lab 15 - additional biochemical tests to identify gram-negative bacteria

further testing is needed to definitively identify gram-negative bactera. what kinds of biochemical testing is done?

• IMViC series

• esculin hydrolysis test

• DNase test

• oxidase test

• nitrate reduction test

most gram-negative bacteria fall under which family?

Enterobacteriaceae, which is enteric gram-negative bacilli

Enterobacteriaceae are separated into what 2 groups?

coliforms, which are lactose-fermenting enteric bacilli (such as Escherichia or Enterobacter)

non-coliforms, which are non-lactose fermenting bacilli (such as salmonella and serratia)

which 2 gram-negative bacteria aren’t a part of Enterobacteriaceae?

Pseudomonas, because although it is a bacillus, it doesn’t ferment carbohydrates and is oxidase positive

Neisseria, because it is a diplococcus that is also oxidase positive

what is the IMViC series tests?

it is a series of tests used to differentiate members of Enterobacteriaceae. it stands for indole, methyl red, voges-proskauer, and citrate utilization

what is the indole test?

it tests an organism’s ability to hydrolyze the amino acid tryptophan and produce indole. it grows the organisms in a broth rich in tryptophan (such as TSB). after incubation, kovac’s reagent is added. if indole is present, it will combine with the reagent to form a distinctive red-colored phase on top of the denser broth

the methyl red and voges-proskauer tests detect the presence of different end products resulting from the metabolism of _

glucose. both tests are performed by inoculating the organism into tubes of MRVP broth which contains glucose

what is the MR test?

the methyl red reagent detects the presence of acids produced by organisms that perform a mixed acid fermentation. a positive MR test is indicated by a red color immediately after adding the reagent

what is the VP test?

barritt’s a and barritt’s b reagents detect the presence of an unusual, non-acidic compound, which is formed by pyruvate

what is the citrate utilization test?

it determines if an organism can use citrate as its only carbon source. the simmons citrate agar slant contains sodium citrate as the sole carbon source, ammonium phosphate as the sole nitrogen source, and a bromthymol blue pH indicator. if citrate is utilized, alkaline end products are released, which increases the pH and changes the indicator color from green to blue

a positive oxidase test is useful in the confirmatory identification of which 2 gram-negative organisms?

pseudomonas and neisseria

bacteria that perform aerobic respiration have have

cytochromes, or heme-containing proteins. together with flavoproteins and ubiquinones, they serve as electron carriers in the ETC

what does a positive oxidase test look like?

the development of a dark purple coloration on the filter

what is the nitrate reduction test?

it determines the ability of different bacteria to reduce nitrate to nitrite, ammonia, or other reduced forms of nitrogen using nitrate reductase or nitrite reductase

after the oxidase test, for pseudomonas fluorescens…

1. is cytochrome oxidase present?

2. observations?

1. yes

2. purple color developed quickly on oxidase strip

after the oxidase test, for neisseria sicca…

1. is cytochrome oxidase present?

2. observations?

1. yes

2. blue/purple color changes on oxidase reagent

after the oxidase test, for escherichia coli…

1. is cytochrome oxidase present?

2. observations?

1. no

2. no color change

after the oxidase test, for micrococcus luteus…

1. is cytochrome oxidase present?

2. observations?

1. yes (most times)

2. produces faint purple color

after the oxidase test, for enterobacter aerogenes…

1. is cytochrome oxidase present?

2. observations?

1. no

2. no color change

after the oxidase test, for pseudomonas aeruginosa…

1. is cytochrome oxidase present?

2. observations?

1. yes

2. deep purple color develops

after the IMViC test, for escherichia coli…

1. indole test result?

2. methyl red test result?

3. voges-proskauer test result?

4. citrate utilization test result?

1. positive

2. positive

3. negative

4. negative

after the IMViC test, for enterobacter aerogenes…

1. indole test result?

2. methyl red test result?

3. voges-proskauer test result?

4. citrate utilization test result?

1. negative

2. negative

3. positive

4. positive

after the IMViC test, for salmonella enterica…

1. indole test result?

2. methyl red test result?

3. voges-proskauer test result?

4. citrate utilization test result?

1. negative

2. positive

3. negative

4. positive

after the bile esculin test, for escherichia coli…

1. growth? appearance?

2. bile tolerant?

3. esculin hydrolysis?

4. interpretation of results?

1. light growth

2. no

3. no

4. not bile tolerant, can’t hydrolyze esculin

after the bile esculin test, for enterobacter aerogenes…

1. growth? appearance?

2. bile tolerant?

3. esculin hydrolysis?

4. interpretation of results?

1. light growth

2. no

3. no

4. not bile tolerant, can’t hydrolyze esculin

after the bile esculin test, for serratia marcescens…

1. growth? appearance?

2. bile tolerant?

3. esculin hydrolysis?

4. interpretation of results?

1. growth (variable)

2. yes (variable)

3. no

4. sometimes grows, but can’t hydrolyze esculin

after the bile esculin test, for staphylococcus epidermidis…

1. growth? appearance?

2. bile tolerant?

3. esculin hydrolysis?

4. interpretation of results?

1. growth

2. yes

3. no

4. bile tolerant but can’t hydrolyze esculin

after the bile esculin test, for enterococcus durans…

1. growth? appearance?

2. bile tolerant?

3. esculin hydrolysis?

4. interpretation of results?

1. growth with black precipitate

2. yes

3. yes

4. bile tolerant, can hydrolyze esculin

after the DNase test, for enterobacter aerogenes…

1. result?

2. observations?

1. negative

2. no clearing around growth; agar is unchanged

after the DNase test, for salmonella enterica…

1. result?

2. observations?

1. negative

2. no clearing around growth; agar is unchanged

after the DNase test, for serratia marcescens…

1. result?

2. observations?

1. positive

2. clear halo around colonies indicating DNA hydrolysis

after the DNase test, for staphylococcus epidermidis…

1. result?

2. observations?

1. negative

2. no clearing around growth; agar is unchanged

after the nitrate reduction test, for escherichia coli…

1. observations after adding sulfanilic acid & a-naphthylamine?

2. what was present in nitrate broth culture?

3. which enzymes are present?

1. red

2. nitrite

3. nitrate reductase

after the nitrate reduction test, for enterobacter aerogenes…

1. observations after adding sulfanilic acid & a-naphthylamine?

2. observations after adding zinc dust?

3. what was present in nitrate broth culture?

4. which enzymes are present?

1. colorless

2. no color change

3. nitrogen gas

4. nitrate reductase and nitrite reductase

after the nitrate reduction test, for pseudomonas fluorescens…

1. observations after adding sulfanilic acid & a-naphthylamine?

2. observations after adding zinc dust?

3. what was present in nitrate broth culture?

4. which enzymes are present?

1. colorless

2. no color change

3. nitrogen gas

4. nitrate reductase and nitrite reductase

after the nitrate reduction test, for staphylococcus epidermidis…

1. observations after adding sulfanilic acid & a-naphthylamine?

2. observations after adding zinc dust?

3. what was present in nitrate broth culture?

4. which enzymes are present?

1. colorless

2. red

3. nitrate

4. neither nitrate/nitrite reductase

after the nitrate reduction test, for enterococcus durans…

1. observations after adding sulfanilic acid & a-naphthylamine?

2. what was present in nitrate broth culture?

3. which enzymes are present?

1. red

2. nitrite

3. nitrate reductase

after the nitrate reduction test, for pseudomonas aeruginosa…

1. observations after adding sulfanilic acid & a-naphthylamine?

2. observations after adding zinc dust?

3. what was present in nitrate broth culture?

4. which enzymes are present?

1. colorless

2. colorless

3. no nitrate/nitrite

4. nitrate reductase & nitrite reductase

what is the function of a reagent?

to initiate/test a chemical reaction

nitrate reduction indicates that the organism is performing _ metabolism

anaerobic

how can you speed up a reaction that is progressing slowly?

by increasing the temperature

in the typical 24-48 hour citrate-positive test, the citrate agar slant is blue in the upper portion but green at the bottom. why?

because of the pH change caused by the bacterial metabolism of citrate

in which metabolic pathway is citrate an important intermediate?

Krebs cycle

what is reduction?

a chemical reaction where a substance gains electrons, decreasing its oxidation state