Lab Exercise 8 - Cultural and Physiological Characteristics of Bacteria I

3 examples of agar plates that test for exoenzymes

lipase

milk agar

starch agar

3 examples of tube media that test for non-exoenzyme metabolic processes

litmus milk

sugar fermentation broth

Kligler’s iron agar

What does litmus milk test for? (4 things)

lactose fermentation

metabolism of proteins

degradation of casein

litmus reduction

What does litmus milk contain?

lactose

casein, other peptones

litmus pH indicator

5 possible reactions of litmus milk

acid production

alkaline production

curd

redox reduction

proteolysis

What is the visible result of litmus milk if lactose fermentation occurs?

acidic pH causes litmus milk to turn pink

What is the visible result of litmus milk of peptone deamination occurs? (alkaline production)

ammonia is released → pH is raised → litmus milk turns blue

color change may be limited to the top of the tube, since peptone deamination is oxygen-dependent

What is the visible result of litmus milk if reduction occurs?

pH indicator loses its color → goes white

a band of color will persist at the top of the media above the white, where O₂ penetrates and re-oxidizes the litmus indicator

What is the visible result of litmus milk of proteolysis occurs?

caseinase is present → casein degrades → milk turns clear (not completely transparent, but not opaque)

What is the visible result of litmus milk if enough acid is produced?

milk proteins denature → curding occurs

curding may be complete or incomplete → lumpy

does not always occur with acid production, but all curding is caused by acid production

phenol red sugar fermentation broth

a medium that tests for sugar fermentation

contents of PR sugar fermentation broth

one sugar: glucose, lactose, or mannitol

indicators: phenol red, a Durham tube

What is the visible result in PR sugar broth if an organism ferments?

phenol red turns yellow

gas formation → bubble collects in Durham tube

note: bubble formation will not always occur, sometimes only yellow color change occurs

What is the visible result in PR sugar broth if an organism can’t ferment?

peptone degradation → ammonia released

phenol red turns cerise (hot pink)

will never experience bubble formation

What does Kligler’s iron agar (KIA) test for?

lactose fermentation

glucose fermentation fermentation

sulfur reduction

What are the carbohydrate sources in KIA?

1.0% lactose (l looks like 1, in the first position → 1.0)

0.1% glucose (l looks like 1, in the second position → 0.1)

other ingredients in KIA

1.0% peptone

a source of ammonia and sulfur via cysteine

indicators in KIA

phenol red: pH indicator

ferric ammonium citrate gives iron: combines with H₂S → forms black precipitate

processes that occur in KIA media

sugar fermentation → acid production → yellow color

peptone deamination NH₃ production → red color remains

What is the visible result in KIA media if an organism ferments lactose?

lactose and glucose are fermented → acid is formed in excess of NH₃

entire tube turns yellow

(1.0% lactose + 0.1% glucose fermented > 1% peptone fermented → yellow)

What is the visible result of KIA media if an organism ferments glucose only?

NH₃ produced from peptone breakdown > acid produced from glucose fermentation

(0.1% glucose < 1.0% peptone)

butt of tube changes to yellow where glucose fermentation occurs

top of tube (past the lower end of the slant) stays red; oxygen can only penetrate so far for peptone deamination to reverse acidification

What is the visible process in KIA media if reversion occurs?

some lactose fermenters → weaker acids → oxidize into neutral end products

tube is yellow throughout, except for tip of the media (not past the lower end of the slant) → red tip

e.g. in 2,3-butanediol lactose fermenters

What is the visible result of KIA media if gas production occurs?

some fermentation forms gaseous end products

gas accumulation → lifting or cracking of media in tube

What is the visible result in KIA media if sulfur reduction occurs?

S reduction → H₂S

H₂S combines with iron → black precipitate that obscures color in the butt of the tube

note: if black appears, acidic environment must also be present

What type of media is a lipase plate? General purpose, selective, differential, or combination?

differential

What does a lipase plate test for?

lipase (an enzyme), which hydrolyzes fats → glycerol and fatty acids

What is the indicator in a lipase plate?

spirit blue

What is the visible result on a lipase plate if lipid hydrolysis occurs?

dark blue zone around growth, no oil surface

What is a positive result in a lipase plate?

blue color

no oily surface, because lipid hydrolysis broke down the fats

(positive result = no fats)

What is a negative result in a lipase plate?

no blue color

oily surface, because lipid hydrolysis did not occur → fats are still present

What type of media is a milk agar plate? General purpose, selective, differential, or combination?

differential

What does a milk agar plate test for?

caseinase (an enzyme), which hydrolyzes casein (a milk protein) → amino acid products

What is a positive result on a milk agar plate?

caseinase is present → breaks down casein → no white color to milk

clearing around the colony

What is a negative result on a milk agar plate?

caseinase is not present → casein remains intact → milk retains its color

no clearing around the colony

What type of media is starch agar? General purpose, selective, differential, or combination?

differential

What does starch agar test for?

amylase (NOT STARCHASE), which breaks down starch into simple sugars

What is the indicator in starch agar?

iodine

added after colony growth

results in purple color when present with starch

What is a positive result on starch agar?

amylase present → starch breakdown → no more starch → no purple around the colony when iodine is added

What is a negative result on starch agar?

no amylase present → starch does not break down → starch remains → purple color around the colony when iodine is added