Eosin Methylene Blue Agar

-Purpose: Eosin methylene blue agar (EMB) is a selective and differential medium used to isolate fecal coliforms.

-Selective: inhibits the growth of gram [+]

-Differential: coliform from non-coliform- if it is coliform it means it ferments lactose which produces a shiny green appearance to the growth. If it's non-coliform-it means that its not inhibited by the dyes but also did not ferment lactose, retains normal color or takes on color of medium

-Contains:

-Digest of gelatin: organic carbon and nitrogen

-Lactose: fermented to acid end-products by coliforms

-Dyes eosin Y: inhibit growth of Gram [+] and reacts w/ vigorous lactose fermenters

Methylene Blue: inhibits growth of Gram [+]

-Gram positive or negative?- Gram [-] because the dyes eosin Y is used partly to inhibit the growth of Gram [+]

-Inhibiting factor?- Dyes eosin Y inhibits the growth of Gram [+]

-Procedure:

-What purpose does the inoculation of the NA plate serve?-as a comparison for growth quality

-Organisms Result:

Enterobacter aerogenes: dark purple w/ metallic green sheen- ferments lactose/or sucrose w/ acid production-coliform

-good growth-gram [-], not inhibited by eosin/ methylene blue- positive result

Staphylococcus epidermidis: colorless- does not ferment lactose or sucrose- non coliform

-no growth-inhibited by EMB- Gram [+]

E. coli: Pink/mucoid- ferments lactose w/ little acid production- coliform

-good growth- Gram [-] and not inhibited by EMB

-Grow or not grow= testing Gram

-Changes color= fermenter

-What purpose do the sugars provide in this medium?-encourage growth of enteric bacteria and to differentiate them based on color reactions when combined with the dyes

Enzyme?-

Phenol Red Broth

-Purpose: to detect carbohydrate fermentation that is indicated by a color change in the medium form orange-red to yellow acid end products

Used to detect ability of an organism to ferment different carbohydrates

FERMENTATION TEST

Differential: used to determine whether a bacteria is capable of fermentation

Contains:

Peptone: Enables microorganisms to grow in broth.

Carbohydrate: lactose, glucose, sucrose

Phenol red: pH indicator [below 6.8=yellow], [above 7.4=pink-magenta], [6.8-7.4=red]

Procedure: Tubes of PR broth contain small inverted Durham tube to trap gas produced

What indicates fermentation?- production of acid [color] and gas production [bubbles]

How is fermentation of carbohydrates detected in this test?- acid production from fermentation of the carbohydrates lowers pH below the neutral range of the indicator makes medium yellow

Lactose is a disaccharide made up of glucose and galactose

Results:

acid/gas=bubbles

acid/no gas=yellow

no reaction [no acid or gas]=bright orange

Alkaline [peptone degradation]=pinkish red

enzyme?-

Methyl Red and Voges- Proskauer Tests

Purpose: used to distinguish between members of the family Escherichia and differentiate them from enterobacter aerogenes

Different bacteria convert dextrose and glucose to pyruvate using different metabolic pathways

MR: If the organism uses the mixed acid fermentation pathway and produces stable acidic end-products, the acids will overcome the buffers in the medium and produce an acidic environment in the medium.

VP: detects organisms that utilize the butylene glycol pathway and produce acetoin. When the VP reagents are added to MR-VP broth that has been inoculated with an organism that uses the butylene glycol pathway, the acetoin end product is oxidized in the presence of potassium hydroxide (KOH) to diacetyl.

Contains:

Peptone: protein w/ nitrogen

glucose: fermentation w/ nitrogen

Phosphate buffer: pH indicator

Differential: between mixed acid fermenters and others- verified by the addition of methy red indicator dye following incubation

What is indicator?- phosphate buffer which turns red when the broth becomes acidic

VP Test: designed to test for organisms that ferment glucose, but quickly convert acid to acetoin, 2,3-Buendol

A positive VP test: is red

A negative VP test: no color change or a copper color

Enzyme: creatine as a catalyst

Starch Hydrolysis

TEST DETECTING HYDROLYTIC ENZYMES

It is a polysaccharide

alpha- D- glucose subunits is the building blocks

The test allows us to determine the presence of which enzymes?- Hydrolyze the starch, alpha- amylase, and oligo-1, 6-glucosidase in the area surrounding the growth

Enzyme functions?- ability to hydrolyze starch by breaking the glycosidic linkages between sugar subunits

These are exoenzymes why are they important?- enzyme secreted from the organism to catalyze reactions outside the cell- Starch is too large to pass through bacterial cell membrane to be of a metabolic value to bacteria it first must split into smaller fragments or individual glucose molecule. Exoenzyme break down starch into individual alpha-glucose subunits

Role of Gram's iodine in the experiment- The reagent iodine is used to detect the presence or absence of starch in the vicinity around bacterial growth.

Iodine reacts w/ starch and produces a blue or dark brown color.

Any Microbial starch hydrolysis will be revealed as a clear zone surrounding the growth

Iodine binds to starch- purple black

clearing around growth- amylase is present [+]

no clearing around growth- no amylase present [-]

Organisms-

E. coli: [-] no clearing around growth

Bacillus cereus: [+] clearing around growth

Urea Hydrolysis Test

Purpose: ability to hydrolyze urea w/ enzyme

Differential: differentiate rapid urease- positive bacteria from slower urease-positive and urease-negative bacteria

Contains:

Urea: a product of decarboxylation of certain amino acids

Peptone: provide nutrients

Potassium phosphate: buffer to resist alkalization

Glucose: provide nutrients

Phenol red: pH indicator yellow

Urea hydrolyzes urea to ammonia which changes the color of phenol red from yellow to pink

Result:

Positive: pink, urea hydrolysis, strong urease production

Negative: No urea hydrolysis, urea is absent, yellow

Urea----[urease +H20]---> 2NH3 + CO2 [ammonia]: Urea can be hydrolyzed to ammonia [NH3] and carbon dioxide [CO2] by bacteria containing the enzyme urease

Ammonia is used by many bacteria as a source of what element?-nitrogen: protects against and acidic environment

Organisms:

Klebsiella pneumoniae: pink [+]- rapid hydrolysis, strong urease production

E. coli: orange [-]- no urea hydrolysis, does not produce urease or cannot live in broth

SIM Medium

Purpose: determination of 3 bacterial activities: sulfur reduction, indole production from tryptophan, motility

Hydrolysis of tryp results in the production of indole w/ the help of tryptophanase enzyme

Production of indole tested and positive result?- Tryptophan hydrollysis in SIM medium can be detected by the addition of Kovac's reagent [DMABA]. DMABA reacts w/ any indole present and produces quinoidal compound- reagent red- fromation of a red color= positive result

Differential:

identify bacteria that are capable of indole production w/ tryptophanase

sulfur reducing members- cyesine desulfurase and thiosulfate

motility

H2S +FeSO4--> H2SO4 +FeS precipitate indicator?- Indicator reaction for H2S production [hydrogen sulfide], a colorless gas, can be detected when it reacts with ferrous sulfate in the medium to produce black precipitate ferric sulfate

Positive result: Black in medium- sulfur reduction- cyesine desulfurase or thiosulfate reductase

Which ingredient could be eliminated if the SIM medium was used strictly for testing motility among sulfur reducers?- tryptophanase- casein/ animal tissue

Which ingredient could be eliminated if the SIM medium was used strictly for testing motility among indole producers?- sodium thiodulfate

Bacteria accomplish sulfur reduction to H2S in 2 ways?- cystein desulfurase [enzyme] and thiosulfate reductase [enzyme]

Sulfur reduction result:

E. Coli: black precipitate present, [+], sulfur reduction-H2S produced

Proteus Mirabilis: black precipitate present, [+], sulfur reduction-H2S produced

Indole Production Result:

E. coli: no red color, [-], organism does not produce tryptophanase and tryptophan not hydrolyized

Proteus mirabilis: red color present, [+], organism produces tryptophanase and hydrolyzes tryptophan into indole and pyruvate

Motility Result:

E. Coli: Blackened medium-not determined

Proteus mirabilis: growth radiating outward from stabline- positive

Triple Sugar Iron Agar

Purpose: It is used to differentiate enterics based on the ability to reduce sulfur and ferment carbohydrates.

Differential: Differentiate bacteria based on the basis of lactose fermentation, glucose fermentation, sucrose fermentation, and sulfur reduction

Provides both anaerobic and aerobic

Contains:

Glucose/lactose/sucrose: sugars

animal proteins: provide carbon/ nitrogen [beef and yeast extract, peptone]

ferrous sulfate [oxidized sulfur]: iron is hydrogen sulfide indicator

sodium thiosulfate [oxidized sulfur]: source of reducible sulfur

Phenol red: pH indicator

Basic results:

Glucose only= red/yellow

all sugars= yellow

Sulfur reduction= black

Fermentation= acidic conditions

Glucose is a good food source so if there is only little it will be faster and easier to see if it can ferment all three [.1% of glucose instead of 1%]

reversion raises pH by producing NH3

Why is it inoculated with a both stab and a streak?- to try not to introduce excessive air and to make sure the bacteria reacts w/ all of the medium

In this test how would you know that your organism only fermented glucose and not the other two sugars?- If the slant produces a red slant and yellow butt after 24 hrs indicates that the bacteria ferments glucose but not lactose and sucrose

Why does all bacteria that ferments lactose or sucrose always ferment glucose as well?- Lactose and sucrose are disaccharides. Glucose is a monomer of both lactose and sucrose

Results:

E. coli: yellow slant/butt= glucose/lactose/sucrose fermented w/ acid accumulation in slant/butt

Morganella morganii: red slant/yellow butt= glucose fermentation w/ acid production, proteins catabolize aerobically [slant] w/ alkaline product- reversion

Pseudomonas aeruginosa: red slant/ no color butt= no fermentation, peptone catabolized aerobically w/ basic products not Enterobacteriaceae

Proteus mirabilis: red slant/ yellow butt= red slant/yellow butt= glucose fermentation w/ acid production, proteins catabolize aerobically [slant] w/ alkaline product- reversion, sulfur reduction

Motility Test

Purpose: Some bacteria have the ability to propel themselves through liquids by means of flagella ( flagellum = singular). These long fibers of protein are found on many bacteria, including most supported by this simulation. However, not all bacteria are able to "swim", even if they have flagella. The purpose of this test is to see if the microbe can "swim" by means of flagella.

Detected: as diffused growth radiating from central stab line

Differential: differentiate between motile bacteria and non-motile bacteria

The concentration agar is reduced from 1.5% to .4% why?- Just enough to maintain its form while allowing movement of motile bacteria

Role of tetrazolium salt?

- to make interpretation easier

-used by bacteria as an electron acceptor

-oxidized form: colorless

-soluble when reduce: red

Procedure indicate that you should stab agar and the move loop up along the same line where you stabbed because?- Lateral movement of the needle will interpretation more difficult to read

Basic result: A positive result for motility indicated when red [reduced] TTC is seen radiating outward from central stab

Organisms Results:

Proteus mirabilis: red diffuse growth radiating outward from stabline- [+] organism is motile

Micrococcus luteus: red growth only along the stab line- [-} organism is non-motile

Coliform

members of Enterobacteriaceae that ferments lactose

Selective Medium

allow certain types of organisms to grow, and inhibit the growth of other organisms

Differential Medium

are used to isolate or identify particular organisms

Hydrolytic Enzyme

reaction that uses water to split complex molecules

Tryptophan

amino acid used in indole production

Reversion

process that takes up to 18 to 24 hrs

as glucose diminishes the organisms located in aerobic region[slant] will begin to break down available amino acids producing ammonia [NH3] and raising pH

Enterics

bacteria of the intestines