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  • 98 EXERCISES FOR THE MICROBIOLOGY LABORATORY

    REFERENCESCollins, C. H., Patricia M. Lyne, J. M. Grange. 1995. Page 117

    in Collins and Liyne's Microbiological Methods, 7th Ed.Butterworth-Heinemann, UK.

    DIFCO Laboratories. 1984. Page 879 in DIFCO Manual, 10th Ed.,DIFCO Laboratories, Detroit, MI.

    Lanyi, B. 1987. Page 55 in Methods in Microbiology, Vol. 19, editedby R. R. Colwell and R. Grigorova, Academic Press Inc., NewYork, NY.

    MacFaddin, Jean F. 2000. Page 412 in Biochemical Tests for Iden-tification of Medical Bacteria, 2nd Ed. Lippincott Williams &Wilkins, Philadelphia, PA.

    Smibert, Robert M. and Noel R. Krieg. 1994. Page 630 in Methods(()tM,...~} for General and Molecular Bacteriology, edited by Philipp

    It ~ __ . I . I . Gerhardt, R. G. E. Murray, Willis A. Wood, and Noel R. Krieg,PI'!f7YW~ American Society for Microbiology, Washington, DC.

    U\d\~(~ {W)"dosi1

    5-13 STARCH HYDROLYSISPhotographic Atlas ReferenceStarch Hydrolysis Test Page 75

    MATERIALS NEEDED FOR THIS EXERCISE

    Per Student Group One starch agar plate Gram iodine (from your Gram stain kit) Recommended organisms:

    Bacillus subtilisStaphylococcus aureus

    PROCEDURE

    Lab One1. Using a marking pen, divide the starch agar plate into

    three equal sectors. Be sure to mark on the bottom ofthe plate.

    2. Label the plate with the organisms' names, your name,and the date.

    3. Spot inoculate two sectors with the test organisms.4. Invert the plate and incubate it aerobically at 35C for

    48 hours.

    Lab Two1. Remove the plate from the incubator and note the loca-

    tion and appearance of the growth before adding theiodine. (Growth that is thinning at the edge may give theappearance of clearing in the agar after iodine is addedto the plate.)

    2. Cover the growth and surrounding areas with Gramiodine. Immediately examine the areas surrounding thegrowth for clearing. (Usually the growth on the agarprevents contact between the starch and iodine so nocolor reaction takes place at that point. Beginning stu-dents sometimes look at this lack of color change andincorrectly judge it as a positive result. Therefore, whenexamining the agar for clearing, look for a halo aroundthe growth, not at the growth itself.)

    3. Record your results in the table provided.

    TA B L E 5 - I 3 AMYLASE T EST RESULTS AND INTERPRETATIONSRESULT INTERPRETATION SYMBOL

    Clearing around growth Amylase is present +

    No clearing around growth No amylase is present -

    OBSERVATIONS AND INTERPRETATIONS

    Using Table 5-13 as a guide, enter your results and interpretations in the table below. (See Photographic Atlas Figure 6-84.)

    OBSERVATIONS AND INTERPRETATIONSORGANISM COLOR RESULT +/- INTERPRETATION

    Uninoculated Sector

    t?~ swbtJM. -(-- ~ -~"'M~ -

    ~

  • Starch Hydrolysis PURPOSE This test is used to differentiate bacteriabased on their ability to hydrolyze starch with the enzymea-amylase or oligo-l,6-glucosidase. It aids in the differ-entiation of species from the genera Corynebacterium,Clostridium, Bacillus, Bacteroides, Fusobacterium, andmembers of Enterococcus .

    PRINCIPLE Starch is a polysaccharide made up ofa-D-glucose subunits. It exists as a mixture of two forms,linear (amylose) and branched (amylopectin), with thebranched configuration being the predominant form. Thea-D-glucose molecules in both amylose and amylopectin arebonded by 1,4-a-glycosidic (acetal) linkages (Figure 6-83).The two forms differ in that the amylopectin contains poly-saccharide side chains connected to approximately every30th glucose in the main chain. These side chains are iden-tical to the main chain except that the number 1 carbon ofthe first glucose in the side chain is bonded to carbon num-ber 6 of the main chain glucose. The bond is, therefore, a1,6-a-glycosidic linkage.

    Starch is too large to pass through the bacterial cellmembrane. Therefore, to be of metabolic value to the bac-teria it must first be split into smaller fragments or individualglucose molecules. Organisms that produce and secrete theextracellular enzymes a-amylase and oligo-l,6-glucosidaseare able to hydrolyze starch by breaking the glycosidic link-ages between the sugar subunits. Although there usually areintermediate steps and additional enzymes utilized, the over-all reaction is the complete hydrolysis of the polysaccharideto its individual a-glucose subunits (Figure 6-83).

    Starch agar is a simple plated medium of beef extract,soluble starch and agar. When organisms that producea-amylase and oligo-Le-glucosidase are grown on starchagar theyhydrolyzethe starch in the medium surroundingthe bacterial growth. Because both the starch and its sugarsubunits are soluble (Clear) in the medium, the reagentiodine is used to detect the presence or absence of starch inthe vicinity around the bacterial growth. Iodine reacts withstarch and produces a blue or dark brown color; therefore,any microbial starch hydrolysis will be revealed as a clearzone surrounding the growth (Figure 6-84).

  • __O-Q-"OCH,0 o-Q-"OC",0 o_Q-"OC"'0 o~Q-"o,", 0 o-Q"OC"' 0 0 __"...,::(__a.;Amylase.OH OH OH OH OH

    H H H H Ha-Amylose

    [1 ,4-a-glucosidic (acetal) linkages]a-D-Glucose

    (many)

    , -S-o

    0-s-

    ::y 0",,-

    _Q-HOCH

    20 -QHOCH20_ -Q-CH2 0 -Q-HOCH20 Q-HOCH20 QHOCH20HOl. a-Amylase- -0 0 0 0 0 0 -- HO OHOH OH OH OH OH Oligo-1 ,6-glucosidase OHH H H H H OH

    f

    Amylopectin[1 ,4-a-glucosidic (acetal) linkages and 1,6-a-glucosidic (acetal) branch linkages]

    Figure 6-83. Starch Hydrolysis by a-Amylase and oligo-l,6-Glucosidase.

    a-D-Glucose(many)

    Sulfur Reduction(SIM Medium)

    PURPOSE The Sulfur Reduction Test is used to differ-entiate members of Enterobacteriaceae, especially the sulfur-reducing Salmonella, Frandsella, and Proteus from thenon-reducing Morganella morganii and Providencia rettgeri .

    Figure 6-84. Starch Hydrolysis Test.A starch agar plate withiodine added to detect amylase activity. Escherichia coli (negative)is above and Bacillus cereus (positive) is below.

    PRINCIPLE The Sulfur Reduction Test, as it appearsin this manual, is performed using SIM medium. SIM mediumalso tests for indole production (page 60) and motility(page 67). It is a semi-solid medium that is formulated withcasein and animal tissue as sources of amino acids, an iron-containing compound, and sulfur in the form of sodiumthiosulfate.

    Sulfur reduction to H2S is an anaerobic activity and canbe accomplished by bacteria in two different ways, depend-ing on the enzymes present. The enzyme cysteine desulfurasecatalyzes the putrefaction of the amino acid cysteine to