Biochemical Characterization Supplemental instruction Fall 2011 Chattahoochee Technical College Picture from http://www.buildinggreentv.com/node/7088 http://steveaoki.dimmak.com/blog/2009/11/useless-information-4-bacteria/ http://www.scienceclarified.com/As-Bi/Bacteria.html Designed by Pyeongsug Kim ©2010 webmaster@science-i.com

Designed by Pyeongsug Kim, ©2010 www.science-i.com Selective media :allows the growth of certain types of organisms, while inhibiting the growth of other organisms. -MSA(Manitol Salt Agar) -EMB(Eosin Methylene Blue) Picture from: http://www2.raritanval.edu/departments/Science/full-time/Weber/Microbiology%20Majors/Chapter6sub/Chapte r6sub_print.html Designed by Pyeongsug Kim, ©2010 www.science-i.com

Designed by Pyeongsug Kim, ©2010 www.science-i.com Differential media :allows one to distinguish between or among different microorganisms based on a difference in colony appearance (color, shape, or growth pattern) on the medium. -MR-VP -MSA(Manitol Salt Agar) -SIM -Phenol red broths -EMB(Eosin Methylene Blue) -Blood agar Picture from: http://www2.raritanval.edu/departments/Science/full-time/Weber/Microbiology%20Majors/Chapter6sub/Chapte r6sub_print.html Designed by Pyeongsug Kim, ©2010 www.science-i.com

Designed by Pyeongsug Kim, ©2010 www.science-i.com Is TSA (Trypticase soy agar ) differential agar or selective agar? Differential Neither!!! (sorry!) TSA and NA media are not differential or selective growth medium. Both Gram-positive and Gram-negative bacteria grow on this medium. Hence, the purpose of this medium is to select bacteria based upon differences in colony morphology. I said wrongfully on Thur.(11/3/11) …Sorry!! Picture from http://elkhorn.unl.edu/epublic/pages/publicationD.jsp?publicationId=1198 Designed by Pyeongsug Kim, ©2010 www.science-i.com

Designed by Pyeongsug Kim, ©2010 www.science-i.com Mannitol Salt Agar (MSA) -selective and differential -ability to tolerate 7% salt concentration and ferment mannitol. -Why Selective? inhibit the growth of Gram negative bacteria (selective for Gram positive) -Why differential? differentiates the salt tolerant organisms on their ability to ferment mannitol. Growth with no mannitol fermentation. Growth with mannitol fermentation. Designed by Pyeongsug Kim, ©2010 www.science-i.com

Designed by Pyeongsug Kim, ©2010 www.science-i.com Mannitol Salt Agar (MSA) -selective and differential A: B: C: Positive(mannitol fermentation) Inhibit growth Negative Designed by Pyeongsug Kim, ©2010 www.science-i.com

Designed by Pyeongsug Kim, ©2010 www.science-i.com EMB(Eosin Methylene Blue) -selective and differential -Why Selective? inhibit the growth of Gram positive bacteria (selective for Gram negative) -Why differential? differentiates coliforms (Lactose fermenters) Fermenters ~ Dark purple(or black) or Green sheen Non-fermeners ~ pink or colorless Designed by Pyeongsug Kim, ©2010 www.science-i.com

Designed by Pyeongsug Kim, ©2010 www.science-i.com EMB(Eosin Methylene Blue) A C B A: B: C: Positive Negative Designed by Pyeongsug Kim, ©2010 www.science-i.com

Designed by Pyeongsug Kim, ©2010 www.science-i.com Hemolytic reaction test -differential test: test for hemolytic reactions  differential for the ability of hemolysis. -used Blood agar -“alpha”, “beta”, “gamma” hemolysis Don’t say “positive” or “negative.” Alpha hemolysis ~ partially break down hemoglobin; a greenish discoloration that surrounds a bacterial colony Beta hemolysis ~ complete lysis of red blood cells and hemoglobin. Clearing of the blood around the colonies. Gamma hemolysis ~ no hemolysis http://www.midlandstech.edu/science/kelleherk/225/labmaterials/sel_diff_media.html Designed by Pyeongsug Kim, ©2010 www.science-i.com

Designed by Pyeongsug Kim, ©2010 www.science-i.com Hemolytic reaction test A B C A: B: C: Alpha hemolysis Gamma hemolysis Beta hemolysis Picture from http://iws2.collin.edu/dcain/CCCCD%20Micro/hemolysis.htm Designed by Pyeongsug Kim, ©2010 www.science-i.com

Designed by Pyeongsug Kim, ©2010 www.science-i.com Extracellular Amylase test -Differential test  differential for the ability to produce extra-cellular amylase to digest starch. -used starch agar -test with gram-iodine (used mordant in Gram stainning)  Iodine is used to test the presence of starch. Black/Blue ~; presence of starch; absence of enzyme (Negative) Light brown ~ absence of starch; presence of enzyme (Positive) Negative Positive Picture from: http://web.fccj.org/~lnorman/unknowns.htm?index=2 Designed by Pyeongsug Kim, ©2010 www.science-i.com

Designed by Pyeongsug Kim, ©2010 www.science-i.com SIM Test -Differential test (media) -Semisolid Hydrogen Sulfide production (from sodium thiosulfate)  Reduce sulfur? Black ~ reduced sulfur or H2S produced (positive). Yellow ~ Not H2S produced (negative) Indole test Indole production from the amino acid, tryptophan use Kovac’s agent  Indole produced? Motility Does the bacterium have one or more flagella that propel the cell? Picture from http://people.rit.edu/~gtfsbi/IntroMicro/20073IntroMicroLab4.htm Designed by Pyeongsug Kim, ©2010 www.science-i.com

Designed by Pyeongsug Kim, ©2010 www.science-i.com SIM Test Kovac’s reagent + + reduced sulfur(H2S produced) produce indole Motile? No Designed by Pyeongsug Kim, ©2010 www.science-i.com

Designed by Pyeongsug Kim, ©2010 www.science-i.com SIM Test Which one contains bacteria that have flagella? Right SIM tube Left SIM tube (sorry!) What type of test? What is the purpose of this test? Which one is positive? Indole test Determine if indole is produced. Left SIM tube Designed by Pyeongsug Kim, ©2010 www.science-i.com

Designed by Pyeongsug Kim, ©2010 www.science-i.com Catalase Test -Differential test -used to identify organisms that produce the catalase(enzyme). Catalase ~ detoxifies hydrogen peroxide by breaking it down into water and oxygen gas (bubble!). Bubble! + Designed by Pyeongsug Kim, ©2010 www.science-i.com

Designed by Pyeongsug Kim, ©2010 www.science-i.com Oxidase Test -Differential test -used to identify to determine if a bacterium produces certain cytochrome c oxidases.  can therefore utilize oxygen for energy production with an electron transfer chain. -Aerobes, facultative aerobes, microphilles -Turn blue-purple Designed by Pyeongsug Kim, ©2010 www.science-i.com

Designed by Pyeongsug Kim, ©2010 www.science-i.com Phenol red(PR) broth Differential test Contain pH indicator phenol red. -Detect fermentation of carbohydrate depending on the type of carbohydrates. PR glucose broth, PR mannitol broth, PR lactose broth, PR sucrose broth or other carbohydrates. A/G Fermentation with acid and gas production A/- Fermentation with acid but no gas production -/- No Fementation Designed by Pyeongsug Kim, ©2010 www.science-i.com

Designed by Pyeongsug Kim, ©2010 www.science-i.com Phenol red(PR) broth a) b) c) d) Red ~ No Fementation Pink ~ No Fementation but degradation of peptone(protein) Yellow/No gas ~ Fementation and no gas production Yellow/ Gas ~ Fementation and gas production Designed by Pyeongsug Kim, ©2010 www.science-i.com

Designed by Pyeongsug Kim, ©2010 www.science-i.com Methyl Red/Voges Proskauer (MR/VP) -used to determine which fermentation pathway is used to utilize glucose. -MR-VP broth + MR test -tests for mixed acid end products from glucose fermentation. -use methyl red. Red ~ mixed-acid produced (positive) Yellow ~ no acid produced (negative) VP test -test for acetoin production from glucose fermentation. - use reagent A & reagent B - Red ~ acetoin produced (positive) Yellow to brown ~ No acetoin produced (negative) Picture from http://web.clark.edu/tkibota/240/Unknowns/MethylRed.htm Designed by Pyeongsug Kim, ©2010 www.science-i.com

Designed by Pyeongsug Kim, ©2010 www.science-i.com Methyl Red/Voges Proskauer (MR/VP) Red Positive! MR(+) A mixed-acid fermentation 3 drops of methyl red. Not red negative MR(-) 15 drops of VP reagent A 3 drops of VP reagent B Red Positive! VP(+) acetoin production After 10-60min. Not red negative VP(-) Designed by Pyeongsug Kim, ©2010 www.science-i.com