Gram Selecting/ Differentiating and Fermentation Tests Flashcards
What does EMB agar test for?
EMB agar selects for Gram-negative bacteria and it differentiates between them based on their ability to ferment the carbohydrate lactose
What does a positive EMB test result:
A) look like
B) indicate
A) colonies will be covered with a black-green metallic sheen and appear purple
B) Positive results indicate vigorous Gram-negative lactose fermenters
What do pink colonies indicate in an EMB test?\
Slow, Gram-negative lactose fermenters
What does a negative EMB test look like and Indicate
If the organism is negative for lactose fermentation, no change will occur.
If the organism is Gram-positive, no growth will be seen
Why do colonies change colour in an EMB/ MAC test?
Lactose fermenters change the colour because fermentation produces lactic acid, which lowers the pH of the agar, activating the pH indicators which change colour.
What does MacConkey agar test for?
MAC selects for Gram-negative organisms and differentiates them based on their ability to ferment the carbohydrate lactose
What does a positive MAC test:
A) Look like
B) Indicate
A) the agar will be red
B) Indicates Gram-negative lactose fermentation
Why can negative MAC results make the agar less-red
If organisms don’t ferment lactose, they will metabolize peptone in the agar instead, which can result in a lighter agar colour
Where are enteric bacteria found
Intestines of warm-blooded animals
What does Phenylethyl Alcohol agar test for?
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
How does PEA inhibit Gram-negative organisms?
PEA increases membrane permeability, allowing K to leak out o the cell and disrupt DNA synthesis
What does the KOH reaction test for?
Gram-negative organisms
What does a stringy, vicious adhesion indicate in a KOH test
Gram-negative organisms
What does Mannitol salt agar test for?
Selects for halophilic (Salt-loving) organisms, differentiates between halophilic mannitol fermenters
What does a positive MS test
A) look like
B) Indicate
Yellow haloes around the colonies indicate a halophilic mannitol fermentor
What does litmus milk test for?
Lactose fermentation, litmus reduction, casein coagulation, casein hydrolysis
What litmus reaction is indicated by a purple top and white bottom?
Litmus is reduced but the pH remains neutral so the broth surface remains purple
What litmus reaction is indicated by a completely pink broth
Lactose fermentation produces lactic acid through the spitting of lactose into glucose and galactose, lowering the pH and tuning the litmus pink
What litmus reaction is indicated by a pink top and white bottom
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
What does an acid curd (lumpiness) indicate?
Acid curds indicate the accumulation of lactic acid, which denatures and precipitates casein.
How is proteolysis (peptonisation) indicated in litmus milk
Purple top, thin whey bottom
Describe proteolysis
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.
What does a blue broth indicate in litmus milk and why does this occur
Alkaline reactions
Indicates partial degradation off casein without the thinning of both associated with proteolysis
What do lysine decarboxylase broths test for
Detect the presence of decarboxylase enzymes based on how the organism breaks down amino acids in the media
What do decarboxylase enzymes do
Remove the carbonyl group -COOH from a specific amino acid, producing carbon dioxide and amines that cells use to synthesize other molecules.
What amine is produces from lysine decarboxylation
Cadaverine, a foul-smelling diamine produces by the putrefaction of animal tissue
Why is mineral oil applied to the top of lysine decarboxylase broth
Mineral oil provides an anaerobic environment to promote glucose fermentation
How is glucose fermentation indicated (LD+)
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
How is glucose fermentation indicated (LD-)
Glucose fermentation produces acids which lower the pH, turning the broth
How is a decarboxylase positive organism indicated
The LD+ tube will be purple, but the LD- tube will be yellow as there are no amino acids present.
How is a darcarboxylase negative organism indicated if glucose fermentation occurred
Both tubes will appear yellow
What does an inconclusive result look like (lysine decarboxylase test)
Both tubes will be purple as glucose fermentation has not occurred, you are unable to determine where the organism has a decarboxylase enzyme
What is TSIA used for
Differential test based on glucose, lactose, and sucrose fermentation and sulfur reduction
Why is TSIA deeper than most agar slants
Provides an aerobic environment along the slant and an anaerobic environment at the butt.
What does a yellow slant and butt indicate (TSIA)
Lactose and/is sucrose fermentation
- doesn’t identify which carbohydrate was fermented or if glucose was fermented
How is glucose-only fermentation identified (TSIA)
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
What does an orange-red butt indicate (TSIA)
Organisms catabolized peptone aerobically only
What does a fully red tube indicate (TSIA)
Organisms did not ferment the carbohydrates, anaerobic and anaerobic peptone utilization occurred
