both to tubes

  • No color change = add zinc

• Results

• SO:

  • Red no zinc (red initial) = Nitratase positive

    • Means nitrate turned to nitrite via nitratase

  • Red with zinc = Nitratase negative

    • Because zinc had to do the job

  • No color w/ zinc = Nitratase positive

    • Because it means nitratase reduced BEYOND nitrite

Methyl Red test (MR)

• Tests for:

  • Production of acid upon glucose fermentation

  • Indicator that organism is mixed acid fermenter

• Reagent + result

  • Methyl red = pH indicator

    • Red for pH under 5 (positive test)

    • Yellow for pH over 5 (negative test)

• MUST vortex

Voges Proskauer Test (VP)

• Tests for:

  • Alcohol production FOLLOWING glucose fermentation

  • Detects ACETOIN = 2-3 butanediol precursor

    • does NOT detect ethanol or 2-3 butanediol

• Reagents AN KOH

  • Alpha naphthol

  • KOH (potassium hydroxide)

• Positive

  • Red color change

  • Negative = yellow brown color

KIA/ Kligler’s Iron Agar slant

• Tests for:

  • Fermentation of glucose and lactose

  • Presence of H2S/ hydrogen sulfide

  • Gas produciton

• Reagents (already in media)

  • Phenol red = pH indicator

    • Red over 7

    • Yellow under 7

      • Different sugar concentrations = color change distribution

  • Iron salt

    • Tests for H2S production

KIA results

• Positive

  • Lactose ferment = yellow THROUGHOUT tube

  • Glucose ferment = RED SLANT, but YELLOW BOTTOM

  • H2S = dark precipitate of iron sulfide (Black color in media)

  • Gas = crack sin media or bubble at bottom

Glucose and lactose in KIA

• Lactose on TOP

• GLucose on BOTTOM

  • Thas why bottom turns yellow if glucose positive

    • Yellow = more acidic remember

• So lactose + glucose ferment = yellow all throughout

• OK? is this making sense

  • Neithe r= red/pink throughout

Miscellaneous tests (day 4 unknown) start

ALL SLANTS SHOULD BE + SIM DEEP SHOULD BE

• Slant = Streaked and stabbed

• SIM deep = SINGLE stap enter and exit through same, why? because of motility

• All incubate at 37

Simmons Citrate test

• Tests for:

  • Utilization of citrate as SOLE CARBON SOURCE

• Reagents

  • NONE

• Positive = Prussian blue

• Negative = green color

Phenylalanine Deamination Test

• Test for:

  • Presence of PHENYLALANSE, breaks down phenylalanine into phenylpyruvic acid/ PPA

• Reagent

  • 10% ferric chloride, detects PPA via color change

• Positive = deep green color change

• Negative = yellow color (of reagent)

SIM Media Deep

• Tests for:

  • SIM

    • S = H2S/hydrogen SULFIDE (s) production

    • I = Indole presence

    • M = motility

• Reagents

  • Chloroform = solubilize media (makes it PERMABLE to Kovac’s reagent)

  • Kovac’s reagent

• Also contains

  • TTC (2,3,5 triphenyl tetrazolium chloride)

SIM breakdown + TTC

• S:

  • Iron salts in media = test H2S

• I

  • Chloroform + Kovac reagent test for INDOLE (more so TRYPTOPHANASE)

    • Indole = due to TRYPTOPHANASE enzyme which converts tryptophan into indole + pyruvic acid

• M/ motility

  • Use SEMI SOLID MEDIA + single inoculation point

    • Less agar = allows for shape but also motile organism to move

• TTC

  • Colorless and soluble when oxidized = no movement

  • Red when reduced

SIM Results

• Positive

  • H2s production = dark precipitate of iron sulfide

  • Tryptophanase/indole = red ring at top

  • Motility = growth radiating from line of inoculation

    • OUTWARD

    • and cloudy in tube

• Negative

  • OPPOSITE OF ALL OF THESE