Microbiology Lab Practical 2

Some bacteria have the enzyme catalase, that breaks down and detoxifies _______ to ___ and ___ partially disabling the antimicrobial effect of this disinfectant.

H2O2 (hydrogen peroxide), H2O, O2

Red blood cells also express ______ --> that's why wounds bubble when exposed to H2O2!!

catalase

Aim of the ________ test is to check for the enzyme that protects bacteria against the toxic by-products of oxygen metabolism --> H2O2

catalase

________(enzyme) is present in most obligate aerobes & facultative anaerobes

catalase

Reagents in the _______ test involve: chromogenic reducing agents; turn blue/black when oxidized

Oxidase

Cytochrome C oxidase (reduced) receives electrons from _______ _______ _______ (oxidized) which turns it black/blue (+ result) (>20 secs!!!!)

chromogenic reducing agent

Any color change after 20 seconds during the _______ test, should be disregarded

Oxidase

Phenol Red Test checks for __________, & if they do, which _______ can be catabolized/used, & if they do, whether or not they produce only _______ alone or along with _______ as by product.

fermentation, sugars, acid, gas

Aim of phenol red test is to determine the ability of an organism to catabolize _____ through __________

sugars, fermentation

The phenol red test has 3 tests in one:

1. Glucose PR- checks for __________ (acid)

2. Sucrose/lactose PR- checks for_______ _____ (i.e., lactase, sucrase)

3. Durham tube – checks for ____ production as _______

1. fermentation

2. carb enzymes

3. gas, byproduct

Reagents in phenol red test include one medium. (has one ______ present, & ___ [indicator])

sugar (glucose, lactose, sucrose, mannitol, etc), pH

In the phenol red test, a pH indicator is used.

Neutral pH is _____

acidic pH is ______

basic pH is ______

red, yellow, pink

If the phenol red medium turns yellow (+), bacteria are using the ______ during _________; if purplish (-), they utilize ______ through _______ respiration.

Red= (-)

sugars, fermentation, proteins, aerobic

The durham tube within the phenol red medium checks for ___ production(indicatesfermentation)

gas (CO2, H2)

If the phenol red test doesn't change colors, it means the bacteria doesn't _________ for that specific _____

ferment, sugar

The methyl red test's aim is to detect organisms capabilities to ferment ______ ___ ______.

mixed acid glucose, acidic (pH-4)

Reagents used in methyl red include a medium with ________ & a __ _______

sugar (glucose), pH indicator

Methyl red (neutral pH = ________ - result, while acidic pH = __________ + result)

yellowish, red

Methyl red medium results:

Red = acidic (__ result) Yellow = neutral/basic (______ result)

Phenyl red medium results:

Red = neutral/basic (___ result) Yellow = acidic (_____ result)

positive, negative

negative, positive

No change (yellow media) = __________ for mixed acid fermentation. Red media = __________ (Methyl Red Test)

negative, positive

Test for detecting organisms ability to ferment glucose to acid, then to acetoin. Checks for production of butanediol.

VP (Voges Proskauer) Test

Reagents for Voges-Proskauer test include a medium with ______ & _________ (VP I) (toxic) & _________ ________ (VP II)

sugar (glucose), alpha-naphtol, potassium hydroxide (KOH)

VP I (a-naphtol) & VP II (KOH) both react with w/ acetoin if present producing a ___________

red acetoin compound

Voges-Proskauer Test Results:

-No change/yellowish color = __________ for fermentation (_______ production)

-Red= __________

negative = no acetoin produced/ no partial mixed acid fermentation, positive = partial mixed acid fermentation

___________ Aims to determine the ability of the bacteria to use sodium citrate as the only source of carbon and inorganic ammonium hydrogen phosphate (NH4H2PO4) as a source of nitrogen. Possible only if the organisms are capable of fermenting citrate.

Citrate Utilization Test

Reagents in Citrate Utilization Test:

1. High concentrations of ______ ______ (only source of _____).

2. pH indicator___________ ______ (some citrate positive organisms will produce ______ when using citrate --> ___ pH)

1. sodium citrate, carbon

2. bromothymol blue, ammonia, basic (pH)

Positive citrate utilization test is _____ & indicates growth. Negative (or positive) result is ________ meaning, likely, no growth.

blue, green

Color is tricky. Some bacteria can grow using ______ but the media will not experience a change in color. ______ is ALWAYS positive. ______ could be positive OR negative.

citrate, blue, green

________ _______ Test is used to determine whether an organism reduces nitrate to nitrite.

Nitrate Reduction

UTI test strips/ lab tests look for the presence of ________. Urine in healthy people will NOT contain it (reason for ________ reduction test)

Nitrites, nitrate

UTIs is mostly caused by enterobacteria; they convert nitrates down to ____

nitrites

People suffering migraines might have a mouth microbiome enriched nitrate ________ bacteria that convert Nitrates to _____ oxide.

reducing, Nitric

Gas gangrene is often caused by Clostridium ______. Causes death of muscle tissue.

perfringens

Nitrate Reduction Test:

Nitrates are used as electron acceptor in the e- transport chain (________ respiration) down to _________ gas

anaerobic, Nitrogen

Why should we care about the Nitrate Reduction Test?:

Production of high levels of ______ by gut bacteria. Bacteria converting _______ into _______, might be toxic.

ammonia, nitrates, ammonia

Aim of Nitrate Reduction Test:

Determining the ability of an organism to transform _______ into ______ by the enzyme Nitrate reductase (NR).

nitrates, nitrites

Nitrate Reduction Test Reagents:

a) medium w/ _______

b) alpha-___________ (NR-A) & ________ acid (NR-B)

c) _____ (chemically catalyzes conversion of Nitrates into Nitrites)

a. Nitrate

b. nitrate reagent A (sulfanilic acid), nitrate reagent B (alpha-Naphthylamine)

3. zinc

Results of Nitrate Reduction Test will turn ____ (color) ONLY if ______ is present w/i tube. (via ___ production)

red, nitrites, gas

Nitrate reduction Steps:

1. Add __ drops of nitrate reagent A, & __ drops of reagent B.

2. If colorless after Step 1: add pinch of ____ dust (mix/let rest 10 min)

8, 8, zinc

a-napthylamine & sulfanilic acid ONLY react with __________ (turns RED +)

nitrites

The zinc dust will _______ the reduction of nitrate to nitrite chemically. Thus, if the nitrate has not been reduced by the organisms, i.e., they are nitrate-negative, it will be reduced by the zinc dust and a ___ color will develop in the incubated medium within 15 min.

catalyze, red

If solution turns red/purple after adding Zn to tube (Nitrate reduction) test, it means the organisms weren't able to convert ______ to ________ (Zn reduces it instead)

nitrate, nitrite

Adding Zn to nitrate reduction test tube and having it turn red/purple is a ______ result. If ________, it wouldn't change color due to LACK of _______ (zn wouldn't react, ______ used up!)

negative, positive, nitrate, nitrite

H. Bile Esculin Test Aim:

Differentiates ______________ _____________ (fecal bacteria) from other gamma _________ species (nonhemolytic/group b) based off presence of the enzyme: _________

Enterococcus faecalis, streptococcus, esculinase

H. Bile Esculin Test Reagents:

a) ___: inhibits growth of gram-positive cocci EXCEPT enterococci

b) ______: plant glucoside that can be metabolized by Enterococcus

c) ______ _____: combines w/ esuletin producing a dark brown precipitate

bile, esculin, ferric citrate

H. Bile Esculin Test Results:

_______: Agar darkens

_______: No color change

Positive, negative

H. Bile Esculin Test Reagent Functions:

a. Bile – inhibits growth of Gram-_______ cocci but enterococci.

b. Esculin – Plant glucoside that can be metabolized by __________

c. Ferric citrate- combines with ______- producing a dark ______ precipitate

positive, Enterococcus, esculetin, brown

_______ catabolism is important in the process of putrefaction (rotting) & food spoilage

Protein

Milk is spoiled by bacteria --> _____ is

the most abundant protein in milk. Degradation

of it will produce foul smell in putrid milk.

casein

Protein catabolism occurs via ____________, ____________ & ________ ______ removal (bacteria produce byproducts omitting smell)

decarboxylation, deamination, side chain

Decarboxylation Products are ______ (pH scale) We use pH indicators to identify.

basic

Why should we care about amino acid decarboxylation?

Pressure ulcers and tissue infections emanating ______. Caused by bacterial infections that produce volatile _____ from amino acid decarboxylation (putrescine /cadaverine)

malodor, amines

Aim of Amino Acid Decarboxylation (Arginine, Ornithine, & Lysine) Test:

Used to identify the presence of a ____________ enzyme

decarboxylase

Amino Acid Decarboxylation Test Reagents:

a. ______, ________ or _____ (also _____ and extracts)

b. __ __________ (acidic=yellow under 5.5pH, basic = purple over 6.8)

c. ___ ______ --> Promotes fermentation -> Low pH = Production of decarboxylases

a. Arginine, ornithine, Lysine, sugars

b. pH indicator: bromocresol purple

c. mineral oil

Amino Acid Decarboxylation Test Results:

Yellow --> _______ for decarboxylation

Purple --> _______ for decarboxylation

negative, positive

Positive Decarboxylase Positive Result

Decarboxylase synthesis is activate producing _______ byproducts turning the tube __________ (color)

Alkaline, purple

Phenylalanine Deaminase Test Aim:

determining presence of deaminase enzyme __________

Phenylalanine deaminase

Phenylalanine Deaminase Test Reagents: Medium

a. (also yeast extract)

b. _______ _______ (reacts in the presence of phenylpyruvic acid --> Green color).

a. Phenylalanine

b. Ferric chloride

Phenylalanine deaminase test Results

Positive for deamination: ____________

Negative for deamination: _____________

green color, no change

C. SIM Test (Amino acid side chain removal) Test Aim:

To characterize gram ________ _________ (salmonella vs shigella) based on motility and amino-acid catabolism capabilities

negative enterobacteria

C. SIM Test (Amino acid side chain removal) Consists of 3 Tests in One:

1. _______ reduction: Determine if bacteria display Cysteine Desulfurase activity (removes sulfur from amino acid cysteine). SH2 can also be produced during anaerobic respiration (in anaerobic environment only)

2. ______

3. _____ formation– Determine if bacteria display Tryptophanase activity. Metabolism of tryptophan (Amino acid found in proteins-peptone) metabolized to indole (Indole detected with Kovac’s reagent pink if indole is present)

Sulfur, Motility, Indole

Indole production (Tryptophan Hydrolysis-SIM) Test Aim:

Identify bacteria with the enzyme _________ (breaks down amino acid Tryptophan to Pyruvate for energy -> waste byproduct generated = Indole) (SIM)

Tryptophanase

_____ production is indicative of amino acid breakdown to _____ for energy by the enzyme Tryptophanase (SIM)

Indole, pyruvate

Reagents for Indole production (SIMTest):

Media with 1% _____

_______ reagent (6-8 drops)

Tryptone, Kovac's

Reagents for Indole production (SIM Test) Results:

___ ring formation at the ___ of the tube

after addition of Kovac’s Reagent = + for indole

red, top

SIM motility test Aim: Identify _____ in bacteria.

Reagents: Media with 0.75% )_____ (Solid enough to prevent diffusion of the media while allowing the motility of bacteria)

Results: Turbidity of tube beyond stabbing point indicates ______ (cannot see the black line crossing)

motility, agar, motility

Urease Test Significance:

-Peptic ulcers! Helicobacter pylori can thrive in stomach thanks to _____ --> produces _____ that neutralizes gastric acids.

Urease, ammonia

Urease Test Significance:

urease-positive bacteria converts ___ to _________ (why piss smells so bad)

urea, ammonia

If your pee stinks like ammonia… You might have a urinary tract infection caused by ____ _______.

Proteus mirabilis

Aim of Urease Test: Determining the ability of an organism to hydrolyze ___ into _______ (strong base = increased pH), through the production of the enzyme urease.

Reagents: medium with urea ____ _____ (orange/peach at _____ pH. Pink at _____ pH)

Results:

_______= Pink (high pH)

________ = no color change (peach)

urea, ammonia,

phenol red, neutral, basic

positive

negative

a-naphthylamine and sulfanilic acid are reagents for the ________.

Nitrate Reduction Test

citrate utilization test may be ________ if green

positive and negative

Purple on paper (positive result) is what metabolic test?

Oxidase test

If bubbles are present (positive), the metabolic test is looking for the enzyme _________ (also in RBC)

catalase

Catalase detoxifies ________ _______ (purple paper)

hydrogen peroxide

Voges Proskauer test is used to identify bacteria able to perform _______ fermentation

Butanediol

RED Methyl red test is a ___________ result

positive

Voges Proskauer uses the following reagent/s ______________ + ________ ________

alpha-naphthol + potassium hydroxide

Phenol Red Test Results

red color= ___________ for fermentation (uses __________ or no glucose)

yellow color= ________ for fermentation (bubble indicates _______)

negative, respiration, positive, gas (production)

What is formed when sulfur production is positive (SIM test)? [C]

black precipitate

What happens when Indole is present? (SIM test A)

Red top forms (positive)

Starch agar results + it uses the enzyme __________

amylase

Test for determining the ability of microorganisms to degrade urea by means of the enzyme urease.

Urease Test

urease test

phenol red

Positive result in phenol red test are ______ in color, AND may or may not have a _________ w/i tube

yellow, bubble

decarboxylation test results

Only _______ color is positive

Decarboxylase enzyme present if:

+ Purple

MINERAL OIL (anaerobic)

- yellow = fermentation but no decarboxylation

Purple(positive) decarboxylase test is either acidic or basic: _______

basic

Yellow(negative) decarboxylase test is either acidic or basic: _______

acidic

Test employed to differentiate among the urea- positive gram-negative bacilli on the basis of the ability of the microorganisms to produce phenyl-pyruvic acid by oxidative deamination.

Phenylalanine Deaminase (PDA) Test

Phenylalanine Deaminase Test:

Green is ___________

Unchanged is _________

positive, negative

Phenylalanine Deaminase Test reagents _______ _________ (FeCl3)

Ferric chloride

Methyl Red Test measures the production of ______ _______ by fermentation of glucose

mixed acids