Microbio LAB QUIZ 3 REVIEW

What is selective media?

Contains chemicals that PREVENT the growth of the desired bacteria without inhibiting the growth of the desired bacteria.

What is enrichment media?

Contains chemicals that ENHANCE the growth of the desired bacteria, other bacteria will grow, but the growth of the desired bacteria will be increased.

What is differential media?

Contains various nutrients that allow us to distinguish one bacteria from another based on how they metabolize.

What is this plate’s name, selective agent, results and everything about it?

Name: Phenylethyl Alcohol Agar Plate(PEA)

• SELECTIVE AGENT: Phenylethyl Alcohol

• Disrupts membrane —> P(phosphorus) leaks out

• —→ stops DNA synthesis

• SELECTS FOR: Gram (+) bacteria

• RESULTS: Growth = Gram (+), No Growth = Gram (-)

What is the plate’s name, selective agent, selects for, differential agents, differentiates between, and everything about it?

Name: Mannitol Salt Agar(MSA)

SELECTIVE AGENT: 7.5% NaCl

SELECTS FOR: salt tolerant bacteria (halophiles)

DIFFERENTIAL AGENTS:

1. Mannitol: fermentable sugar

2. Phenol Red: pH indicator

- Acidic = yellow

- Neutral = red

- Basic = shocking pink (like a fuschia)

DIFFERENTIATES BETWEEN:

- Mannitol fermentors: yellow around the colony(halo)

- Non-mannitol fermentors: red or shocking pink around the colony

Extra info: WHEN AN ORG IS NOT SALT TOLERANT WE CAN’T TELL IF IT IS MANNITOL OR NON-MANNITOL, JUST THAT IT’S NON-SALT TOLERANT

What is the plate’s name, selective agents, select for, differential agents, differentiates between, and everything about it?

Name: MaConkey Agar(MAC) Plate)

SELECTIVE AGENTS:

1. Bile Salts: Inhibits Gram (+) bacteria

2. Crystal Violet: inhibits Gram (+)

SELECTS FOR: Gram (-) bacteria

DIFFERENTIAL AGENTS:

1. Lactose: fermentable sugar

2. Neutral Red: pH indicator

• Neutral or basic = colorless

• Acidic = red

DIFFERENTIATES BETWEEN:

• Lactose fermentors: colonies turn a shade of red (or possibly pink)

• Non-lactose fermentors: colonies remain opaque or colorless

ALWAYS TRUST MaConkey AND IF THERE’S NO GROWTH = GRAM(+) NOTHING ELSE

What is the plate’s name, selective agents, select for, differential agents, differentiates between, everything about it?

Name: Eosin Methylene Blue Agar (EMB) - has a dark jolly rancher purple

SELECTIVE AGENT:

1. Eosin

2. Methylene Blue

SELECTS FOR: Gram (-) bacteria

DIFFERENTIAL AGENTS:

• Lactose: fermentable sugar

• Eosin

• Methylene Blue

1. Lactose fermentors: colonies are dark purple, and leads to a green metallic sheen(metal looking green)

2. Non-lactose fermentors: colonies are colorless or pink

Extra info:

MAC is still more accurate

• Gram (+) if it didn’t grow at all

What is the plate’s name, differentiates between, everything about it?

Name: Blood Agar Plate(BAP) - red, blood color

ENRICHED WITH: 5% defibrinated sheep blood

DIFFERENTIAL AGENT: 5% defibrinated sheep blood

DIFFERENTIATES BETEWEEN:

1. Beta-hemolysis: complete hemolysis (meaning there is a clear zone around the colony).

2. Alpha-hemolysis: partial hemolysis (meaning it is cloudy or green around the colony)

3. Gamma-hemolysis: no hemolysis, no change around colony.

TRICK TO RMB: Beta = bad, Alpha = alright, and Gamma = good.

• Hold up to light to see how to differentiate the results.

STUDY RESULTS FROM LAB MANUAL PAGE AROUND 100 SOMETHING, and extra pictures taken from his review.

What are enzymes and the two different types?

DEFINITION: Are proteins that catalyze biological reactions

1. Endoenzymes: function inside the cell

2. Exoenzymes: function outside the cell(so they’re released from the cell to catalyze reactions out of the cell

What is the plate used here, what kind of test is conducted, media, inoculation guide, incubation, theory, results, and everything else about it?

Name: Casein Hydrolysis Test

EXOENZYME TESTED: Caseinase

MEDIA: Skim milk agar(is like a milk color on plate)

INOCULATION GUIDE: loopful streak

INCUBATION: 37°C for 48 hrs

Theory:

Casein(substrate, white) ——→ caseinase —> amino acids(clear, broke down into this)

RESULTS:

(+): clear around the growth (since you released casein to be around you)

(-): white around the growth

REVIEW TABLE’S LAB RESULT PLATE TOO

What is the plate used here, what kind of test is conducted, media, incoulation, incubation, theory, results and everything else about it?

Name: Starch Hydrolysis Test

EXOENZYME TESTED: amylase

MEDIA: Starch agar plate

INOCULATION GUIDE: loopful streak

*After incubation: add iodine over the plate(this will bind to the starch)

INCUBATION: 37°C for 48 hrs

THEORY:

Starch —→ amylase —> simple sugars(is broken down into this)

*Add iodine: starch = blue/black, simple sugar = yellow

RESULTS:

(+): yellow around growth (like a halo)

(-): blue/black around growth

Extra info:

• BC = postive because it is yellow around

• EC lights up, but not positive

WE ALWAYS LOOK AROUND THE COLONY, LIGHTING UP DOESN’T MATTER.

What is the test name, purpose, application, media, theory and everything else about it?

Name: Catalase Test

PURPOSE: to determine if your organism has the enzyme catalase(meaning it breaks down hydrogen peroxide)

APPLICATION: differentiates GPC (gram (+) cocci)

• (+): Staphylococcus and Micrococcus

• (-): Streptococcus and Enterococcus

MEDIA: TSA plate

INOCULATING GUIDE: loopful streak (could do a quadrant or just one line)

INCUBATION: 37°C for 48 hrs

THEORY:

2H2O2(substrate,hydrogen peroxide) —> catalase —> 2H2O + O2 (bubbles)

*After incubation: use a toothpick to put the bacteria on a slide

• Add 1 drop of H2O2(hydrogen peroxide)

RESULTS:

(+): bubbles

(-): no bubbles

What is the the test name, purpose, media, theory, and everything else about it?

Name: Oxidase Test

PURPOSE: to determine, if your organism has the enzyme cytochrome C oxidase.

MEDIA: TSA plate

INOCULATING GUIDE: loopful streak

INCUBATE: 37°C for 48 hrs

THEORY:

• After incubation, use a toothpick to put the bacteria on a piece of filter paper

• Add 1 drop of oxidase reagent(this will detect cytochrome C oxidase)

RESULTS:

(+): purple (within 30 seconds)

(-): color of the bacteria

What is the test name, purpose, media used, theory and everything about it?

Name: Sugar Fermentation Test

PURPOSE: to determine if your organism ferments in desired sugar into an acid or acid and gas(or not at all).

MEDIA: PR(Phenol Red) _________ broth (blank means it depends on what carbohydrate is there)

• Fermentable sugar (depends on what the sugar is)

• Phenol Red: pH indicator

Acidic = yellow

Neutral = red

Basic = shocking pink

• Durham tube: traps gas(upside down glass tube, red cap, gas accumulates inside tube producing a bubble).

INOCULATION GUIDE: loopful incoculation

INCUBATION: 37°C for 48 hrs

THEORY:

Sugar is fermented —> acid (reacts with Phenol Red)

RESULTS:

(+) For Acid ONLY: yellow tube, no gas bubble

(+) For Acid and Gas: yellow tube, gas bubble

(-) For Acid and Gas: red or shocking pink tube, no gas bubble.

What are the two different lactose fermentors, and what is a late lactose fermentor?

Requires 2 enzymes:

• B-galactoside permease(beta-galactoside permease): allows lactose to enter the cell

• B-galactosidase:(beta-galactosidase): breaks lactose —> glucose and galactose forms.

Late lactose fermentors: ONLY have B-galactosidase.

What is the test name, purpose, media used, inoculation guide, theory and everything about it?

Name: ONPG Test

PURPOSE: to detect B-galactosidase in an organism.

MEDIA: TSA plate(tryptic soy agar, previously grown)

INOCULATION GUIDE:

* Add a heavy inoculum from previously grown plate to test tube with 1mL of water(scrap A LOT of the bacteria)

* Add ONPG Tablet

INCUBATION: 37°C for 48 hrs.

THEORY: ONPG

• Is a synthetic substrate of B-galactosidase, does not require a permease to enter cell.

• ONPG(substrate) —> B-galactosidase(breaks)—> O-nitrophenol(yellow enzyme)

RESULTS:

(+): yellow

(-): clear or foggy

Note:

ONPG does not require permease to enter the cell.

What is the IMVIC test/what does each part of IMVIC stand for?

IMVIC: is a series of tests to differentiate GNRs.

I: Indole Production Test

M: Methyl Red Test

V: Voges-Proskauer Test

C: Citrate Utilization Test

What is the name of the test, purpose, the media, inoculation guide, incubation, theory, results, and everything about it?

Name: Indole Production Test

PURPOSE: to determine if your organism has the enzyme tryptophanase (produces indole)

MEDIA: Tryptone broth —> clear color

INOCULATION GUIDE: loopful

INCUBATION: 37°C for 48 hrs.

THEORY: Tryptophan(substrate) —> tryptophanase—> pyruvic acid + NH3 + indole

*After incubation, add 5 drops of kovac’s reagent

indole + kovacs reagent (yellow)

RESULTS:

*After adding 5 drops of kovacs reagent:

(+): pink on top(has the enzyme tryptophanase)

(-): yellow on top

What is the name of the test, the purpose, media, inoculation guide, incubation, theory, and results?

Name: Methyl Red Test

PURPOSE: to determine if your organism ferments glucose into mixed acids.

MEDIA: glucose supplemented nutrient broth

INOCULATION GUIDE: loopful

INCUBATION: 37°C for 48 hrs.

THEORY: glucose is fermented into —> organic acids

After incubation, add 5 drops of

• Methyl Red: pH indicator

- Acidic: red

- Neutral or basic: yellow

RESULTS:

(+): red

(-): yellow

What is the name of the test, purpose, media, inoculation guide, incubation, theory, and results?

Name: Voges-Proskauer Test

PURPOSE: to determine if your organism ferments glucose into a neutral product called acetoin.

MEDIA: glucose supplemented nutrient broth

INOCULATION GUIDE: loopful

INCUBATION: 37°C for 48 hrs.

THEORY:

• glucose is fermented —> neutral product acetoin

• After incubation, to detect acetoin, add:

1. Alpha-naphthol(8 drops)

2. KOH(4 drops) - potassium hydroxide

RESULTS: NEED TO WAIT AT LEAST 15-30 MIN. AFTER ADDING KOH

(+): red

(-): yellow (didn’t have acetoin)

What is the name of the test, purpose, media used, inoculation guide, incubation, theory and result?

Name: Citrate Utilization Test

PURPOSE: to determine if your organism can use Na citrate as the only carbon source

MEDIA: Simmons citrate agar slant (greenish color)

• Na citrate: only carbon (C) source

• Ammonium phosphate: only (N) nitrogen source

• Bromthymol Blue: pH indicator

- Acidic = yellow

- Neutral = green

- Basic = blue

INOCULATION GUIDE: loopful streak in slant

INCUBATION: 37°C for 48 hrs

THEORY:

• If your organism can use Na citrate and ammonia phosphate as the only C and N source (produces ammonia)

—> NH3(basic) - reacts with bromthymol blue

RESULT:

(+): growth, blue (can use Na citrate)

(-): no growth, green

What is the name of the test, purpose, the media, incoculation guide, incubation, theory, and results?

Name: Gelatin Hydrolysis Test

PURPOSE: To detect the present of gelatinase(breaking down gelatin).

MEDIA: Nutrient broth

INOCULATION GUIDE: loopful

*After incubation(1st incubation): add gelatin strip, and re-incubate.

INCUBATION: 37° for 48 hrs. (2x) HAVE TO DO THIS TWICE

THEORY:

Gelatin(protein, substrate, solid, at room temperature)-→ gelatinase(enzyme) —> amino acids(liquid)

• Gelatin strip is a clear blue plastic(underneath the coat of green), and covered with green gelatin(like a sage green, flexible, on both sides).

RESULTS:

(+): strip is clear blue

(-): strip is green(sage green, same color as the top)

*Basically wherever gelatin is working, gelatin will fall off, so you dip it into the broth to figure it out.

What kind of test is this, purpose, application, it’s media, inoculation guide, incubation, theory, and results?

Name: Urea hydrolysis Test

PURPOSE: to detect the presence of urease(enzyme)

APPLICATION: helps differentiate GNR's (gram (-) rods)

MEDIA: TSA plate (previously grown)

• 1 mL tube of water(sterile water)

• Urea tablet(look like yellow vitamin tablets)

- yeast extract: nutrients

- Urea: substrate

- Phenol red: pH indicator

INOCULATION GUIDE: heavy inoculation into water(pick up about 3-4 colonies to transfer in the test tube)

• Add urea tablet to 1mL of water

INCUBATION: 37°C for 48 hrs.

THEORY:

Urea(substrate in tablet) —> urease —> 2NH3(basic, ammonia) + CO2

RESULTS:

(+): shocking pink(reacts with phenol red)

(-): no color change or yellow(or like a bright yellow, salmon, soft pink/peach color)

What is the name of the test, purpose, application, media, inoculation guide, incubation, theory, and results?

Name: Lysine Decarboxylase Test(LDC):

PURPOSE: to detect the presence of lysine decarboxylase.

APPLICATION: to help differentiate GNRs (Gram negative rods)

MEDIA: Mollers Lysine Decarboxylase media(tea color can identify on practicum by oil on top)

• peptone: nutrients

• glucose: induce fermentation

• lysine: amino acid substrate for lysin decarboxylase

- Bromcresol purple: pH indicator

• Acidic = yellow(like a soft baby yellow)

• Neutral = gray(tea color, isn’t really consistent)

• Basic = purple (vibrant purple)

INOCULATION GUIDE: loopful

• layer with mineral oil: keeps O2 out, induces fermentation (so about 1 mL of oil, will be on top of media and block off O2).

INCUBATION: 37°C for 48 hrs. *MUST DO FULL 48 HOURS

THEORY:

- Decarboxylation will occur if:

• Amino acid substrate is present

• There is a drop in pH: glucose + mineral oil —> induces fermentation

Lysine —→ lysine decarboxylase —→ cadaverine(basic) + CO2

RESULTS:

(+): purple(vibrant, reacts with bromcresol purple)

(-): yellow or no change(gray)

What is the name of the test, purpose, application,it’s media, inoculation guide, incubation, theory, and results including what is H2S production?

Name: Kligler Iron Agar(KIA) Test

PURPOSE: differential screening media based on:

• glucose fermentation

• lactose fermenetation

• H2S (hydrogen sulfide) production

APPLICATION: differentiate GNRs(gram(-) rods)

MEDIA: Kligler Iron Agar Slant

• 0.1% glucose: fermentable sugar, low concentration (K/A) only glucose

• 1% lactose: fermentable sugar, high concentration(yellow, yellow, K/K)

• 0.02% ferrous sulfate: detects H2S —> black color.

MEDIA:

• Phenol red: pH indicator

• Nutrient agar(slant)

INOCULATION GUIDE: loopful streak on slant surface,

• needle stab the butt(then remove)

INCUBATION: 37°C for 48 hrs/

THEORY AND RESULTS;

1. Ferments ONLY glucose:

• Yellow butt: acid reacts with Phenol Red

• Red Slant: after low concentration of glucose is used up, amino acid oxidation occurs —> produces ammonia(basic)

2. Ferments lactose OR lactose and glucose:

• Yellow butt and slant: acid reacts with phenol red, so there is a high concentration of lactose.

3. Does NOT ferment either sugar:

• Red or shocking pink butt and slant(so both red slant and butt, doesn’t produce any acid, so nothing should be yellow).

H2S Production:

(+): black percipitant(like a black product)

• Gas production:

(+): butt lift (like there is a lift in the medium)

Extra info:

• Break in yellow tube, ferments into acid as well as gas(no black = (-) for H2S)

• black in bottom = H2S production

• tube with red top, black middle, yellow bottom ONLY ferments glucose(+) for H2S production.