Microbiology Lab Practicum 2

Blood agar

Detects hemolytic ability in G+ cocci Useful in separating Streptococcus species

Urea hydrolysis

Differentiate organisms based on ability to hydrolyze urea with urease Proteus genus is + (Proteus vulgaris)

Gelatin Hydrolysis

Differentiate pathogenic Staphylococcus aureus from S. epidermidis Bacillus species are positive

flavoprotein

ETC carrier molecule that can bypass the next carrier in the chain and transfer electrons directly to oxygen, which produces hydrogen peroxide, a highly potent cellular toxin

-;-;+;+

Enterobacter is __ for indole; methyl red; voges proskauer; citrate

+;+;-;-

Escherichia is __ for indole; methyl red; voges proskauer; citrate

amino acid decarboxylation medium

Møller's decarboxylase base medium contains peptone, glucose, pH indicator bromocresol purple, and the coenzyme pyridoxal phosphate

1,4-alpha-glycosidic (acetal)

alpha glucose moleculesin both amylose and amylopectin are held together by_____ bonds

catalase test theory

catalase converts hydrogen peroxide into water and gaseous oxygen; the ability to synthesize protective enzymes accounts for an organism's ability to live in the presence of oxygen

nitrate reduction theory

many g- bacteria (incl. Enterobacteriaceae) contain the enzyme nitrate reductase and perform a single step reduction of nitrate to nitrite (NO3- to NO2-). Other bacteria, through multistep denitrification, can enzymatically convert nitrate to molecular nitrogen (N2) or nitrous oxide (N2O) via nitrite.

gamma hemolysis

no hemolysis and appears as simple growth with no change to the medium

alpha hemolysis

partial destruction of RBCs and produces and olive greenish discoloration of the agar around the colonies

starch agar

plated medium of beef extract, soluble starch, and agar

catalase test procdure

put drop of hydrogen peroxide on glass slide; inoculate with toothpick and organism; observe for bubbles

hydrolysis

reactions that split complex molecules with water

limit dextrin

the small, branched molecules that remain after amylase activity because amylase cannot disassemble the glucose subunits around branch points of amylopectin

amino acid decarboxylation application

these media are used to differentiate organisms in the family Enterobacteriaceae and to distinguish them from other g- rods; most frequently used amino acids are arginine, lysine and ornithine

oxidase test application

this test is used to identify bacteria containing the respiratory enzyme cytochrome c oxidase; can identify Neisseria and Moraxella; can be useful in differentiating oxidase negative Enterobacteriaceae from oxidase positive Pseudomonadaceae

respiration

transfers chemical potential energy in glucose into high-energy bonds of ATP using glycolysis, the citric acid cycle, and oxidative phosphorylation in an ETC.

nitrate broth

undefined medium of beef extract, peptone, and potassium nitrate; no color indicator is included; any color change is a result of metabolic rxns and/ or reagents added

gelatin hydrolysis (gelatinase test) application

used to determine the ability of a microbe to produce gelatinases; Staphylococcus aureus (+) can be differentiated from S. epidermidis (-); Serratia and Proteus are + members of Enterobacteriaceae whereas most others in the family are -; Bacillus anthracis and cereus are + as well as Clostridium tetani and perfringens

citrate utilization test application

used to determine the ability of an organism to use citrate as its sole source of carbon; distinguishes between members of the family Enterobacteriaceae and also from other g- rods; enterobacter is +

urea hydrolysis application

used to differentiate organisms based on their ability to hydrolyze urea with the enzyme urease; Proteus genus bacteria are rapid urease +; H. pylori is +

SIM medium

used to differentiate the Enterobacteriaceae esp. + Escherichia and - Enterobacter

MRVP application

used to distinguish between members of the family Enterobacteriaceae and differentiate them from other G- rods

nitrate reduction results

-if after adding reagents A and B yellow turns red immediately, nitrite is present (NO3 -> NO2) -if after adding reagents A and B, stays yellow could either be not used (NO3) or nitrogen gas (NO3->N2) -add zinc dust -if turns red -> nitrite (was NO3) -if stays yellow -> nitrate was used up (was NO2)

amino acid decarboxylation theory

Bromocresol purple is purple at pH 6.8 and above and yellow below 5.2; after inoculation, an overlay of mineral oil is used to seal the medium from external oxygen and promote fermentation. glucose fermentation in the anaerobic medium initially turns yellow bc of the accumulation of acid end products (from Enterobacteriaceae's fermentation); low pH and presence of specific amino acid induces decarboxylase + organisms to produce the enzyme' decarboxylation of the amino acids results in the accumulation of amines (alkaline) and turn the medium purple; if the organism is a glucose fermenter but doesn't produce the appropriate decarboxylase, the medium will turn yellow and remain. If the organism doesn't ferment glucose, the medium won't change color. Purple is the only positive result

Oxidase Test

Identifies bacteria containing the respiratory enzyme cytochrome c oxidase Neisseria is + Differentiates between - Enterobacteriaceae and + Pseudomonadaceae Pseudomona aeruginosa is +

Catalase Test

Identifies organisms that produce the enzyme catalase Differentiates between + Staphylococcus and - Streptococcus Used for identifying Mycobacterium species

No decarboxylation and decarboxylase is absent; -; fermentation occurred, no decarboxylase present; -; decarboxylation occurred with decarboxylase; +

If in an amino acid decarboxylation test the media did not change color, ___ and this is a ___ result. If the media turned yellow ___ and this is a ___ result. If the media turned purple, ___ and this is a ___ result

urea hydrolysis occurred and urease is present; +; urea hydrolysis didn't occur and urease was absent; -; urea hydrolysis didn't occur and urease was absent; -

If urea hydrolysis medium is pink this means ___ and is a ___ test; if urea hydrolysis medium is orange this means ___ and is a ___ test; if urea hydrolysis medium is yellow this means ___ and is a___ test

sulfur reduction (H2S production) and thiosulfate reductase or cytesine desulfurase is present; +; sulfur reduction; no sulfur is reduced; -

In SIM media, blackening means ___ and is a ___ result for the ___ portion of the test; no blackening means ___ and is a ___ result.

motile; +; nonmotile; -; cannot say; cannot say

In SIM media, growth radiating outward from the stab line means the organism is ___ and this is a ___ result; no growth radiating out means the organism is ___ and this is a __ result; blackening of the media means ___ and this is a ___ result.

tryptophanase hydrolyzed tryptophan to indole and pyruvate; +; tryptophanase is absent and no indole is formed; -

In SIM media, red (or green) in the alcohol layer of Kovac's (or other) reagent means ___ and is a ___ result; no color change in this layer means ___ and is a ___ result

casease is present; +; casease is absent; -

In casein hydrolysis, clearing the agar means ___ and this is a __ result; if there is no clearing in the agar, ____ and this is a __ result

gelatinase is present; +; gelatinase is absent; -

In gelatin hydrolysis, liquid gelatin means ___ and this is a ___ test; solid gelatin means ___ and this is a ___ test

alpha or beta- amylase or alpha-1,6-glucodiase is present or simply amylase is present; +; alpha or beta- amylase or alpha-1,6-glucodiase is present or simply amylase is absent; -

In starch hydrolysis (amylase test), if there is clearing around the growth that has been flooded with iodine, that means ____ and this is a ___ result. if there is no clearing, that means ___ and this is a ___ result.

reason methyl red + test is yellow

MR + performs mixed acid fermentation. which overcomes the phosphate buffer in the medium and lowers the pH; methyl red indicator dye shows pH (red at 4.4 and yellow 6.2); red is the only + result

SIM Medium: determination of sulfur reduction, indole production, and motility theory

SIM medium is used for determining sulfur reduction, indole production from tryptophan, and motility; sulfur reduction will appear black and is caused by enzymes cytesine desulfurase or thiosulfate reductase which make sulfide gas and ferric sulfide which is the black precipitate; any blackening is a + sulfur reduction test; bacteria possessing the enzyme tryptophanase can hydrolyze tryptophan to pyruvate, ammonia, and indole. When Kovac's reagent (or other) is added to tube, layer over agar is formed. DMABA reacts with any indole and produces a quinoidal compound that turns the reagent layer red (or green) and indicates a positive reaction and the presence of tryptophanase; no color change is indole negative; motility is observed by cloudiness (+)

streptolysin O

Streptococcus hemolysin that is oxygen labile and expresses maximal activity in anaerobic conditions

streptolysin S

Streptococcus hemolysin that is oxygen stable but expresses itself optimally under anaerobic conditions

Staphylococcus; Streptococcus

___ is catalase + but ___ is catalase -

amylopectin; amylose

____ is different from ____ in the way that it has a 1,6-alpha-glycosidic linkage every 30th glucose

alpha or beta

____-amylase breaks the starch polymer into the monosaccharide alpha-glucose and the disaccharide ___-maltose

starch

a complex polysaccharide compound made of alpha glucose monomers in the linear (amylose) or branched (amylopectin) form

alpha-(1,6)-glucosidase

breaks down the limit dextrin into short, linear oligosaccharides that alpha-amylase can convert to alpha-glucose and alpha-maltose

SIM media

combination differential media; tests for sulfur reduction, indole production, and motility for members of Enterobacteriaceae

methyl red and voges-proskauer tests

combination used for both methyl-red and Voges-Proskauer tests; simple solution containing peptone, glucose, and phosphate buffer; peptone and glucose provide protein and fermentable carbohydrate resp.; potassium phosphate resists pH changes in the medium

beta hemolysis

complete destruction of RBCs and hemoglobin, results in a clearing of the medium around the colonies

casein hydrolysis (casease test) application

cultivation and differentiation of bacteria that produce the enzyme casease; Bacillus subtilis is positive

urea hydrolysis (urease test) theory

decarboxylation of certain amino acids produces urea; it can be hydrolyzed to ammonia and co2 by bacteria possessing the enzyme urease; provides nitrogen in a useable form (ammonia) and acts as a virulence factor to some pathogens by counteracting acid; urease genes are activated when a useable nitrogen source is absent/ if urea is present; urea broth is medium and has trace yeast extract and urea; medium contains buffers strong enough to inhibit alkalinization of medium by all but rapid urease positive organisms; phenol red indicated pH (yellow or orange are below 8.4 and pink is above)

methyl red test

designed to detect organisms capable of performing mixed acid fermentation

iodine

the reagent used in starch hydrolysis to detect the presence or absence of starch in the vicinity around the bacterial growth

citrate utilization test theory

designed to differentiate members of the Enterobacteriaceae, which are all facultative anaerobes; tells about the ability of the organism to use citrate as their sole carbon source and perform citrate fermentation; simmons citrate agar is a defined medium; provides means for bacterial species that possess the enzyme citrate permease to transport citrate into the cell and perform citrate fermentation; bact without citrate permease will not grow on this medium; citrate+ bact hydrolyze citrate into oxaloacetate and acetate using the enzyme citrate lyase; enterobacter is citrate +; bact that survive in the medium and utilize the citrate also convert the ammonium phosphate to ammonia (NH3) and ammonium hydroxide (NH4OH) both of which are alkaline; bromothymol blue dye is pH ind. (basic is blue)

voges-proskauer test

designed to identify organisms that are able to ferment glucose with the production of acetoin and 2,3-butanediol, which have neutral pH; adding vp reagents to the medium oxidizes the acetoin to deacetyl which reacts with guanidine nuclei from peptone to make a red color; a positive result is red

gelatin

detects gelatinase (an exoenzyme) that liquefies the solid medium

blood agar

detects hemolytic ability of Gram+ cocci and is especially useful in separating Streptococcus species

catalase test

detects organismal ability to produce catalase

catalase test, oxidase test, nitrate reduction

differentiate bacteria on their ability to respire

Casein Hydrolysis

differentiate bacteria that produce the enzyme casease

Amino acid decarboxylation (decarboxylase test)

differentiate organisms in the family Enterobacteriaceae and to distinguish them from other G- rods Enterobacter aerogenes is +

Nitrate reduction test

differentiates Enterobacteriaceae (mostly + for nitrate reduction) from other G- rods that either don't reduce nitrate or reduce it to N2 or another produce beyond nitrite

Methyl red and Voges Proskauer tests

distinguish between members of Enterobacteriaceae and differentiate them from other G- rods Escherichia is + for MR Enterobacter is + for VP

Citrate Utilization test

distinguishes between members of the family Enterobacteriaceae and also from other G- rods Enterobacter is +

extracellular enzyme (exoenzyme)

enzyme secreted from the organism to catalyze reaction outside the cell

intracellular enzyme

enzyme that catalyzes its reaction inside the cell

catalase

enzyme that detoxifies the cell by converting hydrogen peroxide H2O2 produced in the ETC to H2O and O2

hydrolytic enzymes

enzymes in hydrolytic reactions

deaminases

enzymes that catalyze the removal of an amino acid's amine group (NH2)

decarboxylases

enzymes that catalyze the removal of an amino acid's carboxyl group (COOH)

nitrate reduction test

examines bacterial ability to use nitrate as the FEA of anaerobic respiration

gelatin hydrolysis (gelatinase test) theory

gelatinases comprise a family of extracellular enzymes produces and secreted by some microorganisms to hydrolyze gelatin; then the cell can absorb amino acids and use them metabolically; gelatin nutrient differs from most other solid media in that the solidifying agent (gelatin) is also the substrate for enzymatic activity; gelatinase positive organisms will liquify the medium; gelatinase activity is very slow; gelatin melts at 28 C so might need to be frozen to observe results

streptolysins

hemolysins produced by streptococci; two forms: type O and type S

utilization media

highly defined formulations designed to differentiate organisms based on their ability to grow when an essential nutrient is strictly limited

oxidase test

identifies the presence of cytochrome c oxidase in the ETC of some aerobic respirers

Starch Hydrolysis

identify Gardnerella vaginalis differentiate members of Bacillus

starch hydrolysis application

identify Gardonella vaginalis; differentiates members of the genera Corynebacterium, Clostridium, Bacillus, Bacteroides, Fusobacterium, and Enterococcus

starch agar, milk agar

identify extracellular enzymes capable of diffusing into the medium and producing a distinguishable halo of clearing around the bacterial growth

organism hemolyzes RBCs completely; beta-hemolysis; organism partially hemolyzes RBCs; alpha-hemolysis; organism doesn't hemolyze RBCs; gamma-hemolysis

in blood agar, if there is clearing around the growth this means ____ and is ____; if there is greening around growth this means ___ and is ___; if there is no change in the medium this means ___ and is ___

catalase is present; +; catalase is absent; -

in catalase test, if organism bubbles, ___ and this organism is catalase ___; if organism doesn't bubble, ___ and this organism is catalase ___

citrate is utilized and citrate permease is present; +; citrate is utilized and citrate permease is present; -; citrate is not utilized and citrate permease is not present; -

in citrate utilization test, a blue color means ___ and is a ___ result; no color change and growth means ___ and is a___ result; no color change or growth means ___ and is a ___ result

red; orange or yellow; organism performs a mixed acid fermentation

in methyl red tests, ___ is the only positive result; both __ and __ are negative results. the positive result indicates ___

cytochrome c oxidase is present; +; cytochrome c oxidase is absent; -

in oxidase test, if the streak becomes dark blue/ purple w/i 20 seconds, this means ___ and is a ___ result; if the streak doesn't change color w/i 20 seconds, this means ___ and is a ___ result

red; organism performs a 2,3-butanediol fermentation and acetoin is produced; no color change; organism does not perform a 2,3-butanediol fermentation and acetoin is not produced

in voges-proskauer tests, a positive orgaism is __ colored and this means ___. a negative organism exhibits ___ and this means ___.

MRVP procedures

inoculate MRVP broth; incubate for 2 days; split broth between 2 tubes; for MR, add drops of MR reagent and observe color; for VP, add VPa and VPb reagents; shake A LOT to oxidize 2,3-butanediol; observe color

gelatin hydrolysis procedure

inoculate nutrient gelatin tubes with organism; incubate for week; freeze; observe if solid or liquid

casein hydrolysis procedure

inoculate skim milk agar with two bacteria; incubate for 2 days; observe clearing

starch hydrolysis procedure

inoculate starch agar plate with two organisms, incubate for 2 days, flood with iodine, observe clearing

urea hydrolysis procedure

inoculate urease broth medium tubes with organisms; incubate for 2 days; observe color

anaerobic respiration

involves the reduction of an inorganic molecule other than oxygen; ex- nitrate reduction

blood agar application

is used for isolation and cultivation of many types of fastidious bacteria; differentiates bacteria based on their hemolytic characterisitics esp. w/i the genera Streptococcus, Enterococcus, and Aerococcus

blood agar theory

several species of G+ cocci produce exotoxins called hemolysins, which destroy RBCs and hemoglobin; this agar allows for differentiation of bacteria based on their ability to hemolyze RBCs; there are 3 types of hemolysins (alpha, beta, and gamma); special hemolysins are produced by streptococci

casein hydrolysis (casease test) theory

some bacteria have the ability to produce and secrete enzymes into the environment that catalyze the hydrolysis of large proteins to smaller peptides or individual amino acids, enabling their uptake across the membrane; when plated milk agar is inoculated with a casease-positive organism, secreted casease will diffuse into the medium and create a zone of clearing where the casein has been hydrolyzed

casease

specific proteolytic enzyme that some bacteria produce to hydrolyze milk protein casein; when the casein is broken down, it becomes clear

SIM procedure

stab inoculate medium w/o breaking bottom; inc for 2 days; observe growth/ blackening; add reagent to observe indole color change

starch hydrolysis (amylase test) theory

starch is too large to pass through bacterial cell membrane so it must first be digested extracellularly by amylase enzymes (alpha or beta) or by alpha-1,6-glucodiase

blood agar procedure

streak inoculate blood agar plate with organisms, incubate for 2 days; observe growth and color

oxidase test procedure

streak organisms on piece of filter paper; add oxidase reagent over streaks; wait 20 seconds; observe colors

streak stab technique

technique that provides optimum conditions for streptolysins in blood agar

citrate utilization test

tells about the ability of organisms to use citrate as their sole carbon source and perform citrate fermentation; contains citrate as the only carbon-containing compound and ammonium ion as the only nitrogen source

starch hydrolysis

tests for amylase

casein hydrolysis

tests for casease

amino acid decarboxylation

tests for decarboxylase; differentiates members of Enterobacteriaceae and distinguishes them from other g- rods

gelatin hydrolysis

tests for gelatinase

urea hydrolysis

tests for urease

urease

the hydrolytic enzymes detected by ___ tests are intracellular and produce identifiable color changes in the medium

oxidase test theory

the oxidase test is designed to identify the presence of cytochrome c oxidase. it is able to do this because cytochrome c oxidase has the unique ability to not only oxidize cytochrome c, but catalyze the reduction of cytochrome c by a chromogenic reducing agent called tetramethyl-p-phenylenediamine; chromogenic reducing agents are chemicals that develop color as they become oxidized; the reducing agent is added directly to bacterial growth on solid media; a color change occurs w/i seconds if the reducing agent becomes oxidized, indicating that cytochrome c oxidase is present

SIM application

used to identify bacteria that are capable of producing indole using the enzyme tryptophanase; is a component of the IMViC tests used to differentiate the Enterobacteriaceae, esp. the positive Escherichia coli and the negative Enterobacter; also used to differentiate sulfur-reducing members of Enterobacteriaceae, esp. members of the genera Salmonella, Shigella, and Proteus, from the negative Morganella morganii and Providencia rettgeri; motility is an important differential characteristic of Enterobacteriaceae

catalase test application

used to identify organisms that produce the enzyme catalase; used to differentiate catalase positive Micrococcus and Staphylococcus from catalase negative Streptococcus, Enterococcus, and Lactococcus; can also be used to identify Mycobacterium species

nitrate reduction application

virtually all members of Enterobacteriaceae perform a one step reduction of nitrate to nitrite. The test differentiates them from g- rods that either do not reduce nitrate or reduce it beyond nitrite to N2 or other compounds