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| Instructions |
| This program is designed to aid in the identification of common bacteria through analysis of various physical characteristics and 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 on-line at microbiology101.com or to download the code and make any modifications you wish, in accordance with the Educational Community License, Version 2.0. |
| 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. 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 "No Data." 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. |
| User Name |
| Enter a user name so that your report does not get mixed up with somebody else's. User names are optional. If you do not enter a user name, "Anonymous" will be used instead. |
| Privacy notice: User names are stored temporarily in an activity log file for debugging purposes. |
| Sample ID |
| Enter a sample ID so that reports for multiple samples don't get mixed up. Sample IDs are optional. If you do not enter a sample ID, "0" will be used instead. |
| 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. |
| Select "Indeterminate" if all of the bacteria have a form that does not match any of the morphologies above. This should be a very rare selection. |
| Select "Variable" if the bacteria belong to more than one of the morphologies above and you are certain that you are working with a pure culture. Some bacteria have a variable form, where members may range in form from cocci to rods, or rods to vibrios. |
| Arrangement |
| Bacteria may connect or group together in a variety of ways. |
| Single bacteria are not joined together, but joined bacteria may also be present. |
| 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. |
| Pallisades occur when two or more bacilli join together along their longer dimension. |
| 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. |
| Lenth |
| The length of a bacterium is measured under the microscope with a micrometer. Best results are achieved by measuring 10-20 bacteria at random and averaging the results. Enter your average measurement in micrometers (μm). |
| Do not enter a lenth if the bacterium is a coccus. Instead, enter the diamter in the Width/Diameter box. |
| Width or Diameter |
| The width or diameter of a bacterium is measured under the microscope with a micrometer. Best results are achieved by measuring 10-20 bacteria at random and averaging the results. Enter your average measurement in micrometers (μm). |
| When working with spiral bacteria, measure the actual width of the bacterial body, not the width of the spiral. |
| Colony Color on Nutrient Agar |
| Many bacteria produce colonies that have distinctive color on various media. Select the closest color to the available options or, if the color occupies a mid-point between the available options, select "Indeterminate". If colonies vary in color, select "Variable". |
| Make sure the colony color was determined on nutrient agar. Other media may produce colonies of a different color. |
| Colony Elevation on Nutrient Agar |
| Colony Elevation is used to describe the shape and extent of the colony's rise above the surface of the media. |
| Flat colonies appear uniform and equal in elevation to the surface of the medium. |
| Colonies with a raised margin appear flat, but rise above the surface of the media at the outer edge. Unlike convex colonies, most of the colony appears flat. |
| Convex colonies gradually rise from the middle of the colony to the edge. |
| Umbonate |
| Raised colonies have an elevation higher than the surrounding medium. |
| Plateau |
| Colonies that grow into the medium noticeably penetrate the surface of the medium. |
| Make sure the colony elevation was determined on nutrient agar. Other media may produce different results. |
| Colony Form (Shape) on Nutrient Agar |
| Colony Form is used to describe the shape of the colony, viewed from above or below. |
| Round colonies are round, but do not necessarily form a perfect circle. |
| Rhizoid colonies form paths on the surface of the medium. |
| Filamentous colonies send out tendrils across the surface of the medium. |
| Irregular colonies form shapes that do not fit in the categories above. |
| Swarming colonies move and converge. |
| Make sure the colony form was determined on nutrient agar. Other media may produce different results. |
| Colony Margin on Nutrient Agar |
| Colony Margin is used to describe the quality of the edge of the colony. |
| Entire (or smooth) edges are uniform. |
| Lobate edges form smooth outgrowths or lobes. |
| Undulate edges are smooth, but wavy. |
| Rhizoid edges form pathways between colonies. |
| Filamentous edges produce tendrils. |
| Irregular (or erose) edges do not fit into the categories above. |
| Make sure the colony margin was determined on nutrient agar. Other media may produce different results. |
| Colony Texture on Nutrient Agar |
| Colony Texture is used to describe the appearance of the surface of the colony. |
| Dry colonies may appear dull or powedery. |
| Rough colonies may have noticeable irregularities in the surface. |
| Mucoid colonies appear smooth and moist. |
| Make sure the colony texture was determined on nutrient agar. Other media may produce different results. |
| Colony Opacity on Nutrient Agar |
| Colony Opacity is used to describe the degree to which the colony passes light. |
| Opaque colonies pass no light. |
| Translucent colonies pass some light but are not clear. |
| Transparent colonies pass light and can been seen through to the underlying medium. |
| Make sure the colony opacity was determined on nutrient agar. Other media may produce different results. |
| Culture Odor on Nutrient Agar |
| The odor of bacteria can be a somewhat subjective characteristic, but is occasionally useful in identification. |
| Be very careful when smelling bacterial cultures. Do not smell any bacterial culture unless you know what you are doing or you have the approval of someone who does. Waft the odor toward you, keeping the plate away from your face. Always assume that unknown bacteria are pathogenic. |
| Cultures with a fruity odor are sometimes described as grapey or wine-like. |
| Cultures with a cheesy odor are sometimes described as smelling like Limburger cheese or dirty socks. |
| Cultures with a fecal odor have the smell of sewage. |
| Make sure the culture odor was determined on nutrient agar. Other media may produce different results. |
| 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. |
| 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. |
| Hydrogen Sulfide |
| 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. |
| Tests for hydrogen sulfide include Kliger's Iron Agar and Triple Sugar Iron Agar. |
| 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 Reduction |
| Litmus Reduction is generally determined through Litmus Milk medium. If litmus is reduced, the color will disappear from the solution, turning it milk white, which is the positive result. |
| 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. |
| Acid from Fermentation of Arabinose |
| Some bacteria can use arabinose to obtain energy. This is usually tested with Phenol Red Broth - a broth consisting of arabinose, peptone, and phenol red (pH indicator). Bacteria that are able to use arabinose produce acidic byproducts cause the broth to change in color from red to yellow, the positive result. If the bacteria are unable to use arabinose, but are able to use the peptone, the color will darken to purple. |
| Acid from Fermentation of Fructose |
| Some bacteria can use fructose to obtain energy. This is usually tested with Phenol Red Broth - a broth consisting of fructose, peptone, and phenol red (pH indicator). Bacteria that are able to use fructose produce acidic byproducts cause the broth to change in color from red to yellow, the positive result. If the bacteria are unable to use fructose, but are able to use the peptone, the color will darken to purple. |
| Acid from Fermentation of Galactose |
| Some bacteria can use galactose to obtain energy. This is usually tested with Phenol Red Broth - a broth consisting of galactose, peptone, and phenol red (pH indicator). Bacteria that are able to use galactose produce acidic byproducts cause the broth to change in color from red to yellow, the positive result. If the bacteria are unable to use galactose, but are able to use the peptone, the color will darken to purple. |
| Acid from Fermentation of Glucose |
| Some bacteria can use glucose to obtain energy. This is usually tested with Phenol Red Broth - a broth consisting of glucose, peptone, and phenol red (pH indicator). Bacteria that are able to use glucose produce acidic byproducts cause the broth to change in color from red to yellow, the positive result. If the bacteria are unable to use glucose, but are able to use the peptone, the color will darken to purple. |
| Acid from Fermentation of Lactose |
| Some bacteria can use lactose to obtain energy. This is usually tested with Phenol Red Broth - a broth consisting of lactose, peptone, and phenol red (pH indicator). Bacteria that are able to use lactose produce acidic byproducts cause the broth to change in color from red to yellow, the positive result. If the bacteria are unable to use lactose, but are able to use the peptone, the color will darken to purple. |
| Acid from Fermentation of Maltose |
| Some bacteria can use maltose to obtain energy. This is usually tested with Phenol Red Broth - a broth consisting of maltose, peptone, and phenol red (pH indicator). Bacteria that are able to use maltose produce acidic byproducts cause the broth to change in color from red to yellow, the positive result. If the bacteria are unable to use maltose, but are able to use the peptone, the color will darken to purple. |
| Acid from Fermentation of Mannitol |
| Some bacteria can use mannitol to obtain energy. This is usually tested with Phenol Red Broth - a broth consisting of mannitol, peptone, and phenol red (pH indicator). Bacteria that are able to use mannitol produce acidic byproducts cause the broth to change in color from red to yellow, the positive result. If the bacteria are unable to use mannitol, but are able to use the peptone, the color will darken to purple. |
| Acid from Fermentation of Mannose |
| Some bacteria can use mannose to obtain energy. This is usually tested with Phenol Red Broth - a broth consisting of mannose, peptone, and phenol red (pH indicator). Bacteria that are able to use mannose produce acidic byproducts cause the broth to change in color from red to yellow, the positive result. If the bacteria are unable to use mannose, but are able to use the peptone, the color will darken to purple. |
| Acid from Fermentation of Rhamnose |
| Some bacteria can use rhamnose to obtain energy. This is usually tested with Phenol Red Broth - a broth consisting of rhamnose, peptone, and phenol red (pH indicator). Bacteria that are able to use rhamnose produce acidic byproducts cause the broth to change in color from red to yellow, the positive result. If the bacteria are unable to use rhamnose, but are able to use the peptone, the color will darken to purple. |
| Acid from Fermentation of Sucrose |
| Some bacteria can use sucrose to obtain energy. This is usually tested with Phenol Red Broth - a broth consisting of sucrose, peptone, and phenol red (pH indicator). Bacteria that are able to use sucrose produce acidic byproducts cause the broth to change in color from red to yellow, the positive result. If the bacteria are unable to use sucrose, but are able to use the peptone, the color will darken to purple. |
| Acid from Fermentation of Trehalose |
| Some bacteria can use trehalose to obtain energy. This is usually tested with Phenol Red Broth - a broth consisting of trehalose, peptone, and phenol red (pH indicator). Bacteria that are able to use trehalose produce acidic byproducts cause the broth to change in color from red to yellow, the positive result. If the bacteria are unable to use trehalose, but are able to use the peptone, the color will darken to purple. |
| Acid from Fermentation of Xylose |
| Some bacteria can use xylose to obtain energy. This is usually tested with Phenol Red Broth - a broth consisting of xylose, peptone, and phenol red (pH indicator). Bacteria that are able to use xylose produce acidic byproducts cause the broth to change in color from red to yellow, the positive result. If the bacteria are unable to use xylose, but are able to use the peptone, the color will darken to purple. |
| Gas from Fermentation of Arabinose |
| Some bacteria can use arabinose to obtain energy and may produce gas during fermentation. A Durham Tube is added to test for the presence of gas, which will be formed if the bacteria have the enzyme formic hydrogen lyase, which allows them to convert pyruvate into hydrogen gas and carbon dioxide gas. Any gas in the tube is the positive result. |
| Gas from Fermentation of Fructose |
| Some bacteria can use fructose to obtain energy and may produce gas during fermentation. A Durham Tube is added to test for the presence of gas, which will be formed if the bacteria have the enzyme formic hydrogen lyase, which allows them to convert pyruvate into hydrogen gas and carbon dioxide gas. Any gas in the tube is the positive result. |
| Gas from Fermentation of Galactose |
| Some bacteria can use galactose to obtain energy and may produce gas during fermentation. A Durham Tube is added to test for the presence of gas, which will be formed if the bacteria have the enzyme formic hydrogen lyase, which allows them to convert pyruvate into hydrogen gas and carbon dioxide gas. Any gas in the tube is the positive result. |
| Gas from Fermentation of Glucose |
| Some bacteria can use glucose to obtain energy and may produce gas during fermentation. A Durham Tube is added to test for the presence of gas, which will be formed if the bacteria have the enzyme formic hydrogen lyase, which allows them to convert pyruvate into hydrogen gas and carbon dioxide gas. Any gas in the tube is the positive result. |
| Gas from Fermentation of Lactose |
| Some bacteria can use lactose to obtain energy and may produce gas during fermentation. A Durham Tube is added to test for the presence of gas, which will be formed if the bacteria have the enzyme formic hydrogen lyase, which allows them to convert pyruvate into hydrogen gas and carbon dioxide gas. Any gas in the tube is the positive result. |
| Gas from Fermentation of Maltose |
| Some bacteria can use maltose to obtain energy and may produce gas during fermentation. A Durham Tube is added to test for the presence of gas, which will be formed if the bacteria have the enzyme formic hydrogen lyase, which allows them to convert pyruvate into hydrogen gas and carbon dioxide gas. Any gas in the tube is the positive result. |
| Gas from Fermentation of Mannitol |
| Some bacteria can use mannitol to obtain energy and may produce gas during fermentation. A Durham Tube is added to test for the presence of gas, which will be formed if the bacteria have the enzyme formic hydrogen lyase, which allows them to convert pyruvate into hydrogen gas and carbon dioxide gas. Any gas in the tube is the positive result. |
| Gas from Fermentation of Mannose |
| Some bacteria can use mannose to obtain energy and may produce gas during fermentation. A Durham Tube is added to test for the presence of gas, which will be formed if the bacteria have the enzyme formic hydrogen lyase, which allows them to convert pyruvate into hydrogen gas and carbon dioxide gas. Any gas in the tube is the positive result. |
| Gas from Fermentation of Rhamnose |
| Some bacteria can use rhamnose to obtain energy and may produce gas during fermentation. A Durham Tube is added to test for the presence of gas, which will be formed if the bacteria have the enzyme formic hydrogen lyase, which allows them to convert pyruvate into hydrogen gas and carbon dioxide gas. Any gas in the tube is the positive result. |
| Gas from Fermentation of Sucrose |
| Some bacteria can use sucrose to obtain energy and may produce gas during fermentation. A Durham Tube is added to test for the presence of gas, which will be formed if the bacteria have the enzyme formic hydrogen lyase, which allows them to convert pyruvate into hydrogen gas and carbon dioxide gas. Any gas in the tube is the positive result. |
| Gas from Fermentation of Trehalose |
| Some bacteria can use trehalose to obtain energy and may produce gas during fermentation. A Durham Tube is added to test for the presence of gas, which will be formed if the bacteria have the enzyme formic hydrogen lyase, which allows them to convert pyruvate into hydrogen gas and carbon dioxide gas. Any gas in the tube is the positive result. |
| Gas from Fermentation of Xylose |
| Some bacteria can use xylose to obtain energy and may produce gas during fermentation. A Durham Tube is added to test for the presence of gas, which will be formed if the bacteria have the enzyme formic hydrogen lyase, which allows them to convert pyruvate into hydrogen gas and carbon dioxide gas. Any gas in the tube is the positive result. |
| 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. |