Biol 351 Lab exam 3

What is TSIA testing for?

Glucose, lactose, and sucrose fermentation, gas production, and H2S (sulfur reduction).

What are the 3 sugars in TSIA and their relative amounts?

Glucose (0.1%), lactose and sucrose (about 1%, 10x more than glucose).

Why is glucose only 0.1% in TSIA?

It runs out quickly, so a glucose-only fermenter's slant reverts to alkaline. The 10x lactose/sucrose keeps the slant acid if the organism uses them.

TSIA indicator for fermentation?

Phenol red (yellow = acid, red/pink = alkaline).

TSIA indicator for H2S?

Ferrous sulfate (reacts with H2S to make a black precipitate).

TSIA recording format?

Slant/Butt/Gas/H2S (example K/A/-/+).

In TSIA, what do K and A mean?

K = alkaline (red/pink). A = acidic (yellow).

TSIA K/A means?

Glucose fermentation only (no lactose or sucrose).

TSIA A/A means?

Ferments glucose AND lactose and/or sucrose.

TSIA K/K means?

No carbohydrate fermentation (peptone used only).

Why does a glucose-only fermenter have a pink slant but yellow butt in TSIA?

Glucose runs out and the aerobic slant uses amino acids (makes NH3, raises pH). The anaerobic butt stays acid.

TSIA A/K reaction?

Should never happen. It means improper inoculation (you must stab AND streak).

What butt reaction do all enterics show in TSIA?

Acid (yellow) butt, because all enterics ferment glucose.

How do you see gas in TSIA?

Cracks, bubbles, or agar lifted off the bottom of the tube.

What does a black precipitate in TSIA mean?

H2S production. Thiosulfate reduction needs acid, so it indicates both sulfur reduction and fermentation.

If black H2S hides the TSIA butt, what color is the butt?

Yellow (acidic), because H2S requires acid.

How do you judge lactose/sucrose fermentation if H2S occludes the butt?

Look at the slant. A yellow slant means lactose and/or sucrose fermented. A red slant means glucose only.

What is LIA testing for?

Lysine decarboxylation, lysine deamination, and H2S (sulfur reduction).

LIA pH indicator?

Bromocresol purple (purple = alkaline, yellow = acid).

LIA H2S indicator?

Ferric ammonium citrate.

LIA purple slant/purple butt (K/K)?

Lysine decarboxylase positive, deaminase negative.

LIA purple slant/yellow butt (K/A)?

Decarboxylase negative, deaminase negative, glucose fermented.

LIA red (burgundy) slant/yellow butt (R/A)?

Lysine deaminase positive, decarboxylase negative, glucose fermented.

What causes the burgundy slant in LIA?

Lysine deamination products react with ferric ammonium citrate.

Which 3 genera are lysine deaminase positive?

Proteus, Providencia, Morganella.

Why is lysine deamination only seen in the LIA slant?

It requires oxygen.

Why does glucose fermentation matter for lysine decarboxylation?

Acid from glucose fermentation induces decarboxylase enzymes. Decarboxylation then makes alkaline products and returns the butt to purple.

Which LIA tube part do you read for decarboxylation?

The butt.

What enzyme does the oxidase test detect?

Cytochrome c oxidase.

Oxidase reagent?

Tetramethyl-p-phenylenediamine.

Positive oxidase result?

Deep purple/blue within 20 seconds.

Why read oxidase within 20 seconds?

The reagent oxidizes in air on its own and turns blue, giving a false positive.

How is the oxidase test performed?

Smear a colony on filter paper, add 1-2 drops of reagent, and watch for a color change within 20 seconds.

What reaction does cytochrome c oxidase catalyze?

Transfers electrons from cytochrome c to O2. The reagent gets oxidized by it and turns purple/blue.

Catalase reagent?

Hydrogen peroxide.

What does catalase do?

Breaks down H2O2 into water and O2 (bubbles).

Positive vs negative catalase?

Positive = bubbles. Negative = no bubbles.

What is the catalase test used to differentiate?

Catalase-positive Staphylococcaceae and Micrococcaceae from catalase-negative Streptococcaceae.

How is catalase performed?

Put a colony on a microscope slide, add 1-2 drops of H2O2, and observe immediately for bubbles.

What three tests does MIO do?

Motility, indole production, ornithine decarboxylation.

Why is MIO semisolid?

So motile organisms can move through the medium and make it cloudy.

Motile in MIO?

The entire medium is cloudy (stab line not visible).

Non-motile in MIO?

Clear medium with the stab line visible.

Order to read MIO?

Motility and ornithine first, then add Kovac's reagent for indole.

Why must ornithine decarboxylation be read before Kovac's?

Kovac's reagent changes the tube's appearance and would interfere with reading the other tests.

Ornithine decarboxylase positive in MIO?

Purple/grayish purple.

Ornithine decarboxylase negative in MIO?

Yellow.

Why does ornithine decarboxylation need glucose fermentation?

Acid from fermentation (yellow) triggers decarboxylase. Decarboxylation makes putrescine, which raises pH to purple.

MIO pH indicator?

Bromocresol purple.

Indole production reaction?

Tryptophanase cleaves tryptophan into indole + pyruvate + ammonia.

Reagent for indole?

Kovac's reagent.

Positive indole result?

Red ring/layer at the top after adding Kovac's.

What is Simmons citrate testing for?

Ability to use citrate as the sole carbon source.

Citrate medium nitrogen source?

Ammonium phosphate (sole nitrogen source).

Citrate pH indicator?

Bromothymol blue.

Citrate positive vs negative?

Positive = blue (pH 7.6). Negative = green (no change).

What must bacteria have to use citrate?

Citrate permease (to transport citrate into the cell).

Why is citrate positive blue?

Citrate use (and ammonium phosphate use) makes the medium alkaline, which turns bromothymol blue blue.

What is malonate used to differentiate?

Escherichia from Enterobacter.

How does malonate affect the TCA cycle?

It competitively inhibits succinate dehydrogenase (succinate -> fumarate step).

What happens to an organism that cannot use malonate?

Its TCA cycle is inhibited and it cannot grow.

Malonate positive vs negative?

Positive = dark blue (pH 7.6). Negative = green or slightly yellow.

Why is malonate positive blue?

The organism uses malonate and the medium turns alkaline, turning bromothymol blue blue.

Phenol red broth positive vs negative?

Positive = yellow (below pH 6.8). Negative = red/pink.

What causes yellow in phenol red broth?

Acid from carbohydrate fermentation lowers the pH.

What causes pink in phenol red broth?

Deamination of peptone amino acids makes ammonia, which raises the pH.

What is the Durham tube for?

Detecting gas production (bubble or pocket). Used with glucose.

What does each phenol red broth contain?

One specific carbohydrate (glucose, lactose, or sucrose, etc.). Label them because they look the same.

Urea broth indicator?

Phenol red.

Positive vs negative urease?

Positive = pink. Negative = orange/yellow.

Urease reaction?

Urea is hydrolyzed to ammonia + CO2. Ammonia raises the pH.

Which genera are rapid urease positive?

Proteus, Morganella, Providencia.

Nitrate reduction: reagent A and B?

A = sulfanilic acid. B = alpha-naphthylamine.

Nitrate test step 1?

Add reagent A and B and mix. Red means nitrate was reduced to nitrite (positive, done).

Nitrate: no color after A and B?

Incomplete. Either no reduction or reduced past nitrite, so add zinc.

Nitrate: what does zinc do?

Artificially reduces any remaining nitrate to nitrite.

Nitrate: red after adding zinc?

Negative. Nitrate was still present, so the organism did not reduce it.

Nitrate: no color change after zinc?

Positive. Nitrate was reduced past nitrite to other nitrogen compounds.

Nitrate to nitrite pathway?

NO3- to NO2- by nitrate reductase (single step).

Full denitrification pathway?

NO3- to NO2- to NO to N2O to N2.

What does gas in the nitrate Durham tube (non-fermenter) mean?

The organism reduced nitrate to N2 gas (test completed).

What is MSA selective and differential for?

Selective for Staphylococcus (high salt). Differential for mannitol fermentation.

Why is MSA selective?

7.5% NaCl dehydrates and kills most bacteria. Staph tolerates it.

Why is MSA differential?

Mannitol fermentation makes acid, which turns phenol red yellow.

MSA yellow means?

Mannitol fermented, possible pathogenic Staphylococcus aureus.

MSA red/pink means?

No mannitol fermentation, Staphylococcus other than S. aureus.

MSA no or poor growth means?

Inhibited by NaCl, not Staphylococcus.

What is BEA used for?

Presumptive ID of enterococci and the Streptococcus bovis (Group D) group.

BEA selective vs differential component?

Bile is selective. Esculin is differential.

BEA reaction?

Esculin is split into glucose + esculetin. Esculetin + Fe3+ (ferric citrate) makes a dark brown/black precipitate.

BEA positive vs negative?

Positive = dark brown/black. Negative = no change from uninoculated tube.

What is MacConkey selective for?

Gram-negative bacteria (bile salts and crystal violet inhibit Gram-positives).

What is MacConkey differential for?

Lactose fermentation.

MacConkey indicator?

Neutral red (colorless above pH 6.8, pink/red below).

MacConkey positive for lactose fermentation?

Pink/red colonies.

MacConkey negative for lactose fermentation?

Colorless colonies (still growth, just no lactose fermentation).

No growth on MacConkey means?

Not a Gram-negative (streak lines visible, no colonies).

Why is lactose fermentation pink on MAC?

Acid lowers the pH below 6.8 and neutral red turns pink.

Gram stain order of reagents?

Crystal violet, iodine, ethanol (decolorizer), safranin.

Gram-positive cell wall and result?

Thick peptidoglycan retains crystal violet-iodine, so cells are purple.