Microbiology101.com Unknown Bacteria Assistant

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Instructions

This program is designed to aid in the idenitification of common bacteria through analysis of various biochemical test results. The intended audience is an introductory microbiology class engaged in the identification of unknown bacteria. This program is free. You are welcome to use it through this link on my web site or to download the code and make any modifications you wish, in accordance with the GNU General Public License.

Download the most recent version here: uba.v124.tar.gz. Also see the Administrator's Notes.

This program compares the test results you enter to bacterial profiles which have been assembled from a number of sources (primarily Bergey's Manual). The main consideration for producing a good match is the quality of the data you enter. A large number of good quality test results will produce the best match. When a test has not been performed or has produced a result you are uncertain how to interpret, leave the test set to "Indeterminate." This is always better than guessing the result.

Once you have entered the data for each test, select a matching algorithm and click the "Click to Continue" button. You will receive a report of matches and a summary of the test data that you entered. Any match above 80% is a strong match and a good candidate for your bacteria. If it is not the actual bacteria, it is very likely to be within the same genus. Matches below 80% should be suspect, unless the number of comparison factors is less than 10. Some bacteria are better described than others. A well described bacterium may have established results for more than 20 tests, while a poorly described bacterium may have less than 10. The less well known a bacterium is, the less reliable a match the program produces. This is reflected in the "valid comparison factors" noted along with the percent match. Even a 99.9% match is not always trustworthy when the number of comparison factors is small.

General Information
User Name
Sample ID

Visual Inspection
Bacterial Morphology
Bacterial Arrangement
Bacterial Length
Bacterial Width
Colony Color
Colony Elevation
Colony Form
Colony Margin

Stains
Acid-Fast Stain
Endospore Stain
Gram Stain

Tube Tests
Gelatin Hydrolysis
IMViC Tests
Indole
Methyl Red
Voges-Proskauer
Simmons Citrate
Kligler's Iron Agar
Litmus Milk
Motility
Nitrate
Phenol Red Tests
Arabinose
Fructose
Galactose
Glucose
Lactose
Maltose
Mannitol
Mannose
Rhamnose
Sucrose
Trehalose
Xylose
Rapid Urea Hydrolysis
Optimal Temperature
Thioglycolate

Other Tests
Catalase
Oxidase

Plate Tests
Blood Agar
Milk Agar
Spirit Blue Agar
Starch Agar

Antibiotic Resistance
Ampicillin
Ciprofloxacin
Erythromycin
Penicillin
Tetracycline
Vancomycin

General Information

User Name

Enter a user name so that your report does not get mixed up with somebody else's.

Sample ID

Enter a sample ID so that reports for multiple samples don't get mixed up.

Visual Inspection

Bacterial Morphology

Bacteria come in five basic shapes, or morphologies, which are determined by inspection under the microscope.

* A coccus is sphere-shaped.
* A bacillus is rod shaped.
* A spirillum is spiral shaped and has flagella at each pole.
* A spirochete is spiral shaped and has a single flagellum.
* A vibrio is comma-shaped.

Bacterial Arrangement

Bacteria may connect or group together in a variety of ways.

* Single bacteria do not group together.
* Pairs are usually end to end, but may be side to side when bacteria pallisade.
* Tetrads are groups of four, generally found in the case of cocci or pallisading bacilli.
* Chains are groups of more than two bacteria joined end to end.
* Clusters are groups of more than two bacteria joined in an irregular structure.

Bacterial Morphology

The length of a bacteria is measured under the microscope. Choose the range that your measurement fits.

Bacterial Morphology

The width of a bacteria is measured under the microscope. Choose the range that your measurement fits.

Stains

Gram Stain

Bacteria can be divided into to large groups based on the ability of their cell walls to retain the stain crystal violet. Gram Positive bacteria retain crytal violet when exposed to a decolorizing agent due to their thick peptidoglycan cell walls. Gram Negative bacteria do not retain the crystal violet and lose their color. A red stain (safranin or fuschin) is then added to re-stain the gram negative bacteria. In a successful Gram stain, Gram positive bacteria are blue and Gram negative bacteria are red.

Endospore Stain

Spores are stained by steam-staining with malachite green. A decolorizing agent is then applied, removing the malachite green from vegetative bacteria and a red counterstain is applied. The end result is that spores are stained green while vegetative bacteria are stained red. The presence of any green-stained bodies is a positive result. Lack of any green stained bodies is either a negative result if the sample was properly stressed or may be an indeterminate result if the sample was not stressed.

Acid-Fast Stain

Bacteria with cell walls that contain mycolic acid or high levels of lipids are difficult to Gram stain, but can be successfully stained using the Ziehl-Neelsen or acid-fast method. Carbolfuschin is used as the primary stain and is fixed with steam. After decolorizing, methylene blue is added as a counter stain. A positive result is red/purple and a negative result is blue.

Tube Tests

IMViC Tests

Indole Test (tryptophanase)

This test determines whether bacteria posess enzymes in the category known as tryptophanase. These bacteria are able to break down tryptophan into indole, pyruvate, and ammonium. A positive result is a deep red color and a negative result is yellow.

Methyl Red test

The Methyl Red test determines whether a mixed or single acid use pathway is used in the use of glucose. A red color is positive and indicates that the mixed acid pathway is used. A yellow color is negative.

Voges-Proskauer test

This test determines whether the 2,3-butanediol use pathway is used in the use of glucose. The test detects the presence of acetoin which is a precursor of 2,3-butanediol. A positive result is red or pink mixed with brown. A negative result is greed to yellow mixed with brown.

Simmons Citrate test (citrase)

This test determines whether bacteria posess the enzyme citrase, which breaks down citrate into acetic acid and oxaloacetic acid. If so, oxaloacetic acid is hydrolyzed, resulting in pyruvic acid and carbon dioxide, which produces an alkaline reaction that changes the medium from green to blue. A positive result is blue; a negative result is green.

Kligler's Iron Agar (use of glucose)

Kligler's Iron Agar is used to determine several different properties of bacteria. Bacteria that are capable of useing glucose will quickly use up the small amount present (usually in the first few hours.) They then have two remaining nutrient options: lactose and amino acids. If the bacteria are able to use glucose but not lactose, acidic byproducts are produced for a short time before basic products are produced in the deamination of amino acids. If the bacteria are able to use lactose, acidic byproducts are produced. Bacteria that are not capable of using lactose will deaminate amino acids, producing basic byproducts. Bacteria that are capable of reducing sulfur will produce hydrogen sulfide - visible as a black precipitate. Note that bacteria that produce hydrogen sulfide will make the medium turn black. This can obscure the yellow color.

Result	Description
A/A	Slant and butt are yellow
K/A	Slant is red and butt is yellow
K/K	Slant is red and butt is red
K/NC	Slant is red, butt is unchanged
A/A,G	Slant and butt are yellow, gas produced
K/A,G	Slant is red and butt is yellow, gas produced
K/A,G,H2S	Slant is red and butt is yellow with black precipitate, gas produced
K/A,H2S	Slant is red, butt is yellow with black precipitate
A/A,H2S	Slant is yellow and butt is yellow with black precipitate

Rapid Urea Hydrolysis (urease)

This test is used to determine if bacteria posess the enzyme urease, with which urea is broken down, producing ammonia, a weak base. A positive result is pink; a negative result is yellow to orange.

Motility

This test is used to determine whether bacteria posess flagella that allow them to migrate away from the inoculated area. A positive result is turbidity throughout the tube; a negative result is clear in areas away from the inoculation site.

Litmus Milk (lactose use)

This is a multi-part test. If lactose is used, the solution will turn pink, which is the positive result. If protein is digested, alkaline byproducts will form, turning the solution blue, which is the positive result. If casein is digested, a curd will form, which is the positive result. If casein protein is entirely digested into its component amino acids, the solution will clear to a light brown color, which is the positive result. If litmus is reduced, the color will disappear from the solution, turning it milk white, which is the positive result.

Result	Description
A	Pink
Alk	Blue
AC	Pink, curd(s) present
ACR	Pink top, white bottome, curd(s) present
ACGR	Pink top, white bottom, solid
AlkP	Clear brown top, brown bottom
AlkP-R	White

Gelatin test (gelatinase)

This test is used to detect whether bacteria posess the enzyme gelatinase, with which gelatin is broken down into smaller peptides and amino acids. The positive result is gelatin that is liquid below 32 degrees Celsius.

Phenol Red Tests

Phenol Red Broth - Glucose

Some bacteria can use glucose to obtain energy. This is tested with a broth consisting of glucose, peptone, and phenol red (pH indicator). A Durham Tube is added to test for the presence of gas, which will be formed if the bacteria have the enzyme formic hydroen lyase, which allows them to convert pyruvate into hydrogen gas and carbon dioxide gas. If the bacteria are able to use glucose, acidic byproducts cause the broth to change in color from red to yellow. If the bacteria are unable to use glucose, but are able to use the peptone, the color will darken to purple.

Result	Description
A/G	Broth is yellow and gas is present in the Durham Tube
A/-	Broth is yellow but no gas is present in the Durham Tube
K	Broth has darkened to purple and no gas is present
-/-	No reaction

Phenol Red Broth - Sucrose

Some bacteria can use sucrose to obtain energy. This is tested with a broth consisting of sucrose, peptone, and phenol red (pH indicator). A Durham Tube is added to test for the presence of gas, which will be formed if the bacteria have the enzyme formic hydroen lyase, which allows them to convert pyruvate into hydrogen gas and carbon dioxide gas. If the bacteria are able to use sucrose, acidic byproducts cause the broth to change in color from red to yellow. If the bacteria are unable to use sucrose, but are able to use the peptone, the color will darken to purple.

Result	Description
A/G	Broth is yellow and gas is present in the Durham Tube
A/-	Broth is yellow but no gas is present in the Durham Tube
K	Broth has darkened to purple and no gas is present
-/-	No reaction

Phenol Red Broth - Mannose

Some bacteria can use mannose to obtain energy. This is tested with a broth consisting of mannose, peptone, and phenol red (pH indicator). A Durham Tube is added to test for the presence of gas, which will be formed if the bacteria have the enzyme formic hydroen lyase, which allows them to convert pyruvate into hydrogen gas and carbon dioxide gas. If the bacteria are able to use mannose, acidic byproducts cause the broth to change in color from red to yellow. If the bacteria are unable to use mannose, but are able to use the peptone, the color will darken to purple.

Result	Description
A/G	Broth is yellow and gas is present in the Durham Tube
A/-	Broth is yellow but no gas is present in the Durham Tube
K	Broth has darkened to purple and no gas is present
-/-	No reaction

Phenol Red Broth - Galactose

Some bacteria can use galactose to obtain energy. This is tested with a broth consisting of galactose, peptone, and phenol red (pH indicator). A Durham Tube is added to test for the presence of gas, which will be formed if the bacteria have the enzyme formic hydroen lyase, which allows them to convert pyruvate into hydrogen gas and carbon dioxide gas. If the bacteria are able to use galactose, acidic byproducts cause the broth to change in color from red to yellow. If the bacteria are unable to use galactose, but are able to use the peptone, the color will darken to purple.

Result	Description
A/G	Broth is yellow and gas is present in the Durham Tube
A/-	Broth is yellow but no gas is present in the Durham Tube
K	Broth has darkened to purple and no gas is present
-/-	No reaction

Phenol Red Broth - Fructose

Some bacteria can use fructose to obtain energy. This is tested with a broth consisting of fructose, peptone, and phenol red (pH indicator). A Durham Tube is added to test for the presence of gas, which will be formed if the bacteria have the enzyme formic hydroen lyase, which allows them to convert pyruvate into hydrogen gas and carbon dioxide gas. If the bacteria are able to use fructose, acidic byproducts cause the broth to change in color from red to yellow. If the bacteria are unable to use fructose, but are able to use the peptone, the color will darken to purple.

Result	Description
A/G	Broth is yellow and gas is present in the Durham Tube
A/-	Broth is yellow but no gas is present in the Durham Tube
K	Broth has darkened to purple and no gas is present
-/-	No reaction

Phenol Red Broth - Maltose

Some bacteria can use maltose to obtain energy. This is tested with a broth consisting of maltose, peptone, and phenol red (pH indicator). A Durham Tube is added to test for the presence of gas, which will be formed if the bacteria have the enzyme formic hydroen lyase, which allows them to convert pyruvate into hydrogen gas and carbon dioxide gas. If the bacteria are able to use maltose, acidic byproducts cause the broth to change in color from red to yellow. If the bacteria are unable to use maltose, but are able to use the peptone, the color will darken to purple.

Result	Description
A/G	Broth is yellow and gas is present in the Durham Tube
A/-	Broth is yellow but no gas is present in the Durham Tube
K	Broth has darkened to purple and no gas is present
-/-	No reaction

Phenol Red Broth - Mannitol

Some bacteria can use mannitol to obtain energy. This is tested with a broth consisting of glucose, peptone, and phenol red (pH indicator). A Durham Tube is added to test for the presence of gas, which will be formed if the bacteria have the enzyme formic hydroen lyase, which allows them to convert pyruvate into hydrogen gas and carbon dioxide gas. If the bacteria are able to use mannitol, acidic byproducts cause the broth to change in color from red to yellow. If the bacteria are unable to use mannitol, but are able to use the peptone, the color will darken to purple.

Result	Description
A/G	Broth is yellow and gas is present in the Durham Tube
A/-	Broth is yellow but no gas is present in the Durham Tube
K	Broth has darkened to purple and no gas is present
-/-	No reaction

Phenol Red Broth - Lactose

Some bacteria can use lactose to obtain energy. This is tested with a broth consisting of lactose, peptone, and phenol red (pH indicator). A Durham Tube is added to test for the presence of gas, which will be formed if the bacteria have the enzyme formic hydroen lyase, which allows them to convert pyruvate into hydrogen gas and carbon dioxide gas. If the bacteria are able to use lactose, acidic byproducts cause the broth to change in color from red to yellow. If the bacteria are unable to use lactose, but are able to use the peptone, the color will darken to purple.

Result	Description
A/G	Broth is yellow and gas is present in the Durham Tube
A/-	Broth is yellow but no gas is present in the Durham Tube
K	Broth has darkened to purple and no gas is present
-/-	No reaction

Reduction of nitrate

This test is used to determine whether bacteria posess the enzyme nitratase, which is used to reduce nitrate to nitrite and other nitrogen compounds. Three compounds are used to verify the results of this test. A red broth following the addition of N1 and N2 reagents is a positive result. A red broth following addition of zinc is a negative result. A colorless broth following addition of zinc is a positive result.

Thioglycolate

This test determines how bacteria use oxygen.

Result	Description
Obligate aerobe	Broth is turbid at the top
Microaerophile	A band of broth is turbid just below the top band
Facultative anaerobe	Broth is turbid throughout
Obligate anaerabe	All broth is turbid below the top, clear band

Other Tests

Oxidase test (cytochrome oxidase)

This test determines whether bacteria posess the enzyme cytochrome oxidase, which transfers electrons from the electron transport chain to oxygen, reducing it to water. A positive result is a dark blue/purple.

Catalase test

This test determines whether bacteria posess the enzyme catalase, which breaks down hydrogen peroxide into water and oxygen gas. The production of gas bubbles indicates a positive result.

Plate Tests

Milk Agar test

This test is used to determine whether bacteria posess the enzyme casease, which is used to hydrolyze the milk protein casein. Clearing of the area surrounding the colonies is a positive result.

Starch hydrolysis (amylase, oligo-1,6-glucosidase)

This test is used to determine whether bacteria posess the enzymes amylase and/or oligo-1,6-glucosidase, which secreted and hydrolize starch external to the bacteria. A positive result is a clear region surrounding the colony following the addition of iodine.

Spirit Blue Agar test (lipase)

This test is used to detect whether bacteria posess the enzyme lipase, with which triglycerols are broken down into glycerol and long chain fatty acids. The positive result is a clear region surrounding the colony.

Hemolysis test (hemolysins)

This test is used to detect whether bacteria posess enzymes known as hemolysins, which are used to lyse red blood cells and break down hemoglobin. A greenish area surrounding the colony is a positive result for alpha-hemolysin. A clear area surrounding the colony is a positive result for beta-hemolysin. No change indicates a negative result, also known as gamma hemolysis.

Antibiotic Resistance Tests

Penicillin Resistance

This test determines whether bacteria are resistant to the antibiotic penicillin. A clear area around the antibiotic disc is a positive result.

Tetracycline Resistance

This test determines whether bacteria are resistant to the antibiotic tetracycline. A clear area around the antibiotic disc is a positive result.

Erythromycin Resistance

This test determines whether bacteria are resistant to the antibiotic erythromycin. A clear area around the antibiotic disc is a positive result.

Ampicillin Resistance

This test determines whether bacteria are resistant to the antibiotic ampicillin. A clear area around the antibiotic disc is a positive result.

Ciprofloxacin Resistance

This test determines whether bacteria are resistant to the antibiotic ciprofloxacin. A clear area around the antibiotic disc is a positive result.

Vancomycin Resistance

This test determines whether bacteria are resistant to the antibiotic vancomycin. A clear area around the antibiotic disc is a positive result.

Choosing an Algorithm

Three matching algorithms are available:

Strict: This method matches every test with an equal weight and does the worst job at matching unless key test results are wrong, in which case its ability to match on less diagnostic tests can still provide the correct answer.
Weighted: This method weights the most diagnostic tests for each bacteria, ensuring that the most important tests contrbute more to the match, which generally provides the best results.
Differential: This method uses the binary tree outlined in Bergey's. This is the best method when test results are accurate and bacteria are good matches for their profile test results. Many test results are ignored in this method, as the process only continues down the tree until a match is made.

Display Mode

The default display mode (With color highlighting) uses color highlighting in the match rankings and in detailed match profiles. Color highlighting is useful for on-screen viewing or if you have a color printer. The alternate display mode (Without highlighting) is produces a better printout on non-color printers.