APPENDIX I: BIOCHEMICAL TESTS

Cellular and colonial morphologies can only partially characterize bacteria. Identification
of a bacterium also requires a determination of its biochemical and immunological properties.
The action of bacteria on organic and inorganic compounds is widely employed for classification.
A variety of tests must be conducted to identify bacteria especially since different strains of the
same genus and species show biochemical variations. The results of a series of biochemical
tests will increase the probability of correctly identifying unknown bacteria. Listed below are the
biochemical tests available in the lab that can be used for characterizing microorganisms. These
tests make legitimate identification possible. Many other physiological activities of bacteria are
also used for identification.

I. Carbohydrates Catabolism (Sugar Fermentation)

Procedure and Background

Different organisms can utilize different sugars as their primary source of carbon.
Inoculate the tube with a loop full of bacteria collected with your wire directly from the colony.
Be sure to use sterile technique in opening your plate, flaming your loop, and flaming your
tube at appropriate times. Incubate the tube 2 - 4 days at 37oC [or as instructed]

Reading Sugar Ferments

Results may be read as early as 18 hours after inoculation at room temperature. In a clinical
laboratory, tubes are held for two to three weeks to be sure of results in the case of slow fermenters.
An uninoculated control tube should be included with the tests for accurate comparison. The
important things to make note of include turbidity, color change, and gas production shown in the
Durham tube. The results can be read as one of the following:

No change (NC)
Growth MUST be present to assure proper inoculation took place. It is indicated by
turbidity in the tube but neither a change in color nor gas in the Durham tube should
occur. If no growth is present then the medium will not be turbid, the test is not valid, and
it must be repeated.

Acid Production (A+)

A variety of acid end-products are produced by bacteria from fermentable
carbohydrates. Ordinarily, no attempt is made to identify the acid constituents, but simply
to demonstrate the production of acid. Color changes in a pH indicator in the medium are
used to give qualitative evidence of acid production.

Gas Production (G+)

Gaseous end products such as CO2 and H2 are often formed during
fermentation of carbohydrates. Gas production is detected in the Durham tube (a small,
inverted vial). It is initially filled with medium because of the vacuum in the autoclave but
as gas is released during inoculation it collects in the vial. A floating Durham tube or one
with any gas present represents fermentation regardless or the color, which sometimes
fades with very strong positive tests.

Production of Acid but not Gas (A+G-)

Growth is present within the tube with a change in the color of the medium to pink.
No gas bubble is present in the Durham tube. This result indicates that the organism
ferments the sugar substrate but only produces acidic as an end-product. As previously
mentioned, it is not possible for an organism to ferment a sugar substrate and produce
gas only.
 Production of Acid and Gas (A+G+)
Growth is present within the tube. The color of the medium has changed to pink and
a gas bubble is present in the Durham tube. False positive results may occur regarding
the gas reading if the Durham tube has stuck to the side of the tube or if small air
bubble has been introduced by tipping the tube.

*Reminder
Occasionally, a significant gas bubble will be present when there is no indication of
pH change. This is usually because the indicator has been used up or degraded, leaving
no pink color. A few drops of fresh Andrades indicator will show that acid has indeed
been formed. TAs can provide this but if they are not available it can be found in the prep
room refrigerator in a large dark brown glass bottle. Seen below from left to right is an
un-inoculated tube, an inoculated tube with no fermentation, an inoculated partial/weak
positive, and an inoculated strong positive tube. Gas production in this image is difficult to
read.

II. Methyl Red Test (Homolactic / Heterolactic Glucose Fermentation)

Procedure and Background

Metabolism of glucose by bacteria causes varying amounts of acid production. Some
bacteria are homolactic fermenters and produce small amount of acid (lactic) while others
are heterolactic fermenters and produce not only more acid but many types of acids (e.g.
lactic, butyric, formic, proprionic, etc.) when fermenting glucose. The methyl red test roughly
determines the amount of acid produced from glucose metabolism. Significant acid
production suggests heterolactic fermenters of glucose are present while minimal acid
production suggests that they are homolactic. Inoculate a Clark-Lubs broth (buffered
peptone glucose broth). Incubate the tube 2 - 4 days at 37oC [or as instructed].

Reading the Methyl Red Test

Test for the degree of acid production by adding a few drops of methyl red indicator to the
tube and mixing gently. Methyl red can both be found on the bookshelf next to the chalkboard
if not at your bench. A red color indicates that the hydrogen ion concentration, relative pH, in
the medium is below 5.0 and is regarded as a positive test because the bacteria are mixed
acid / heterolactic fermentors. A yellow color indicates that the pH of the medium is above
5.0 and is regarded as a negative test meaning the bacteria are homolactic fermentors.

III. Voges-Proskauer Test (Acetoin Production from Glucose Fermentation)

Procedure and Background

Some bacteria produce a metabolic by-product called acetoin during glucose
fermentation. The Voges-Proskauer test assays for this acetoin production. Inoculate a
 Clark-Lubs broth (buffered peptone glucose broth) and incubate the tube 2 - 4 days at 37oC
[or as instructed]

Reading the Voges-Proskauer Test

To test for the production of acetoin, add 10-15 drops of Voges-Proskauer Reagent A
(alpha-naphthol solution) and 10-15 drops of Voges-Proskauer Reagent B (40% KOH) to the
culture. These solutions can both be found on the bookshelf next to the chalkboard. Shake
the culture well at intervals. The appearance of a pink color within a few minutes indicates
the presence of acetoin (a positive test). The color should deepen with time.

IV. Gelatin Liquefaction (Presence of Gelatinase Enzyme)

Procedure and Background

Some bacteria have enzymes that can break down the protein gelatin, causing it to lose
its solidifying properties. These enzymes are referred to as gelatinases. Inoculate a gelatin
deep by stabbing a loop full of the organism of interest into the deep. Incubate the tube 2 - 4
days at 37oC [or as instructed]

Reading the Gelatin Liquefaction Test

A positive test is one in which the gelatin has become liquefied. Occasionally, the
temperature of a warm laboratory will cause the gelatin to melt. If actual liquefaction has
occurred, placing of the tube in a coldwater bath will not cause re-solidification of the
medium. If the medium readily solidifies, temperature rather than bacterial action was the
cause of the liquefaction.

V. Indole Test (Presence of Tryptophanase Enzyme)

Procedure and Background

Some organisms are capable of breaking down the amino acid tryptophan because they
possess the enzyme tryptophanase. This enzyme hydrolyzes tryptophan into indole, pyruvic
acid, and ammonia as seen below. Presence of the enzyme is determined by assaying for
 indole. Inoculate a tryptone broth with the bacterium of interest and incubate 2 - 4 days at
37oC [or as instructed].

Credit Wikipedia
http://en.wikipedia.org/wiki/File:Indole.PNG

Reading the Indole Test

Add 10-15 drops of Kovacs reagent (p-dimethyl amino benzaldehyde dissolved in
isoamyl alcohol and hydrochloric acid), so as to form a layer of reagent on top of the medium
and mix gently. Kovacs reagent can both be found on the bookshelf next to the chalkboard if
not at your bench. A red color indicates that the bacteria possess tryptophanase (a positive
result) which means the bacterium can breakdown tryptophan and form indole. Avoid
breathing reagent vapors.

VI. Citrate Test (Citrate Utilization)

Procedure and Background

Some bacteria can utilize citrate as a sole source of carbon and energy. These bacteria
grow well on a Simmons citrate agar, which contains the organic acid citrate as a carbon
source and ammonium ions as a nitrogen source. This medium also contains the pH
indicator bromothymol blue that will turn from green to blue if an organism utilizes the citrate
and releases the alkaline metabolic by-product (OH).

Inoculate a Simmons citrate agar slant with a loopful of the organism of interest by spreading
the liquid in a zig-zag pattern along the surface of the slant paying attention to use proper
sterile technique. Incubate 2 - 4 days at 37oC [or as instructed]

Reading the Citrate Test

A change in the medium from green to blue indicates that the bacterium is able to utilize
citrate as its sole carbon and energy source. The blue color is the result of the production of
alkaline metabolic by-products that cause the pOH indicator to change from green to blue.
 There will be no growth or color change for those bacteria incapable of using citrate as a
carbon source.

VII. Catalase Test (Presence of Catalase Enzyme)

Procedure and Background

Most bacteria which use oxygen as the final hydrogen acceptor first form the toxic bi-
product hydrogen peroxide (O2+H2 à H2O2). To deal with this toxic compound, these
bacteria possess the enzyme catalase which quickly converts hydrogen peroxide into water
and oxygen (2H2O2 à 2H2O+O2). Most obligate anaerobes, microorganisms which can
only grow in less than atmospheric levels of oxygen, are considered catalase negative
because they lack the enzyme. Most aerobes which require oxygen and facultative
organisms that can function either way do possess the enzyme and are, therefore, catalase
positive. The exception to this generalization is the facultative lactic acid bacteria
Streptococcus which are catalase negative.
Place a small observable amount of the bacteria from a well-isolated colony directly on a
clean microscope slide. Place a drop of 3% hydrogen peroxide directly on these bacteria.
Observe.

Reading the Catalase Test

Evolution of oxygen, as indicated by vigorous bubbling, is interpreted as a positive result.
Do not test peroxide directly on a colony on a plate because ingredients of media such yeast
extracts and blood will themselves produce a positive catalase test and could confuse the
results.
VIII. Oxidase Test (Presence of Cytochrome aa3)

Procedure and Background

The oxidase test is used to identify the presence of a particular enzyme found in the
electron transport chain of certain bacteria. This enzyme, cytochrome oxidase (aa3) , can
breach down oxidase reagent yielding colored products. The two main groups of bacteria
which contain cytochrome aa3 in their electron transport chains are Neisseria and
Pseudomonas. Bacteria cultured on blood agar, Mueller-Hinton agar, or TSY agar can be
used to perform this test. Place a piece of filter paper in an empty half of a Petri dish and
moisten it with oxidase reagent. With a sterile loop, pick up a little of the colony and rub it
onto the filter paper in a short streak.

Reading the Oxidase Test

A streak which quickly turns deep purple as is indicated at the top of the drawing below
indicates that the organism possesses cytochrome aa3. Other lines may present from contact
with the filter paper, as shown at the bottom of the drawing, but their lack of color deepening
suggest that they are negative results. Discard the filter paper in the biohazard receptacle
and quickly rinse remaining oxidase reagent to avoid staining the glass Petri dish.

IX. Urease Test (Presence of Urease Enzyme)

Procedure and Background

The urease test assays for whether a bacterium possesses the enzyme urease. This
enzyme will hydrolyze urea leading the production of ammonia and CO2. The urease test
media contains the pH indicator phenol red. This indicator is pinkish yellow at pH’s above 8.4
and bright pink at pH’s below. Inoculate a urea agar slant with a loopful of the organism of
interest. Incubate at 25°C for approximately one week.

Reading the Urease Test

Urease activity will be indicated by the agar turning a bright pink color. If the agar color
remains unchanged, the organism does not possess this enzyme.
X. Motility Test (Presence of Flagella)

Procedure and Background

Some bacteria possess flagella that allow them to move in their environment. These
specialized structures can be detected by performing a specialized flagella staining technique
or by directly assaying for motility using motility agar. Motility agar contains a reduced agar
concentration that allows the bacteria to move more easily within the agar and a dye
(triphenyltetrazolium chloride) which allows bacterial movement to be detected more easily.
Inoculate a motility agar deep with a loopful of the organism of interest. Stab straight down
into the agar and pull the loop up directly along the same path. Incubate the tube 2 - 4 days
at 37oC [or as instructed]

Reading the Motility Test

If the bacteria are motile, a purplish haze will be seen throughout the deep as the
bacteria have migrated away from the streak line. This would be considered a positive
motility test. If the bacteria are not motile, a purple feather-pattern will be visible. These are
bacteria growing directly along the streak line. This would be considered a negative motility
test.

XI. Triple Sugar Iron Test (TSI)

Procedure and Background

As previously discussed, different microorganisms depend of different carbohydrates as
their carbon source for metabolism. The TSI media contains lactose, glucose, and sucrose
as well as ferrous sulfate (iron). The phenyl red pH indicator reacts with acid bi-products and
helps illustrate which of these sugars are being fermented. Collect some of the unknown
bacterium from a colony on your plate and either create a turbid saline suspension or apply
directly to the test. Stab your inoculated loop into the agar and then zigzag across the
surface of the slant making sure to use sterile technique.

Reading the TSI Test

The test can be a bit tricky to read. Pay special attention to what part(s) of the slant have
changed color thus indicating fermentation in that area. Also, note gas production in the form
of cracks and hydrogen sulfate production in the form of black precipitate.

No change (NC)
An unchanged red slant red butt combination with no black precipitate or gas
produced indicates no change. This is an unlikely result unless dealing with certain
extremophiles. Confirm proper inoculation for accurate results.

Acid Production (A+)

The phenyl red pH indicator will show where in the slant acid is being released from
fermentation thus indicating which sugars are being utilized. A red slant / yellow
bottom combination suggests glucose fermentation indicates glucose fermentation only.
A completely yellow tube suggests definite glucose fermentation but also lactose and/or
sucrose. A yellow slant / red bottom suggests lactose and/or sucrose but not glucose
fermentation.

Gas Production (G+)

Gaseous end products such as CO2 and H2 are often formed during
fermentation of carbohydrates. This may be evident as bubbles, cracks, or a complete
lifting of the agar from the bottom of the tube.

Hydrogren Sulfide Production (H2S)

Some organisms use thiosulfate as a terminal electron acceptor which is reduced to
hydrogen sulfide and binds with the sulfate forming a ferrous sulfide black precipitate.

*Reminder
If the color of the media is completely black along a slant and/or bottom than you can
assume that area is acid positive as well. Hydrogen sulfide precipitate could not be
present if fermentation had not taken place.

Credit: microbesinfo.com