Microbiology final lab practical

carbohydrate fermentation: sugar

1. lactose

2. dextrose

3. sucrose

carbohydrate fermentation: pH indicator

phenol red

carbohydrate fermentation: durham tube

used to measure gas collection

carbohydrate fermentation: interpreting results

+ test:

yellow = + acid; no gas (+A)

yellow + gas = acid & gas (+AG)

- test:

red = neutral + cannot produce gas (-)

Triple iron sugar agar slant

dextrose - 0.1%

lactose - 1%

sucrose 1%

Triple iron sugar agar slant: indicator

phenol red

Triple iron sugar agar slant process

streak slant & stab butt

Triple iron sugar agar slant: interpreting results

red butt & slant = -

yellow butt & red slant = + for glucose only

yellow butt & yellow slant = + for lactose/sucrose

black = produces H2S

Oxidase

1. to determine if organism produces cytochrome oxidase

2. uses TSA plate

Oxidase test

add 2-3 drops of p-amino

Oxidase: interpreting results

maroon/purple after 30 sec = +

no change after 30 sec = -

Indole

to determine if an organism produces tryptophanase

tryptophane = indole + pyruvic acid

media: SIM

indole: test

add 5 drops of kovac's (reacts with indole)

indole: interpreting results

red ring on top = +

no change = -

methyl red

does organism convert glucose to mixed acids

(lactic, formic, acetic)

methyl red: indicator

methyl red

methyl red: media

MRVP broth

methyl red: test

add 5 drops of methyl red

methyl red: interpreting results

cherry red = + (ferments acid)

no change/orange = -

nitrate reduction

to nitrite using nitrate broth

nitrate reduction: test

add 5 drops of nitrate A --> mix

add 5 drops of nitrate B

nitrate reduction: interpreting results

red = + NO2

no change = add pinch of zinc

if tube turns red = - for reduction

if tube does not change = super reducer --> NH3

vogues proskaur

does organism convert glucose to non-acid products

vogues proskaur: test

1. add VPA = alpha mapthol

2. add VPB = KOH

3. let sit for 30 minutes

vogues proskaur: interpreting results

red after 30 mins = +

no change = -

citrate

does organism produce enzymes citrate and citrate permease

use citrate for carbon

citrate: indicator

bromthymol blue - look for alkalinity

citrate: interpreting results

has both enzymes; blue = +

green = -

urease

urea-uses urease-> CO2 + NH3 + H20

urease: indicator

phenol red; higher pH = pink

urease: media

urease broth

urease: interpreting results

yellow = acid

pink = base

H2S production

does organism have cysteine desulferase

H2S production: indicator

FeS = will form a black precipitare

H2S production: media

SIM tube

catalase

H2O2 --> H2O + O2

catalase: interpreting results

aerobic = +

facultative = will look negative but produce delayed bubbles

carbohydrate fermentation: will turn dextrose, lactose, & sucrose yellow

Escherichia coli

TSI-A yellow butt & red streak

Proteus vulgaris

TSI-A red butt & streak

Pseudomonas aeroginoas

TSI-A yellow butt & streak

Escherichia coli

oxidase: produces cytochrome oxidase

Pseudomonas aeroginosa

indole: will form red ring at top

Proteus vulgaris

methyl red: will form mixed acids

Proteus vulgaris

voges proskaur: will form non-acid products

Bacillus cereus

will produce H2S

Proteus vulgaris

Citrate: will turn blue

Pseudomonas aeroginosa

Urease: will use urease

Proteus vulgaris

Catalase: produces bubbles/aerobe

Proteus vulgaris

moist heat mode of action

destroys proteins = denatures enzymes

moist heat methods

1. boiling

2. autoclaving: 15 minutes of steam under pressure

3. pasteurization: low heat to kill organisms but not to destroy quality

May survive moist heat if organism

has a spore