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Isolation of Pseudomonas Species

from Soil

The purpose of this experiment is to attempt to
isolate a
Pseudomonas species from soil samples around campus.

In this experiment, we will be using a technique called the
enrichment culture method to try and isolate
Pseudomonas species from soil samples around campus. It is a well known
fact that members of the Pseudomonas species are found in soil and water,
so we should be able to isolate the species using a liquid enrichment media.
The liquid medium, which will be pre-made for you, will contain 50 ml of
basal salts broth, supplemented with 5 ml of 2.5% mandelic acid. Basal salts
broth is a minimal medium, which means that it provides the very minimum
essential nutrients for an organism. Basal salts broth is made up of the
following: 800 ml of de-ionized water, 100 ml of 0.5M sodium pyrophosphate
(Na4 O7P4), 100 ml 1.0M monobasic potassium phosphate (KH2PO4), 1 ml of
0.1M calcium chloride (CaCl2), 1 ml of 1.0M magnesium sulfate (MgSO4), 2
grams of ammonium sulfate((NH4)2SO4), and 10 ml of 1% glucose.

Mandelic acid is a compound that can be used by members of the

Pseudomonas species as a sole source of carbon and energy aerobically. By
growing the cultures at 30°C, we can encourage the growth of more types of
Pseudomonas species, such as P. fluorescens. After isolating a bacterium,
you will use several diagnostic tests to identify the species: Gram stains, a
litmus milk test, a nitrate reduction test, and you will also do a 4-way streak
on two types of Pseudomonas media. Pseudomonas F media is specially
formulated to enhance fluorescein production, while Pseudomonas P media
is specially formulated to enhance pyocyanin production. This experiment
will take several weeks to complete. Days 1 and 2 will be during week 2,
days 3 and 4 during week 3, and so on. In addition to this isolation
experiment, you will also be performing other experiments as we move
through the course.

Litmus Milk Test

Litmus milk is a special kind of media used to detect lactose fermentation.
Litmus milk is made up of 100 grams/liter of skim milk powder, 0.5
grams/liter litmus, and 0.5 grams/liter of sodium sulfite. Milk is made up of
lactose, casein, lacto-albumin, and lactoglobulin. Bacterial enzymes that
digest the units that compose milk cause a change of pH in the medium,
which causes the litmus in the medium to change color. At a pH of 8.3 or
above, litmus is blue or purple and at a pH of 4.5 and below, the litmus is red
or pink.

Microorganisms generally metabolize milk substrates in one of the following

ways: lactose fermentation, gas production, litmus reduction, curd formation,
proteolysis, or alkaline reaction. Organisms capable of using lactose as a
carbon source utilize the insoluble enzyme β-galactosidase, and degrade
lactose into glucose + galactose, the glucose + galactose into pyruvic acid,
and the pyruvic acid into lactic acid. The presence of lactic acid is detected
easily in litmus milk because it causes the pH to drop to around 4.

In litmus reduction reactions, the oxidation of lactose produces lactic acid,

butyric acid, carbon dioxide, and hydrogen gas. Fermentation is a process
involving oxidations that occur in the absence of molecular oxygen, and are
seen as the removal of hydrogen from a substrate. When in its oxidized
state, the litmus is purple; when it accepts hydrogen from a substrate, it
becomes reduced and turns white, or milk-colored.

The biochemical activities of different microorganisms grown in litmus milk

may result in the formation of two distinct types of curds, or clots. An acid
curd forms when lactic acid or other organic acids cause the precipitation of
casein as calcium caseinate to form an insoluble clot. A rennet curd is
formed when the microbe produces rennin, an enzyme that acts on casein to
form paracasein, which in the presence of calcium ions is converted to
calcium paracasein, forming an insoluble curd. If you have difficulty
distinguishing between the two types of curds, try inverting the test tube.
An acid curd will not move, but a rennet curd is semi-solid, and will move
when the tube is tilted.

Because some organisms cannot utilize lactose fermentation to obtain

energy, they use other ways to gain energy. In proteolysis, or peptonization,
organisms hydrolyze casein using proteolytic enzymes. The digestion of
proteins is accompanied by the evolution of large amounts of ammonia,
which results in an alkaline pH in the medium. In the upper portions of the
test tube, the litmus turns deep purple, while the medium begins to lose
body and produces a translucent, brown color towards the bottom of the test
tube as hydrolysis occurs.

Reaction Condition of culture Interpretation

Slight acid
Indicator pale pink Glucose utilized but not lactose

Acid production Indicator pink Glucose and lactose utilized

Acid clot Medium clotted, indicator Casein precipitated by acid. This

pink type of clot will dissolve if treated
with NaOH

Indicator color is reduced

Result of very rapid growth of the
Reduction of white (leuco) form very
organism and lowering of oxygen
indicator often accompanies the
content in the bottom of the tube
formation of an acid clot

Medium clotted, whey Precipitation of casein by rennin.

Rennet clot
formed, indicator varies This clot does not dissolve in NaOH

partial degradation of casein into

Indicator shows no change
Alkaline Reaction shorter polypeptide chains; release
or turns deeper blue
of alkaline end products

Indicator deep purple in

Proteolytic enzymes hydrolysing
upper portion, changing to
Proteolysis casein; ammonia liberated from
brown and whey-like in
lower part
Nitrate Reduction Test
The identification of some bacteria is aided by determining if the organism
can reduce nitrate (NO3) to nitrite (NO2), or another nitrogenous compound
such as ammonia (NH3) or nitrogen gas (N2).

In order to determine if a bacterium can reduce nitrate, the test organism is

inoculated into nitrate reduction broth, such as indole nitite medium, or
another medium that contains large amounts of nitrate in the form of KNO3.
After incubating your culture for 24 hours, α-naphthylamine and sulfanilic
acid are added. These two compounds react with nitrite and turn the broth
red, which indicates that nitrate was reduced. If there is no color change,
then nitrite is not present; it could be that the unknown culture did not
reduce nitrate, or it could be that it reduced nitrate to nitrite, but then
further reduced the nitrite into ammonia or nitrogen gas. To differentiate
between these two reactions, a few grains of zinc are added. Zinc catalyzes
the reduction of nitrate to nitrite. Therefore if the addition of zinc turns the
broth red, then nitrate must still be present in the culture, meaning the
organism did not reduce it to nitrite – the zinc did. If after the addition of
zinc there is still no color change, the result is positive. This result simply
means that there was no nitrate, no nitrite, and that the nitrate was reduced
to nitrite which was reduced to ammonia or nitrogen gas. To summarize, see
the table below:

Addition of
Addition of
Solution A and Result
Solution B

Turns red n/a Reduces Nitrate

The zinc converted

the nitrate to nitrite –
No color change turns red
nitrate not reduced by
Nitrate was reduced
to nitrite and then
No color
No color change further reduced to
nitrogen gas or

Results of Testing
The gram stain of Pseudomonas aeruginosa will show rod shaped, gram
negative results. In the litmus milk test, Pseudomonas aeruginosa should
display rapid peptonization. In the nitrate reduction test, Pseudomonas
aeruginosa should reduce nitrate.
Please refer to the diagram on the following page for a representation of how
this experiment will be carried out.
Day 1
1. Inoculate one flask (labeled BSM1, for Basal Salts w/Mandelic acid,
primary culture) with a pea-sized amount of soil. Gently swirl.
2. Incubate for 24 hours at 30°C on a shaking table
Day 2
1. Examine for growth. If no growth is present, return to incubator for an
additional 24 hours.
2. If growth is present, aseptically transfer your primary culture into a
sterile 50-ml conical tube, and centrifuge at 1,000X for 2 minutes to
spin down any soil that is suspended in the broth; centrifuging at a low
speed should not spin down the bacteria.
3. Remove the conical tube from the centrifuge, using caution not to re-
suspend the soil; transfer 1 ml of this primary culture into a flask with
fresh media and swirl (the second flask will be labeled BSM2, indicating
that it consists of the same media, but that it is the secondary culture.)
4. Incubate for 24 hours at 30°C on shaking table.
5. Prepare and examine a Gram stain from the primary culture.
6. Refrigerate the primary broth culture
Day 3
1. If no growth is present in the primary broth culture, get a new flask of
BSM1 and repeat the steps from Day 1. If growth is present from your
primary culture, proceed with the steps indicated for Day 2.
2. If growth is present in the secondary broth culture, perform a four-way
streak inoculation on an agar enrichment plate, labeled BSMA (Basal
Salts w/Mandelic acid + Agar).
3. Incubate inverted at 30°C for 24 hours
4. Prepare and examine a Gram stain from the secondary broth culture
5. Refrigerate the secondary broth culture
Day 4
1. Examine the primary plate for discrete colonies. From a discrete
a. Prepare a Gram stain
b. Perform a four-way streak on a fresh BSMA plate. (This is so that
you can be sure you have a pure colony.)
2. Incubate the secondary agar plate for 24 hours at 30°C
3. Refrigerate the primary agar culture
Day 5
1. Examine the secondary agar plate. If the colonies look similar, from a
discrete colony:
a. Prepare a Gram stain
b. Inoculate a trypticase soy agar (TSA) slant.
2. Incubate the agar slant for 24 - 48 hours at 30°C
3. Refrigerate the secondary agar plate culture
Day 6
1. Prepare and examine a Gram stain from the TSA slant
2. Using the TSA slant culture, inoculate appropriately labeled tubes of
nitrate broth and litmus milk by loop inoculation.
3. Incubate for 24 - 48 hours at 30°C
4. Inoculate a Pseudomonas F plate and a Pseudomonas P plate from the
TSA slant. Incubate for 24 – 48 hours at 30° C.
Day 7
1. Examine litmus milk tube for results
2. For nitrate reduction test, add 5 drops of solution A (sulfanilic acid) to
the test tube, followed by 5 drops of solution B (α-naphthylamine) and
observe for color change
3. If no color change occurs, add a few granules of zinc, and observe for
color change.


Rosenberg, S.L. Regulation of the Mandelate Pathway in Pseudomonas

aeruginosa. JOURNAL OF BACTERIOLOGY, Dec. 1971, Vol. 108, No. 3. pp.

Cappuccinno, James, and Natalie Sherman. Microbiology: A Laboratory

Manual. Seventh Edition. Benjamin Cummings: San Francisco. 2005.