BIOL 351 Lab Exam 3

biochemical tests

differentiate and sometimes identify microorganisms based on specific biochemical characteristics

A mixed culture must be what before they can be tested and identified?

isolated and cultivated

selective media

designed to enhance isolation by inhibiting growth of some organisms while encouraging growth of others

differential media

contains indicators to expose differences between organisms

coliforms

subgroup of Enterobacteriaceae that produce acid and gas from lactose fermentation

most prominent member of the coliform group

E. coli

Most coliforms are normal inhabitants of the…

human intestinal tract

The presence of coliforms in the environment may be evidence of…

fecal contamination

TRUE OR FALSE: The media used for biochemical tests may be any combination of selective, differential, defined or undefined instead of one singular type.

True

Nutritional components of media used for biochemical tests are typically selected to…

obtain optimal growth

inhibitors make the medium…

selective

inhibitors are designed to…

prevent growth of specific groups of microorganisms while allowing other organisms to grow

substrates are almost always…

what makes the medium differential

substrates have differing abilities to…

perform a specific chemical reaction that differentiates organisms

indicators make the expected reaction…

visible

indicators are frequently…

a dye that changes color as pH changes

indicators can also be…

chemicals that react with products of a reaction and produce a color change

TRUE OR FALSE: A fundamental feature of media used for biochemical tests is whether it’s positive or negative.

True

List of all fundamental features of media used for biochemical tests

• Type of media (Selective, Differential, Defined or Undefined)

• Nutritional components

• Inhibitors

• Substrates

• Indicators

• Positive or Negative

fermentation is the preferred source of

metabolic activity

fermentation

process of extracting energy from oxidation of organic compounds (e.g., carbohydrates like glucose, sucrose, lactose)

organisms capable of fermenting glucose will…

exhaust glucose supply before utilizing sucrose or lactose

fermentation produces…

acid byproducts lowering the pH

What kind of gas do many types of fermentation products produce?

hydrogen gas

When do microbes utilize peptones or amino acids?

when carbohydrates are depleted or when microbes are incapable of carbohydrate fermentation

Microbes utilizing peptones or amino acids results in…

NH₃ production, raising pH

Some different capabilities of microbes:

• only ferment glucose

• ferment glucose and sucrose and/or lactose

• cannot ferment any carbohydrates

Triple Sugar Iron Agar (TSIA)

differentiates bacteria on basis of sugar fermentation

list of sugar fermentation sources

• glucose

• sucrose/lactose

• sulfur reduction

• gas production

TSIA slant is a…

shallow slant with deep butt

TSIA slate provides…

aerobic and anaerobic growth environments

TSIA slant is inoculated by…

stab in butt followed by fishtail streaks of slant

Carbohydrates in TSIA slant

glucose, sucrose, lactose

Sources of oxidized sulfur in TSIA slant

sodium thiosulfate (and ferrous sulfate)

Fermentation indicator in TSIA slant

phenol red

Sulfur reduction indicator in TSIA slant

Ferrous sulfate

When TSIA is inoculated with a glucose ONLY fermenter, fermentation of glucose…

produces acid → lowers pH and turns medium yellow throughout

When TSIA is inoculated with a glucose ONLY fermenter, glucose concentration is…

very low (0.1%) and so is depleted quickly

When TSIA is inoculated with a glucose ONLY fermenter, as glucose diminishes…

organisms in aerobic region (slant) begin to break down available amino acids → produce NH₃ → raise pH and turn media pink

Organisms breaking down available amino acids only occurs in the aerobic region, or the slant, because of…

the anaerobic conditions in the butt

When TSIA is inoculated with an organism capable of fermenting glucose AND lactose and/or sucrose, fermentation of glucose…

lowers pH and turns medium yellow throughout

When TSIA is inoculated with an organism capable of fermenting glucose AND lactose and/or sucrose, because lactose and/or sucrose concentration are 10X higher than glucose…

greater acid production keeps slant and butt yellow

When TSIA is inoculated with organisms incapable of carbohydrate fermentation…

organism utilizes peptone and amino acids → alkalinizes medium and turns it red (appears as inoculated)

In TSIA, gas is produced by…

fermentation of carbohydrates

In TSIA, gas production appears as…

fissures in medium or will lift agar off of bottom of tube

In TSIA, some gas production is…

more obvious than others

In TSIA, to find evidence of gas production, look for…

cracks or bubbles in agar

In TSIA, hydrogen sulfide (H₂S) may be produced by…

reduction of thiosulfate in medium or by breakdown of cysteine in peptone

In TSIA, ferrous sulfate indicator reacts with hydrogen sulfide to form…

black precipitate

What conditions must exist for thiosulfate reduction?

Acidic conditions

In TSIA, black precipitate indicates…

BOTH sulfur reduction and fermentation

In TSIA, if black precipitate obscures color of butt, it MUST be…

yellow

What is the format to record results for TSIA?

slant/butt/gas/H₂S

To record the pH result of TSIA, what letters are used for yellow color versus pink/red, respectively?

A for Acidic, K for Alkaline

To record gas or H₂S production in TSIA, what symbols are used?

(+) or (-)

Record the TSIA results for the tube in the picture.

K/A/-/+

Lysine Iron Agar (LIA) is used to…

differentiate enterics based on ability to decarboxylate or deaminate lysine

LIA is a combination medium that detects…

ability to decarboxylate or deaminate lysine and to reduce sulfur

LIA is prepared as…

a slant with a deep butt (aerobic and anaerobic)

pH indicator in LIA

bromocresol purple

Sulfur reduction indicator in LIA

ferric ammonium citrate

lysine decarboxylation in LIA

glucose fermentation produces acids → lowers pH and turns medium yellow → induces production of decarboxylase enzymes

In LIA, subsequent decarboxylation of lysine will…

alkalinize agar and return it to purple

LIA results should be interpreted based on…

color of butt

yellow butt in LIA

(+) glucose fermentation, (-) lysine decarboxylation

purple butt in LIA

(+) lysine decarboxylation, (+) glucose fermentation

In LIA, if organism produces lysine deaminase…

deamination produces compounds that react with ferric ammonium citrate and produce red (burgundy/maroon) color

What 3 genera of bacteria are the only ones that can perform lysine deamination?

• Proteus

• Providencia

• Morganella

In LIA, lysine deamination reaction requires…

oxygen → only seen in slant

red slant and yellow butt in LIA

(+) lysine deamination

purple slant in LIA

(-) lysine deamination

In LIA, purple result is recorded as…

K for Alkaline

In LIA, yellow result is recorded as…

A for Acidic

In LIA, red result is recorded as…

R for deamination

What is the result recording format for LIA?

slant color/butt color

LIA result: Purple slant/purple butt

K/K, lysine deaminase (-), lysine decarboxylase (+)

LIA result: Purple slant/yellow butt

K/A, lysine deaminase (-), lysine decarboxylase (-), glucose fermentation

LIA result: Red slant/yellow butt

R/A, lysine deaminase (+), lysine decarboxylase (-), glucose fermentation

oxidase test

identify bacteria containing cytochrome c oxidase

Glycolysis and Krebs cycle result in…

accumulation of reduced coenzymes (NADH and FADH₂)

To continue oxidizing glucose, coenzymes NADH and FADH₂ must be converted to…

previous oxidized state via electron transport chain (ETC)

Important ETC molecules are…

flavoproteins, iron-sulfur proteins, cytochromes

oxidase test procedure

bacterial colony is transferred to paper and saturated with reagent

oxidase test results can be viewed through…

a dramatic color change occurring within 20 seconds

oxidase test positive result

reducing agent becomes oxidized → indicates that cytochrome oxidase c is present (+)

oxidase test negative result

reducing agent is NOT oxidized → no cytochrome oxidase c is present (-)

In oxidase test, cytochrome c oxidase transfers e- from…

cytochrome c to O₂

In oxidase test, when cytochrome c is present, what reduces it?

tetramethyl-p-phenylenediamine

In oxidase test, the redox reaction results in…

oxidized tetramethyl-p-phenylenediamine

What color is oxidized tetramethyl-p-phenylenediamine?

deep purple/blue

Blue color from oxidase test on sample

positive result

No color from oxidase test

negative result

catalase test

identify organisms that produce enzyme catalase

Catalase test is most commonly used to differentiate…

members of catalase-positive Staphylococceae and Micrococcaceae from catalase-negative Streptococcaeceae

In the catalase test, what type of microbes utilize ETC?

aerobes and facultative anaerobes

ETC is capable of accepting and donating electrons as conditions require…

molecules in ETC alternate between oxidized and reduced states, passing electrons down chain to final electron acceptor

Usual final electron acceptor of ETC

O₂

In catalase test, an alternate pathway uses…

one carrier molecule, flavoprotein, that bypasses carriers and passes electrons directly to oxygen

In catalase test, the alternate pathway produces what two highly potent toxins?

• hydrogen peroxide [H₂O₂] (catalase)

• superoxide radicals [O₂^-] (superoxide dismutase)

catalase test procedure

bacterial colony is transferred to microscope slide and saturated with hydrogen peroxide