biol 3201 miller lab 5

selective medium

medium that allows only certain microorganisms to grow and inhibits the growth of other microorganisms

differential medium

medium that contains substances that cause some bacteria to take on an appearance that distinguishes them from other bacteria

test to media types

selective:

differential:

selective & differential: EMB

EMB as a selective media

growth of Gram negative bacteria goes unaffected

growth of Gram positive bacteria is affected by the incorporation of eosin and methylene blue dyes in the media

EMB as a differential media - metallic green sheen colonies

Gram negative bacteria that vigorously ferment lactose

EMB as a differential media

metallic green sheen colonies

purple colonies

pink/colorless colonies

EMB as a differential media - purple colonies

Gram negative bacteria that moderately ferment

lactose

EMB as a differential media - pink/colorless colonies

Non-lactose-fermenting bacteria

what is the common confusion when interpreting EMB colony results

colorless colonies are confused with purple colonies

media itself is purple

in the colorless colonies you can see through

in the purple colonies you can't see through

organisms break dow carbs by

oxidation or fermentation

carb breakdown by oxidation or fermentation produces

acid

phenol red carb test reveal acid through

color change in pH indicator from phenol red to yellow

red at alkaline pH values

yellow at acidic pH values

*Gram(+) test

phenol red carb tube is used for

Gram positive cocci and rods

OF: O/F carb tubes are used to determine

whether an organism produces acid through fermentation

or only through oxidation

OF: how are O/F carb tubes and phenol red carb tubes similar

any carb cane added to the medium

OF: how are O/F carb tubes different from phenol red carb tubes

contain semisolid agar rather than liquid

OF: the semisolid agar in O/F carb tubes serves what purpose

helps to create anaerobic environment in the F (fermentation) tube necessary for fermentation reactions

OF: how do you inoculate for both F and O tubes

stab the F tube with an inoculating needle containing your culture

OF: what is added to inoculated F tube

sterile mineral oil

added to top of tube to ensure an anaerobic environment

OF: what is added to inoculated O tube

no oil is added

OF: acid production in O/F carb tubes is indicated by

color change from green to yellow

OF: yellow organisms in both O and F tubes

fermentation of carbs

OF: yellow organism in only O tube

do not carry out fermentation

produce acid only through the oxidative pathway (respiration)

OF: what is required for proper results of O/F carb tube results

both an O and F tube must be used with each culture tested

MR: examples of genera that ferment glucose to produce organic acids

Escherichia

Salmonella

Proteus

MR: genera that ferment glucose to produce what kinds of organic acids

lactic

acetic

succinic

formic acids

MR: what is produced in mixed acid fermentation

organic acids: lactic, acetic, succinic, formic acids

CO2

H2

ethanol

MR: what lowers a MRVP broth to >5.0

sufficient acid production

MR: methyl red test tests for

mixed acid fermentation

MR: MRVP medium

a glucose broth that is buffered with peptone and dipotassium phosphate

MR: what pH indicator in used in methyl red test

methyl red

MR: red, methyl red test result

acid is present

a positive result

MR: what does a positive/red, methyl red test result indicate

organism carried out mixed acid fermentation

VP: bacteria do not carry out mixed fermentation and rather

ferment glucose to produce limited amounts of some organic aids and more neural end product 2,3-butanediol

VP: bacteria produce

some organic aids and more neural end product 2,3-butanediol

VP: bacteria that carry out butanediol fermentation

Klebsiella

Serratia

some Bacillus

VP: some Bacillus produce butanediol when grown on

glucose

VP: results to produce (-MR) test

produces butanediol and is (+)VP

VP: end product detection

2,3-butanediol is detected once converted to acetoin

VP: how is acetoin created

oxidation of 2,3-butanediol

reactions with reagents VP1 (alpha-naphthol) and VP2 (KOH)

- reagents are added to a 3 to 5 day old culture grown in MR-VP medium and vigorously shaken to oxidize the 2,3-butanediol to acetoin

- the tube is allowed to stand at room temperature for 30 minutes

VP: reagents

VP1 (alpha-naphthol) and VP2 (KOH)

VP: medium type

MR-VP medium

VP: reaction

2,3-butanediol is oxidized to acetoin

VP: acetoin is indicated by

color change from pink to red if present

CT: some bacteria are capable of using __ as a sole carbon source

citrate

CT: where is citrate oxidatively metabolized

Krebs cycle

CT: bacteria that can cleave citrate to produce oxaloacetate and pyruvate

Klebsiella aerogenes (previously Enterobacter aerogenes)

Salmonella typhimurium

CT: bacteria that can cleave citrate produce

oxaloacetate and pyruvate

CT: oxaloacetate and pyruvate are fermented to produce

end products like;

- formate

- acetate

- lactate

- acetoin

- CO2

CT: citrate test medium contains

citrate

salts

CT: use of salt in citrate test medium

serve as a sole nitrogen source for growth

CT: organisms that degrade citrate use

ammonium salts from nitrogen

CT: organisms that degrade citrate produce

ammonia

CT: ammonia production in citrate test results in

alkaline medium

pH indicator turns from dark green to deep Prussia blue

CT: pH indicator in citrate test

bromothymol blue

CT: bromothymol blue color

dark green

CT: what does the color change in bromothymol blue indicator to deep Prussian blue indicate

utilization of citrate

SIM stands for

sulfur, indole, motility

SIM tests

hydrogen sulfide production (sulfur reduction)

*SIM-HS:

indole production (tryptophan degradation)

*SIM-IP

motility

*SIM-M

SIM-HS: bacteria that reduce elemental sulfur produce

hydrogen sulfide (H2S)

SIM-HS: bacteria that reduce elemental sulfur produce get their energy from

coupling reaction (elemental sulfur --> H2S) to the oxidation of Kreb's cycle intermediates (acetate and succinate)

SIM-HS: SIM tube determines

if a microorganism can reduce elemental sulfur

SIM-HS: SIM medium contains

ferrous ammonium sulfate and sodium thiosulfate

SIM-HS: SIM indicators

ferrous ammonium sulfate and sodium thiosulfate

SIM-HS: SIM indicators indicate

the production of hydrogen sulfide (H2S)

SIM-HS: organisms that reduce sulfur in SIM media produce

H2S gas

SIM-HS: H2S gas produced by reducing sulfur in SIM media interacts with

ferrous ammonium sulfate and sodium thiosulfate

forms ferrous sulfide

SIM-HS: SIM ferrous sulfide indicator

black precipitate

SIM-IP: bacteria that can degrade tryptophan produce

indole

ammonia

pyruvic acid

SIM-IP: purpose of pyruvic acid from tryptophan degradation

used for various metabolic purposes

SIM-IP: enzyme responsible for cleavage of tryptophan

tryptophanase

SIM-IP: tryptophan degradation by enzyme is detected by

Kovac's reagent

SIM-IP: Kovac's regent that is produced from tryptophan degradation by enzyme forms

deep red color if indole is present

SIM-IP: broth used in indole production test

tryptone broth 1%

SIM-IP: tryptone broth 1% contains high amounts of

tryptophan

SIM-IP: tryptone is derived by

pancreatic digestion of the protein

SIM-M: major organelles of motility in bacteria

flagella

SIM-M: flagella function

allow cells to move toward nutrients in the environment

or more away from harmful substances (EX acids)

SIM-M: chemotaxis

cell movement that occurs in response to chemical stimulus (acid)

SIM-M: flagellum structure

rigid, helical

extends 10microns out from cell

very thin (>0.2microns)

flagella vs flagellum

flagella - plural

flagellum - singular

SIM-M: flagella under a microscope

they are so thin they are below that resolution of the light microscope, unless stained by special techniques

SIM-M: flagella cause bacterial cells to. move because

they rotate, in a way similar to a screw on a boat engine that rotates to properly a boar through the water

SIM-M: motility can be determined

1 microscopically

2 inoculating SIM semisoft agar medium

SIM-M: microscopically viewing motility uses a

wet mount

SIM-M: microscopically viewing motility procedure

drop of viable cells are placed on microscope slide

covered with glass

slide is observed with phase-contrast microscope

SIM-M: microscopically viewing motility microscope type

phase-contrast

SIM-M: what confirms motility under phase-contrast microscope

raid, swimming movement of cells

SIM-M: Brownian motion

movement caused by currents under cover glass

due to molecular bombardment of cells causing cells to shake/wiggle about

- do not move in an vectorial way

cans can appear to move because currents can be created under cover glass when pressure is exerted by focusing oil immersion lens or by went mount drying out

SIM-M: true swimming motility must be differentiated from

Brownian motion of cells

SIM-M: ways cells can move that seem like motility movement

Brownian motion

- cause cells to shake/wiggle about; no vectorial movement

due to currents can be created under cover glass when pressure is exerted by focusing oil immersion lens

- causes cells to sweep across the field

by went mount drying out

- causes cells to sweep across the field

SIM-M: SIM agar concentration

0.4%

medium does not inhibit bacteria from swimming through the medium

SIM-M: SIM viewing motility procedure

organism in inoculated by stabbing the semisolid agar with inoculating needle

SIM-M: SIM viewing motility procedure: motile (+)result

when inoculated, the organism will swim away from line of inoculation into the uninoculated surrounding medium

causes medium to turn turbid

SIM-M: SIM viewing motility procedure: motile (-)result

nonmotile bacteria will be found only along the line of inoculation

SIM-M: arrangement of flagella

swarming motility: some extremely motile bacteria are

able to move rapidly and chase nutrients that they can metabolize for growth

swarming motility bacteria

Proteus mirabilis

swarming motility: Proteus mirabilis on a plate

once incubated, the bacteria will start to move out in every direction

during movement each bacterium absorbs nutrients and increases in size

after a certain distance, they divide and the progeny continue to move out

causes formation of swarms (concentric rings) on plate

swarming motility: Proteus mirabilis on a plate produces

swarms of concentric rings