Proteins, Amino Acids, and Enzymes II: The IMViC Tests

Harley−Prescott:

Laboratory Exercises in

Microbiology, Fifth Edition



IV. Biochemical Activities

of Bacteria



25. Proteins, Amino Acids,

and Enzymes II: The IMViC

Tests



Why Are the Following Bacteria

Used in This Exercise?

In this exercise the student will learn how to perform the

IMViC series of tests that distinguish between different enteric (pertaining to the small intestine) bacteria. To illustrate

the various IMViC reactions, the authors have chosen four

enteric bacteria. Enterobacter aerogenes (Gr. aer, air) is a

facultatively anaerobic gram-negative rod that has peritrichous flagella. It is a motile lactose fermenter. E. aerogenes

is widely distributed in nature, occurring in fresh water, soil,

sewage, plants, vegetables, and animal and human feces. It is

indole negative, MR negative, VP positive, and Simmons citrate positive. Escherichia coli (Gr. colon, large intestine) is

a facultatively anaerobic gram-negative rod that is motile

with peritrichous flagella or nonmotile. It is a lactose fermenter. E. coli occurs as normal flora in the lower part of the

intestine of warm-blooded animals. It is indole positive, MR

positive, VP negative, and Simmons citrate negative. Klebsiella oxytoca is a facultatively anaerobic gram-negative rod.

It is nonmotile and a lactose fermenter. K. oxytoca occurs in

human feces and clinical specimens, soil, water, grain, fruits,

and vegetables. It is indole positive, often MR negative, VP

positive, and Simmons citrate positive. Proteus vulgaris (L.

vulgaris, common) is a gram-negative facultatively anaerobic rod that occurs in the intestines of humans and a wide variety of animals, in manure, and in polluted waters. It has

peritrichous flagella, is motile, and does not ferment lactose.

P. vulgaris is indole positive, MR positive, VP negative, and

sometimes Simmons citrate positive.



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found in the intestines of humans and other mammals

belong to the family Enterobacteriaceae. These bacteria are short, gram-negative, nonsporing bacilli. They

can be subdivided into lactose fermenters and nonfermenters. Examples include pathogens (Salmonella and

Shigella, lactose nonfermenters), occasional pathogens

(Klebsiella and Escherichia, lactose fermenters; and

Proteus, lactose nonfermenter), and normal intestinal

microbiota (Enterobacter, lactose fermenter).

The differentiation and identification of these enteric bacteria can be accomplished by using the

IMViC test (indole, methyl red, Voges-Proskauer,

and citrate; the “i” is for ease of pronunciation).



Indole Production

The amino acid tryptophan is found in nearly all proteins. Bacteria that contain the enzyme tryptophanase

can hydrolyze tryptophan to its metabolic products,

namely, indole, pyruvic acid, and ammonia. The bacteria use the pyruvic acid and ammonia to satisfy nutritional needs; indole is not used and accumulates in the

medium. The presence of indole can be detected by the

addition of Kovacs’ reagent. Kovacs’ reagent reacts

with the indole, producing a bright red compound on

the surface of the medium (figures 25.1, 25.2). Bacteria

producing a red layer following addition of Kovacs’

reagent are indole positive; the absence of a red color

indicates tryptophan was not hydrolyzed, and the bacteria are indole negative.



Methyl Red Test

Medical Application

The following medically important bacteria are MRϩ: Escherichia coli (opportunistic urinary tract infections), Salmonella typhi (typhoid fever), Shigella dysenteriae (bacterial dysentery), and Yersinia pestis (plague). The following

is MRϪ: Enterobacter aerogenes (urinary tract infections).

Bordetella pertussis (whooping cough) is citrate negative whereas all other Bordetella species are citrate positive. The enteric bacteria such as Klebsiella pneumoniae

(pneumonia) and Enterobacter are citrate positive and can

be distinguished in the clinical laboratory from the opportunistic pathogen Escherichia coli (urinary tract infections)

which is citrate negative.



Principles

The identification of enteric (intestinal) bacteria is of

prime importance in determining certain food-borne

and waterborne diseases. Many of the bacteria that are



154



Biochemical Activities of Bacteria



All enteric bacteria catabolize glucose for their energy

needs; however, the end products vary depending on

the enzyme pathways present in the bacteria. The pH

indicator methyl red (see appendix E) detects a pH

change to the acid range as a result of acidic end products such as lactic, acetic, and formic acids. This test is

of value in distinguishing between E. coli (a mixed

acid fermenter) and E. aerogenes (a butanediol fermenter). Mixed acid fermenters such as E. coli produce a mixture of fermentation acids and thus acidify

the medium. Butanediol fermenters such as E. aerogenes form butanediol, acetoin, and fewer organic

acids. The pH of the medium does not fall as low as

during mixed acid fermentation. As illustrated in figure 25.3, at a pH of 4, the methyl red indicator turns

red—a positive methyl red test. At a pH of 6, the indicator turns yellow—a negative methyl red test.



Voges-Proskauer Test

The Voges-Proskauer test (named after Daniel Voges,

German physician, and Bernhard Proskauer, German



Harley−Prescott:

Laboratory Exercises in

Microbiology, Fifth Edition



IV. Biochemical Activities

of Bacteria



© The McGraw−Hill

Companies, 2002



25. Proteins, Amino Acids,

and Enzymes II: The IMViC

Tests



Figure 25.1 Indole Test. The tube on the left is indole negative and the tube on the right is indole positive.

Biochemistry within bacteria

tryptophanase



indole + pyruvic acid + ammonia



Tryptophan

Biochemistry within tubes

Indole + p-dimethylaminobenzaldehyde



HCl

amyl alcohol



Rosindole dye

(cherry red compound)



Kovacs’ reagent

Kovacs’ reagent



Indole –



Figure 25.2 Indole Test With KEY Tablets. The development

of a red color within a few minutes after adding 10 drops of Kovacs’

reagent—a positive test (tube on the left). Negative reactions are

colorless (tube on the right).



hygienist, in the early twentieth century) identifies bacteria that ferment glucose, leading to 2,3-butanediol

accumulation in the medium. The addition of 40%

KOH and a 5% solution of alpha-naphthol in absolute

ethanol (Barritt’s reagent) will detect the presence of

acetoin—a precursor in the synthesis of 2,3-butanediol.

In the presence of the reagents and acetoin, a cherry-red

color develops. Development of a red color in the culture medium 15 minutes following the addition of Barritt’s reagent represents a positive VP test; absence of a

red color is a negative VP test (figure 25.4).



Indole +



Citrate Utilization Test

The citrate utilization test determines the ability of

bacteria to use citrate as a sole carbon source for their

energy needs. This ability depends on the presence of a

citrate permease that facilitates transport of citrate

into the bacterium. Once inside the bacterium, citrate is

converted to pyruvic acid and CO2. Simmons citrate

agar slants contain sodium citrate as the carbon source,

NH4+ as a nitrogen source, and the pH indicator (see

appendix D) bromothymol blue. This test is done on

slants since O2 is necessary for citrate utilization. When

bacteria oxidize citrate, they remove it from the

medium and liberate CO2. CO2 combines with sodium

(supplied by sodium citrate) and water to form sodium

carbonate—an alkaline product. This raises the pH,

turns the pH indicator to a blue color, and represents a

positive citrate test; absence of a color change is a

negative citrate test (figure 25.5). Citrate-negative cultures will also show no growth in the medium.



Procedure

Indole Production Test

First Period

1. Label each of the SIM deep tubes with the name of

the bacterium to be inoculated (E. coli, P. vulgaris,



Proteins, Amino Acids, and Enzymes II: The IMViC Tests



155



Harley−Prescott:

Laboratory Exercises in

Microbiology, Fifth Edition



IV. Biochemical Activities

of Bacteria



© The McGraw−Hill

Companies, 2002



25. Proteins, Amino Acids,

and Enzymes II: The IMViC

Tests



Figure 25.3 Methyl Red Test. (a) Escherichia coli, MR+. (b) Enterobacter aerogenes, MR–.

Biochemistry within bacteria



Acetyl-CoA,

2,3-butanediol,

ethanol,

lactic and

formic

acids



E. aerogenes



Glucose



2 pyruvate



CO2 + H2 (pH = 6.0)



Succinic,

lactic,

acetic,

formic

acids



E. coli



CO2 + H2 (pH = 4.0)



Biochemistry within tubes

Methyl

red

indicator



(a)



(b)



Methyl

red +



Methyl

red –



Figure 25.4 Voges-Proskauer Test. (a) Enterobacter aerogenes, VP+. (b) Escherichia coli, VP–.

Biochemistry within bacteria



CO2

Glucose + 1/2 O2



CO2



␣-acetolactate



2 pyruvate



acetoin



2,3-butanediol



Biochemistry within tubes



Acetoin + ␣-naphthol



40% KOH

diacetyl + creatine (pink complex)

absolute

alcohol



Barritt’s reagent



(a)



156



Biochemical Activities of Bacteria



(b)



VP+



VP–



Harley−Prescott:

Laboratory Exercises in

Microbiology, Fifth Edition



IV. Biochemical Activities

of Bacteria



25. Proteins, Amino Acids,

and Enzymes II: The IMViC

Tests



© The McGraw−Hill

Companies, 2002



Figure 25.5 Citrate Test. (a) Enterobacter aerogenes; blue color is positive. (b) Escherichia coli; green color is negative.

Biochemistry within bacteria

Sodium Citrate permease

citrate

citrase



Pyruvic

+ oxaloacetic acid + CO2

acid



Biochemistry within tubes

Excess sodium from sodium citrate + CO2 + H2O



Na2CO3 (alkaline) pH



(a)



(b)



Citrate +



Citrate –



and E. aerogenes), your name, and date. (If SIM

medium is unavailable, tryptic soy broth is a good

substitute for testing indole production.)

2. Using aseptic technique (see figure 14.3),

inoculate each tube by a stab inoculation or with a

loopful of culture.

3. Incubate the tubes for about 24 hours at 35°C.



Second Period

1. Remove the tubes from the incubator and while

wearing disposable gloves, add 0.5 ml (about 10

drops) of Kovacs’ reagent to each tube, and shake

the tube gently. A deep red develops in the

presence of indole. Negative reactions remain

colorless or light yellow.

2. Based on your observations, determine and record

in the report for exercise 25 whether or not each

bacterium was capable of hydrolyzing the

tryptophan.



2. Using aseptic technique, inoculate each tube with

the appropriate bacterium by means of a loop

inoculation.

3. Incubate all tubes at 35°C for 24 to 48 hours. For

slow fermenters, it may take four to five days.



Second Period

1. Transfer h of each culture into an empty test tube

and set these aside for the Voges-Proskauer test.

2. To the i of the culture remaining in each tube,

add 0.2 ml (about 4 to 5 drops) of methyl red

indicator. Carefully note any color change (a red

color is positive).

3. Based on your observations, determine and record

in the report for exercise 25 whether or not each

bacterium was capable of fermenting glucose,

lowering the pH of the medium.



Voges-Proskauer Test

Second Period



Methyl Red Test

First Period

1. Label each of the MR-VP broth media tubes with

the name of the bacterium (E. coli, E. aerogenes,

and K. oxytoca) to be inoculated, your name, and

date.



1. Use the h aliquot from the methyl red test. While

wearing disposable gloves, add 0.6 ml of Barritt’s

solution A and 0.2 ml of solution B to each

culture, and shake vigorously to aerate.

(Alternatively, about 15 drops of reagent A

followed by 5 drops of reagent B works fairly



Proteins, Amino Acids, and Enzymes II: The IMViC Tests



157



Harley−Prescott:

Laboratory Exercises in

Microbiology, Fifth Edition



IV. Biochemical Activities

of Bacteria



25. Proteins, Amino Acids,

and Enzymes II: The IMViC

Tests



well and avoids pipetting.) Positive reactions

occur at once or within 20 minutes and are

indicated by the presence of a red color.

2. Based on your observations, determine and record

in the report for exercise 25 whether or not each

bacterium was capable of fermenting glucose,

with the production of acetylmethylcarbinol.



Citrate Utilization Test

First Period

1. Label each of the Simmons citrate agar slants

with the name of the bacterium (E. coli, E.

aerogenes, and K. oxytoca) to be inoculated, your

name, and date.

2. Using aseptic technique, inoculate each bacterium

into its proper tube by means of a stab-and-streak

inoculation.

3. Incubate these cultures for 24 to 48 hours at 35°C.



Second Period

1. Examine the slant cultures for the presence or

absence of growth and for any change in color

from green to blue.



158



Biochemical Activities of Bacteria



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2. Based on your observations, determine and record

in the report for exercise 25 whether or not each

bacterium was capable of using citrate as an

energy source. The development of a deep blue

color is a positive test.



HINTS AND PRECAUTIONS

(1) Incubate the SIM agar deeps for only 24 hours prior

to adding Kovacs’ reagent because the indole may be

further metabolized if the incubation time is prolonged.

This could result in false negatives for some bacteria

that produce indole from tryptophan. (2) The indole

production test also can be performed by adding Kovacs’ reagent to a tryptic soy broth culture. (3) Use no

more than five drops of methyl red. If more is added,

this may impart a red color to the medium that is unrelated to specific metabolic end-products.



Harley−Prescott:

Laboratory Exercises in

Microbiology, Fifth Edition



IV. Biochemical Activities

of Bacteria



Laboratory Report



© The McGraw−Hill

Companies, 2002



25. Proteins, Amino Acids,

and Enzymes II: The IMViC

Tests



25



Name: ———————————————————————

Date: ————————————————————————

Lab Section: —————————————————————



Proteins, Amino Acids, and Enzymes II: The IMViC Tests

1. Based on your observations, record whether or not each bacterium was capable of hydrolyzing the tryptophan

to produce indole.

Bacterium



Color of Reagent Layer



Indole + or –



E. coli



________________________



___________________________



E. aerogenes



________________________



___________________________



P. vulgaris



________________________



___________________________



2. Based on your observations, record whether or not each bacterium was capable of fermenting glucose, with

the production of either acids or acetylmethylcarbinol.

Methyl Red Test



Voges–Proskauer Test



Bacterium



Medium Color



+ or –



Medium Color



+ or –



E. coli



____________



___________



____________



____________



E. aerogenes



____________



___________



____________



____________



K. oxytoca



____________



___________



____________



____________



3. Based on your observations, record whether or not each bacterium was capable of using citrate as an energy

source.

Bacterium



Presence or Absence of Growth



Color of Medium



Citrate Use + or –



E. coli



_______________________________



________________



__________________



E. aerogenes



_______________________________



________________



__________________



K. oxytoca



_______________________________



________________



__________________



159



Harley−Prescott:

Laboratory Exercises in

Microbiology, Fifth Edition



IV. Biochemical Activities

of Bacteria



25. Proteins, Amino Acids,

and Enzymes II: The IMViC

Tests



© The McGraw−Hill

Companies, 2002



Review Questions

1. What is the component in the SIM deep tubes that makes this medium suitable to detect the production of

indole by bacteria?



2. What organic molecule is necessary to detect mixed acid fermentation by bacteria?



3. Why did you shake the MR-VP culture?



4. Can a bacterium that ferments using the 2,3-butanediol pathway also use the mixed acid route? Explain your

answer.



5. Why is a chemically defined medium necessary for the detection of citrate utilization by bacteria?



6. Complete the following table.



Test



Significant Ingredients



Indole



____________



________________________________________________________________________



Methyl red



____________



________________________________________________________________________



Voges–Proskauer



____________



________________________________________________________________________



Citrate



160



Medium



____________



________________________________________________________________________



Biochemical Activities of Bacteria



Harley−Prescott:

Laboratory Exercises in

Microbiology, Fifth Edition



IV. Biochemical Activities

of Bacteria



© The McGraw−Hill

Companies, 2002



26. Proteins, Amino Acids,

and Enzymes III: Casein

Hydrolysis



E X E RC I S E



26



Proteins,Amino Acids, and Enzymes III:

Casein Hydrolysis

SAFETY CONSIDERATIONS

Be careful with the Bunsen burner flame and the water

baths. No mouth pipetting. Keep all culture tubes upright in a test-tube rack or in a can.



Pronunciation Guide

Bacillus subtilis (bah-SIL-lus SUB-til-us)

Escherichia coli (esh-er-I-ke-a KOH-lee)

Pseudomonas aeruginosa (soo-do-MO-nas a-ruh-jinOH-sa)



Materials per Student

24- to 48-hour tryptic soy broth cultures of

Escherichia coli (ATCC 11229), Bacillus

subtilis (ATCC 6051), and Pseudomonas

aeruginosa (ATCC 10145)

tubes of plate count agar (tryptone glucose yeast

agar)

inoculating loop

boiling water bath

sterile petri plates

water bath set at 48° to 50°C

water bath set at 35°C

Bunsen burner

test-tube rack

wax pencil

sterile skim milk

5-ml pipette with pipettor



Learning Objectives

Each student should be able to

1. Understand the biochemical process of

deamination

2. Determine the ability of some bacteria to secrete

proteolytic enzymes capable of hydrolyzing the

protein casein by performing a casein hydrolysis

test

3. Explain what a zone of proteolysis indicates



Suggested Reading in Textbook

1. Protein and Amino Acid Catabolism, section 9.9.

2. Dairy Products, section 41.6.



Why Are the Above Bacteria Used

in This Exercise?

In this exercise, the student will learn how to perform a casein hydrolysis test to detect the presence of proteolytic enzymes. Thus, the authors have chosen three bacteria that

have been used in prior exercises. Escherichia coli will produce a negative reaction; Bacillus subtilis and Pseudomonas

aeruginosa will produce positive reactions.



Principles

Casein is a large milk protein incapable of permeating

the plasma membrane of bacteria. (Its presence is the

reason milk is white.) Therefore, before casein can be

used by some bacteria as their source of carbon and

energy, it must be degraded into amino acids. Bacteria

accomplish this by secreting proteolytic enzymes that

catalyze the hydrolysis of casein to yield amino acids

(figure 26.1), which are then transported into the cell

and catabolized.

When milk is mixed with plate count agar, the casein in the milk makes the agar cloudy. Following inoculation of the plate count agar, bacteria that liberate

proteases (e.g., caseinase) will produce a zone of proteolysis (a clear area surrounding the colony). Clearing of the cloudy agar (a positive reaction) is the result of a hydrolytic reaction that yields soluble amino

acids (figure 26.2b). In a negative reaction, there is no

protease activity, and the medium surrounding the

bacterial colony remains opaque (figure 26.2b)



161



Harley−Prescott:

Laboratory Exercises in

Microbiology, Fifth Edition



IV. Biochemical Activities

of Bacteria



© The McGraw−Hill

Companies, 2002



26. Proteins, Amino Acids,

and Enzymes III: Casein

Hydrolysis



Figure 26.1 Proteolytic Hydrolysis.

Protease

H



O



H



O



H



H



O



C



C



O

N



C



H



R1



N



C



H



C



C



R2



+ H2O



N



C



H



R1



C



+

O–



H3N+



R2



Peptide

bond

Peptide



Procedure

First Period

1. Melt the tubes of plate count agar by placing them

in the boiling water bath. After melting, place the

tubes in the 48° to 50°C water bath for 10

minutes.

2. With a wax pencil, mark the bottom of a petri

plate into three sections: label one E. coli, the

second, B. subtilis, and the third, P. aeruginosa.

Add your name and date to the plate.

3. Pipette 2 ml of warm (48° to 50°C) sterile skim

milk into the petri plate. Add the melted agar and

mix thoroughly by moving the plate in a circular

motion. Allow this medium to gel on a cool, level

surface.

4. As shown in figure 14.3, aseptically spotinoculate (figure 26.2a) each third of the petri

plate with the appropriate bacterium as per the

label. Place a loopful of culture on the center of

each section and spread it in a circular fashion to

cover an area about the size of a dime or less (5 to

18 mm in diameter).

5. Incubate the plate in an inverted position at 35°C

for 24 to 48 hours.



Carboxyl

component



Amino

component



Figure 26.2 Procedure for Determining Casein

Hydrolysis. (a) Spot inoculation of a plate count agar with milk

plate. (b) Plate exhibiting two zones of proteolysis.



Plate count agar



0.5 cm



(a)



Zones of

proteolysis



Bacterial growth

(no proteolysis)



(b)



Second Period

1. Examine the plate count agar for the presence or

absence of a clear zone (zone of proteolysis)

surrounding the growth of each of the bacterial

test organisms. You can see the clear zones best

against a black background.

2. Based on your observations, determine and record

in the report for exercise 26 which of the bacteria

were capable of hydrolyzing the casein. Also,

measure the zone of hydrolysis for each colony.



162



Biochemical Activities of Bacteria



HINTS AND PRECAUTIONS

Aseptic technique must be followed because possible

contaminating microorganisms from the air or body

might be capable of hydrolyzing casein and thus lead to

erroneous results.