The unknown microbe from sample 1A5 underwent a series of biochemical tests which identified it as Pseudomonas aeruginosa. A gram stain showed it was a gram negative bacillus that was motile and catalase positive. Further tests for cytochrome oxidase, growth on selective and differential media, and production of pyocyanin pigment eliminated other Pseudomonas species, confirming the microbe as P. aeruginosa.

1. 1A5
Allison August
April 16, 2015
LIFE 206 Unknown Lab Report
1A5

2. 1A5
STEP # 1
NAME OF TEST
Gram Stain
MEDIUM Glass slide
INCUBATION (if applicable)
No incubation
SPECIAL PROCEDURE(S) (if applicable) Gram stain procedure with heat fixing
REAGENT(S) USED & AMOUNT (if
applicable)
Crystal violet, grams iodine, decolorizer,
safranin
APPEARANCE OF RESULTS Pink, rod shaped (bacillus)
RESULT (positive or negative, and for
what)
Gram negative
INTERPRETATION (what do the results
mean?)
The result of a gram negative gram stain
confirms that this microbe is within the
Pseudomonas family.
BACTERIA OR SPECIES ELIMINATED
All gram positive bacteria and gram negative
bacteria except Pseudomonas can be
eliminated because our unknown is
Pseudomonas.
WHY ELIMINATED?
All gram positive bacteria and all gram
negative bacteria except Pseudomonas can be
eliminated because our unknown is
Pseudomonas so all others are irrelevant.
POSSIBILITIES REMAINING
Pseudomonas

3. 1A5
STEP # 2
NAME OF TEST
Motility Test
MEDIUM
Motility agar deep with TTC
INCUBATION (if applicable)
28 degrees Celsius, 2 days
SPECIAL PROCEDURE(S) (if applicable)
Stab test with inoculating needle using a
heavy inoculum
REAGENT(S) USED & AMOUNT (if
applicable)
Motility agar deep with TTC
APPEARANCE OF RESULTS
Growth around the stab line radiated outward
in all directions
RESULT (positive or negative, and for
what)
Motile positive
INTERPRETATION (what do the results
mean?)
The result of motile positive means that this
microbe is within the Pseudomonas family
because it has flagella.
BACTERIA OR SPECIES ELIMINATED
All motile negative bacteria can be eliminated
because our unknown is Pseudomonas.
WHY ELIMINATED?
All motile negative bacteria can be eliminated
because our unknown is Pseudomonas.
POSSIBILITIES REMAINING Pseudomonas

4. 1A5
STEP # 3
NAME OF TEST
Catalase Test
MEDIUM
Glass slide
INCUBATION (if applicable)
No incubation
SPECIAL PROCEDURE(S) (if applicable)
Dry smear on glass slide and place a few
drops of H2O2 on top. Observe.
REAGENT(S) USED & AMOUNT (if
applicable)
Hydrogen peroxide (H2O2)
APPEARANCE OF RESULTS
Bubbles (O2) being released
RESULT (positive or negative, and for
what)
Catalase positive
INTERPRETATION (what do the results
mean?)
The result of catalase positive means that this
microbe is within the Pseudomonas family
because it is aerobic. It can breakdown
H2O2.
BACTERIA OR SPECIES ELIMINATED
All catalase negative bacteria can be
eliminated because our unknown is
Pseudomonas. Species eliminated because
they do not produce the enzyme catalase.
WHY ELIMINATED?
All catalase negative bacteria can be
eliminated because our unknown is
Pseudomonas which is aerobic not anaerobic.
All others do not breakdown H2O2 and could
be toxic to cells.
POSSIBILITIES REMAINING Pseudomonas

5. 1A5
STEP # 4
NAME OF TEST
Cytochrome oxidase test
MEDIUM Glass slide
INCUBATION (if applicable)
No incubation
SPECIAL PROCEDURE(S) (if applicable)
On a glass slide, use deionized water to wet
the disk. Place a heavy inoculum on the disk
and observe for three minutes.
REAGENT(S) USED & AMOUNT (if
applicable)
TMPD or DMPD disk
APPEARANCE OF RESULTS
The reagent is a dark-blue to maroon color
(oxidized)
RESULT (positive or negative, and for
what)
Oxidase positive
INTERPRETATION (what do the results
mean?)
The test reagent, TMPD dihydrochloride acts
as an artificial electron donor for the enzyme
oxidase. The oxidized reagent forms the
colored compound indophenol blue. The
cytochrome system is usually only present in
aerobic organisms that are capable of using
oxygen as the terminal electron acceptor.
BACTERIA OR SPECIES ELIMINATED
All oxidase negative bacteria can be
eliminated because our unknown is
Pseudomonas. Possibly able to eliminate P.
syringae.
WHY ELIMINATED?
The reagent oxidized so all oxidase negative
bacteria can be eliminated because our
unknown is Pseudomonas.
POSSIBILITIES REMAINING
Pseudomonas

6. 1A5
STEP # 5
NAME OF TEST
Pseudomonas F agar
MEDIUM
Differential, King B
INCUBATION (if applicable)
No incubation
SPECIAL PROCEDURE(S) (if applicable)
Split plate in half with 1A5 and 1E10; view
under regular lighting and UV light
REAGENT(S) USED & AMOUNT (if
applicable)
King B agar
APPEARANCE OF RESULTS
Regular lighting: thick fluorescent green/ blue
smear
UV lighting: minimal fluorescent
RESULT (positive or negative, and for
what)
Growth fluorescein positive
INTERPRETATION (what do the results
mean?)
A Growth fluorescein positive means it
contains fluorescein and can grow in room
temperature conditions. Pseudomonas F agar
contains magnesium and potassium salts that
enhance the production of fluorescein (under
UV).
BACTERIA OR SPECIES ELIMINATED
All growth fluorescein negative bacteria can
be eliminated because our unknown is
Pseudomonas. Possibly able to eliminate P.
putida and P. syringae.
WHY ELIMINATED?
All growth fluorescein negative bacteria can
be eliminated because our unknown is
Pseudomonas. Others could not survive on an
agar with these conditions.
POSSIBILITIES REMAINING
Pseudomonas

7. 1A5
STEP # 6
NAME OF TEST
Gelatinase test
MEDIUM Gelatin
INCUBATION (if applicable)
25- 28 degrees Celsius for two days
SPECIAL PROCEDURE(S) (if applicable)
Stab nutrient gelatin medium with heavy
inoculum
REAGENT(S) USED & AMOUNT (if
applicable)
Nutrient gelatin medium
APPEARANCE OF RESULTS Inoculated tube is liquefied
RESULT (positive or negative, and for
what)
Gelatinase positive
INTERPRETATION (what do the results
mean?)
Gelatin hydrolysis test is used to detect the
ability of an organism to produce gelatinase
(proteolytic enzyme) that liquefy
gelatin. Hydrolysis of gelatin indicates the
presence of gelatinases which is positive.
BACTERIA OR SPECIES ELIMINATED
Eliminated P. cichorii and P. putida with
possibly eliminating P. fluoescens.
WHY ELIMINATED?
If gelatinase negative, it did not hydrolyze.
POSSIBILITIES REMAINING P. aeuruginosa, P.chlororaphis, P. fluorescens,
P. syringae

8. 1A5
STEP # 7
NAME OF TEST
Arginine dihydrolase test (ADH)
MEDIUM
Nitrate broth
INCUBATION (if applicable) 25-28 degrees Celsius for two days
SPECIAL PROCEDURE(S) (if applicable)
Stab test and poor sterile mineral oil on top of
slant
REAGENT(S) USED & AMOUNT (if
applicable)
Mineral oil
APPEARANCE OF RESULTS Hot pink ADH medium
RESULT (positive or negative, and for
what)
ADH positive
INTERPRETATION (what do the results
mean?)
The hot pink is a pH indicator phenol red.
1A5 resulting in ADH positive means the
bacterium can use amino acid arginine as a
source of carbon and energy for growth. The
mineral oil is used to cut off oxygen to
stimulate anaerobic conditions. Unknown is
capable of producing the enzyme arginine
dihydrolase.
BACTERIA OR SPECIES ELIMINATED P. cichorii, P. syringae
WHY ELIMINATED?
The above are ADH negative.
POSSIBILITIES REMAINING
P. aeruginosa, P. chlororaphis, P. flourescens,
P. putida

9. 1A5
STEP # 8
NAME OF TEST
Nitrate/ nitrite reduction
MEDIUM
Nitrate broth
INCUBATION (if applicable) 28 degrees Celsius for two days
SPECIAL PROCEDURE(S) (if applicable)
Using a heavy inoculum to broth then
incubated. Add 12 drops of Nitrate B and
Nitrate A, watch. If nothing, add a small
amount of powdered zinc.
REAGENT(S) USED & AMOUNT (if
applicable)
Nitrate and nitrite broth and zinc
APPEARANCE OF RESULTS
No color change after adding nitrate A and B;
then, no color change after adding powdered
zinc but there were small bubbles
RESULT (positive or negative, and for
what)
Nitrate positive
Nitrite positive
INTERPRETATION (what do the results
mean?)
The nitrate broth is a differential medium used
to distinguish between bacteria and their
ability or inability to reduce nitrate to nitrite
via the enzyme nitrate reductase which 1A5 is
positive for. Secondly, 1A5 is positive for
nitrite reductase because this unknown can
further reduce the nitrite to ammonia or
nitrogen gas.
BACTERIA OR SPECIES ELIMINATED
P. cichorii, P. fluorscens, P. putida, P.
syringae
WHY ELIMINATED? The above are negative for nitrate reductase
and negative for nitrite reductase.
POSSIBILITIES REMAINING P. aeruginosa, P. chlorophis

10. 1A5
STEP # 9
NAME OF TEST
Growth Temperature
MEDIUM
TSA agar
INCUBATION (if applicable)
One plate at room temperature and another
plate at 37 degrees Celsius for two days
SPECIAL PROCEDURE(S) (if applicable) Streak for growth not isolation
REAGENT(S) USED & AMOUNT (if
applicable)
TSA agar
APPEARANCE OF RESULTS
Growth on room temperature plate and 37
degrees Celsius plate
RESULT (positive or negative, and for
what)
Positive growth
INTERPRETATION (what do the results
mean?)
1A5 has growth on both plates in different
temperature extremes means that this microbe
grows in a certain temperature range.
BACTERIA OR SPECIES ELIMINATED P. cichorii, P. syringae
WHY ELIMINATED?
We can eliminate those Pseudomonas that do
grow at or above 37 degrees Celsius.
POSSIBILITIES REMAINING
P. aeruginosa, P. chlororaphis, P. flourescens,
P. putida

11. 1A5
STEP # 10
NAME OF TEST
Pseudosel agar
MEDIUM
Selective and differential
INCUBATION (if applicable)
Room temperature for two days
SPECIAL PROCEDURE(S) (if applicable)
Streak for growth not isolation; view under
regular and UV lighting
REAGENT(S) USED & AMOUNT (if
applicable)
Cetrimide and Pocyanin in agar
APPEARANCE OF RESULTS Regular lighting: green/ yellow growth
UV lighting: light green/ white glow
RESULT (positive or negative, and for
what)
Pyocyanin positive and positive for growth
INTERPRETATION (what do the results
mean?)
Since 1A5 showed growth it means that the
cetrimide (a quaternary ammonium
compound) did not inhibit the growth of this
particular Pseudomonas being the selective
medium that it is. Also, the agar is differential
containing magnesium chloride and potassium
sulfate that stimulates the blue-green pigment
shown in 1A5 called pyocyanin.
BACTERIA OR SPECIES ELIMINATED
P. chlororaphis, P. flourescens, P. putida,, P.
cichorii, P. syringae
WHY ELIMINATED?
According to the chart, all the above are not
positive for pyocyanin. Some did have growth
(P. chlororaphis, P. putida) but no pyocyanin.
POSSIBILITIES REMAINING
P. aeruginosa,

12. 1A5
STEP # 11
NAME OF TEST
Pseudomonas Isolation Agar (PIA)
MEDIUM
Selective and differential
INCUBATION (if applicable) Room temperature for two days
SPECIAL PROCEDURE(S) (if applicable) Streak for growth not isolation; view under
regular and UV lighting
REAGENT(S) USED & AMOUNT (if
applicable)
Irgasan (triclosan) and pyocyanin in agar
APPEARANCE OF RESULTS
Regular lighting: thick growth with dark green
smear
UV lighting: minimal fluorescein
RESULT (positive or negative, and for
what)
Positive for growth and positive for pyocyanin
INTERPRETATION (what do the results
mean?)
1A5 grew on this selective and differential
medium which eliminates many Pseudomonas
that cannot grow on this agar. The pigment of
pyocyanin was increased to differentiate
pseudomonads more easily. With the selective
agent Irgasan it prevents growth of certain
gram positive and gram negative bacteria,
however, some pseudomonads are resistant
yet can still grow on the medium.
BACTERIA OR SPECIES ELIMINATED
P. chlororaphis, P. flourescens, P. putida,, P.
cichorii, P. syringae
WHY ELIMINATED?
The above are not positive for pyocyanin.
Some did have growth (P. chlororaphis, P.
putida, P. flourescens) but no pyocyanin.
POSSIBILITIES REMAINING P. aeruginosa

13. 1A5
Unknown 1A5
Pseudomonas aeruginosa

14. 1A5
1st Streak for isolation TSA plate of 1E10
1st Streak for isolation TSA plate of 1A5

15. 1A5
2nd Streak for isolation TSA plate of 1A5
Gram Stain (negative and bacillus rods) of 1E10

16. 1A5
Catalase positive test on 1E10

17. 1A5
Motile positive test on 1E10

18. 1A5
Gram Stain (negative and bacillus rods) of 1A5
TSA 37 degrees
1E10 growth
1A5 growth

19. 1A5
TSA Room Temperature
1E10 growth
1A5 growth
Pseudomonas F agar
Regular Lighting: UV lighting:
1E10 yellow growth with white spots under bright yellow with white spots
1A5 thick fluorescent blue/green growth did not light up blue/green

20. 1A5
Pseudomonas Isolation Agar (PIA)
Regular lighting: UV lighting:
1E10 no growth no fluorescein
1A5 thick growth- purple with green smear very minimal fluorescence

21. 1A5
Pseudosel agar
Regular lighting: UV lighting:
1E10 no growth no fluorescent
1A5 green/ yellow growth light green white glow

22. 1A5

23. 1A5

24. 1A5

25. 1A5

26. 1A5