Starch Agar Protocol

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Created: Thursday, 01 November 2012
Author • Archana Lal
• Naowarat Cheeptham

Information History

In 1812, the Russian chemist Gottlieb Kirchhoff hydrolyzed starch into

glucose by boiling its suspension with sulphuric acid. In 1814, J. J. Colin
and H. F. Gaultier de Claubry showed that iodine develops blue color with
starch (6). In 1815, S. S. Stromeyer confirmed this test. In 1833,
Payen and Persoz (12) isolated a white, water soluble substance from
germinating barley by ethanol precipitation. The substance was capable
of hydrolyzing starch and was named diastase. In 1883, Duclaux
introduced the custom of designating an enzyme by the substrate on
which its action was first observed and adding the suffix, "-ase." In
1835, the Swedish chemist Jons Berzelius called the hydrolysis process
"catalysis" and demonstrated that the germinating barley extract
catalyzed starch hydrolysis more efficiently than sulphuric acid
(11; http://www.scienceclarified.com/everyday/Real-Life-Physics-Vol-3-
Biology-Vol-1/Enzymes-How-it-works.html). The α-amylases were named
by Kuhn in 1925, because the hydrolysis products are in the alpha
configuration (17).

Starch agar was first developed by Vedder (13) as a suitable medium for
cultivating Neisseria. Before the development of starch agar, salt-free
veal agar (salt-free veal broth with 2% agar, neutralized to
phenolphthalein with or without 5% defibrinated rabbit blood (15)) was
used to grow cultures of Neisseria. However, one of the disadvantages of
veal agar was that transfers had to be made every 2 to 3 days in order to
keep the cultures alive.

Starch agar was developed as a medium on which Neisseria could remain

alive for a longer period (20 to 40 days, initially in an incubator at 35 to
37oC for several days and then at room temperature) without the need of
frequent transfers. Vedder (13) developed starch agar that consisted of
beef infusion agar (beef infusion with 1.5% to 1.75% agar) to which 1%
of cornstarch was added. Other kinds of starch (tapioca, potato, and
wheat) in the beef infusion agar were also tested to determine whether
starch from any source was equally suitable. Neisseria grew on all of
these starch agars better than on the ordinary media but the most
luxuriant growth was observed with cornstarch. Cornstarch appeared to
be more suitable for other organisms as well.

Vedder noticed that Neisseria grew densely on this medium after 24

hours of incubation and continued to increase growth for up to 3 or 4

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 days, after which there was no apparent increase in the density of the
growth. He also found the starch agar suitable for routine use as most of
his stock cultures grew as well or better than on plain agar. In addition,
the cultures grown on starch agar were suitable for antigen preparation
as starch did not have any adverseeffect on the cultures and the growth
was dense. Furthermore, it is known that Neisseria are fastidious,
capnophilic, and susceptible to cool temperatures, drying, and fatty acids
(http://www.life.umd.edu/classroom/bsci424/PathogenDescriptions/Neiss
eria.htm). Hence, Neisseria requires complex media that are normally
prewarmed to 35 to 37oC. Soluble starch is at times added to neutralize
fatty acid toxicity to enhance Neisseria growth
(http://www.life.umd.edu/classroom/bsci424/PathogenDescriptions/Neiss
eria.htm). Neisseria grows best in a moist atmosphere supplemented
with CO2.

Some other organisms that did not grow well on other media grew
rapidly on the starch agar (13). A number of strains of tubercle bacilli
and one of the possible leprae bacilli grew freely on the starch agar
medium (13). Dextrose starch agar was used by Wilkins, Lewis, and
Barbiers (16) in an agar dilution procedure to test the activity of
antibiotics against Neisseria species. Presently, starch agar is no longer
used for cultivating Neisseria but with pH indicators it is used to isolate
and presumptively identify Gardnerella vaginalis.

Starch agar and modified related media are also used to detect certain
bacterial contamination in food industry. For instance, a modified
selective medium containing sodium glutamate, starch, and phenol red
was invented to select forPseudomonas, both amylase positive and
amylase negative species, (8, 10) and Aeromonas in milk and dairy
products (7). In another study, starch ampicillin agar, which contains the
antibiotic ampicillin for selection and uses starch fermentation as
differentiation, was created for the isolation of Aeromonas spp. from food
products (4).

Purpose

Starch agar is a differential medium that tests the ability of an organism

to produce the extracellular enzymes (exoenzymes) α-amylase and oligo-
1,6-glucosidase that are secreted out of the bacteria and diffuse into the
starch agar. These enzymes hydrolyze starch by breaking the glycosidic
linkages between glucose subunits and allow the products of starch
hydrolysis to enter the cell.
Starch agar is also used in differentiating members of various genera
which have both amylase-positive and amylase-negative species,
including Streptococcus, Clostridium,
Corynebacterium, Fusobacterium, Enterococcus,
Pseudomonas, and Bacillus (8, 10).

Theory

Starch, a polysaccharide, is made up of α-D-glucose subunits bonded to

each other by α-glycosidic linkages. It exists as a mixture of linear α-
amylose form and branched amylopectin form, the latter being

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 predominant (8). Alpha-amylose is a linear polymer of several thousand
α-D-glucose linked by 1,4-α-glycosidic bonds. The amylopectin is larger
than amylose. It consists mainly of α-D-glucose linked by 1,4-α-
glycosidic bonds but is a branched molecule with 1,6-α-glycosidic branch
points every 24 to 30 glucose residues (14).

FIG 1 Structure of starch molecule showing the 1,4-α-glycosidic and 1,6-

α-glycosidic linkages.

Polymers such as starch molecules are too large to be transported into

the bacterial cells through the plasma membrane. Some bacteria
produce and secrete the extracellular enzymes α-amylase and oligo-1,6-
glucosidase and hydrolyze starch molecules outside the cell by breaking
the glycosidic linkages between glucose subunits. The resulting dextrin,
maltose, or glucose molecules are more readily transported into the
bacterial cell to be used inmetabolism.

Enzymatic hydrolysis of starch occurs at the α-1,4- and α-1,6-glycosidic

linkages that hold the starch polymer together. Alpha-amylase
hydrolyzes the α-1,4-glycosidic linkages of starch. It attacks the interior
of polysaccharide chains resulting in the formation of a mixture of
fragments of 5 to 9 units of the alpha configuration (10). Amylase
completely splits amylose into glucose subunits. The enzyme oligo-1,6-
glucosidase acts on 1,6-branch points as well as α-1,4-glycosidic linkages
in starch. It cleaves glucose units from the nonreducing ends of the
polysaccharide starch and results in the formation of linear or branched
dextrins and maltose. Dextrins and maltose are transported into the
bacteria and are hydrolyzed by specific intracellular enzymes (10).

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 FIG 2 Starch hydrolysis by α-amylase and oligo-1,6-glucosidase. Alpha-
amylase hydrolyzes α-1,4-glycosidic linkages of starch whereas oligo-
1,6-glucosidase acts on 1,6-branch points as well as α-1,4-
glycosidic linkages in amylopectin.

When bacteria capable of producing α-amylase and oligo-1,6-glucosidase

are grown on starch agar, they secrete these enzymes into the
surrounding areas and hydrolyze the starch (8). To detect the hydrolysis
of starch, Gram’s iodine (I2KI solution or Lugol's iodine) is used. Gram’s
iodine reacts with starch to form a dark blue, purple, or black complex
depending upon the concentration of iodine. The α-linkages give the
amylose chain a spiral conformation which is responsible for a soluble
dark blue starch-iodine complex with iodine reagent (10) and when
triiodide ions in the iodine reagent slip into this spiral structure, the
complex becomes blue. If this spiral conformation disintegrates, the blue
color is lost. Highly branched chains of amylopectin form a red insoluble
complex with iodine because they do not coil effectively (10). Upon
hydrolysis of amylopectin, the first dextrin formed is erythrodextrin,
which gives a color progressing from blue to violet to red-brown after the
addition of iodine. With further hydrolysis, the iodine color is not
produced because of the formation of colorless achroodextrins.

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 FIG 3 Diagrammatic representation of complete hydrolysis of starch
showing all the intermediates and their reaction to iodine.

RECIPE (18)

Starch agar composition (g/liter)

Beef extract 3g

Soluble starch 10 g

Agar 12 g

Distilled water 1 liter

Suspend the first three ingredients in 1 liter of distilled water. Mix

thoroughly. Heat with frequent agitation and carefully bring to just
boiling. Do not allow to boil as excessive boiling may hydrolyze the
starch (5). Autoclave at 121oC for 15 min at 15 psi. Final pH of the
medium should be 7.5 + 0.2 at 25oC. After sterilization, pour the melted
medium into sterilized petri plates (approximately 20 to 30 ml per plate)
and let it solidify before use. Prepared medium is light amber to slightly
opalescent. The prepared starch agar plates become opaque if
refrigerated (10). The prepared medium can be dispensed into screw-

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 cap tubes and stored for up to 2 weeks. After 2 weeks the starch changes
and reddish purple spots may develop upon addition of iodine (1). The
stored medium in the tubes should be melted in a boiling water bath,
poured into individual plates, and brought to room temperature before
use.
Starch agar medium is also commercially available as premixed
dehydrated powder from biological supply companies. The
manufacturer’s instructions should be followed to prepare the plates. This
medium can also be purchased as premade agar plates from biological
supply companies.

PROTOCOL

Inoculation. Use a fresh (16- to 18-hour) pure culture of test bacteria

as an inoculation source. Pick a single isolated colony and either single
streak or spot inoculate the surface of the agar medium. A single starch
agar plate can be divided into four quadrants for four different
inoculations, except when using motile organisms.

Incubation. Incubate plates for 24 to 48 hours or longer (3 to 5 days)

(3) at 35 ± 2oC in an incubator.

Starch hydrolysis test. After proper incubation, flood the surface of

the agar with Gram’s iodine solution. Record results immediately as the
blue-black color formed with starch may fade (1) giving a false-positive
result of absence of starch. Appearance of a clear zone surrounding the
bacterial growth indicates starch hydrolysis (+) by the organism due to
its production of the extracellular enzymes. The zone will start out
yellow (from the iodine) and becomeprogressively lighter yellow and then
clear. The lack of a clear zone surrounding the growth indicates that
starch is present and has not been hydrolyzed (-) and the organism did
not produce the extracellular enzymes.

Flooding plates with iodine reagent does not contaminate the

plates. Plates can be incubated further and retested if
necessary (9). (Please refer to the Tips & Comments section).

Iodine reagent should be a rich yellow-gold to brownish color and stored

in a dark bottle to avoid any light exposure. It should be tested with
known positive and negative cultures before use.

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 FIG 4 Growth of Bacillus subtilis on starch agar plate before the addition
of iodine solution (A) and after the addition of iodine solution (B). After
the addition of iodine the clearing surrounding the bacterial growth
indicates starch hydrolysis (+).

FIG 5 Growth of Escherichia coli on a starch agar plate before the

addition of iodine solution (A) and after the addition of iodine solution
(B). After the addition of iodine the dark blue or black color surrounding
the bacterial growth (lack of a clear zone) indicates absence of starch
hydrolysis (-).

SAFETY

The ASM advocates that students must successfully demonstrate the

ability to explain and practice safe laboratory techniques. For more
information, read the laboratory safety section of the ASM Curriculum
Recommendations: Introductory Course in Microbiology and
the Guidelines for Biosafety in Teaching Laboratories.

COMMENTS AND TIPS

By placing a white sheet under the plate, the yellow zone going clear will
be more easily observed.

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 If reincubation is anticipated, do not flood the plate with the iodine
reagent. Apply a few drops of iodine reagent to a small area of the agar
around the growth. Then if needed, the plate can be reincubated and
retested. There are two reasons for this variation: (i) iodine, as an
antiseptic, may kill or inhibit further growth of the bacteria and (ii)
flooding may cause the bacterial growth to lift off of the agar surface.

This protocol can lead to a class discussion on the biological and

ecological significance of amylase production or other related metabolic
exoenzymes. As questions to the students, (i) what is the purpose of the
beef extract in the medium? (ii) If starch agar were prepared without
beef extract, what would you expect to happen to the organisms you
have tested?

Microbial amylase has many important applications in industry, including

food processing (e.g., high fructose corn syrup production),
pharmaceuticals (e.g., digestive aids), and textiles (e.g., laundering
products and detergents) (2). The isolation of novel microbial
amylases (e.g., that tolerate cold, alkaline conditions, etc.) is an ongoing
discoveryprocess. Instructors are encouraged to consider such issues for
challenge problems, scientific inquiry, or libraryresearch papers.
TABLE 1 Suggested BSL1 bacteria to use for starch agar
protocol
Organism ATCC numbers Resultsa
Bacillus subtilis ATCC 6051 or ATCC 465 (++)
Bacillus cereus ATCC 2 or ATCC 13061 (+)
Staphylococcus epidermidis ATCC 155 or ATCC 14990 (-)
Escherichia coli ATCC 23724 or ATCC 33876 (-)
Serratia marcescens ATCC 264 or ATCC 43862 (-)
a
(++), strong positive reaction for starch hydrolysis; (+),
positive reaction for starch hydrolysis; (-), negative reaction for starch
hydrolysis.

REFERENCES

1. Allen PW. 1918. A simple method for the classification of bacteria as

to diastase production. J. Bacteriol. 3:15–17.
2. Madigan MT, Martinko JM, Stahl DA, Clark DP. 2012. Brock
biology of microorganisms, 13th ed. Benjamin Cummings, San
Francisco, CA. http://www.scribd.com/doc/74592238/Biology-of-
Microorganisms-13th-Ed-M-Madigan-Et-Al-Pearson-2012-BBS.
3. Collins CH, Lyne PM, Grange JM. 1995. Collins and Lyne's
microbiological methods, 7th ed, p 117. Butterworth-Heinemann,
Oxford, United Kingdom.
4. Corry JEL, Curtis GDW, Baird RM. 2003. Handbook of culture media
for food microbiology, 2nd ed. Elsevier Science, Amsterdam,
The Netherlands.
5. Cowan ST. 1974. Cowan & Steel's manual for the identification of
medical bacteria, 2nd ed, vol 12, p 148, 162. Cambridge University
Press, Cambridge, England.
6. Delly JG. 2004. The literature of classical microchemistry, spot tests,
and chemical microscopy.
http://www.modernmicroscopy.com/main.asp?article=69&page=2.

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 Accessed 29 March 2012.
7. Flint S, Hartley N. 1996. A modified selective medium for
the detection of Pseudomonas species that cause spoilage of milk and
dairy products. Int. Dairy J. 2:223–230.
8. Leboffe MJ, Pierce BE. 2010. Microbiology: laboratory theory and
application, 3rd ed. Morton Publishing Company, Englewood, CO.
9. Lennette
EH, Balows A, Hausler WJ, Jr, Shadomy HJ. 1985. Manual of clinical
microbiology, 4th ed, p 918–919, 945. American Society for
Microbiology, Washington, DC.
10. MacFaddin JF. 2000. Biochemical tests for identification of medical
bacteria, 3rd ed, p 412–423. Lippincott Williams and
Wilkins, Philadelphia, PA.
11. Marini I. 2007. Two hydrolytic enzymes and an epistemological-
historical approach. School in Science Issue
4. http://www.scienceinschool.org/repository/docs/issue4_enzymes.pdf.
12. Payen A, Persoz JF. 1833. Memoire sur la diastase, les principaux
produits de ses reactions et leur applicationsaux arts industriels. Annales
de chimie et de physique 53:73–92.
13 . Vedder EB. 1915. Starch agar, a useful culture medium. J. Infect.
Dis. 16:385–388.
14. Voet D, Voet JG. 1990. Biochemistry, p 256. John Wiley & Sons,
New York, NY.
15. Warden CC. 1915. Studies on the Gonococcus, III. J. Infect.
Dis. 16(3):426–440.
16. Wilkins JR, Lewis G, Barriers AR. 1956. Streptonivicin, a new
antibiotic. III. In vitro and in vivo
evaluation. Antibiot. Chemother. 6:149–156.
17. Worthington Biochemical Corporation. 2012. Amylase alpha,
I.U.B.: 3.2.1.1, 1,4-α-D-Glucanohydrolase. http://www.worthington-
biochem.com/AA/default.html. Accessed 10 May 2012.
18. Zimbro
MJ, Power DA, Millwer SM, Wilson GE, Johnson JA. 2009. Difco and
BBL manual: manual of biological culture media, 10th ed, p 879–
880. Becton Dickinson and Co., Sparks, MD.

REVIEWERS

This resource was peer-reviewed. Participating reviewers:

Thomas Edison dela Cruz

University of Santo Tomas, Manila, Philippines

Anne Hanson
University of Maine, Orono, ME

Roxana B. Hughes
University of North Texas, Denton, TX

Min-Ken Liao
Furman University, Greenville, SC

Karen A. Reiner

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 Andrews University, Berrien Springs, MI

Patricia Shields
University of Maryland, College Park, MD

Erica Suchman
Colorado State University, Ft. Collins, CO

Jeremy Martin O. Torres

Ateneo de Manila University, Quezon City, Philippines

2012 AD HOC PROTOCOL REVIEW COMMITTEE

Sue Katz Amburn

Rogers State University, Claremore, OK

Benita Brink
Adams State University, Alamosa, CO

Elaine Brunschwig
Cuyahoga Community College, Cleveland, OH

Laura Cathcart
University of Maryland, College Park

Deborah V. Harbour
College of Southern Nevada, Las Vegas, NV

Jackie Reynolds
Richland College, Dallas, TX

Kate Rodgers
Virginia Tech, Blacksburg, VA

CONTRIBUTORS

The following contributed to the Comments and Tips section at the ASM
Conference for Undergraduate Educators2012.

Participating contributors:

Jason C. Baker
Missouri Western State University, Saint Joseph, MO

Sarah Boomer
Western Oregon University, Monmouth, OR

Joseph Caruso
Florida Atlantic University, Boca Raton, FL

William Courctiesne
University of Nevada, Reno, NV

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 Michael Hanophy
St. Joseph’s College, Brooklyn, NY

Zoe Hawk
Arizona Western College, Yuma, AZ

Geoffrey Holm
Colgate University, Hamilton, NY

Norrenna Hubbard
Hondros College, West Chester, OH

Tamara McNealy
Clemson University, Clemson, SC

Elizabeth Mitchell
Central Piedmont Community College, Charlotte, NC

Jeffrey Pommerville
Glendale Community College, Glendale, AZ

S. N. Rajagopal
University of Wisconsin, Lacrosse, WI

Jackie Reynold
Richland College, Dallas, TX

Harlan Scott
Howard Payne University, Brownwood, TX

Lisa Spring
Central Piedmont Community College, Charlotte, NC

Jacqueline Washington
Nyack College, Nyack, NY

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