life 206_labwriteup_1a5
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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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