MCB3020L General Microbiology Laboratory

Lab 5: Biochemical Testing of Isolated Bacterial Cultures

General Microbiology Laboratory Manual 2021

Purpose: The purpose of this lab session is to determine the biochemical properties of isolated
pure bacterial cultures using various enzymatic assays. Students will also interpret previously
obtained results for these assays.

INTRODUCTION: BIOCHEMICAL TESTING OF BACTERIA

Bacteria occur with a limited number of cellular shapes (a gradient from the coccus shape
to bacillus shape), cellular arrangements (single, doubled, clumped, and in organized or
filamentous chains), and with either thin or thick peptidoglycan cell walls (as determined by
Gram staining). The simple morphology (coccus, rod, spiral or filament) and the Gram stain
reaction are only two taxonomic characteristics of bacteria. Many species have nearly identical
morphologies. For example, a Gram negative rod could be Escherichia coli, Enterobacter
aerogenes, or any of hundreds of other species. Bacteria compete for similar resources through
niche partitioning and chemical defenses.
Because of this situation, the bacteriologist must rely on getting as many other traits of the
bacterium as possible in order to identify it. The most common traits used to distinguish different
bacterial species are those determining the type of enzymatic activities present for a given
bacterium. These properties can be determined through biochemical testing and enzymatic
surveys. For proper identification of unknown bacterial cultures, it is important that biochemical
tests are performed upon pure cultures so the results of such tests reflect the biophysical
properties of one single species of bacteria and not several. When the results of enough
biochemical tests have been determined, a unique “fingerprint” for each bacterium can be
determined that helps to tell it apart as a unique species or genus compared to other bacteria.
Bacterial cultures were initially isolated from various environments near and around
Florida International University. Gram staining procedures have been conducted, gross colony
morphology has been characterized, and cellular shape and configuration has been recorded.
Samples will be used to inoculate into the following biochemical tests: within OF Media (with
and without mineral oil), upon and within Kligler’s Triple Iron Sugar (TSI) Agar slant, upon a
Urea Agar plate, within a Litmus Milk Broth, upon a BHI Starch Agar plate, upon and within a
Nutrient Gelatin slant, within a Nitrate broth, and within a Methyl Red/Voges-Proskauer broth
media.

DIFFERENTIATION MEDIUM
Properties of different types of media can be used to determine the biochemical properties
and further isolate your bacterial sample. Listed below are some general rules for medium
preparation for a selective media but treat these as guidelines only. Ideally, growth medium
should be both selective and differential, however, in order to narrow down choices and ensure a
further isolated bacterial sample, you should concentrate on making a selective medium that you
believe will still foster the growth of your bacterial isolate. Take into consideration the following
guidelines:
A. Carbon and energy sources can be at high concentrations (1-3%) but for bacteria that
live in low nutrient environments, this could be inhibitory.

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B. Nitrogen source. Is your suspected microbe a prototroph or auxotroph? Nitrogen is

present in the environment, and can potentially be used by bacteria, in many forms. Inorganic
forms range from the most reduced (NH4+) to the most oxidized (NO3-). Other nitrogen sources
are organic, for example amine, imine and azole nitrogen sources in amino acids and nucleotides.
What source will you use? Understand your reasons.
C. Phosphorus and Sulfur sources. These are the easiest: all microbes can use sulfate as a
sulfur source, and phosphate as the phosphorus source. These can be added as simple sulfate and
phosphate salts (what are those?).
D. Trace mineral sources. Remember that life requires elements such as zinc, manganese,
calcium, nickel, cobalt, etc., at micro concentrations (“micronutrients”). How do these get into
the medium?

BIOCHEMICAL TESTS
Gelatin Hydrolysis. Proteolysis, an enzymatic process, is detected with nutrient gelatin
medium. In this medium, the solidification agent is the protein gelatin instead of agar, otherwise
the medium has the same ingredients as nutrient agar. After growth in this medium, it is tested
for liquefaction as gelatin is solid below 28°C. The incubation of bacteria can occur at any
temperature above or below 28°C. After growth, the tubes are placed in an ice bath to test for
liquefaction (gelatin breakdown). Make sure your organism has shown growth before testing for
liquefaction.

Results of a gelatin hydrolysis test: (A) positive test result showing liquefaction of gelatin (representing as gelatin in
liquid form) after cooling to 0°C in an ice bath for 10 minutes, and (B) negative test result showing gelatin
remaining solid after 10 minutes in an ice bath. Note: The red color is not indicative of a result for this test and is
only used to visualize the liquefaction within the slant. (Image http://asmscience.org/visuallibrary)
Kligler's Triple Sugar Iron (TSI) Agar. Several facts about your organism can be
determined by observing the nature of growth on Kligler's Triple Sugar Iron Agar. The medium
is prepared as a “deep”, which means the surface has only a small slant at the top. It is inoculated
by streaking the surface and then stabbing the agar deep down to the bottom (as shown in the

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diagram below). In doing so, one inoculates down into the anaerobic-oxygen limited zone as well
as on the aerobic surface zone. The medium contains a complex nitrogen source so most
organisms can grow on it.
The medium contains 0.1% glucose and 1% lactose and the dye phenol red. When
analyzing results, growth of bacteria within the tube should be observed first. Obligate aerobes
will only grow on the surface of the slant whereas facultative organisms will grow both on the
surface and in the deep. Sometimes aerobes will grow partly down into the deep if oxygen has
diffused there, but usually not to the bottom of an inoculation that has reached the bottom of the
tube.
The color of the both the slant and deep of the tube should then be analyzed to determine if
glucose, lactose, and sucrose has been fermented. If the bacteria can utilize only glucose (and not
lactose), only a small reaction (acid) will be produced around the growth in the deep and the
slant should remain pink. For facultative bacteria the slant is yellow (aerotolerant fermenters). If
the organism can utilize lactose then the whole tube should turn yellow (acid production from
lactose) because there is ten times as much lactose than glucose. Some bacteria produce alkaline
end-products from the metabolism of nitrogenous nutrients. This will be seen as a change in the
color from red to pink. Phenol red turns a pink color in alkaline conditions. This reaction
generally occurs under aerobic conditions.
Additionally, one can detect certain byproducts of these enzymatic reactions from this test.
Fermentation that results in gas production should result in a splitting of the agar in the deep.
Furthermore, the production of hydrogen sulfide can be observed in this medium. Many bacteria
are able to reduce sulfur in certain components of the medium (sulfur amino acids or thiosulfate)
to hydrogen sulfide. If they can do that, the hydrogen sulfide produced will react with the iron
(ferrous sulfate) in the medium to produce a black precipitate, ferrous sulfide. If the medium
turns black, particularly in the deep, it indicates the production of hydrogen sulfide.

Diagram illustrating streaking and stabbing techniques of Kligler’s Triple Sugar Iron (TSI) Agar medium using an
inoculating wire. (Image http://microbeonline.com)

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Examples of common reactions by multiple bacterial species are shown below visually and
also in summary table format.

Kligler Triple Sugar Iron (TSI) agar test: left to right showing control (red), Pseudomonas sp. testing for no sugar
fermentation (red), Morganella morganii testing for glucose fermentation only (yellow deep, red top), Escherichia
coli testing for complex sugar fermentation of lactose and/or sucrose (complete yellow), Salmonella typhimurium
testing for complex sugar fermentation of lactose and/or sucrose (complete yellow) and gas production (air gaps in
deep), Proteus mirabilis testing for hydrogen sulfide production anaerobically (black deep) and glucose only
fermentation (red top, yellow deep), and Citrobacter freundii testing for hydrogen sulfide production anaerobically
(black deep) and complex sugar fermentation of lactose and/or sucrose (yellow top). (Images from
http://asmscience.org/visuallibrary)

Summary of common reactions amongst example bacterial isolates; note that sometimes gas production isn’t
observed if bacteria not incubated long enough under anaerobic conditions (Image http://microbeonline.com).

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OF Medium. This is formally known as Hugh-Liefson medium and tests whether glucose
is metabolized oxidatively, fermentatively, or both. Some bacteria only produce acid under
aerobic conditions while others only under anaerobic conditions. Facultative bacteria will
produce acid under both aerobic and anaerobic conditions. Other, non-glucose utilizing bacteria
can also grow on the medium but will produce neutral or basic end products. The OF medium is
a semi-solid (agar) medium containing peptone, glucose and the dye bromo-thymol blue. This
dye is yellow in acid conditions and blue in alkaline conditions. It therefore appears green
(optical illusion) at neutral conditions. It is important that the medium be autoclaved just prior to
use.
For this test two tubes are required and both are inoculated with the isolate. After
inoculation, sterile mineral oil is layered over the medium surface of only one tube. This tube is
the anaerobic tube. Oxygen can diffuse into the other. After incubation, the tubes are observed
for their color and presence or absence of growth. If no growth occurs, this test is invalid. Note
that this medium provides only one fermentable sugar, glucose. Although bacteria have the
ability to ferment many sugars, and this varies from one isolate to another, if they can ferment
any sugar at all, it is glucose. In addition to the color change, you should be able to detect gas
production because, in this semisolid medium, if your unknown produces gas it should “crack”
the agar.

Oxidative Fermentation (OF) Media test, all images left test tube anaerobic with mineral oil cap; Left to Right:
Control (green), Alcaligenes faecalis inoculation testing slightly basic (blue) under aerobic conditions, Pseudomonas
aeruginosa inoculation testing slightly acidic (yellow) under aerobic conditions, Escherichia coli inoculation testing
fermentatively acidic (yellow) under aerobic and anaerobic conditions, and Klebsiella pneumoniae testing
fermentatively acidic (yellow) under aerobic and anaerobic conditions. (Images from
http://asmscience.org/visuallibrary)

Litmus Milk. This media contains simple sterilized skim milk with litmus added. Bacteria
can produce different reactions with milk proteins: coagulation, peptidase clearing (proteolysis,
or breaking down, of milk proteins) or no reaction. Additional reactions observed include the
production of acidic (pink) conditions, alkaline (blue) conditions, and/or gas (frothing). When
observing Litmus milk reactions, it is important to keep test tubes level and undisturbed to avoid
potential mixing of separated or coagulated proteins and acid/alkaline layers.

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Litmus milk test results based upon reaction with or proteolysis of milk proteins in the test tube solution. Note that
colors may not appear exactly as visualized above and will vary by bacterial species, even those capable of the same
category of fermentation. (Image and text from http://microbiologyinfo.com/litmus-milk-test)

Starch Hydrolysis. The ability of bacteria to produce amylase enzyme can be determined
from starch containing agar. The isolate is streaked onto either nutrient agar or BHI agar
containing soluble starch. After growth, the plate is flooded with Gram's iodine which causes
starch to turn dark blue or purple in color. If the bacteria produced amylase that has digested the
starch, there will be a clear zone around the colonies.

Bacterial growth (A, left) on BHI starch agar plates of Bacillus subtilis (top) and Escherichia coli (bottom), and after
flooding plate with Iodine (B, right). Positive reaction for starch hydrolysis is represented by a clearing zone around
bacterial growth that can extend through entire depth of agar plates. Areas without bacterial growth show as dark
purple due to reaction of iodine with starch and air (Image: http://asmscience.org/visuallibrary)

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Urease. The ability of bacteria to produce urease is tested with urea agar which contains
peptone and glucose with 2% urea. The enzyme urease hydrolyses urea to carbon dioxide and
ammonia. The medium contains phenol red as a pH indicator (yellow in acid, red near pH 7, pink
in alkaline conditions). A positive test results in the medium becoming pink after growth of the
bacterium on the agar. The urease agar test can be prepared on an agar plate or within a test tube
as a slant.
Examples of various urease test results are shown below.

Urease agar slant reaction test results for aerobic and anaerobic growth. From left to right: control (no reaction), all
pink (positive for urease enzyme hydrolyzing Urea and production of alkaline/basic reaction under both aerobic and
anaerobic conditions), pink surface (positive for Urease enzyme hydrolysis only under aerobic conditions), and
yellow (no urease enzyme present, but bacterial growth causes acidic conditions and potential gas formation as
indicated by yellowing of butt) (Image: http://asmscience.org/visuallibrary)

Nitrate Reduction. Some bacteria can reduce nitrate to nitrite, which is an activity found
in situations when oxygen is absent or limiting. In such cases, nitrate replaces oxygen as the
terminal electron acceptor in electron transport (respiration). Further, some organisms can
continue to reduce nitrite to either ammonia (one pathway) or to di-nitrogen (another pathway).
Nitrate reduction is tested in nitrate broth which is nutrient broth to which 0.2% KNO3 is added.
The broth is dispensed into the tube that also contains a Durham tube within it. A second tube—
your control tube, will also contain a Durham tube within it, but it will only consist of a nutrient
broth without the addition of the 0.2% KNO3. After growth in the media is obtained, the tubes
are tested (mixed with a drops of nitrite test reagents- sulfanilic acid and alpha-napthylamine) for
the presence of nitrite and the Durham tube is examined for the presence of gas (N2). A positive
test result is indicated by a color change of the nitrate reagent from a pale yellow or brown to a
red or pink color. If bacteria can only reduce nitrate to nitrite, a color change will occur after the
two reagents are added. However, nitrites can be further reduced to ammonia which would not

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be reflected in the first steps. Therefore, if there is no color change occurring after the addition of
nitrite test reagents, zinc powder must be added. Zinc powder reacts with nitrate to produce a
color change. Thus, if tested bacteria can reduce nitrate to ammonia, no color change will occur
after the addition of zinc, thereby indicating a positive result for the overall test due to the
breakdown of nitrate to ammonia (both nitrate reductase and nitrite reductase enzymes present).
If there is a red or pink color observed after the addition of zinc, nitrates are still present thereby
indicating a negative result for the overall test.

A B C

(A) Nitrogen reduction test results for Escherichia coli, positive for reducing nitrate to nitrite- as indicated by red
reaction after both reagents added (B) Neisseria lactamica after the addition of both reagents. No color change
indicates reduction of nitrate can’t yet be determined (C) Neisseria lactamica after the addition of Zinc shows the
bacteria is not capable of reducing Nitrate to Nitrite. Neither bacteria produces Di-Nitrogen gas, as no Durham Tube
shows gas production within either Nitrate test tube (Image: http://www.asmscience.org/visuallibrary)

Catalase and Oxidase Tests. Two easily performed tests (catalase test and oxidase test)
which do not require incubation are related to the use of oxygen. These tests can be performed on
your bacterial sample immediately and are key indicators used to classify bacteria into broad
groups in dichotomous keys or determine which identification test kits to utilize.
In the catalase test a portion of the colony is taken and placed in a drop of 3% H 2O2. The
generation of bubbles (O2 gas) within a minute is a positive test. Cells growing in the presence of
oxygen must protect themselves from super-oxide, a powerful oxygen radical that oxygen
tolerant cells rapidly convert to hydrogen peroxide and water (by super-oxide dismutase).
Catalase is an iron protein which avidly binds a molecule of hydrogen peroxide; then when it
comes upon the next H2O2 it reacts one with the other to form oxygen and water, thereby
eliminating hydrogen peroxide:
H2O2 + H2O2 ======> 2 H2O + O2 ↑

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Bacterial samples exposed to hydrogen peroxide. Positive test result (top) indicated by bubbling as hydrogen
peroxide is cleaved into water and oxygen gas. Negative test result (bottom) indicates the bacteria tested do not have
the catalase enzyme (Image: http://asmscience.org/visuallibrary)

The oxidase test tests for the presence of cytochrome-C (Cyt-C). If a microbe has Cyt-C it
will be able to oxidize the oxidase test reagent (tetramethyl-paraphenylenediamine) to a blue
color. The test reagent in the reduced state is colorless, as indicated in the image below.

Presence of the cytochrome-C enzyme in bacterial samples results in a blue/purple color change (left), while those
lacking cyt-C remain colorless (Image: http://asmscience.org/visuallibrary)

To do this test, a part of the colony is placed into a drop of the test reagent. A positive test is seen
by the change in the color of the bacteria to dark blue. Cytochrome C oxidase is important in the
electron transport chain during respiration of many organisms, including humans. Bacteria that
disrupt this change can cause serious respiratory infections. However, not all respiratory bacteria
have Cyt-C. Many bacteria lacking Cyt-C possess other functional cytochrome electron transport
systems in their respiratory pathways. A simplified diagram of the cytochrome C oxidase
reaction is shown below.

Diagram of cytochrome C pathway in bacteria (Image http://www.asmscience.org/visuallibrary)

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Methyl Red Test

The methyl red (MR) test detects whether bacteria produce enough fermentation of
glucose to lower the pH of a culture below 4.5 via production of stable acids. Certain members
of the Enterobacteriaceae and Actinobacteria are all capable of producing stable acids through
glucose fermentation. A positive result is a color change to red (or orange) through the methyl
red indicator dye. The MR test is performed on a broth with bacterial growth at both 24 hours
and 48 hours following incubation so that one can determine whether any acid produced within
24 hours of initial incubation remains stable over time.

Methyl red (MR) test results for two bacteria, positive (A left), and negative (B right). (Image from
http://www.microbiologyinfo.com)

Voges-Proskauer Test
The Voges-Proskauer (VP) test is named after two 19th century scientists who discovered
that the addition of potassium hydroxide to certain bacterial cultures resulted in a red reaction.
This reaction was later shown to be the result of acetyl-methyl carbinol production during
glucose fermentation. The VP test is often performed alongside the MR test because both involve
reactions following glucose fermentation, because both reactions are performed with reagents
applied to bacteria growing in the same medium (MR broth), and because the results of both tests
are diagnostic for many bacterial genera.

Voges-Proskauer test results, positive (left), and negative (right). (Image from http://www.microbiologyinfo.com)

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EXPERIMENTAL PROTOCOL
In this lab you will confirm you have obtained a pure culture and perform multiple
biochemical tests aimed at characterizing the metabolism of your pure cultures which will be
used to help determine bacterial identities by the end of the semester.
In the next lab session, the following biochemical tests can be observed for results
following incubation without further manipulation or the addition of reagents: OF Media,
Kligler’s TSI agar, Urea agar, and Litmus milk broth. Tests requiring the addition of reagents
after incubation include:

• BHI starch agar, which must be flooded with iodine to determine if bacteria have the
amylase enzyme necessary to digest starch.
• Nutrient gelatin slant, which must be placed into an ice bath for at least 10 minutes
before observing whether liquefaction of gelatin has occurred.
• Nitrate broth, which must have nitrate reduction reagents added in sequence to
determine whether denitrification has occurred. Zinc powder may need to be added as
an additional step.
• Methyl red and Vogues-Proskauer broth, which must have reagents added in
sequence to determine which byproducts have occurred due to glucose fermentation.
Equipment:
1. Micropipettes and sterile tips
2. Inoculating loops
3. Bunsen burner
4. Glass spreaders sterilized in alcohol
5. Glass slides

Materials (per group):

1. 2 x test tubes OF medium (3 mL in a 13x100 mm tube)
2. 1 tube sterile mineral oil (2 mL in a 13x100 mm tube)
3. 1 Kligler's Iron Agar deep slant (10 mL in 16x150 mm tube)
4. 1 Nutrient gelatin deep slant (2 mL in a 13x100 mm tube)
5. 1 Litmus milk tube (3 mL in a 13x100 mm tube)
6. 1 Urea agar plate
7. 1. BHI starch agar plate
8. 1 Nitrate broth tube (3 mL in a 13x100 mm tube)
9. 1 Nitrate broth tube without nitrate (control)
10. 1 MR-VP broth (5 mL)
11. Catalase and oxidase test reagents

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12. Gram staining reagents

PART A: BIOCHEMICAL TESTING INOCULATION

Procedure:

1. Analyze the colony morphology of your isolation streaking plate from last laboratory
session. Does this match your previous colony?

2. Gram stain an isolated colony from the tail of your isolation streak of each plate to
confirm your sample is a pure culture before proceeding to biochemical testing. Obtain
40x and 100x objective pictures of all samples.

3. OF Medium Test. Inoculate OF Medium by removing OF test tubes from water bath
individually. Place a loopful of bacteria into the OF test tubes when they are just cool to
the touch. Vigorously roll the test tube in your hand and set aside to cool and solidify.
Repeat for the second OF test tube, but this time place enough sterile mineral oil over the
surface to create an anaerobic environment.
4. Kligler’s Iron Agar. Inoculate the KIA slant with an inoculating needle rather than a
loop. First streak the slant surface with a sterile loopful of your bacterial pure culture,
then stab the inoculum deep down to the bottom of the test tube with your needle.
Remove the needle carefully so that the agar reseals the deep portion.
5. Urea Agar. Create a lawn-like streaking of bacteria across the entire surface of the agar.
You can do this by taking a loopful of bacteria and spreading over the entire surface
using a sterile cotton swab. Rotate the plate at a 90 degree angle and repeat this process to
ensure the proper inoculation of the agar plate.
6. BHI Starch Agar. As for the urea agar, create a lawn-like of bacteria over the entire
surface as noted above.
7. Nutrient Gelatin. Streak the surface of the gelatin tube using a sterile loopful of bacteria
from your pure culture. After incubating, the gelatin will liquefy and your bacterial
sample will distribute throughout the medium.
8. Nitrate Reduction Broth. Place a sterile loopful of bacteria from your pure culture into
both the test tube containing nitrate broth and the control tube. Be sure to label each test
tube before beginning as they physically appear the same.
9. Litmus Milk Broth. Place a sterile loopful of bacteria from your pure culture into the
litmus milk test tube, and ensure your sample is well mixed.
10. Incubate all media at 30ºC - 37 ºC (depending on culture source) for 48 hours. To
conserve space, place all test tubes together in a small test tube rack and label each test
tube separately. Properly label and stack all agar plates and incubate inverted.
(Stockroom will remove after 48 hours and place plates in the refrigerator)

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11. Methyl Red and Vogues-Proskauer Test. Inoculate each of your MR-VP media with
enough bacteria from your pure culture into the broth to make a visibly turbid solution
and ensure your sample is mixed well. Incubate broth for 48 hours at 30-37°C depending
on bacterial sample.
(Stockroom will remove after 48 hours and place broth in the refrigerator)

12. Catalase Test. To perform the catalase test, use a sterile loop to remove part of your
bacterial culture and place it onto a flame-sterilized slide. Add a drop of catalase reagent
(3% H2O2) and observe for bubbles. If positive, bubbles should appear within a couple of
minutes.

13. Oxidase Test. To perform the oxidase test, place a drop of oxidase test reagent on a slide.
With a sterile loop, remove part of your bacterial pure culture colony and place it into the
reagent. If the cells turn blue within one minute, it is a positive test result. Alternatively
use the oxidase test reagent on a cotton swab and touch the colony being tested with the
reagent-swab.

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CLASS ACTIVITY WORKSHEET

Section: --
Group: --
Environment: --
Sample Picture Description
Original colony (from last lab) Colony Morphology:
Size:
Shape:
Color:
Margins:
Elevation:
Consistency:
Transparency:
Other:

Gram stain (from last lab) Cellular Morphology:

Size:
Shape:
Arrangement:
100x objective Color:
Staining characteristics:
Other:

Isolated streak on nutrient agar Colony Morphology:

plate Size:
Shape:
Color:
Margins:
Elevation:
Consistency:
Transparency:
Other: Does this match the
previous colony?

Gram stain of isolated streak 40x objective Cellular Morphology:

Size:
Shape:
Arrangement:
Color:
100x objective Staining characteristics:
Other: Does this match the
previous colony?

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