1A5

Allison August April 16, 2015

LIFE 206 Unknown Lab Report1A5

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

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 RESULTSGrowth 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

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

1A5

STEP # 4

NAME OF TEST Cytochrome oxidase testMEDIUM 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

STEP # 5

1A5

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 RESULTSRegular lighting: thick fluorescent green/ blue smearUV 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

STEP # 6NAME OF TEST Gelatinase test

1A5

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

STEP # 7

NAME OF TEST Arginine dihydrolase test (ADH)

1A5

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

STEP # 8

NAME OF TEST Nitrate/ nitrite reduction

MEDIUM Nitrate broth

1A5

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 positiveNitrite 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

STEP # 9

NAME OF TEST Growth Temperature

MEDIUM TSA agar

1A5

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 RESULTSGrowth 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

STEP # 10

NAME OF TEST Pseudosel agar

MEDIUM Selective and differential

1A5

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 growthUV 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,

STEP # 11

NAME OF TEST Pseudomonas Isolation Agar (PIA)

MEDIUM Selective and differential

1A5

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 smearUV 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

1A5

Unknown 1A5

Pseudomonas aeruginosa

1A5

1st Streak for isolation TSA plate of 1E10

1st Streak for isolation TSA plate of 1A5

2nd Streak for isolation TSA plate of 1A5

1A5

Gram Stain (negative and bacillus rods) of 1E10

Catalase positive test on 1E10

1A5

Motile positive test on 1E10

1A5

Gram Stain (negative and bacillus rods) of 1A5

TSA 37 degrees 1E10 growth1A5 growth

1A5

TSA Room Temperature1E10 growth1A5 growth

Pseudomonas F agarRegular Lighting: UV lighting:

1E10 yellow growth with white spots under bright yellow with white spots1A5 thick fluorescent blue/green growth did not light up blue/green

1A5

Pseudomonas Isolation Agar (PIA)Regular lighting: UV lighting:

1E10 no growth no fluorescein 1A5 thick growth- purple with green smear very minimal fluorescence

1A5

Pseudosel agarRegular lighting: UV lighting:

1E10 no growth no fluorescent 1A5 green/ yellow growth light green white glow

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