Lab Medias

General Purpose Media

support growth of a variety of microorganisms; good for maintaining cultures

Selective Media

Use inhibitors to prevent growth of certain organisms

Differential Media

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

Common Indicators

dyes, reagents, blood cells, culture conditions

Combination Media

Selective and differential; have both inhibitors and indicators

Complex Media

exact chemical composition is not known; nutrients are supplied as extracts or digests from natural/organic sources (box cake mix)

Defined Media

Have a known chemical composition for every component (homemade cake)

Phenylethyl Alcohol (PEA)

Selective only; inhibitor: Phenylethyl Alcohol—> prevents growth of gram negatives

often used to select for gram positive species in patient samples

Blood Agar (BA)

Differential only; indicator: red blood cells —> differentiates microbes based on hemolysis patterns

Hemolysis Patterns

Beta: Best (complete hemolysis)

Alpha: Average (partial hemolysis)

Gamma: Garbage (no hemolysis)

MacConkey Agar (MAC)

Selective and Differential; inhibitor: crystal violet + bile salts —> inhibits growth of gram positives; indicator: neutral red —> differentiates lactose fermenters from non-fermenters by color change when acid is produced

used for identification of gram negative enterics (enterobacteriaceae)

Eosin Methylene Blue (EMB)

Selective and Differential; inhibitor: eosin + methylene blue —> prevents growth of gram positives; indicator: eosin + methylene blue —> differentiates lactose fermenters from non-fermenters with color change based on type of fermentation

used to screen for coliforms

Motility Media

soft agar that tests for motility through turbidity

flagella = motility

inoculated in a single stab —> motile organisms will grow throughout the media, non-motile organisms will remain confined to the path of the inoculation

Litmus Milk

Differential; indicators: lactose, casein and other peptones, and litmus pH indicator

Lactose: fermentation produces acid —> milk proteins are denatured —> curdling/solidifying of milk

Litmus Milk: color change to pink when acid is present in an oxidized state; if reduced, milk will turn white

Casein/peptones: if caseinase is present, casein is degraded —> milk will turn “clear”

Phenol Red (PR) Sugar Fermentation Broth

Differential; indicators: phenol Red and Durham tube

Phenol Red: fermentation turns the phenol Red yellow

Durham tube: gas by-products of fermentation make a bubble in the tube

No fermentation: peptone degredation releases ammonia —> phenol Red turns pink

Kligler’s Iron Agar (KIA)

differential; indicators: 1% lactose, 0.1% glucose, 1% peptone, phenol red, iron

1.0% lactose + 0.1% glucose > 1.0% peptone (1.1 > 1) —> agar turns yellow

gas —> lifting/cracking of agar

glucose: glucose only fermentation —> not enough acid produced to balance NH3 from peptones —> top red, butt yellow (0.1 < 1)

2,3 butanediol fermentation —> not as stable as mixed acid —> reversion back to neutral/alkaline pH (mostly yellow, barely any red at top)

iron: reduction of H2S (acidic environment) combines with iron to form a black precipitate (butt black, top red)

Lipase Plate

differential; indicator: spirit blue

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

(+) = blue (-) = no color

Milk Agar Plate

differential; indicator: casein (milk protein)

if the microbe produces caseinase, casein is hydrolyzed —> loses white color

(+) = clear around colony

(-) = no clearing around colony

Starch Agar

differential; indicator: iodine

enzyme amylase in microbe will break down starch

add iodine to colony after growth

(+) = purple

(-) no difference

Biochemical Tests

tests that yield results within minutes

Catalase Test

Differential; indicator: 3% hydrogen peroxide (H2O2)

tests for catalase - converts hydrogen peroxide into water and oxygen

+ : bubbles

- : no reaction

Oxidase Test

differential; indicator: dimethyl-p-phenylenediamine hydrochloride —> in the presence of cytochrome c oxidase, reagent is oxidized and turns dark blue/black

Nitrate Broth

differential; indicator: Nitrate I and II, also Zn powder

If nitrate is reduced to nitrite, then Nitrate I and II will react with NO2 —> brick red color (nitrate reductase is present and nitrate was respirated)

If nitrate is not used and is residual, added Zn powder will catalyze the rxn (red result AFTER Zn addition indicates that nitrate was not used —> no nitrate reductase in organism)

if there is no result when Zn is added, then the nitrate reductase reduced nitrate all the way to N2 (nitrate reductase is present and nitrate was respirated)

Urea Broth

differential; indicator: phenol red

also contains urea —> detects urease

urea —> 2 Nh3 and CO2 —> increases pH —> phenol red color change

pH > 8.1 —> cerise

neutral pH —> red

acidic pH —> yellow

Gelatin Test

differential; indicator: gelatin

tests for gelatinase

+ : liquid/less solid media

- : solid media

gelatinase present —> hydrolysis of gelatin —> media no longer solid

Phenylalanine Slant

differential; indicator: 10% FeCl3

also contains phenylalanine

tests for phenylalanine deaminase —> phenylalanine —> PPA + NH3 —> add 5-10 drops FeCl3 reagent (reacts with PPA)

+ : deep green color (phenylalanine is deaminated)

- : no change (no phenylalanine deaminase)

pee pee in the pool turns green

SIM Test

differential; indicators; Fe —> FeS, Kovac’s reagent, motility media

black precipitate: H2S produced by the bacterium reacts with Fe in medium to produce FeS

pink color at top: positive for tryptophanase (tryptophan broken —> produces indole)

turbidity away from stab: bacteria is motile

MR-VP Broth

differential; indicators: methyl red and VP I and II

MR and VP tests must be done separately

methyl red —> high acid concentration (pH < 4.4) retains red color —> mixed-acid fermentation

VP I (alpha naphthol) —> intensifies red color

VP II (KOH) —> produces an alkaline condition that favors acetoin oxidation —> diacetal and O2 is made —> presence of O2 produces a red color

acetoin intermediate is produced by 2,3 butanediol fermenters

Tryptone Broth

differential; indicator: kovac’s reagent

detects tryptophanase

tryptophanase hydrolyzes tryptophan —> indole, pyruvate, and NH3 —> add 3-5 drops of kovac’s reagent —> extracts indole to the top —> indole + kovac’s reagent = cerise

Simmons Citrate Slant

differential; indicator: bromothymol blue

detects utilization of citrate as sole carbon source for growth

+ : blue or growth

- : no growth

growth is the primary indicator of citrate indication

if it’s blue it grew

IMViC

set of 4 tests that can be used to differentiate E. coli from Enterobacter aerogenes

• indole (EC +, EA -)

• methyl-red (EC +, EA -)

• voges-proskauer (EC -, EA +)

• citrate (EC -, EA +)

Mannitol Salt Agar (MSA)

combination media; inhibitor: 7.5% NaCl (selects for staphylococcus spp.), indicator: phenol red

contains 1% w/v D-mannitol (fermentable by Staph aureus and Staph saprophyticus)

D-mannitol fermented—> acid produced —> phenol red turns yellow

+ : yellow color

- : stays red

Plasma Test

differential; indicator: rabbit plasma

tests for coagulase —> forms fibrin clots in plasma

+ : solid

- : liquid/not solid

DNase Test

differential; indicator: methyl green (forms a green complex with dsDNA)

also contains ~2% w/v intact dsDNA

tests for DNase —> breaks down DNA into nucleotides

+ : clearing of DNA-dye complex around colony

- : no clearing