Roth 0039 User guide

1

GELRITE

®

0039

Gellan Gum for Microbiological Applications

GELRITE® is a trademark of Merck & Co., Inc. (Rahway, NJ) Kelco Division, U.S.A.

Developed especially for use as a gelling agent for microbiological media, GELRITE® gellan gum is a highly­purified, natural anionic heteropolysaccharide that forms rigid, brittle, agar-like gels at approximately half the
use level of agar, in the presence of soluble salts.

1,2,3

GELRITE® is the ideal gelling agent for a wide range of gelling applications, for the following reasons:

Advantages of GELRITE® Compared to Agar

 GELRITE® gellan gum may be used at approximately half the use level of agar
 GELRITE® , produced by a tightly-controlled fermentation process, has consistent product quality.

GELRITE® is unaffected by the vagaries of natural conditions which affect the basic properties of agar.

 GELRITE® gels are remarkably clear in comparison to those formed with agar.
 Gels prepared with GELRITE® set faster than those made with agar. In microbiological applications this

reduces plate preparation time.

 Gels prepared with GELRITE® are stable at high temperatures. In microbiological media, this supports

incubation required by thermophilic microorganisms.

 GELRITE® contains no contaminating matters (e.g., phenolic compounds) as those found in agar that are

toxic to certain sensitive organisms.

Ease of Processing with GELRITE® Media

 GELRITE® gellan gum disperses and hydrates easily in either hot or cold deionized water, forming

viscous solutions in cold distilled water.

 In the presence of soluble salts, GELRITE® can be used to provide high gel strength at low GELRITE®

concentrations (normally at approximately half the concentration required for agar).

 At high temperatures, the low viscosity of GELRITE® solutions facilitates pipetting, pumping, and pouring

upon cooling, GELRITE® solutions gel quickly and uniformly.

 GELRITE® is able to withstand normal autoclaving conditions.
 GELRITE® is generally resistant to enzymatic degradation.
 GELRITE®itself is chemically inert to most biological growth media additives (additive must be heated to

just above GELRITE® gel point before incorporation).

1

For further explanations of GELRITIE`gellan gum as an agar replacer, refer to Kelco Applications Bulletin CD-33.

2

U.S. patents 4, 326, 052 and 4, 326, 053.

3

GELRITE® gellan gum may be marketed as an in vitro diagnostic device (U.S. Food & Drug Administration review under

21 U.S.C. 510(k)).

Instructions for use

2

Quality of Microbiological Media Prepared with GELRITE®

 GELRITE® gellan gum gels have proven to be a suitable growth matrix for a wide variety of

microorganisms, including those traditionally cultured on agar plates as well as other species not easily
grown on other substances.

 GELRITE® gels are exceptionally clear, making them an excellent analytical tool.
 GELRITE® gels have essentially the same shelf life as agar gels.

Chemical and Physical Properties

Chemical composition: polysaccharide comprising glucuronic acid, rhamnose and glucose.
Physical state: dry powder
Solids: 85-95%
Color: white

Media can be formulated with 0.6-0.8 % GELRITE® gellan gum and 0.10 % MgSO4*7H2O to achieve gels
strengths ranging from 225-500 g/cm² (Marine Colloid gel tester).
GELRITE® gellan gum is a linear polysaccharide comprising glucuronic acid, glucose, rhamnose, and
O-acetyl moieties. Recent research suggests that the GELRITE® gellan gum tetrasaccharide repeating unit
has the structure: 1 3)ß-D-Glcp-(14)-ß-D-GlcpA-(14)-ß-D-Glcp-(14)--L-Rhap-(1

Characteristics of GELRITE® Gels

Gel structure is integral to choosing suitable cultural media that are used to isolate pure bacterial cultures, to
characterize colonial morphology, to perform microbiological tests, and to enumerate microbes. Traditionally,

agar has been used as the matrix for solid media. In the presence of
soluble salts, GELRITE®gellan gum has a gel structure that effectively
supports microbial growth (refer to bulletin CD-27) and is viewed as an
excellent alternative to agar as a growth medium.
Gels prepared by autoclaving at 121 °C and 15 psi for 15 minutes. Gel
strength determined using a Marine Colloids Gel Tester with small
plunger at slow speed.

Figure 1. Response surface curve for gels showing gel strength as a function of
GELRITE® gellan gum and magnesium concentrations.

Gel Strength-Gel strength is a useful parameter in determining appropriate gel structure for support of
microbial growth. The gel strength of GELRITE® gels is highly dependent on the type of salt added, the
GELRITE® concentration (as shown in Figure 1), and the soluble salt concentration (as shown in Figures 2
and 3). Figure 1 is a response surface methodology (RS) curve which emphasizes the GELRITE® and
MgSO4*7H2O relationship that results in varied gel strengths. It should be noted that a gel strength of
250-450 g/cm² is a commonly found gel strength range of agar in microbiological media. The appropriate
GELRITE® and MgSO4*7H2O concentrations that yield this gel strength may be estimated from the RS
curve. It should be noted that frequently a nutrient medium containing simple salts does not require
additional salt to form an effective gel, e.g., Brain Heart Infusion.
Magnesium is the preferred ion in microbiological media applications. It produces in most cases a thermally
reversible gel.

1

O'NEILL, M. A. and others. "Structure of the acidic extracellular gelling polysaccharide produced by Pseudomonas elodea."
Carbohydrate Research, vol. 124, no. 1 (1983) 123-133.
JANNSON, RE. and others. "Structural studies of gellan gum, an extracellular polysaccharide eleborated by Pseudomonas elodea."
Carbohydrate Research, vol. 124, no. 1 (1983) 135-139.

3

Effect of Salts

GELRITE® gellan gum requires the presence of either monovalent or divalent cations for gelation.
GELRITE® gellan gum shows unique versatility in its gel characteristics: Gels can be modified as desired
by changing the concentration and type of cation in the GELRITE® media.
As shown in Figures 2 and 3, divalent cations such as magnesium or calcium have a much more profound
effect on gel strength than do monovalent ions such as sodium or potassium. Of the cations tested, calcium
exerts the most profound effect on the gel strength of GELRITE® . In fact, gels prepared with calcium do not
remelt under normal sterilization conditions.
Gelled media prepared using GELRITE® and a small amount of CaCI, (0.1 %) are remarkably stable at 80
°C for at least 10 days. There is no syneresis in this gel system, whereas the agar gel and the
MgSO4-mediated GELRITE® gel show severe syneresis under these conditions. This feature is especially
suitable to the culturing of many thermophiles.1

GELRITE® requires both a heating cycle and the presence of cations for gelation to occur GELRITE®
requires heating to approximately 100 °C to achieve complete solubility in the presence of ions. The
GELRITE® solution will then remain essentially non-viscous until, upon cooling, it reaches its gel setting
point, at which time gelation occurs very rapidly, much more rapidly than agar for example. The gel setting
temperature is a function of the GELRITE® and cation concentrations and can vary from 35 to >5O °C at 1
% GELRITE® concentration, as illustrated in Figures 4 and 5. The gel setting temperature will sometimes
increase
10-15 °C if the GELRITE® solutions are allowed to stand in a water bath for more than 30-40 minutes. The
GELRITE® media can be kept fluid for longer periods of time by increasing the temperature of the water bath
by 15 °C above the stated gel set temperature for a particular medium.
Note that for all cations used, gel setting temperature increased with increasing cation concentration, even
though gel hardness increases to a maximum then decreases.

* Figures 2-5:

Gels were prepared with a 1 % GELRITE® gellan gum solution using varying amounts of NaCI, KCI, CaCl2*2H2O or
MgCl2*6H2O Solutions were autoclaved 15 minutes at 15 psi. Gel hardness was measured at 25 °C using the
INSTRON 1122 Universal Testing Machine.

1

For information on GELRITE® as a gelling agent in media for thermophilic microorganisms, see: Lin, CC and L.E. Casida, Jr. Applied
and Environmental Microbiology, vol. 47, no. 2 (Feb. 1984) pp. 427-429.

*Figure 3.
Effect of Monovalent Salts on Gel Hardness 1 % GELRITE Gels

*Figure 2.
Effect of Divalent Salts on Gel Hardness 1 % GELRITE Gels

*Figure 5.
Effect of Divalent Salts on Gel Set Point 1 % GELRITE Gels

*Figure 4.
Effect of Monovalent Salts on Gel Set Point 1 % GELRITE Gels

4

Effect of Temperature

As illustrated in Figure 6, GELRITE® gellan gum solutions demonstrate thermally reversible viscosity
changes. The viscosity decreases sharply with increasing temperature, but returns to its original value upon
cooling. When prepared in ion-free water, GELRITE® solutions may be heated and cooled without gelation
occurring.

1

GELRITE® has good thermal stability and is able to
withstand a normal autoclaving cycle (121 °C, 15
psi, 15 minutes) without losing significant gel
strength. When compared to agar, its thermal
stability is remarkably similar during the initial
autoclaving cycle, which is most significant since
most solid media undergo only one such
sterilization cycle.

Compatibility with Nutrient Additives

GELRITE® gellan gum is completely compatible with nutrient additives commonly used with agar gels.
GELRITE® remains inert to most additives with the exception of soluble salts (see Figures 1 and 2).

Optical Clarity

GELRITE® gellan gum gels are optically clear as noted in Table 1. As demonstrated by the percentage
transmittance, GELRITE® gels are optically as clear as or clearer than compatible agar gels.

Table 1. Light Transmittance of Gelled Media Prepared with Agar and with GELRITE® gellan gum
MgCl2*6H2O

Medium

Agar

(%)

GELRIT

E®

(%)

Wavelength

(nm)

%

Transmittance*

Difco Nutrient Broth

1.5 - 530

52.5

Difco Nutrient Broth

-

0.8

530

73.0

Brain Heart Infusion

1.5 - 530

42.5

Brain Heart Infusion

-

0.75

530

55.7

Trypicase Soy Broth

1.5 - 530

54.9

Trypicase Soy Broth

-

0.75

530

73.2

Potato Dextrose Broth

1.5 - 535

63.0

Potato Dextrose Broth

-

0.75

535

66.5

* % transmittance was measured on a Spectronic 20. The blank was broth control for each medium. All media were autoclaved for is

minutes at 121 °C and 15 psi. % transmittance was measured after gelation. The wavelengths selected gave lowest transmittance for
each medium.

Conformation

Preliminary X-ray fiber diffraction studies2 of gellan gum suggests that a three-fold helical conformation exists
in the solid state. The O-acetyl substituents on gellan gum appear to affect the packing of these helices into
crystalline domains. The most crystallinity is seen in the absence of O-acetyl groups; this may relate to the
formation of rigid, brittle gels from solution.

1

Viscosity of a 1 % GELRITE® solution in deionized water, measured at 60 rpm on a Brookfield LVT viscometer using the appropriate
spindle.

2

CARROLL, W; M.J. MILES; and V.J. MORRIS (ARC Food Research Inst., Norwich) "Fibre-diffraction studies on the extracellular
polysaccharide from Pseudomonas elodea." International Journal of Biological Macromolecules, Vol. 4 (Dec. 1982) pp. 432-433.

*Figure 6.
Effect of Temperature on Viscosity 1 % GELRITE Solution

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Synergism with Other Polymers

For industrial gelling applications other than those in the microbiological media area, GELRITE® gellan gum
exhibits a useful synergism with other polymers.
Synergism studies show evidence of gel strength enhancement when GELRITE® is blended with gelatin or
gum arabic. At 0.5 % total gum concentrations in standard tap water1, 1:1 blends of GELRITE® and Gelatin
or gum arabic show respectively an approximate 60 % and 40 % increase in gel strength relative to 0.5 %
GELRITE® alone.

Storage and Handling

Storage and handling procedures should follow the normal practices recognized as desirable for naturally
derived polymeric gelling agents. The gel strength of the dry powder is retained, even after prolonged
storage at 50 °C (122 °F) and will recover from freezing. However, as with any polysaccharide, such
changes in temperature may tend to reduce its stability and should be avoided. GELRITE® gellan gum
should be stored tightly closed, in a cool ( <60° F) dry place for maximum shelf life.

Toxicity

GELRITE®gellan gum caused no oral toxicity in rats fed the pure product at a single dose of 5000 mg/kg.
From the results of eye irritation tests, GELRITE® is not considered to be an eye irritant. During dust
inhalation tests, no toxic symptoms were exhibited by rats exposed to concentrations averaging 6.09 mg/liter
for a 4-hour period. The lungs of these necropsied rats appeared normal.
Mutagenicity tests show GELRITE® to be negative in the Ames test. No special precautions are required to
handle GELRITE® gellan gum. Other studies on the safety of gellan gum are in progress. Please contact
your Kelco technical service representatives should you require further information.

Gellan Gum Thermal-Reversible Gelling Agent as an Agar Replacer

The gels formed by GELRITE® gellan gum in the presence of soluble salts have been tested and found to be
a suitable growth matrix for most clinical and nonclinical organisms. The gel characteristics necessary to
function as a matrix for solid media include thermal-reversibility2, optical clarity, and compatibility with
nutrient additives. (Refer to Bulletin CD-32 for further product descriptions.)
Some benefits of using GELRITE® are:

 wet enough to support growth

but

 dry enough to separate colonies

AND

 fine enough to prevent migration of organisms within the gel

but

 coarse enough to allow diffusion of macromolecular nutrients.

1

Standard tap water is formulated to represent a typical water supply containing 1,000 ppm sodium chloride and 147 ppm calcium
chloride dihydrate.

2

Under certain conditions gels can be made

6

GELRITE® Comparison with Agar

Traditionally, agar has been used as a matrix for solid media suitable for isolating pure cultures. The agar
used has been clear and smooth in texture so that characterization of colonial morphology by shapes,
textures, pigmentations, etc., could be readily apparent. All tests using GELRITE® as the gelling component
for nutrient media have shown that GELRITE® rapidly sets into a smooth gel that is as clear as or clearer
than agar, thus facilitating characterization of organisms.

1-2

The gel strength is dependent on the type of soluble salt added, the GELRITE® concentration, and the salt
concentration. (Refer to Bulletin CD-32 for graphic and tabular data.) Some GELRITE® salt combinations
appear to be more suitable growth matrices for particular microorganisms. GELRITE® gels with MgSO4*7H2O
are extremely effective. Gels made with a 0.75 % concentration of GELRITE® and a 0.1 % concentration of
MgSO4*7H20 are comparable to gels made with 1.5 % agar. These gels range from approximately 225-500
g/cm2 as measured on an Instron 1122.

Preparation of GELRITE® Nutrient Broth Gels

Procedure
Method 1 (Preferred)

1. To make 1 liter of nutrient broth containing GELRITE® as the solidifying agent, add and dissolve the
following ingredients to 1000 ml distilled water in the order listed:

8.0 g Nutrient Broth (dehydrated powder)

1.0 g MgSO4*7H2O

Then add 8.0 g GELRITE® with vigorous stirring to disperse.

2. Heat to boiling while stirring to dissolve the GELRITE®.

3. Autoclave the above mixture for 15 minutes at 121 °C, 15 psi.

4. Gently swirl the mixture for 15 seconds. (CAUTION should be taken when handling a superheated
mixture to avoid burns.)

5. Cool to 60 °C

6. Pour into petri dishes. (Note that GELRITE® gels set rapidly, thus allowing more plates to be poured in a
shorter period of time.)

Method 2

1. Using same amounts of components as in METHOD 1, add GELRITE® to distilled water.

2. Bring mixture to boil.

3. Add nutrient broth and magnesium salt.

4. Autoclave under conditions in METHOD 1 and continue from step 4 in METHOD 1.

The resulting gel strength obtained from nutrient broth, using 0.8 % w/w GELRITE® as the gelling agent, is
700 ±150 g/cm2, which is comparable to the same system gell with 1.5 % agar. The gel setting point of the
GELRITE® system is 41 °C ± 2 °C.

1

The U.S. Food & Drug Administration has determined under Section 510(k), that GELRITIE gellan gum is substantially equivalent to

agar.

2

Gellan gum is covered by U.S. patents non-thermal-reversible. US-4326052 and US-4326053.

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Preparation of GELRITE® Eosin Methylene Blue (EMS) Gels

Procedure
Method 1 (Preferred)

1. To make 1 liter of Eosin Methylene Blue (EMB) containing GELRITE® as the solidifying agent, add and
dissolve the following ingredients to 1000 ml distilled water in the order listed:

22.5 g EMB (dehydrated powder, without agar)

1.0 g MgSO4*7H2O

Then add 8.0 g GELRITE® with vigorous stirring to disperse.

2. Heat to boiling while stirring to dissolve the GELRITE®.

3. Autoclave the mixture for 15 minutes at 121 °C, 15 psi.

4. Gently swirl the mixture for 15 seconds. (CAUTION should be taken when handling a superheated
mixture to avoid burns.)

5. Cool to 60 °C.

6. Pour into petri dishes. (Note that GELRITE® gels set rapidly, thus allowing more plates to be poured in a
shorter period of time.)

Method 2

1. Using same amounts of components as in METHOD 1, add GELRITE® to distilled water.

2. Bring mixture to boil.

3. Add EMB and magnesium salt.

4. Autoclave under conditions in METHOD 1 and continue from step 4 in METHOD 1.

The resulting gel strength obtained from EMB, using GELRITE® as the gelling agent, is 475 ± 75 g/cm2,
which is comparable to the same system gelled with 1.5 % agar. The gel setting point of the GELRITE®
system is 42 °C ±2 °C.

Preparation of GELRITE® Salmonella Shigella (SS) Gels

Procedure

1. To make 1 liter of Salmonella Shigella (SS) media, containing GELRITE® as the gelling agent, dissolve

46.5 g SS media (Dehydrated powder without agar) in 1,000 ml distilled water. Then add 6.0 g GELRITE®
with vigorous stirring to disperse.

2. Heat with repeated stirring and boil for one minute.

3. Cool to 60 °C.

4. Pour into petri dishes. (Note that GELRITE® gels set rapidly, thus allowing more plates to be poured in a
shorter period of time.)

The resulting gel strength obtained from SS, using GELRITE® as the gelling agent, is 425 ±110 g/cm2, which
is comparable to the same system gelled with 1.5 % agar. The gel setting point of the GELRITE® system is
53 °C ±2 °C.

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Preparation of GELRITE® Tryptic Soy Gels

Procedure
Method 1 (Preferred)

1. 1.To make 1 liter of Tryptic Soy Media containing GELRITE® as the solidifying agent, add and dissolve
the following ingredients to 1000 ml distilled water in the order listed:

27.0 g Tryptic Soy Media (Dehydrated powder without agar)

1.0 g MgSO4*7H2O
Then add 8.0 g GELRITE® with vigorous stirring to disperse.

2. Heat to boiling with stirring to dissolve the GELRITE®.

3. Autoclave the above mixture for 15 minutes at 121 °C, 15 psi.

4. Gently swirl the mixture for 15 seconds. (CAUTION should be taken when handling a superheated
mixture to avoid burns.)

5. Cool to 60 °C (except for blood plates, which must be cooled to 50 °C prior to blood addition to avoid
hemolysis).

6. Pour into petri dishes. (Note that GELRITE® gels set rapidly, thus allowing more plates to be poured in a
shorter period of time.)

Method 2

1. Using same amounts of components as in METHOD 1, add GELRITE® to distilled water.

2. Bring mixture to boil.

3. Add Tryptic Soy medium and magnesium salt.

4. Autoclave under conditions in METHOD 1 and continue from step 4 in METHOD 1.

The resulting gel strength obtained from Tryptic-Soy gels, using GELRITE® as the gelling agent, is 770
±110 g/cm2, which is comparable to the same system gelled with 1.5 % agar. The gel setting point of the
GELRITE® system is 52 °C ±2 °C.

Preparation of GELRITE® Tryptic Soy/Blood Gels

The present method for the preparation of blood plates made using GELRITE® involves preparing two
solutions and combining them after sterilization. The gel setting point of the GELRITE® -tryptic soy/blood
mixture is 48 °C ±1 °C, therefore it is critical to maintain the temperature at 50 °C or above once the
solutions are combined. A batch process is outlined below giving the details for the preparation of a 200 ml
volume. Because of the critical temperature of the gel setting point, it is difficult to prepare volumes greater
than 200 ml using a batch process. However, in a multifeed, continuous system, this difficulty would be
circumvented.

Procedure

1. Dissolve 5.4 g tryptic soy medium in 190 ml of distilled water.

2. Divide the solution into two parts. One portion should contain 130 ml volume (solution A) and the other

portion should be 60 ml in volume (solution B).

3. Add 1 drop of a 1-10 dilution of POURITE1 to solution A. To solution B, add 2 gms GELRITE®.

4. Autoclave the above solutions for 15 minutes at 121 °C, 15 psi.

5. Gently swirl the solutions for 15 seconds. (CAUTION should be taken when handling a superheated

mixture to avoid burns.)

6. Cool solution A to 50 °C and aseptically add 10 ml of defibrinated sheep blood which has been warmed

to 25 °C. Mix well to insure even distribution of blood.

7. While mixing solution A, immediately add solution B, which has been previously cooled to 70 °C.

8. Pour into petri dishes. (Note that GELRITE® gels set rapidly, thus allowing more plates to be poured in a

shorter period of time.)

The resulting gel strength obtained for tryptic soy/blood plates, using GELRITE® as the gelling agent, is
360 ±20 g/cm2, which is comparable to the same system gelled with 1.5 % agar.

1

Trademark of: American Scientific Products, Division of American Hospital Supply Corp.

9

Preparation of GELRITE® Triple Sugar Iron (TSI) Gels

Procedure

1. To make 1 liter of Triple Sugar Iron (TSI) medium containing GELRITE® as the solidifying agent dissolve

46.5 g TSI medium (Dehydrated powder without agar) in 1,000 ml distilled water. Then add 6.0 g
GELRITE® with vigorous stirring to disperse.

2. Heat with repeated stirring and boil for one minute to dissolve completely.

3. Distribute into test tubes and autoclave for 15 minutes at 121 °C, 15 psi.

4. After autoclaving, slant tubes to prepare deep butts.

The resulting gel strength obtained from TSI, using GELRITE® as the gelling agent, is 350 ±25 g/cm2, which
is comparable to the same system gelled with 1.5 % agar. The gel setting point of the GELRITE® system is
49 °C ±2 °C.

Preparation of Plant Tissue Culture Gels

Procedure

1. To make 1 liter of gelled media combine:

2 g GELRITE® and all other components

1

in 1,000 ml of distilled water.

2. Heat with repeated stirring and boil for one minute to dissolve completely.

3. Distribute into test tubes and autoclave for 15 minutes at 121 °C, 15 psi.

4. Allow to cool.

Preparation of GELRITE® Chocolate Gels

Procedure:

1. To make one liter of chocolate medium containing GELRITE® as the solidifying agent, first add 25 gms of
Heart Infusion broth, and 7.5 gms of GELRITE® to 1000 ml distilled water. Stir the GELRITE® suspension
vigorously to disperse any lumps.

2. Autoclave both aliquots at 121 °C, 15 psi for approximately 15 minutes.

3. Place in a water bath at 80 °C.

4. Add 50 ml of defribinated sheep blood to the Heart Infusion GELRITE® and allow to remain at 80 °C for
15 minutes.

5. Dispense into sterile petri plates. The resulting gel strength obtained with Chocolate GELRITE® medium
is 360 gm/cm2 ±30 gm/cm2, which is comparable to the same system gelled with approximately 1.5 %
agar.

Preparation of GELRITE® Egg Yolk Gels

Procedure:

1. To prepare 1 liter of egg yolk medium containing GELRITE® as the solidifying agent, first divide the
distilled water into two 500 ml aliquots.

2. To one, add 25 gms of Heart Infusion broth and a magnetic stirring bar. To the other, add 7.5 gms of
GELRITE®. Stir the GELRITE® suspension vigorously to disperse any lumps.

3. Autoclave both aliquots at 121 °C, 15 psi for approximately 15 minutes.

4. Cool the Heart Infusion broth to 50 °C and the GELRITE® to 70 °C.

5. Add 5 ml of egg yolk to the Heart Infusion broth with mixing (magnetic stirrer).

6. Add the GELRITE® to this mixture with constant stirring (magnetic stirrer).

7. Dispense into sterile petri plates.

The resulting gel strength obtained with egg yolk medium solidified with GELRITE® is 200 gms/cm2 which is
comparable to the same system gelled with approximately 1.5 % agar.

1

Should there be any components that cannot be autoclaved, these can be added after autoclaving. Add as liquids while the
GELRITE® gellan gum solution is between 55-60 °C.

10

Preparation of GELRITE® TCBS Gels

Procedure

1. To make 1 liter of TCBS medium containing GELRITE® as the solidifying agent, add and dissolve the
following ingredients to 1,000 ml distilled water in the order listed.

Sodium Thiosulfate 10.0 gm
Sodium Citrate 10.0 gm
Oxgall 5.0 gm
Sodium Cholate 3.0 gm
Sucrose 20.0 gm
Pancreatic Digest of Casein, U.S.P. 5.0 gm
Peptic Digest of Animal Tissue, U. S. P. 5.0 gm
Yeast Extract 5.0 gm
Sodium Chloride 10.0 gm
Iron Citrate 1.0 gm
Thymol Blue 0.04 gm
Bromothymol Blue 0.04 gm
Then add 7.5 g GELRITE® 7.5 gm
with vigorous stirring to disperse.

2. Heat with stirring and boil for one minute.

3. Cool to 50-55 °C and pour into plates.

The resulting gel strength obtained with TCBS using GELRITE® as the gelling agent, is 400 ±40 gm/cm2,
which is comparable to the same system gelled with approximately 1.5 % agar.

Evaluation of GELRITE® as an Agar Replacer

Screening tests conducted by Merck Sharp & Dohme Research Laboratories (MSDRL) and by Kelco
Research and Development indicate that GELRITE® gels are suitable as agar replacers for performing
biochemical and enzymatic tests. The process of enumerating microbes during tests for such things as
ecological factors and quality control previously has been limited to agar gels. The GELRITE® gels readily
accommodate this process.

An evaluation of the suitability of GELRITE® as an agar replacer for clinical isolates was conducted. Varied
nutrient broths compared GELRITE® gels with microbiological-grade agar as the solidifying agent.
Approximately 50 clinical organisms with differing growth requirements were tested as indicated in Table I.

Table II identifies nonpathogenic organisms that were tested by Kelco Research and Development
laboratories. Once again, test results indicated that GELRITE® gels were comparable or superior to agar in
setting, optical clarity, and various gel strengths that could be attained by adjustments of salt and/or
GELRITE® concentrations.

The overall performance of the GELRITE® gels was favorably compared to that of the microbiological-grade
agar. GELRITE® gels were considered to be superior to agar in their setting properties, clarity, and
adjustability of gel strength.

11

Table I.

Clinical lsolates6 Culture Media Used in Evaluation of GELRITE® TM gum vs Agar

Organism

Media

Escherichia coli

CL 1552

Trypticase Soy® (TS), Blood, McConkey (McC), SS, Bismuth,
Sulfite, EMB, TSI

Shigella dysenteriae

CL 1726

TS, Blood, McC, SS, Bismuth Sulfite, Brilliant Green, EMB, TSI

Shigella flexneri

CL 1719

TS, Blood, McC, SS, Bismuth Sulfite, Brilliant Green, EMB, TSI

Shigella boydii

CL 1727

TS, Blood, McC, SS, Bismuth Sulfite, Brilliant Green, EMB, TSI

Shigella sonnei

CL 1705

TS, Blood, McC, SS, Bismuth Sulfite, Brilliant Green, EMB, TSI

Edwardsiella tarda

CL 2076

TS, Blood, McC, TSI

Klebsiella pneumoniae

CL 1697

TS, Blood, McC, TSI

Klebsiella ozaenae

CL 904

TS, Blood, McC, TSI

Klebsiella oxytoca

CL 1060

TS, Blood, McC, TSI

Enterobacter cloacae

CL 1779

TS, Blood, McC, EMB, TSI

Enterobacter aerogenes

CL 1548

TS, Blood, McC, EMB, TSI

Enterobacter agglomerans

CL 1387

TS, Blood, McC, EMB, TSI

Hafnia alvei

CL 136

TS, Blood, McC, EMB, TSI

Serratia marcescens

CL 1520

TS, Blood, McC, TSI

Serratia liquefaciens

CL 1977

TS, Blood, McC, TSI

Citrobacter freundii

CL 1663

TS, Blood, McC, TSI

Citrobacter diversus

CL 1519

TS, Blood, McC, TSI

Proteus vulgaris

CL 1190

Blood, McC, TSI, Nutrient (N)

Proteus mirabilis

CL 1772

Blood, McC, TSI, N

Morganella morganii

CL 1555

Blood, McC, TSI, N

Providencia rettgeri

CL 1192

TS, Blood, McC, TSI

Providencia alcalifaciens

CL 787

TS, Blood, McC, TSI

Providencia stuartii

CL 1443

TS, Blood, McC, TSI

Salmonella typhimurium

CL 1866

TS, Blood, McC, SS, Bismuth Sulfite, Brilliant Green, EMB, TSI

Salmonella enteritidis

CL 1362

TS, Blood, Mcc, SS, Bismuth Sulfite, Brilliant Green, EMB, TSI

Salmonella typhi

CL 1935

TS, Blood, SS, Bismuth Sulfite, Brilliant Green, EMB, TSI

Arizona hinshawii

CL 2075

TS, Blood, McC, TSI

Yersinia enterocolitica

CL 1626

TS, Blood, McC, SS, Bismuth sulfite, Brilliant Green, EMB, TSI

Acinetobacter calcoaceticus

CL 1658

TS, Blood, McC, TSI

Haemophilus influenzae

CL 1826

TS, Chocolate

Haemophilus parainfluenzae

CL 1824

TS, Chocolate

Pseudomonas aeruginosa

CL 1560

TS, Blood, McC, TSI

Pseudomonas fluorescens

CL 1542

TS, Blood, McC, TSI

Pseudomonas maitophilia

CL 2016

TS, Blood, McC, TSI

Staphylococcus aureus

CL 1500

TS, Blood, McC, Egg Yolk(EY), Phenyl Ethyl Alcohol(PEA)

Staphylococcus epidermidis

CL 1803

TS, Blood, McC, TSI, EY, PEA

Staphylococcus saprophyticus

CL 1941

TS, Blood, McC, TSI, EY, PEA

Streptococcus pyogenes

CL 1925

TS, Blood

Streptococcus agalactiae

CL 1342

TS, Blood

Streptococcus faecalis

CL 1776

TS, Blood

Streptococcus pneumoniae

CL 1842

Blood, BHI

Streptococcus Group C

CL 909

TS, Blood

Streptococcus Group G

CL 1929

TS, Blood

Flavobacterium odoratum

CL 1435

TS, Blood, McC, TSI

Moraxella species

CL 387

TS, Blood, McC, TSI

Campylobacter fetus subsp. jejuni

CL 2320

TS, Blood

Vibrio cholerae

CL 2020

TS, Blood, EY, PEA

Clostridium perfringens

CL A144

BHI, Blood, EY, PEA

Peptostreptococcus anaerobius

CL A107

BHI, Blood, EY, PEA

Bacteroides fragilis

CL A1 77

BHI, Blood

6

All isolates are alphanurnerically identified by MSDRL classifications

12

Table II.

Nonpathogenic Isolates and Culture Media Used in
Evaluation of GELRITE® gellan gum vs Agar

Organism

Media

Agromyces ramosus

ATCC 25173

Trypticase GELRITE®

Arthrobacter globiformis

ATCC 8010

Trypticase Soy GELRITE®

Aureobasidium pullulans

NRRL Y-3861

Potato Dextrose
GELRITE®

Azotobacter vinelandii

ATCC 9047

Burk's GELRITE®

Beijerinckia lacticogenes

ATCC 19361

Burk's GELRITE®

Erwinia carotovora

ATCC 8061

Nutrient GELRITE®

Nocardia salmonicolor

ATCC 21243

Nutrient GELRITE®

Trichoderma longibrachiatum

ATCC 13631

Potato Dextrose
GELRITE®

Zoogloea ramigera

ATCC 25935

Trypticase Soy GELRITE®

All plates were made according to procedures identified in this application bulletin.

Technical Service

Kelco has a highly qualified staff ready to serve you at all times. If you have a question or would like to
receive samples and technical literature, write or call your Kelco office in San Diego.

The information contained herein is, to Kelco's best knowledge, true and accurate, but all recommendations
or suggestions are made without guarantee, since we can neither anticipate nor control the different
conditions under which this information and our products are used. Therefore, Kelco does not warrant the
application of this information to particular situations. Further Kelco disclaims all liability with regard to its
customers' infringement of third party patents. Kelco recommends that its customers apply for licenses under
any relevant patents. No statement herein or by our employees shall be construed to imply the nonexistence
of relevant patents nor as a recommendation or inducement to infringe said patents. It is Kelco~ policy,
however, to assist our customers and to help in the solution of particular problems which may arise in
connection with applications of Kelco products.

D

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::

Carl Roth GmbH & Co.
Schoemperlenstr. 1-5
76185 Karlsruhe

Tel: [phone]

Fax: [phone]

GELRITE® 500 g 0039.1

1 kg 0039.2

Carl Roth GmbH + Co. KG

Schoemperlenstraße 3-5 • 76185 Karlsruhe • P.O. Box 100121 • 76231 Karlsruhe
Phone: [phone] • Fax: [phone] • [e-mail] • www.carlroth.com

The company is a limited partnership with headquarters in Karlsruhe, reg. court Mannheim HRA 100055. Roth Chemie GmbH, with headquarters in Karlsruhe,
reg. court Mannheim HRB 100428, is the personally liable partner. Managing Director: André Houdelet. Sales tax identification number: DE 143621073. sse 07/2021