Biochemical Test Media for Lab Unknown Identification

http://www.microbelibrary.org/ASMOnly/details.asp?id=1247&Lang= p 1/8

Biochemical Test Media for Lab Unknown Identification—Part 1 Resource Type: Visual: ImagePublication Date: 8/29/2002 Figure 1 Figure 2 Figure 3 Figure 4 Figure 5

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Authors Jackie Reynolds Math and Science Richland College Dallas, Texas 75243USAEmail: jackiesr@dcccd.edu Most bacteria of medical importance can be grown on artificial culture media. Culture media can be nonselective or selective. Nonselective media allow a wide variety of bacteria to grow (e.g., nutrient agar or blood agar). Selective media allow only certain organisms to grow because they have specific inhibitors added to the media (e.g., the bile salts in MacConkey agar). Additionally, culture media may also be differential, which allows groups of biochemically related bacteria to be distinguished from other groups of bacteria. This series of images illustrates some common biochemical media reactions for identifying bacteria. Although some bacteria can be identified by visual observation using microscopy, definitive identification usually requires further tests, many of them biochemical. Diagnostic laboratories use various biochemical media to isolate and identify bacteria from clinical specimens. These images can be used for practice questions on quizzes, lab practical reviews, or as guides for students as they are reading their own tests in lab. Figure 1. Simmon citrate. This differential test determines the ability of an organism to use citrate as its sole carbon source and is used to differentiate the Enterobacteriaceae. The uninoculated medium is green. The bromothymol blue pH indicator changes to blue when an organism is able to metabolize the citrate and produce alkaline by-products. Positive: Klebsiella pneumoniae and Enterobacter cloacae. Negative: Escherichia coli. Figure 2. Phenylalanine deaminase. This differential media identifies bacteria which possess the enzyme phenylalanine deaminase. One of the primary uses is to differentiate Proteus and Providencia from the rest of the Enterobacteriaceae. The medium contains phenylanine which, in the presence of the enzyme phenylalanine deaminase, is reduced to phenylpyruvic acid. This by-product can be detected by the addition of an oxidizing agent, which will turn the media green if the acid is present. Yellow indicates a negative result. Figure 3. Amino acid decarboxylase. This differential test determines an organism's ability to decarboxylate an amino acid. An amino acid (either lysine or ornithine) is added to the broth with a pH indicator. After inoculation, the broth is sealed with mineral oil to promote fermentation. Fermentation causes an accumulation of acid products, which in turn induces the decarboxylase enzyme (if present). Decarboxylation produces alkaline end products, indicated by a purple color. Yellow indicates a negative test.

a pH indicator determines if an organism is able to ferment mannitol. The addition of zinc dust will reduce any remaining nitrate. This differential test distinguishes organisms that can precipitate casein. lactose.asp?id=1247&Lang= p 2/8 Figure 4. Positive complete (full reduction—clear): Pseudomonas aeruginosa.org/ASMOnly/details. Negative growth (salt intolerance): Micrococcus. If gas by-products are produced a bubble will be present in the small. Phenol red broth. Figure 7. Litmus milk—casein precipitation. Sometimes the accumulation of acid following lactose fermentation can lead to the precipitation of casein. If nitrite is produced. Following incubation in a nitrate broth. while others reduce the produced nitrite even further to nitrogen gas. or sucrose) and phenol red (pH indicator) are added to a peptone medium with a small inverted tube to trap any produced gas. If the broth remains clear. no gas: Staphylococcus aureus. which is a positive indicator for mannitol fermentation.microbelibrary. This is called a "positive complete. Figure 5. further testing is needed to determine if no reduction occurred or if the nitrite was further reduced. forming an acid clot. it selects for organisms that can grow in a high salt content. Positive mannitol fermentation (yellow): S. If an organism can metabolize the sugar. . Because the medium contains 7. This differential test determines the ability of an organism to ferment sugars. causing the broth to turn pink. This indicates a negative test for nitrate reduction. Negative mannitol fermentation (no change in color): S." Positive for nitrite (red): Escherichia coli. Lactose fermentation causes a lowering of the pH. the broth will turn red. Yellow indicates an acidic pH change. acid is produced and the indicator turns yellow. the organism reduced the nitrite all the way down to another nitrogenous compound. which in turn causes the litmus to change from purple to pink. Additionally. If the broth remains clear after the addition of the zinc dust. inverted tube. Litmus milk—lactose fermentation. This differential test determines the ability of an organism to reduce nitrate. Positive growth (salt tolerance): Staphylococcus. sulphanilic acid and α-naphthylamine are added. Biochemical Test Media for Lab Unknown Identification—Part 2 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 Enlarged View Labeled View Enlarged View Enlarged View Enlarged View Figure 6. This differential test distinguishes organisms that can ferment lactose. Negative (pink): Acinetobacter calcoaceticus. Mannitol salt agar (MSA). Figure 8. Glucose fermentation Positive (yellow). epidermidis. Some organisms reduce nitrate to nitrite. aureus. A sugar (glucose. MSA is both selective and differential and is used to differentiate Staphylococcus species from each other and from Micrococcus species.Biochemical Test Media for Lab Unknown Identification http://www.5% salt. coagulase negative staphylococci. Nitrate reduction test.

org/ASMOnly/details. The lower half of the tube allows for anaerobic conditions. Pour plate technique. Positive (yellow). Figure 9. This single tube test determines if an organism is oxidative or fermentative. no gas: Staphylococcus aureus. The bacterial sample is streaked onto an agar plate containing tributyn. Sucrose fermentation Positive (yellow). This technique is used for bacterial enumeration and determines the bacterial count in a milliliter or gram of a specimen. If the bacteria does not produce and secrete lipase. Figure 13.Biochemical Test Media for Lab Unknown Identification http://www. Negative: Escherichia coli.asp?id=1247&Lang= p 3/8 Positive (yellow). Figure 12. there will be a zone of clearing surrounding the sample. while the upper half contains aerobic conditions. This differential test determines an organism's ability to produce amylase. Negative (no change): Pseudomonas aeruginosa. Each colony represents one colony forming unit (CFU). gas: Escherichia coli. This differential test determines if an organism produces the secreted enzyme lipase. a zone of clearing will surround the inoculation. Close-up of pour plate. Negative (no change): Escherichia coli and Pseudomonas aeruginosa. Positive: Serratia marcescens. The CFU/ml or CFU/g is then calculated using a standard formula. acid is . no gas: Staphylococcus aureus. a triglyceride hydrolyzed by the enzyme lipase. Spirit blue lipase test. gas: Proteus vulgaris and Escherichia coli. Lactose fermentation Positive (yellow). If the bacteria secretes lipase. gas: Proteus vulgaris. The bacterial sample is incubated on an agar plate containing starch and iodine (as an indicator).microbelibrary. Negative (no change): Proteus vulgaris and Pseudomonas aeruginosa. Uni-OF glucose media is supplemented with glucose as the carbohydrate source and a pH indicator. After incubation. Positive (yellow). the agar will remain opaque. Carbohydrates may be metabolized by one of two processes: oxidation (aerobic) or fermentation (anaerobic). Figure 10. Unified-Oxidation-Fermentation (Uni-OF) glucose test (a variation of the OxidationFermentation Hugh-Leifson Base). Negative: Escherichia coli. If an organism is able to metabolize the glucose in the condition present. Starch agar. Biochemical Test Media for Lab Unknown Identification—Part 3 Figure 11 Figure 12 Figure 13 Enlarged View Enlarged View Labeled View Figure 11. colonies appearing on the agar are counted. If the organism produces amylase. Positive: Bacillus subtilis.

Figure 17. green in lower half): Bacillus subtilis. Oxidative metabolism: (yellow in upper half. the agar will remain opaque.org/ASMOnly/details. there will be a zone of clearing surrounding the sample. Methylene blue tributyrin lipase test. A color of blue or green indicates that metabolism did not occur. Fermentative metabolism (yellow in both halves): Vibrio cholerae. This test determines if an organism is oxidative or fermentative. Hugh-Leifson glucose medium is supplemented with glucose as the carbohydrate source and a pH indicator. Oxidative metabolism indicated by yellow in the aerobic tube and green in the anaerobic tube. If the bacteria does not produce and secrete lipase. The bacterial sample is streaked onto an agar plate containing tributyrin.Biochemical Test Media for Lab Unknown Identification http://www. This differential test determines if an organism produces the secreted enzyme lipase. A color of blue or green indicates that metabolism did not occur. Figure 14. Positive: Serratia marcescens. Fermentative metabolism indicated by yellow in both tubes. Oxidation-fermentation Hugh-Leifson base. Figure 16. while the other tube is incubated under aerobic conditions. Example: Bacillus subtilis. Example: Vibrio cholerae. Figure 15. If the bacteria secretes lipase. Negative: Escherichia coli. One tube is covered with vaspar wax for anaerobic conditions. Represents uninoculated control or a negative reaction (no change).microbelibrary. . Carbohydrates may be metabolized by one of two processes: oxidation (aerobic) or fermentation (anaerobic). Example: Pseudomonas. Negative (green in both halves): Pseudomonas. Blue or green in both halves indicates that the organism cannot metabolize glucose. Biochemical Test Media for Lab Unknown Identification—Part 4 Figure 14 Figure 15 Figure 16 Figure 17 Enlarged View Enlarged View Enlarged View Labeled View Figures 14–16. An organism with oxidative metabolism will display yellow in the upper half of the tube and green in the lower half. acid is produced and the medium turns yellow. If an organism is able to metabolize the glucose in the condition present. An organism with fermentative metabolism will display yellow in both halves of the tube. a triglyceride hydrolyzed by the enzyme lipase. Blue or green in both tubes indicates that the organism cannot metabolize glucose.asp?id=1247&Lang= p 4/8 produced and the media turns yellow.

asp?id=1247&Lang= p 5/8 Legend written by: Kristen Catlin American Society for Microbiology Washington.org/ASMOnly/details. 20036 kcatlin@asmusa.microbelibrary.C. D.Biochemical Test Media for Lab Unknown Identification http://www.org Figure 1 Figure 2 Figure 3 Figure 4 .

org/ASMOnly/details.asp?id=1247&Lang= p 6/8 Figure 5 Figure 6 Figure 7 Figure 8 .Biochemical Test Media for Lab Unknown Identification http://www.microbelibrary.

Biochemical Test Media for Lab Unknown Identification http://www.org/ASMOnly/details.asp?id=1247&Lang= p 7/8 Figure 9 Figure 10 Figure 11 Figure 12 .microbelibrary.

asp?id=1247&Lang= p 8/8 Figure 13 Figure 14 Figure 15 Figure 16 Figure 17 .Biochemical Test Media for Lab Unknown Identification http://www.org/ASMOnly/details.microbelibrary.

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