Starch Hydrolysis

98
EXERCISES
FOR
THE
MICROBIOLOGY
LABORATORY
5-13
Lab Two
STARCH HYDROLYSIS
Photographic Atlas Reference Starch 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 subtilis Staphylococcus au reus
PROCEDURE
1. Remove the plate from the incubator and note the location and appearance of the growth before adding the iodine. (Growth that is thinning at the edge may give the appearance of clearing in the agar after iodine is added to the plate.) 2. Cover the growth and surrounding areas with Gram iodine. Immediately examine the areas surrounding the growth for clearing. (Usually the growth on the agar prevents contact between the starch and iodine so no color reaction takes place at that point. Beginning students sometimes look at this lack of color change and incorrectly judge it as a positive result. Therefore, when examining the agar for clearing, look for a halo around the growth, not at the growth itself.) 3. Record your results in the table provided.
REFERENCES Collins, 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, edited by R. R. Colwell and R. Grigorova, Academic Press Inc., New York, NY. MacFaddin, Jean F. 2000. Page 412 in Biochemical Tests for Identification of Medical Bacteria, 2nd Ed. Lippincott Williams & Wilkins, Philadelphia, PA. Smibert, Robert M. and Noel R. Krieg. 1994. Page 630 in Methods for General and Molecular Bacteriology, edited by Philipp .I . Gerhardt, R. G. E. Murray, Willis A. Wood, and Noel R. Krieg, American Society for Microbiology, Washington, DC.
Lab One 1. Using a marking pen, divide the starch agar plate into three equal sectors. Be sure to mark on the bottom of the 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.
(()tM,...~}
It
PI'!f7YW~
~ __ . I
U\
d\~(~
TA B L E 5 - I 3
RESULT
{ W)"dosi1
RESULTS
INTERPRETATION
AMYLASE
T EST
Amylase is present
AND
INTERPRETATIONS
SYMBOL
Clearing around growth 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
ORGANISM COLOR RESULT
AND
+/-
INTERPRETATIONS
INTERPRETATION
Uninoculated Sector
~"'M~
~
t?~
swbtJM.
-(-
Starch Hydrolysis
PURPOSE This test is used to differentiate bacteria
based on their ability to hydrolyze starch with the enzyme a-amylase or oligo-l,6-glucosidase. It aids in the differentiation of species from the genera Corynebacterium, Clostridium, Bacillus, Bacteroides, Fusobacterium, and members of Enterococcus .
PRINCIPLE Starch is a polysaccharide made up of

a-D-glucose subunits. It exists as a mixture of two forms, linear (amylose) and branched (amylopectin), with the branched configuration being the predominant form. The a-D-glucose molecules in both amylose and amylopectin are bonded by 1,4-a-glycosidic (acetal) linkages (Figure 6-83). The two forms differ in that the amylopectin contains polysaccharide side chains connected to approximately every 30th glucose in the main chain. These side chains are identical to the main chain except that the number 1 carbon of the first glucose in the side chain is bonded to carbon number 6 of the main chain glucose. The bond is, therefore, a 1,6-a-glycosidic linkage. Starch is too large to pass through the bacterial cell membrane. Therefore, to be of metabolic value to the bacteria it must first be split into smaller fragments or individual glucose molecules. Organisms that produce and secrete the extracellular enzymes a-amylase and oligo-l,6-glucosidase are able to hydrolyze starch by breaking the glycosidic linkages between the sugar subunits. Although there usually are intermediate steps and additional enzymes utilized, the overall reaction is the complete hydrolysis of the polysaccharide to 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 produce a-amylase and oligo-Le-glucosidase are grown on starch agar theyhydrolyzethe starch in the medium surrounding the bacterial growth. Because both the starch and its sugar subunits are soluble (Clear) in the medium, the reagent iodine is used to detect the presence or absence of starch in the vicinity around the bacterial growth. Iodine reacts with starch and produces a blue or dark brown color; therefore, any microbial starch hydrolysis will be revealed as a clear zone 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 H

a-Amylose
[1 ,4-a-glucosidic (acetal) linkages]
a-D-Glucose (many)
-S-o
0-s-
0",,-
- -0
_Q-
HOCH
0 2
-Q
0
HOCH OH
::y
0_ 2 0
-Q-
CH
2 0 0
-Q-
HOCH
0
2
0
OH
OH
OH
HOCH QHO OH
0
l.
a-Amylase

0 --
Oligo-1 ,6-glucosidase
QHOCH2 HOOH OH OH
0
Amylopectin
[1 ,4-a-glucosidic (acetal) linkages and 1,6-a-glucosidic (acetal) branch linkages] Figure 6-83.
a-D-Glucose (many)
Starch Hydrolysis by a-Amylase and oligo-l,6-Glucosidase.
Sulfur Reduction (SIM Medium)

PURPOSE The Sulfur Reduction Test is used to differentiate members of Enterobacteriaceae, especially the sulfurreducing Salmonella, Frandsella, and Proteus from the non-reducing Morganella morganii and Providencia rettgeri . PRINCIPLE The Sulfur Reduction Test, as it appears in this manual, is performed using SIM medium. SIM medium also tests for indole production (page 60) and motility (page 67). It is a semi-solid medium that is formulated with casein and animal tissue as sources of amino acids, an ironcontaining compound, and sulfur in the form of sodium thiosulfate. Sulfur reduction to H2S is an anaerobic activity and can be accomplished by bacteria in two different ways, depending on the enzymes present. The enzyme cysteine desulfurase catalyzes the putrefaction of the amino acid cysteine to
Figure 6-84.
Starch Hydrolysis Test. A starch agar plate with iodine added to detect amylase activity. Escherichia coli (negative) is above and Bacillus cereus (positive) is below.

Starch Hydrolysis

Uploaded by

Copyright:

Available Formats

Starch Hydrolysis

Uploaded by

Document Information

Original Description:

Copyright

Available Formats

Share this document

Share or Embed Document

Sharing Options

Did you find this document useful?

Is this content inappropriate?

Copyright:

Available Formats

Starch Hydrolysis

Uploaded by

Copyright:

Available Formats

98

Photographic Atlas Reference Starch Hydrolysis Test Page 75

MATERIALS NEEDED FOR THIS EXERCISE

Clearing around growth No clearing around growth

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.)

PRINCIPLE Starch is a polysaccharide made up of

__ 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 H

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

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

Sulfur Reduction (SIM Medium)

You might also like