biochemical test2

BEA test:

Negative:

no darkening of the medium after 48 hours (not a member of group D Streptococcus or Enterococcus)

\***Malonate test:

In the Krebs cycle, \___ oxidizes succinate to fumarate. Malonate is structurally similar enough to succinate that it can replace it in this reaction. Why is this important?

succinate dehydrogenase

Competitive inhibition of succinate dehydrogenase and the buildup of succinate in the cell shuts down the TCA cycle and will kill the organism unless it can utilize malonate as its sole carbon source.

\***Oxidase test:

In aerobic respiration, the enzyme \___ transfers electrons from cytochrome c to the final electron acceptor, \___

cytochrome c oxidase

oxygen

what is the reagent used for the oxidase test

oxidase

What is the purpose of the oxidase test?

Testing for the Cytochrome C oxidase respiratory enzyme

How is the oxidase test performed?

Place loopful of culture on filter paper; add drops of tetramethyl-p-phenylenediamine; observe for color change within 30 seconds

Oxidase test:

Why should you read the results within 30 seconds?

The reagent used for this test is unstable and may oxidize independently shortly after they become moist

Postive oxidase test

dark blue/purple within 20 seconds

negative oxidase test

no color change to blue/purple within 20 seconds

\***Oxidase test:

The chromogenic reducing agent that is added to the filter paper when performing the oxidase test is called \___, which is colorless in its \___ state.

tetramethyl-p-phenylenediamine

reduced

reagent used for catalase test

hydrogen peroxide

positive catalase test

bubbles

negative catalase test

no bubbles

Why is the catalase test used?

to test for catalase activity and the ability to breakdown hydrogen peroxide.

How is the catalase test performed?

Place a drop of hydrogen peroxide on a slide; add a loopful of culture to the slide; observe for bubbles

\***Catalase test:

This test is used to differentiate between members of the catalase positive \___ and the catalase negative \___

Micrococcaceae

Streptococcaceae

\***Catalase test:

What is the role of flavoprotein in the electron transport chain?

Flavoprotein is an ETC carrier molecule that can bypass the next carrier in the chain and transfer electrons directly to oxygen, which produces hydrogen peroxide.

\***Catalase test:

Two highly potent byproducts that are produced as a byproduct of this reaction are \___ and \___. The enzymes responsible for eliminating these cellular are \___ and catalase, respectively.

superoxide radical

hydrogen peroxide

superoxide dismutase

MAC positive for growth:

good growth

organism is not inhibited by CV or bile \= gram negative

MAC negative for growth:

no colonies, streak lines visible

no growth/poor growth

organism is inhibited by CV and bile \= gram positive

MAC positive for lactose fermentation:

pink to red growth

MAC negative for lactose fermentation:

growth is colorless

What does MAC do?

Test for lactose fermentation and the ability for organisms to grow in the presence of bile salts and CV

Is MAC selective or differential? Why?

Selective and differential. It is selective because it inhibits the growth of gram-positive bacteria due to the presence of CV and bile salts. It is differential because it contains indicators to differentiate lactose fermenters from lactose non-fermenters.

\***What pH indicator is added to MacConkey agar?

Neutral red dye

\***MacConkey agar is a selective media that inhibits growth of some organisms due to the presence of crystal violet and \___. Which group of organisms is inhibited by the presence of these selective components?

bile salts

Gram positive bacteria

\***MacConkey agar differentiates bacteria on the bases of what metabolic activity?

Lactose fermentation

Positive malonate:

blue

malonate utilized

Negative malonate:

green

malonate not utilized

Why is the malonate test used?

To determine if an organism utilizes malonate as its sole carbon source.

Malonate test:

What causes the color change?

If the organism utilizes malonate, it will alkalinize the medium and change the indicator from green to deep blue.

\***True or False? Malonate broth, which contains a high concentration of sodium malonate, yeast extract, and a very small amount of glucose, is an example of a highly defined, differential medium.

True

positive citrate:

blue

or no color change, but there is growth

citrate is utilized

pH of the media is alkaline

negative citrate:

green and no growth

citrate is not utilized

pH of the media is acidic

Why is the citrate test used?

To determine if an organism utilizes citrate at its sole carbon source.

Citrate test:

Determine the chemical reaction that takes place.

The enzyme citrate permease transports citrate into the cell and performs citrate fermentation. The enzyme citrate lyase hydrolyzes citrate into oxaloacetate and acetate. Oxaloacetate is then converted to pyruvate.

Citrate test:

What causes the color change?

Bacteria that utilize citrate convert the ammonium phosphate to ammonia and ammonium hydroxide, which alkanalize the agar, making the pH go up, changing the medium from green to blue.

\***Citrate test:

Bacterial species that are able to utilize citrate as their sole carbon source have an enzyme that allows them to transport citrate into the cell, where it is hydrolyzed to \___ and \___

oxaloacetate and acetate.

\***citrate test:

A highly defined formulation designed to differentiate organisms based on their ability to grow when an essential nutrient is strictly limited is called a \___

utilization media

BEA test:

Positive:

medium is darkened within 48 hours (member of group D Streptococcus or Enterococcus)

BEA test:

Why is this test used?

To determine an organisms ability to hydrolyze esculin with the enzyme esculinase producing D-glucose and Esculetin, in the presence of bile salts.

BEA test:

What is the color change?

Dark brown/black precipitate which darkens the medium.

BEA test:

Determine the chemical reaction that takes place.

Esculetin reacts with ferric ions in the medium to produce a dark brown to black precipitate in the medium.

BEA test:

Is this test selective or differential? Why?

Selective and differential. It is selective because it encourages the growth of Group D streptococci from non-Group D streptococci. It is differential because it allows us to distinguish between different microbes based on their ability to hydrolyze esculin in the presence of bile.

\***BEA test:

Complete the biochemical reaction shown below including enzymes and products of the reaction:

Esculin -\> \____ + \____

Esculin -\> (Esculinase on arrow) beta-D-Glucose + Esculetin

\***BEA test:

The purpose of the bile esculin test is to presumptively identify which group of microorganisms?

Group D Streptococcus or Enterococcus

Nitrate test:

After incubation, 5 drops of sulfanilic acid and Naphthylamine have been added and mixed. Result: no change

negative for nitrate reduction to nitrite

addition of zinc is necessary

Nitrate test:

After incubation, 5 drops of sulfanilic acid and Naphthylamine have been added and mixed. Result: red

positive for nitrate reduction to nitrite

Nitrate test:

after addition of zinc

result: no color change

positive for reduction of nitrate to something other than nitrite

Nitrate teset:

after addition of zinc:

result: red

negative reaction

Nitrate test:

Describe how this test is performed and the reagent used.

Inoculate with pure culture and incubate. Examine Durham tube, if there is a bubble and the organism is a non-fermenter, it reduced nitrate to N2 and the test is complete. If there is no bubble, add 8 drops of sulfanilic acid and 8 drops of Naphthylamine and mix. If red, organism reduces nitrate to nitrite and the test is complete. If not red, add zinc. If red after addition of zinc, organism does not reduce nitrate. If not red, organism reduces nitrate to something other than nitrite.

Nitrate test:

what is the chemical pathway from nitrate to nitrite?

Nitrous acid forms and reacts with sulfanilic acid to form diazotized sulfanilic acid, which reacts with alpha-naphthylamine to form p-sulfovenzene-azo-alpha-naphthylamine.

Nitrate test: