isolating and identifying microorganisms is very important

Unknowns Project

Christine Kelly

4/26/2015

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Introduction:

Isolating and identifying microorganisms is very important. There are many ways to

determine the differences in microorganisms. They can be determined by their shape and staining

properties, as well as the way they are grouped. They can also be determined by their reactions to

different biochemical testing. Using these methods can be very important in discovering the type

of organism present.

Understanding the procedure in isolating and identifying bacteria can help in many

different fields to include medical and biological research fields. The medical field can use

identification to assure proper treatments are given to patients. Also “many of these tests are

designed to identify Gram-negative organisms, since there is a larger percentage of pathogen”

(Kerr & McHale, 2003) that are Gram-negative. The rise in antibiotic resistance makes this even

more important. In biological research, soil and water microbiology play important parts in our

everyday lives and in many discoveries. Some of these discoveries have led to new antibiotics in

our constant battle to keep bacteria at bay within our bodies.

Morphologies of bacteria play an important role in isolation and identification. The

ability to determine different morphologies is the first step in isolation. This is followed by gram

staining to determine if you have a gram positive or gram negative bacterium. The ability to

determine these things help lead to biochemical tests which will further identify the bacteria

present.

Methods and Materials:

2 Gram Positive

Mannitol Fermentation

Fermentation of Mannitol

Staphylcoccus aureas

No Fermemtnation of Mannitol

Staphylococcus epidermidis

Streptococcus agalactiae

Streptococcus salivarius

Micrococcus luteus

Bacillus subtilis

Starch Agar

Amalayse production


Bacillus subtilis

No Amylase Production

Staphylococcus epidermidis


Micrococcus luteus

Nitrate Reduction

No Nitrate Reduction


NO3 to NO2

Bacillus subtilis

Urea

Urease Production

Staphylococcus Epidermidis

No Urease Production


Micrococcus luteus

TSI

Glucose Fermentation only

Micrococcus luteus

Glucose and Lactose fermentation


Confirmation

Glucose and Lactose Fermentation tubes

3Gram Negative

Baird Parker

Growth, brown colonies, no clearing

Proteus vulgaris

No growth

Alcaligenes faecalis

Enterobacter aerogenes

Escherichia coli

Psuedomonas aeruginosa

Salmonella typhimurium

Serratia marcescens

Shigella flexneri

Oxidative-Fermentation Metabolism

Fermentation


Escherichia coli


Serratia marcescens

Shigella flexneri

Oxidative

Psuedomonas aeruginosa

Non saccharolytic

Alcaligenes faecalis

Phenylalanine Slant

Phenylalanine catabolism by phenylalanine deaminase


Confirmation

Glucose fermentation and Lactose fermentation

No catabolism

Escherichia coli


Serratia marcescens

Shigella flexneri

Sucrose fermentation Ferments Sucrose

Escherichia coli

Serratia marcescens

No fermentation


Shigella flexneri

Citrate

Citrate

Utilizes Citrate


Does not utilize citrate

Shigella flexneri

Does not utilize Citrate

Escherichia coli

Utilizes Citrate

Serratia marcescens

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Results:

UNKNOWNS RESULTS FORM

Student: Christine Kelly Unknown #: 3

Instructor: Dr. Paz Identification: Streptococcus agalactiae

MORPHOLOGICAL CHARACTERISTICS

Cell Shape & Arrangement: Coccus in chains Colony Growth on TSAYE: Small, cream and circular

Gram’s Reaction: Gram positive reaction

Special Stains (optional): Isolation Temperature: 25 C

PHYSIOLOGICAL CHARACTERISTICS

Media 1:

Mannitol Fermentation

Observations

There was no change in the color of the tube and no gas present.

Interpretation

There was no fermentation of mannitol sugar.

Staphylococcus aureas ruled out

Incubation Temperature/Time

25 C / 48 hours

Used as a confirmation test?

No

Media 2:

Amylase

Observations

There was no growth on the plate and no clearing.

Interpretation

No Amylase was produced.

Streptococcus salivarius and Bacillus subtilis ruled out


25 C/ 48 hours


No

Media 3:

Urea

Observations

The tube remained yellow in color.

Interpretation

There was no production of

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urease.

Staphylococcus epidermidis ruled out


25 C / 48 hours


No

Media 4: TSI Observations

Both the slant and the bottom of the tube turned yellow.

Interpretation

Lactose and glucose were both fermented.

Indicates

Streptococcus agalactiae is the unknown.


25 C / 48 Hours


No

Media 5:

Glucose Fermentation

Observations

The tube changed from a blue to a more greenish color.

Interpretation

Glucose fermentation occurred.


25 C/ 48 hours


Yes

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UNKNOWNS RESULTS FORM

Student: Christine Kelly Unknown #: 3

Instructor: Dr. Paz Identification: Enterobacter aerogenes

MORPHOLOGICAL CHARACTERISTICS

Cell Shape & Arrangement: bacilli, chained together Colony Growth on TSAYE: small, circular, cream

Gram’s Reaction: Gram Negative

Special Stains (optional): Isolation Temperature: 25 C

PHYSIOLOGICAL CHARACTERISTICS

Media 1: Baird Parker Observations

There was no growth observed on the plate

Interpretation

No coagulase, no tellurite reduction.

Proteus vulgaris ruled out


25 C / 48 Hours


No

Media 2: Oxidative Fermentation metabolism

Observations

Both tubes yellowed

Interpretation

Fermentation took place in both aerobic and anaerobic tubes.

Pseudomonas aeruginosa and Alcaligenes faecalis ruled out.


25 C / 48 hours


No

Media 3:Phenylalanine Slant Observations

The tube showed growth, and greening.

Interpretation

The presence of green mean Phenylalanine was catabolized by Phenylalanine deaminase.

Indicates


25 C / 48 hours


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Yes or No

Enterobacter aerogenes as the gram negative bacteriaMedia 4: Glucose Fermentation Observations

The tube turned very yellow and had gas.

Interpretation

Glucose was fermented confirming Enterobacter

aerogenes


25 C / 48 hours


Yes

Media 5: Lactose Fermentation Observations

This tube was yellow and had the presence of gas

Interpretation

Lactose was fermented confirming Enterobacter

aerogenes


24 C / 48 hours


Yes

Discussion:

Isolating the organisms there was a problem getting the gram negative organism to separate

appropriately. There were no problems isolating the gram positive organism. Both organisms were

viewed under the microscope. The gram positive organism was observed to be coccus and in chains

indicating it would be a Streptococcus species. The flow chart for the gram positive organism was made

prior to the gram staining.

The gram positive flow chart was chosen to gain a direct way to identification without using

many tests. There were no issues with the tests. After gram-staining it was known to be a Streptococcus

species, so the tests performed as expected given the result of Streptococcus agalactiae.

The gram negative bacteria were harder to isolate. This may have been because the

morphologies on both the gram positive and gram negative bacteria were very close in appearance.

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Once separated the gram negative bacteria became much more cooperative. The tests proceeded as

expected. Again the shortest path to identification was used. The gram negative flow chart started with

eliminating one possibility with the Baird Parker plate. The oxidative fermentation metabolism would

have eliminated two as the bacteria. There was no need to go past the phenylalanine slant due to the

positive result of the bacterium catabolizing phenylalanine by phenylalanine deaminase. The catabolism

of phenylalanine identified the bacterium as Enterobacter aerogenes.

The knowledge gained in performing these tests can be carried on to many different fields

involving microbiology to include the medical profession as well as research based professions. The

understanding of gram staining and biochemical testing in isolation and identification are really required

knowledge for anyone working in the microbiology field.

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Works CitedKerr, T. J., & McHale, B. B. (2003). Applications in General Microbiology. Winston-Salem: Hunter

Textbooks Inc. .

isolating and identifying microorganisms is very important

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