Gram Selecting/ Differentiating and Fermentation Tests

Question 1

What does EMB agar test for?

Answer 1

EMB agar selects for Gram-negative bacteria and it differentiates between them based on their ability to ferment the carbohydrate lactose

Question 2

What does a positive EMB test result:
A) look like
B) indicate

Answer 2

A) colonies will be covered with a black-green metallic sheen and appear purple
B) Positive results indicate vigorous Gram-negative lactose fermenters

Question 3

What do pink colonies indicate in an EMB test?\

Answer 3

Slow, Gram-negative lactose fermenters

Question 4

What does a negative EMB test look like and Indicate

Answer 4

If the organism is negative for lactose fermentation, no change will occur.
If the organism is Gram-positive, no growth will be seen

Question 5

Why do colonies change colour in an EMB/ MAC test?

Answer 5

Lactose fermenters change the colour because fermentation produces lactic acid, which lowers the pH of the agar, activating the pH indicators which change colour.

Question 6

What does MacConkey agar test for?

Answer 6

MAC selects for Gram-negative organisms and differentiates them based on their ability to ferment the carbohydrate lactose

Question 7

What does a positive MAC test:
A) Look like
B) Indicate

Answer 7

A) the agar will be red

B) Indicates Gram-negative lactose fermentation

Question 8

Why can negative MAC results make the agar less-red

Answer 8

If organisms don’t ferment lactose, they will metabolize peptone in the agar instead, which can result in a lighter agar colour

Question 9

Where are enteric bacteria found

Answer 9

Intestines of warm-blooded animals

Question 10

What does Phenylethyl Alcohol agar test for?

Answer 10

Selects for Gram-positive organisms by inhibiting the growth of Gram-negative organisms
- note, PEA doesn’t kill Gram-negative organisms, it just stops their growth

Question 11

How does PEA inhibit Gram-negative organisms?

Answer 11

PEA increases membrane permeability, allowing K to leak out o the cell and disrupt DNA synthesis

Question 12

What does the KOH reaction test for?

Answer 12

Gram-negative organisms

Question 13

What does a stringy, vicious adhesion indicate in a KOH test

Answer 13

Gram-negative organisms

Question 14

What does Mannitol salt agar test for?

Answer 14

Selects for halophilic (Salt-loving) organisms, differentiates between halophilic mannitol fermenters

Question 15

What does a positive MS test
A) look like
B) Indicate

Answer 15

Yellow haloes around the colonies indicate a halophilic mannitol fermentor

Question 16

What does litmus milk test for?

Answer 16

Lactose fermentation, litmus reduction, casein coagulation, casein hydrolysis

Question 17

What litmus reaction is indicated by a purple top and white bottom?

Answer 17

Litmus is reduced but the pH remains neutral so the broth surface remains purple

Question 18

What litmus reaction is indicated by a completely pink broth

Answer 18

Lactose fermentation produces lactic acid through the spitting of lactose into glucose and galactose, lowering the pH and tuning the litmus pink

Question 19

What litmus reaction is indicated by a pink top and white bottom

Answer 19

Lactose fermentation turns the broth top pink, litmus reduction turns the broth bottom white as litmus is being used as an electron acceptor for anaerobic fermentation, only the parts of the tube untouched by oxygen are reduced

Question 20

What does an acid curd (lumpiness) indicate?

Answer 20

Acid curds indicate the accumulation of lactic acid, which denatures and precipitates casein.

Question 21

How is proteolysis (peptonisation) indicated in litmus milk

Answer 21

Purple top, thin whey bottom

Question 22

Describe proteolysis

Answer 22

Proteolysis occurs when organisms derive their energy from hydrolyzing milk proteins into amino acids. This releases ammonia and raises the pH, allowing the broth to thin and transform into whey.

Question 23

What does a blue broth indicate in litmus milk and why does this occur

Answer 23

Alkaline reactions

Indicates partial degradation off casein without the thinning of both associated with proteolysis

Question 24

What do lysine decarboxylase broths test for

Answer 24

Detect the presence of decarboxylase enzymes based on how the organism breaks down amino acids in the media

Question 25

What do decarboxylase enzymes do

Answer 25

Remove the carbonyl group -COOH from a specific amino acid, producing carbon dioxide and amines that cells use to synthesize other molecules.

Question 26

What amine is produces from lysine decarboxylation

Answer 26

Cadaverine, a foul-smelling diamine produces by the putrefaction of animal tissue

Question 27

Why is mineral oil applied to the top of lysine decarboxylase broth

Answer 27

Mineral oil provides an anaerobic environment to promote glucose fermentation

Question 28

How is glucose fermentation indicated (LD+)

Answer 28

Glucose fermentation produces acids which lower the pH of the broth which turns on the bacterial gene that encodes for decarboxylase enzymes, in turn decarboxylating the available amino acids, resulting in alkaline products which raise the pH and turn the broth purple

Question 29

How is glucose fermentation indicated (LD-)

Answer 29

Glucose fermentation produces acids which lower the pH, turning the broth

Question 30

How is a decarboxylase positive organism indicated

Answer 30

The LD+ tube will be purple, but the LD- tube will be yellow as there are no amino acids present.

Question 31

How is a darcarboxylase negative organism indicated if glucose fermentation occurred

Answer 31

Both tubes will appear yellow

Question 32

What does an inconclusive result look like (lysine decarboxylase test)

Answer 32

Both tubes will be purple as glucose fermentation has not occurred, you are unable to determine where the organism has a decarboxylase enzyme

Question 33

What is TSIA used for

Answer 33

Differential test based on glucose, lactose, and sucrose fermentation and sulfur reduction

Question 34

Why is TSIA deeper than most agar slants

Answer 34

Provides an aerobic environment along the slant and an anaerobic environment at the butt.

Question 35

What does a yellow slant and butt indicate (TSIA)

Answer 35

Lactose and/is sucrose fermentation

- doesn’t identify which carbohydrate was fermented or if glucose was fermented

Question 36

How is glucose-only fermentation identified (TSIA)

Answer 36

Glucose-only fermentation produces a red slant and yellow butt
- organisms in the aerobic slant hydrolyze the amino acids and produce ammonium, raising the pH through a process called reversion

Question 37

What does an orange-red butt indicate (TSIA)

Answer 37

Organisms catabolized peptone aerobically only

Question 38

What does a fully red tube indicate (TSIA)

Answer 38

Organisms did not ferment the carbohydrates, anaerobic and anaerobic peptone utilization occurred