MCB3020C UCF Wilson Lab midterm

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102 Terms

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lab attire

lab coats, gloves, goggles, long pants, long hair should be tied back, closed-toe shoes

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aseptic technique

limits the presence and spread of potentially harmful organisms

prevents exposure of yourself and others to potentially harmful bacteria

reduces the posibility of contaminating your sample

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two-tube transfer

1. Sterilize loop

2. Uncap tubes

3. Sterilize tubes

4. Transfer from culture to fresh medium

5. Sterilize tubes

6. Recap tubes

7. Sterilize loop

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staining

useful for identifying morphology, size, and arrangement of the bacterial sample

simple, differential, structural

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catioinic

basic dyes, + charged chromophores

+ ion exhibits color

methylene blue or crystal violet

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anionic

acidic dyes, - charged chromophores

- ion exhibits color

acid fuchsin, congo red, nigrosin

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negative stain

stain doesn't penetrate the cell

- negatively charged stain repelled by - charge at cell surface

- transparent cells against a colored background

good for examining morphology and size of bacterial cells

- cell are NOT heat fixed, so distortion of the bacterial cells is minimized

useful for organisms that don't stain easily

nigrosin: black anionic dye

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simple stain

one dye used to stain all cells present

- we will use crystal violet (cationic dye)

useful in distinguishing different morphologies, arrangement, and relative sizes

colors the cells and provides contrast when using bright field microscopy

+ charge of the dye interacts with the - charge on bacterial cell wall

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smear preparation [from a broth]

1. aseptically transfer 1 loopful of culture to a clean slide and spread to maximum thinness

2. allow to air dry

3. heat fix by holding over microincinerator for 10 seconds - USE CLOTHESPIN!!

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smear preparation [from slant or plate]

1. add 1 loopful of water to the slide FIRST

2. aseptically collect bacterial sample from plate or slant

- just touch the loop to the bacterial growth on the plate, slant, mix well to suspend

3. allow to air dry

4. heat fix by holding over microincinerator for 1o seconds - USE CLOTHESPIN!!

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microscope

know the parts

exercise 2 slide 14

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illumination [bright-field microscopy]

light source

condensor: directs light towards the objective lens in bright field microscopy

iris diaphragm: adjusts the diameter of the cone of light so that it just fills the objective lens

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magnification [bright-field microscopy]

ocular: 10x

objectives: 4x, 10x, 40x & 100x

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resolution [bright-field microscopy]

resolution is the smallest distance between two objects which can be seen as separate

resolving power: 0.2 mm

average microbe: 1.0 mm

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oil immersion

100x = oil immersion

MUST use oil

oil has the same refractive index as glass

-oil stops refraction of light between specimen and lens

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resolution

d(resolution) = lamda/(2NA)

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cocci

spherical bacteria

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bacilli

Rod shaped bacteria

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differential stain

detects differences between organisms through the use of many dyes and reagents

- gram stain

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structural stain

confirm structural characteristics of cells

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Hans Christian Gram (mid 1880s)

developed the gram stain

danish physician and scientist

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gram stain

used crystal violet to stain bacterial cells

when cleaning up sample with ethanol - found some bacterial cells would not retain purple

- considered the stain a "failure"

now the most widely used & important stain in bacteriology

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peptidoglycan layer

differentiates bacteria into 2 groups based on the thickness of the peptidoglycan cell wall

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primary dye [gram stain]

crystal violet

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mordant [gram stain]

Gram's iodine

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decolorizer [gram stain]

95% ethanol

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secondary dye/counterstain [gram stain]

safranin

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gram negative

stain pink

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gram positive

stain purple

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acid-fast staining: Ziehl-Neelsen Method

differential stain

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acid-fast microorganisms

have high mycolic acid content in their cell walls - waxy and resists staining

steam is used to force the primary dye to penetrate the waxy cell wall

fastness = to retain the dye when challenged by a decolorizer

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primary dye [acid-fast stain]

carbol fuchsin

[need steam]

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decolorizer [acid-fast stain]

acid alcohol

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methylene blue [acid-fast stain]

secondary dye/ counterstain

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acid-fast

cerise or pink/violet because of the high wax content they retain the dye after decolorization

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acid-fast negative

blue because the cells will readily decolorize and be counterstained with methylene blue

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acid fast genera

Mycobacterium and Nocardia

-M. tuberculosis: causative agent of tb

-M. leprae: causative agent of Hansen's disease (leprosy)

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spore staining: Schaeffer-Fulton Method

structural stain used to detect dormant forms of bacteria [endospores]

- these structures can be released called - free spores

- to force the primary dye into the resistant endospore steaming is used

- stained spores are then resistant to decolorization

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primary dye [spore staining]

malachite green

- steam

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decolorizer [spore staining]

water

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secondary dye / counterstain [spore staining]

safranin

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endospores

spores are green inside pink bacterial cells

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free spores

small green oval bodies [free]

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if no spores

only pink cells will be observed

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genera of spore forming bacteria

Bacillus: gram + rod

- B. anthracis

Clostridium: gram + rod

- C. botulinum

- C. tetani

-C. difficile

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pure culture

contains a single microbial species

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environmental & patient samples

almost always have a mix of microbes

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pure culture techniques

aim to dilute bacterial samples in order to separate individual cells that can grow into isolated colonies when plated

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growth medium and agar

a variety of different types of media are available

- general purpose

- selective

- differential

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general purpose medium

used when trying to isolate microbes from a mixed culture

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solid media

are often preferred when trying to obtain pure cultures

-agar is solidifying agent derived from seaweed

microbes are unable to metabolized agar

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pour plate

this method can be used in both a quantitative and non-quantitative way depending on the dilution method used

- by diluting individual cells are played which can grow into colonies

- culture is diluted in molten agar (> 40 degrees C)

- once diluted agar is mixed well it is poured into an empty sterile petri dish and allowed to solidify (40 C)

- colonies can be found on top of agar, embedded in agar, and underneath agar

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spread plate isolation

brother culture diluted in a series of tubes - serial dilution

- quantitative or non-quantitative as the dilution is performed with an inoculating loop

- colonies will only form on the surface of the agar

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streak plate technique

method to separate a single species from a mixed species population

- sample is plated across the surface of the agar in 3 zones

- isolation of 3 mixed species

- primary streak

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general purpose media

support growth of variety of microorganisms

- good for maintaining cultures

- includes: nutrient agar (NA), tryptic soy agar (TSA), brain heart infusion (BHI)

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selective media

use inhibitors to prevent growth of certain organisms

- includes: phenyl ethyl agar (PEA), eosin methylene blue (EMB), MacConkey agar (mac)

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differential media

allow growth of many microbes, but differentiation is seen by indicators that detect changes that have occurred

- dye, reagents, blood cells, culture conditions

- includes: EMB, blood agar, macconkey agar

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combination media

are both selective and differential

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complex media

undefined, exact chemical composition not know [box cake mix]

- usually have nutrients supplied as extracts or digests from natural sources

- BHI, TSA

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defined

known chemical composition for each component [homemade cake]

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phenylethyl Alcohol Agar (PEA)

- selective only

- inhibitor: phenyethyl alcohol

- prevents growth of gram -

- often used to select for gram + species in patient samples

- staphylococci

- get rid of contaminants like E. coli

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blood agar (BA)

- differential only

-indicator: red blood cells

- TSA base w/ 5% sheep's red blood cells added

- blood agar can be useful in differentiating hemolysis patterns of microorganisms

-lysis of red blood cells

- beta: best

- alpha: average

- gamma: trash

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macconkey agar (Mac)

- selective and differential = combination

- indicator: neutral red

- differentiate lactose fermenters from non-fermenters

- inhibitor: crystal violet and bile salts

- prevent growth of gram +

- used for identification of gram - enterics

- enterobacteriaceae

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eosin methylene blue (EMB)

- selective and differential = combination

-inhibitors: eosin & methylene blue

- prevents growth of gram +

- indicator: eosin & methylene blue

- differentiate lactose fermenters from non-fermenters

- used to screen for coliforms

-fecal colliforms

- can also be used when testing for UTI's

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testing for the motility of bacteria

- organisms that have flagella are motile

- not all species produce flagella

- motility can be tested using motility media

- a motility is detectable due to the use of a soft agar (decreased agar concentration)

- a dye indicator may or may not be used to aid in the detection of motility

-motility media are inoculated by a single line stab

- motile: organisms can swim through the soft agar and spread away from inoculation stab line

- non-motile: organisms remain confirmed to the path of inoculation by the soft gel of the medium

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viable plate count

- method for diluting a broth culture so that a small sample of it can be plated

- individual cells are isolated

- spread plate technique used

- when colonies grow they can be counted to determine the original concentration of the starting sample

- viable cells: are those that can replicate and form colonies

- this method does not detect dead cells

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important formulas

- dilution factor: a/a+b

- a: volume being transferred

- b: volume being added to

- total tube dilution: the total dilution of the original sample in each of the tubes

- you must multiply the combined dilution of the previous tube by the dilution factor of your transfer

- plate dilution: amount plated (in mL)/ 1mL

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standard plate count

Following incubation: count the plate that seems to have between 30 and 300 colonies

calculate the dilution factors (DF): between the counted plate and the original culture

- multiply the plate count by the reciprocal/inverse of the dilution factors

- results are reported as CFU/mL

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biochemical tests

agar plate tests for exoenzymes:

- lipase

- milk agar

- starch agar

tube media tests for other metabolic processes:

- litmus milk

- sugar fermentation

- kligler's iron agar

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litmus milk

contains:

- lactose

- casein and other peptones

- litmus pH indicator

tests for:

- fermentation of lactose

- metabolism of proteins

- degradation of casein

- litmus reduction

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litmus milk: fermentation

fermentation of lactose produces acids that reduce the pH of the litmus milk solution

- litmus milk turns pink at acidic pH

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litmus milk: peptone deamination

some bacteria may instead deaminate peptones, releasing ammonia into solution

- this rases the pH of the litmus milk, turning the solution blue

peptone deamination is an oxygen dependent process, so the color change may be limited to the top of the tube

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litmus milk: reduction (redox)

litmus milk only functions as a pH indicator in an oxidized redox states - loses its color when reduced

- if the bacterial culture produces a reducing environment, then the litmus milk will go white as the litmus indicator is reduced

a band of color will persist at the top of the media, where oxygen can penetrate and re-oxidize the litmus indicator

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litmus milk: proteolysis

some bacteria produce the caseinase enzyme

- if casein is degraded, the milk will turn clear

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litmus milk: curding

if enough acid is produced from lactose fermentation, milk proteins may become denatured

- some curds completely solidify the milk, or may incompletely curdle the milk, leading to a lumpy consistency

curding does not always occur with acid production

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phenol red (PR) sugar fermentation broth

contains one sugar: glucose, lactose, or mannitol

indicators: phenol red and a durham tube

- detect fermentation of included sugar

if an organism ferments:

- phenol red turns yellow

- gas formation: bubble collects in durham tube

if an organism can't ferment:

- peptone degradation releases ammonia

- phenol red indicator turning a shade of "hot pink" called cerise

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Kligler's Iron Agar (KIA)

tests for: lactose fermentation, glucose fermentation, sulfur reduction

carbohydrates sources: 1.0% lactose, 0.1% glucose

other ingredients: 1.0% peptone (source of ammonia & sulfur via cysteine)

indicators:

- phenol red = pH indicator

- iron from ferric ammonium citrate - combines with H2S to form a black precipitate

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KIA media: lactose fermenters

acid can be from fermentation of glucose (0.1%) and lactose (1.0%)

NH3 is released by the breakdown of peptone (1%) in the presence of oxygen

if both glucose and lactose are fermented, acid products will be produced in excess of NH3, and the entire tube will turn yellow

1% lactose + 0.1% glucose > 1% pepton (1.1% - 1%)

fermentation can result in the formation of gaseous end products which can lead to lifting or cracking of the media in the tube

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KIA media: glucose only

glucose fermentation only: amount of NH3 produced by breakdown of peptone exceeds the amount of acid produced by glucose fermentation

breakdown of peptone will only take place in the presence of oxygen

deamination of peptones keep only the upper part of the slant at neutral/alkaline pH

butt of the tube will stay yellow due to the glucose fermentation, as oxygen can not penetrate far enough into the butt for peptone deamination to reverse acidification

0.1% glucose < 1.0% peptone

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KIA media: reversion

some lactose fermenting bacteria produce weaker, less stable acids that can be oxidized into neutral end products

- leads to reversion of part of the slant back to neutral/alkaline pH

often observed with Enterobacter aerogenes, which uses the 2,3-butanediol pathway for lactose fermentation

- 2,3-butanediol products are not as stable as the products of the mixed acid fermentation pathway [lactate, acetate, succinate & formate], and can readily be neutralized in aerobic environments

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KIA media: sulfur reduction

reduction of sulfur produces H2S which combines with iron in the medium to form a black precipitate

- precipitate will obscure color in the bottom of the tube

H2S will only form as a byproduct of sulfur reduction in an acidic environment

- black precipitate indicates that fermentation and acid production have occurred

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lipase plate

differential media

tests for the enzyme lipase

- hydrolyzes fats > glycerol and fatty acids

indicator: spirit blue

hydrolysis of lipids will produce a dark blue zone around the growth with no oily surface

- + = blue color

- - = no color

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milk agar plate

differential media

tests for enzyme caseinase

- hydrolyzes casein (milk production) > amino acid products

casein gives milk its white color loses its color when broken down - clearing

- + = clear around colony

- - = no clearing around colony

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starch agar

differential media

test for the enzyme amylase

- breaks down starch into simple sugars

add iodine after colony growth as the indicator

- iodine + starch = purple

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tests that yield results within minutes

catalase

oxidase

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tube media tests for other metabolic processes

nitrate broth

gelatin

urea broth

phenyalanine

SIM

IMViC

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fermentation vs respiration

conversion of organic molecules to an energy source (ATP)

glucose > pyruvate via glycolysis

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respiration

final electron acceptor is an inorganic molecule

- oxygen > aerobic respiration

- nitrate, sulfate > anaerobic respiration

general pathway for respiration

glycolysis > kreb's cycle > electron transport chain

transfer of electrons to molecules with a more positive potential

we are indirectly testing for the presence of an ETC

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catalase test

during aerobic metabolism, reactive oxygen species (ROS) may be produced:

- superoxide radicals

- hydrogen peroxide (H2O2)

some bacteria have enzymes capable of breaking down these toxins (aerobic & facultative anaerobes)

- superoxide dismutase (SOD)

- catalase

detects catalase

- converts hydrogen peroxide > water & oxygen

- 2 H2O2 > 2H2O + O2

add 3% hydrogen peroxide to the edge of the colony

- oxygen product will be seen as bubbles coming from the sample

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cytochromes C oxidase

main function is to remove electrons from cytochrome c (oxidize) and transfer them to oxygen (reduces)

- cytochrome c > cytochrome c oxidase > oxygen

however, it can also reduce cytochrome c by oxidizing a chromogenic reducing agent

- these reducing agents develop color when they become oxidized

- dimenthyl-p-phenylenediamine

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oxidase test

detects cytochrome oxidase

- transfers electrons to final electron acceptor - oxygen

indicator: dimethyl-p-phenylenediamine hydrochloride (an aromatic amine)

in the presence of cytochrome c oxidase, reagent is oxidized and turns dark blue to black

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nitrate respiration

utilizes nitrate (NO3) as final inorganic electron acceptor

requires enzyme nitrate reductase to convert nitrate to nitrite

some bacteria have other enzymes to reduce nitrate all the way down to N2 (denitrification)

others can reduce nitrate to ammonium for incorporation (assimilatory nitrate reduction)

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nitrate broth

detects nitrate reductase which reduces nitrate to nitrite (NO3 > NO2)

nitrate I (sulfanilic acid) and nitrate II (dimethyl-alpha-napthylamine) react with NO2 > brick red color

if nitrate is not used and is residual, then zinc powder will catalyze the reaction

- a red result after Zn addition indicates that nitrate was not used = the organism did not have the enzyme

- if there is no reaction after zinc is added that means it was reduced all the way to nitrogen gas no zinc was used

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urea broth

detects urease

- degrades urea > 2 ammonia (NH3) and carbon dioxide (CO2)

- increases pH - more alkaline

contains urea and the pH indicator phenol red

- as pH increases > 8.1 > cerise

- neutral pH > red

- as pH decreases > yellow

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gelatin

detects with presence of gelatinase

- catalyzes the hydrolysis of gelatin > amino acids

- gelatin solidifies in low temperatures

- need ice bath

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phenyalanine slant

medium detects production of phenylalanine deaminase

- phenylalanine > phenylpyruvic acid (PPA) + ammonia (NH3)

add 5-10 drops of 10% ferric chloride (FeCl3) reagent

- in the presence of PPA, FeCl3 appears a deep green color

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SIM

s: sulfide

- H2S produced by bacterium reacts with Fe in medium to produce FeS

i: indole

- add 3-5 drops of Kovak's reagent

m: motility

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fermentation

final electron acceptor is an organic molecule

- does not produce as much energy as respiration

pyruvate > general end products are organic acids and gases (CO2, H2)

- mixed acid fermentation

- 2,3-butanediol fermentation

can also be used as a general term for the breakdown of polysaccharides into monomers that can be fermented

ex: lactose > glucose & galactose > pyruvate

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MR-VP broth

methyl red (MR) and Vogues-Proskauer (VP) test must be done separately

methyl red (MR): tests for mixed acid fermenters

add 3-4 drops of methyl red reagent

- detects high acid production, lowering pH to 4.4 or less

a low acid concentration does not allow retention of the red color

vogues-proskauer (VP): tests for 2,3-butanediol fermenters

- glucose > acetoin > 2,3-butanediol

2,3-butanediol fermenters produce the acetoin intermediate

- acetoin can interact with VP I and VP II (Barritt's reagents)

add 10 drops of VP I (alpha napthol) to intensify the red color

add 10 drops of VP II (KOH), produces an alkaline condition that favores acetoin oxidation > diacetal and O2 is mad, presence of O2 produces a red color

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tyrptone broth

detects tryptophanase

- hydrolyzes tyrptophan > indole, pyruvate and ammonia (NH3)

add 3-5 drops of Kovak's reagents (DMABA and HCI dissolved in amyl or butyl alcohol)

- reagent is nonpolar & extracts indole to top

- indole + kovac's regeant = cerise