LABORATORY #8
Preliminary Identification of Enterobacteriaceae
Laboratory #8
Preliminary Identification of Enterobacteriaceae
Skills= 35 points
Objective:
At the completion of this laboratory, the student will be able to:
1. Demonstrate and recognize the possible reactions of various Enterobacteriaceae on MacConkey agar and
TSI slants.
2. Discuss the chemical composition of the TSI slant.
3. Interpret color changes within the TSI slant.
4. Demonstrate and recognize the IMViC reactions of various Enterobacteriaceae.
5. Discuss the principles of the IMViC tests.
6. Demonstrate the reactions of motility, urease, decarboxylase, nitrate reduction, and phenylalanine deaminase
reactions with Escherichia coli, Proteus mirabilis/vulgaris, Salmonella species, and Citrobacter freundii.
7. Discuss the reactions of motility, urease, decarboxylase, nitrate reduction, citrate, and phenylalanine
deaminase reactions.
Materials:
MacConkey cultures of:
Escherichia coli
Proteus mirabilis/vulgaris
Salmonella species
Citrobacter freundi
Klebsiella pneumoniae
TSA cultures of :
Klebsiella pneumoniae- for Indole
Escherichia coli- for Indole
Chocolate cultures of :
Neisseria species
3 Triple Sugar iron (TSI) slants
2 Motility tubes
2 Urease slants
2 Clean test tubes
2 Lysine decarboxylase tubes
2 Decarboxylase blanks
2 Nitrate tubes
2 Phenylalanine tubes
Mineral oil
Alpha-naphthylamine (Nitrate B)
Sulfanilic acid (Nitrate A)
Zinc dust
MLAB 2434 – Laboratory 8 – Page 4
LABORATORY #8
Preliminary Identification of Enterobacteriaceae
10% Ferric chloride
1 Indole/Kovac’s dropper (p-dimethylaminobenzaldehyde)
2 Methyl red-Voges-Proskauer broth (MR-VP)
2 Citrate slants
Methyl red reagent
5% alpha-naphthol (Barrett's A/VP A)
40% KOH (Barrett's B/VP B)
References:
1. Mahon and Manuselis, Textbook of Diagnostic Microbiology,Third Edition, Chapter 20
2. Mahon and Manuselis, Textbook of Diagnostic Microbiology,Fourth Edition, Chapter 9
3. Engelkirk, P. G., & Duben-Engelkirk, J. (2008). Laboratory Diagnosis of Infectious
Diseases: Essentials of Diagnostic Microbiology. Baltimore, MD: Lippincott Williams
& Willkins.
4. BD DMACA Indole package Insert, 2010.
Principles:
Gram-negative bacilli belonging to the family Enterobacteriaceae are the most frequently encountered microorganisms in the clinical microbiology laboratory. Many members of this group are indigenous to the
gastrointestinal tract. Members of the Enterobacteriaceae may be recovered from infections of virtually every
anatomical site.
Gram-stain morphology is neither helpful in separating members of the Enterobacteriaceae from other gramnegative bacilli nor in making species identifications. The morphology of colonies growing on blood agar is
also of limited diagnostic usefulness because most species appear as dull gray, dry to mucoid colonies. Some
species of Proteus may grow diffusely over the agar surface as a thin film, a phenomenon called swarming.
MacConkey Agar
MacConkey agar is a differential plating medium for the selection and recovery of the Enterobacteriaceae and
related enteric gram-negative bacilli.
The bile salts and crystal violet inhibit the growth of gram-positive bacteria and some fastidious gram-negative
bacteria.
Lactose is the sole carbohydrate. Lactose-fermenting bacteria produce colonies that are varying shades of red,
due to the conversion of the neutral red indicator dye (red below 6.8) from the production of mixed acids.
Colonies of non-lactose-fermenting bacteria appear colorless or transparent.
Procedure:
1. Observe the colonies of Escherichia coli, Salmonella species, and Citrobacter freundii on the
MacConkey's agar.
2. Record results on the report form by using the shorthand “LF” for lactose fermenter or “NLF” for lactose
nonfermenter.
MLAB 2434 – Laboratory 8 – Page 5
LABORATORY #8
Preliminary Identification of Enterobacteriaceae
TSI Slants
Although a preliminary identification of the Enterobacteriaceae is possible, based on biochemical reactions on
primary isolation media, further species identification requires the determination of additional metabolic
characteristics that reflect the genetic code and unique identity of the carbohydrates. (It is common for
laboratory microbiologists to refer to all carbohydrates as sugars, although certain substrates are not sugar in the
chemical sense.)
The term fermentation refers to an oxidation-reduction metabolic process that takes place in an anaerobic
environment, with an organic substrate serving as the final hydrogen (electron) acceptor in place of oxygen. In
bacteriologic test systems, this process is detected by visually observing color changes of pH indicators as acid
products are formed. All tests used to measure an organism's ability to enzymatically degrade a sugar into acid
products may not always be fermentative; we will study other processes in subsequent exercises.
Triple sugar iron agar (TSI) is virtually indispensable in further identification of gram-negative bacilli. The
sugars present in TSI are lactose, sucrose and dextrose. Lactose is present in 10 times the concentration of
dextrose; the ratio of sucrose to dextrose is also 10:1.
Ferrous sulfate and sodium thiosulfate are added as a H2S detector. H2S production is a two-step process. In the
first step, H2S is formed from sodium thiosulfate. Because the H2S is a colorless gas, the ferrous sulfate is
necessary to visually detect H2S production. In some cases, the butt of the TSI slant will be completely black,
obscuring the yellow color from carbohydrate fermentation. Because H2S production requires an acid
environment, even if the yellow color is not seen, it is safe to assume glucose has been fermented.
Also included in the TSI media is phenol red. Phenol red is the pH indicator. Utilization of peptones results in
the release of ammonia, which increases the pH due to the alkaline bi-product production. This results in a deep
red color. When the pH is below 6.8, a yellow color is produced. Uninoculated media is red because the pH is
buffered at 7.4.
The agar is poured on a slant. This configuration results in essentially two reaction chambers within the same
tube. The slant portion, exposed throughout its surface to atmospheric oxygen is aerobic; the lower portion,
called the butt or the deep, is protected from the air and is relatively anaerobic.
Procedure:
1. Inoculate three (3) TSI slants with Escherichia coli, Salmonella species, and Citrobacter freundii.
a. TSI tubes are inoculated with a long straight wire. A well-isolated colony recovered on an agar
plate is touched with the end of an inoculating needle which is stabbed into the butt of the tube;
but not all the way to the bottom of the tube.
b. Upon removing the inoculating wire from the butt of the tube, streak the slant surface with a back
and forth motion. This motion is referred to as fishtailing.
2. Cap loosely and incubate in a non-CO2 incubator at 35°C for 18-24 hours.
3. Interpret and record results based on the below interpretation.
MLAB 2434 – Laboratory 8 – Page 6
LABORATORY #8
Preliminary Identification of Enterobacteriaceae
Interpretation of Reactions:
The glucose (dextrose) concentration is one tenth of the concentration of lactose and sucrose. When glucose
only is fermented, the small amount of acid produced is oxidized rapidly in the slant, which will remain or
revert to an alkaline pH with a red color. In contrast, the acid reaction is maintained in the butt because it is
under lower oxygen tension.
The concentration of lactose and sucrose are much greater, so the large amount of acid produced by the
fermentation of either cannot be oxidized back to an alkaline pH, so the slant turns yellow.
If the butt or slant color is obscured by blackening due to the production of H2S by the organism, note that this
reaction requires acid conditions for the thiosulfate reduction; so the black precipitate in the medium is an
indication of fermentation and sulfur reduction. If the black precipitate obscures the color of the butt, glucose
fermentation has occurred.
Reaction patterns can be written using shorthand, in which the slant result is written first, followed by the butt
reaction, separated by a slash. The letter “K” or “”ALK” indicates alkaline. The letter “A” indicates acid. The
letter “G” means that gas was produced during glucose fermentation. For example, an organism that ferments
glucose in the slant and butt would be written “A/A.” If the organism produces H2S that would be written “H2S
+
.” If the organism produces gas, The “G” can be added to the butt reaction. For example, “A/AG.”
Reaction
(slant/butt)
Interpretation
K/A
Glucose fermentation with acid production.
Proteins catabolized.
Glucose and lactose and / or sucrose
fermentation
No fermentation
Gas production
Red/ yellow
A/A
Yellow/ yellow
Red/ Red
Gas bubbles in butt, medium
sometimes split
Explanation
K/K
G
H2S +
Blackening of the butt
Hydrogen sulfide produced
Motility
Principle:
Bacterial motility is an important characteristic in making a final species identification. Bacteria move by
means of flagella, the number and location of which vary with the different species. Flagellar stains are
available for this determination but are not commonly used.
Motility media are widely used to visually observe the path of movement of the organism through the agar. The
agar concentrations are 0.4% or less to allow for free spread of the organisms.
Procedure:
1. Inoculate two tubes of motility, one each of Escherichia coli and Klebsiella pneumoniae. The colony to
be tested is picked with an inoculating needle and carefully stabbed once only approximately 2/3's of the
depth of the medium. The stab is made straight in and out.
MLAB 2434 – Laboratory 8 – Page 7
LABORATORY #8
Preliminary Identification of Enterobacteriaceae
2.
3.
The tube is incubated with loose caps at 35°-37°C for 18-24 hours.
Record results as “Pos” or “Neg” for the test organisms.
Interpretation:
The motility test is interpreted by making a macroscopic examination of the medium for a diffuse zone of
growth flaring out from the line of inoculation. Nonmotile organisms grow only along the line of the
inoculation, while motile organisms spread out from the line of inoculation. Always interpret motility by
comparing the tube to an uninoculated motility tube
Urease
Principle:
Urease is an enzyme possessed by many species of microorganisms that can hydrolyze urea with the release of
ammonia. The ammonia reacts in solution to form ammonium carbonate resulting in alkalization and an
increase in the pH of the medium with a color change of the indicator to bright pink. Many enteric bacteria
possess the ability to metabolize urea, but members of Proteus, Morganella, and Providencia are considered
rapid urease-positive organisms.
Procedure:
1. Heavily streak two urea slants with P. mirabilis/vulgaris, and E. coli.
2. Incubate slants with loose caps for 18-24 hours.
3. Record results based on interpretation below.
Interpretation:
Organisms that hydrolyze urea rapidly may produce positive reactions (bright pink or red color) within one or
two hours. These organisms should be recorded as “Pos” on the report form. Less active species may take three
or more days. Those organisms that are negative for urease activity should be reported as “Neg” on the report
form.
Decarboxylase
Principle:
Decarboxylases are a group of substrate-specific enzymes that are capable of attacking the carboxyl (COOH)
portion of amino acids, with the formation of alkaline-reacting amines. This reaction, known as
decarboxylation, forms CO2 as a second product. Each decarboxylase enzyme is specific for an amino acid.
Lysine, ornithine, and arginine are the three amino acids routinely tested in the identification of
Enterobacteriaceae.
Moeller decarboxylase medium is the base commonly used for determining the decarboxylase capabilities of the
Enterobacteriaceae. This medium contains a small amount of glucose for the production of acid, which is
required for the decarboxylase reaction. Each specific amino acid is added to its respective tubes. During the
initial stages of incubation, the tube turns yellow due to the fermentation of the glucose and the subsequent
production of acid by-products; if the amino acid is then decarboxylated, alkaline amines are formed and the
medium reverts to its original purple color. A control tube, consisting of only the base without the amino acid,
MLAB 2434 – Laboratory 8 – Page 8
LABORATORY #8
Preliminary Identification of Enterobacteriaceae
must also be set up in parallel. The control tube must turn and remain yellow in order for the results of the
decarboxylase tests to be valid. The control tube determines the viability of the organism.
For this decarboxylation test, two tubes will be used, both contain glucose, but only one of which contains
lysine, the amino acid being tested.
Procedure:
1. Inoculate one (1) lysine decarboxylase tube and one (1) decarboxylase control with Escherichia coli by
touching the inoculating loop to one colony and inserting it to the inside of the tube while tilted.
2. Inoculate one (1) lysine decarboxylase tube and one (1) decarboxylase control with Proteus
mirabilis/vulgaris by touching the inoculating loop to one colony and inserting it to the inside of the tube
while tilted.
3. Overlay each set of tubes with 1 mL of mineral oil and incubate with caps tightened at 35°-37°C for 1824 hours.
4. Record results based on interpretation below.
Interpretation:
The control tube must be yellow in order for the results of the decarboxylases to be valid. The tests are bright
purple if positive for decarboxylation; yellow if negative. For positive test results, record as “Pos” in report
form. For negative test results, record as “Neg” in report form.
Phenylalanine Deaminase
Principle:
Phenylalanine is an amino acid which upon deamination forms a keto acid, phenylpyruvic acid. Of the
Enterobacteriaceae, only members of the Morganella, Proteus and Providencia genera possess the deaminase
enzyme necessary for this conversion. The presence of phenylpyruvic acid is determined by the addition of 10%
ferric chloride, with the development of a green color.
Procedure:
1. Streak two slants of phenylalanine with colonies of E. coli and P. mirabilis respectively.
2. Incubate at 35-37°C for 18 to 24 hours.
3. At the end of this incubation, add 4 or 5 drops of ferric chloride to the agar surface. As the reagent is
added, rotate the tube to dislodge the surface colonies.
4. Record results based on interpretation below.
Interpretation:
The immediate appearance of an intense green color indicates the presence of phenylpyruvic acid and a positive
test. For positive test results, record as “Pos” in report form. For negative test results, record as “Neg” in report
form.
Nitrate Reduction
Principle:
The capability of an organism to reduce nitrates to nitrites is an important characteristic used in the
identification and species differentiation of many groups of microorganisms. All Enterobacteriaceae, except
MLAB 2434 – Laboratory 8 – Page 9
LABORATORY #8
Preliminary Identification of Enterobacteriaceae
certain biotypes of Enterobacter agglomerans and Eriwinia, demonstrate nitrate reduction. The test is also
helpful in identifying numbers of the Haemophilus, Neisseria, and Branhamella genera.
Organisms demonstrating nitrate reduction have the capability of deriving oxygen from nitrates to form nitrites
and other reduction products.
NO32- + 2e- + 2H → NO2 + H2O
Nitrate
Nitrite
The presence of nitrites in the test medium is detected by the addition of alpha-naphthylamine and sulfanilic
acid, with the formation of a red diazanium dye.
Procedure:
1. Inoculate two tubes of nitrate medium: one with a loopful of E. coli and one with a loopful of Neisseria
species.
2. Incubate with caps loose at 35°
-24 hours.
3. At the end of the incubation, add 1 ml each of sulfanilic acid (NitrateA) and alpha naphthylamine(
Nitrate B)-in that order. If no red color develops at this point, add a very small amount of zinc dust.
4. Record results based on interpretation below.
Interpretation:
The development of a red color within 30 seconds after adding the test reagents indicates the presence of nitrites
and represents a positive reaction for nitrate reduction. If no color develops after adding the test reagents, this
may indicate either that nitrates have not been reduced (a true negative reaction), or that they have been reduced
to products other than nitrites, such as ammonia, molecular nitrogen, nitric oxide (NO) or nitrous oxide (N2O),
and hydroxylamine. Since the test reagents detect only nitrites, the latter process would lead to a false negative
reading. Thus it is necessary to add a very small quantity of zinc dust to all negative reactions. Zinc ions reduce
nitrates to nitrites, and the development of a red color after adding zinc dust indicates the presence of residual
nitrates and confirms a true negative reaction.
IMViC
IMViC= Indole, Methyl-red, Voges-Prokauer, Citrate
Indole Test
Principle:
Indole is one of the metabolic degradation products of the amino acid tryptophan. Bacteria that possess the
enzyme tryptophanase are capable of hydrolyzing and deaminating tryptophan with the production of indole,
pyruvic acid, and ammonia. Indole production is an important characteristic in the identification of many
species of micro-organisms, being particularly useful in separating E. coli (positive) from members of the
Klebsiella-Enterobacter group (mostly negative).
The indole test is based on the formation of a red color complex when indole reacts with the aldehyde group of
MLAB 2434 – Laboratory 8 – Page 10
LABORATORY #8
Preliminary Identification of Enterobacteriaceae
p-paraminobenzaldehyde (Kovac’s reagent).
Procedure:
1. Grasp the middle of the Indole dropper with the thumb and forefinger and squeeze gently to break the
ampule inside the dropper. Break ampule close to the center one time only. The opened ampule is good
for one day only.
2. Tap the bottom of the dropper on the tabletop a few times.
3. Inoculate a cotton-tipped applicator with one colony of E. coli. Add one drop of indole reagent to the
cotton-tipped applicator and observe for a blue-green color within two (2) minutes.
4. Repeat step #3 using Klebsiella pneumoniae.
5. Record results based on interpretation below.
Interpretation:
The development of a blue to blue-green color is indicative of a positive result. No color or a pink color
indicates a negative result. Record as either “Pos” or “neg” in report form.
Methyl Red
Overview:
Enterics can metabolize glucose either by mixed acid fermentation(MR) or the butylene glycol pathway(VP).
The methyl red (MR) and Voges-Proskauer (VP) tests are able to detect end products from glucose
fermentation. These tests detect different end products from different pathways.
Principle:
Methyl red is a pH indicator with a range between 6.0 (yellow) and 4.4 (red). The pH at which methyl red
detects acid is considerably lower than the pH for other indicators used in bacteriologic culture media. Thus, in
order to produce a color change, the test organisms must produce large quantities of strong acids (lactic, acetic,
formic) from glucose via the mixed acid fermentation pathway. Since many species of the Enterobacteriaceae
may produce sufficient quantities of strong acids that can be detected by methyl red indicator during the initial
phases of incubation, only those organisms that can maintain this low pH after prolonged incubation (48 to 72
hours), overcoming the pH buffering system of the medium, can be called methyl-red-positive.
Procedure:
1. Inoculate a tube of MR-VP broth with Escherichia coli, and another MR-VP broth with Klebsiella
pneumonia. This medium also serves for the performance of the Voges-Proskauer test.
2. Incubate the broth at 35°-37°C for 24 to 48 hours.
3. At the end of this time, pour 1/4 of the broth into a small, clean test tube and add twenty (20) drops of
the methyl red reagent directly to original broth. Save the remainder of the broth for the VogesProskauer Test below.
4. Record results based on interpretation below.
Interpretation:
The development of a stable red color in the surface of the medium is indicative of sufficient acid production to
lower the pH to 4.4, and is a positive test. Since other organisms may produce lesser quantities of acid from the
test substrate, an intermediate orange color between yellow and red may develop. This does not indicate a
positive test. Record as either “Pos” or “neg” in report form.
MLAB 2434 – Laboratory 8 – Page 11
LABORATORY #8
Preliminary Identification of Enterobacteriaceae
Voges-Proskauer Test
Principle:
Pyruvic acid, the pivotal compound formed in the fermentative degradation of glucose, is further metabolized
via a number of metabolic pathways depending on the enzyme system possessed by different bacteria. One such
pathway results in the production of acetoin (acetyl-methyl carbinol), a neutral-reacting end product. Organisms
such as members of the Klebsiella-Enterobacter group produce acetoin as the chief end product of glucose
metabolism, and form less quantity of mixed acids. In the presence of atmospheric oxygen and 40% potassium
hydroxide, acetoin is converted to diacetyl, and alpha-naphthol serves as a catalyst to bring out a red color
complex.
Procedure:
1. To a small, clean test tube containing 1/4 (approximately 2 ml) of the MR-VP broth from the above
methyl red test, add 0.6 ml (12 drops) of the alpha-naphthol solution “VP A” followed by 0.2 ml (4
drops) of 40% KOH, “VP B”. It is essential that the reagent to be added in this order.
2. Shake the tube gently to expose the medium to atmospheric oxygen and allow the tube to remain
undisturbed for 10 to 15 minutes.
3. Record results based on interpretation below.
Interpretation:
A positive test is the development of a red color after 15 minutes following addition of the reagents. The test
should not be read after standing over one hour because negative Voges-Proskauer cultures may produce a
cooper-like color. Record as either “Pos” or “Neg” in report form.
Citrate Utilization
Principle:
Some bacteria can obtain energy in a manner other than the fermentation of carbohydrates by utilizing citrate as
the sole source of carbon; this characteristic is important in the identification of many members of the
Enterobacteriaceae.
The utilization of citrate in citrate medium is detected by the production of alkaline by-products. This
utilization produces ammonia (NH+), leading to alkalinization of the medium. Bromthymol blue, yellow below
pH 6.0 and blue above pH 7.6, is the indicator.
Procedure:
1. A small amount of Escherichia coli is inoculated onto the slant of the citrate agar using an inoculating
needle. Do not stab the slant. Repeat using Klebsiella pneumonia.
2.
3. Record results based on interpretation below.
Interpretation:
A positive test is the development of a deep blue color within 24 to 48 hours. A positive test may also be read
in the absence of a blue color if there is visible colonial growth along the inoculation streak line. A positive
interpretation from reading the streak line can be confirmed by incubating the tube for an additional 24 hours,
when a blue color usually develops. Record as either “Pos” or “Neg” in report form.
MLAB 2434 – Laboratory 8 – Page 12
LABORATORY #8
Preliminary Identification of Enterobacteriaceae
MLAB 2434 – Laboratory 8 – Page 4
LABORATORY #8
Preliminary Identification of Enterobacteriaceae
Name:________________________
Date:________________________
Lab #8: Identification of Enterics
Report Form
Points= 35
(1 point each)
TSI Slant
Organism
Reaction on
MacConkey
(LF/NLF)
Slant
(A/K)
Butt
(A/K)
Gas
(G)
H2S Production
(H2S)
Shorthand
Escherichia coli
Salmonella species
Citrobacter freundii
MLAB 2434 – Laboratory 8 – Page 5
LABORATORY #8
Preliminary Identification of Enterobacteriaceae
Lab #8
Report Sheet (con’t)
Motility
Urease
Decarboxylase
Lysine
Control
Nitrate
Phenylalanine
Indole Methyl- VogesCitrate
Red
Prokauer
Escherichia coli
Proteus
mirabilis/vulgaris
Klebsiella
pneumoniae
Neisseria species
MLAB 2434 – Laboratory 8 – Page 4
LABORATORY #8
Preliminary Identification of Enterobacteriaceae
Name: ___________________
Date:____________________
Lab #8: Study Questions
Points= 32
Instructions:
The student may use the course textbook, course lecture notes, or Internet to answer the
following question.
1. List at least three (3) metabolic processes that TSI reactions determine. ( 3 pts.)
2. What two (2) processes do MacConkey reactions determine? (1 pt.)
3. Define fermentation. ( 1 pt)
4. What does it mean when the butt of a TSI slant is yellow and the slant is neutral? (1 pt.)
5. Why is it important to incubate a TSI slant with a loose cap? (1 pt.)
6. Using your textbook and lecture notes, what could be the possible organisms in a TSI slant
with the following characteristics?
(1 pt.)
Slant: pink
Butt: yellow
Blackening in the agar; cracking or bubbling in the agar
MLAB 2434 – Laboratory 8 – Page 5
LABORATORY #8
Preliminary Identification of Enterobacteriaceae
7. Using your textbook and lecture notes, what could be the possible organisms in a TSI slant
with the following characteristics?
(1 pt.)
Slant: yellow
Butt: yellow
No blackening; cracking or bubbling in the agar
8. Which carbohydrates are fermented if both the slant and the butt are yellow? (1.5 pts.)
9. What causes a slant to turn red or alkaline? (1 pt.)
10. What two things could it mean if the TSI slant and butt exhibits no color change? (2 points)
11. Using your textbook and lecture notes, list two (2) gram negative bacilli which are lactose
positive (ferment lactose). List two (2) which are lactose negative. (4 pts.)
12. List the tests in the IMViC reactions and briefly explain the principles of each. (8 pts.)
MLAB 2434 – Laboratory 8 – Page 6
LABORATORY #8
Preliminary Identification of Enterobacteriaceae
13. Why must there be a clear red color in the methyl red tube instead of orange or yellow to be
called positive? (1 pt.)
14. Explain why organisms which are methyl-red positive are usually Voges-Proskauer
negative. (1 pt.)
15. When inoculating motility media, why is it necessary to stab straight in and then straight
out? (1 pt.)
16. Why must the control tube in the decarboxylase test be yellow in order for the results to be
accurate? (1 pt.)
17. In your own words, explain why zinc dust must be added to each negative nitrate reduction
test. (1 pt.)
18. Which are the only Enterobacteriaceae that are phenylalanine deaminase positive? (1.5 pts.)
MLAB 2434 – Laboratory 8 – Page 7