Plant Tissue Culture Protocols

Pathology Media

 
Fungal and Bacterial Detection Media
Media Preparation and Storage
Media Composition Table

 


 

Fungal and Bacterial Detection Media
Prod. No. Name Product Use Preparation
B 1662 Bacteria screening medium 523 Use to screen explants for bacterial contamination during culture initiation. 32.15 g/L; boil, autoclave 15 min. at 121°C
C 1176 Corn meal agar Cultivation of phytopathological fungi. To encourage the production of chlamydospores by Candida albicans, add 1% Tween 80 (Prod. No. P 4780). 17.0 g/L; boil, autoclave 15 min. at 121°C
C 1551 Czapek-Dox broth Cultivation of fungi and bacteria capable of utilizing inorganic nitrogen; useful for soil microorganisms. 35.0 g/L; boil, autoclave 15 min. at 121°C
L 1900 Luria broth base Cultivation of bacteria used in molecular biology studies. 15.5 g/L; boil, autoclave 15 min. at 121°C
L 2025 Luria agar base 30.5 g/L; boil, autoclave 15 min. at 121°C
M 6409 Malt extract broth Isolation and detection of yeasts and molds. 15.0 g/L; boil, autoclave 15 min. at 121°C
M 6907 Malt extract Agar 33.6 g/L; boil, autoclave 15 min. at 121°C
N 4019 Nutrient Agar General purpose culture medium for cultivation of nonfastidious organisms. Medium base for use as a selective, differential or enriched medium when supplemented. Recommended for the examination of water. 31.0 g/L; boil, autoclave 15 min. at 121°C
N 7519 Nutrient broth 8.0 g/L; boil, autoclave 15 min. at 121°C
O 3506 Oatmeal agar Cultivation of fungi, particularly macrospore formation. 72.5 g/L; boil, autoclave 15 min. at 121°C
P 2182 Potato dextrose agar Cultivation of bacteria, fungi, yeast and molds. 39.0 g/L; boil, autoclave 15 min. at 121°C
P 6685 Potato dextrose broth 24.0 g/L; boil, autoclave 15 min. at 121°C

 


 

Media Preparation and Storage

   
C 1176
C 1551
L 1900
L 2025
M 6409
M 6907
N 4019
N 7519
O 3506
P 2182
P 6685
 
Corn meal agar
Czapek-Dox broth
Luria broth base, Miller
Luria agar base, Miller
Malt extract broth
Malt extract agar
Nutrient agar 1.5%
Nutrient broth
Oatmeal agar
Potato dextrose agar
Potato dextrose broth
 

 


 

Corn Meal Agar (Product No. C 1176)

Corn meal agar is recommended for the culture of phytopathological fungi and is also recommended for the production of chlamydospores by Candida albicans. The basic steps for preparing this product are listed below:

  1. Suspend 17.0 g of powder in 1 liter of distilled water (W 3500) and heat to boiling or until the medium is completely dissolved. Add 1% polyoxyethylenesorbitan monooleate (Tween 80) (Product No. P 4780) to encourage chlamydospore production.
  2. Sterilize the medium in a validated autoclave at 1 kg/cm2 (15 psi), 121 °C. The medium should attain a temperature of 121 °C for at least 15 minutes. Refer to the Sterilization of Media Protocol.
  3. Dispense the medium as desired.

STORAGE

All medium preparations should be stored at 0-5 °C. Store dry medium at room temperature (below 30 °C). The dehydrated powder should be light yellow, homogeneous and free-flowing. Deterioration of powdered medium may be recognized by: 1) granulation, clumping, or particulate matter throughout the powder; 2) pH change; 3) inability to promote growth when properly used.

 


 

Czapek Dox Broth (Product No. C 1551)

Czapek Dox Broth is a defined liquid medium, designed for the cultivation of fungi and bacteria capable of utilizing inorganic nitrogen and is especially useful for the cultivation of various soil bacteria and fungi and many saprophytic aspergilli. This medium is prepared with only inorganic sources of nitrogen (Sodium Nitrate) and a chemically defined carbon source (Glucose). This product is a modification of the Czapek formula of Dox according to Thom and Raper (1945). The basic steps for preparing this product are listed below:

  1. Suspend 35.0 g of powder in 1 liter of distilled water and heat to boiling or until the medium is completely dissolved.
  2. Sterilize the medium in a validated autoclave at 1 kg/cm2 (15 psi), 121 °C. The medium should attain a temperature of 121 °C for at least 15 minutes. Refer to the Sterilization of Media Protocol.
  3. Dispense the medium as desired. The prepared medium may have a slight precipitate.

STORAGE

All medium preparations should be stored at 0-5 °C. Store dry medium at room temperature (below 30 °C). The dehydrated powder should be white, homogeneous and free-flowing. Deterioration of powdered medium may be recognized by: 1) granulation, clumping, or particulate matter throughout the powder; 2) pH change; 3) inability to promote growth when properly used.

 


 

Malt Extract Broth (Product No. M 6409)
Malt Extract Agar (Product No. M 6907)

Malt Extract Broth and Agar are used for the isolation and detection of yeasts and molds. The carbohydrates present in these media are well suited to support the growth of many fungi. The basic steps for preparing these products are listed below:

  1. Suspend the appropriate amount of powder in 1 liter of distilled/deionized water. Dissolve the agar medium by heating to a boil. Avoid overheating this product as it might result in a softer agar gel and increased darkening of the medium.
  2. Sterilize the medium in a validated autoclave at 1 kg/cm2 (15 psi), 121 °C. The medium should attain a temperature of 121 °C for at least 15 minutes. Refer to the Sterilization of Media Protocol.
  3. Dispense the medium as desired (before or after autoclaving).

STORAGE

All medium preparations should be stored at 0-5 °C. Store dry medium at room temperature (below 30 °C). The dehydrated powder should be off-white, homogeneous and free-flowing. Deterioration of powdered medium may be recognized by: 1) granulation, clumping, or particulate matter throughout the powder; 2) pH change; 3) inability to promote growth when properly used.

 


 

Nutrient Broth (Product No. N 7519)
Nutrient Agar 1.5% (Product No. N 4019)

Sigma Nutrient Broth and Agar 1.5% are general purpose microbiological media for less fastidious organisms. The basic steps for preparing these products are listed below:

  1. Suspend the appropriate amount of powder in 1 liter of distilled/deionized water. Dissolve the agar medium by heating to a boil.
  2. Sterilize the medium in a validated autoclave at 1 kg/cm2 (15 psi), 121°C. The medium should attain a temperature of 121 °C for at least 15 minutes. Refer to the Sterilization of Media Protocol.
  3. Dispense the medium as desired (before or after autoclaving).

STORAGE

All medium preparations should be stored at 0-5 °C. Store dry medium at room temperature (below 30 °C). The dehydrated powder should be light tan, homogeneous and free-flowing. Deterioration of powdered medium may be recognized by: 1) granulation, clumping, or particulate matter throughout the powder; 2) pH change; 3) inability to promote growth when properly used.

 


 

Oatmeal Agar (Product No. O 3506)

Sigma Oatmeal Agar is recommended for the cultivation of fungi, particularly for macrospore formation.The basic steps for preparing this product are listed below:

  1. Suspend 72.5 g of powder in 1 liter of distilled/deionized water and heat to boiling until the medium is completely dissolved. Agitate the medium to obtain a uniform suspension.
  2. Sterilize the medium in a validated autoclave at 1 kg/cm2 (15 psi), 121 °C. The medium should attain a temperature of 121 °C for at least 15 minutes. Refer to the Sterilization of Media Protocol.
  3. Dispense the medium as desired. Swirl the medium during dispensing to obtain a uniform mixture.

STORAGE

All medium preparations should be stored at 0-5 °C. Store dry medium at room temperature (below 30 °C). The dehydrated powder should be beige, non-homogeneous and slightly lumpy. The prepared medium will be off-white to opaque with non-homogeneous particles present. Deterioration of powdered medium may be recognized by: 1) granulation, clumping, or particulate matter throughout the powder; 2) pH change; 3) inability to promote growth when properly used.

 


 

Potato Dextrose Broth (Product No. P 6685)
Potato Dextrose Agar (Product No. P 2182)

Potato Dextrose Broth and Agar are standard media for culturing bacteria, fungi, yeast and molds. The basic steps for preparing these products are listed below:

  1. Suspend the appropriate amount of powder in 1 liter of distilled/deionized water. Dissolve the agar medium by heating to a boil.
  2. Sterilize the medium in a validated autoclave at 1 kg/cm2 (15 psi), 121 °C. The medium should attain a temperature of 121 °C for at least 15 minutes. Refer to the Sterilization of Media Protocol.
  3. Dispense the medium as desired (before or after autoclaving).

STORAGE

All medium preparations should be stored at 0-5 °C. Store dry medium at room temperature (below 30 °C). The dehydrated powder should be light beige, homogeneous and free-flowing. Deterioration of powdered medium may be recognized by: 1) granulation, clumping, or particulate matter throughout the powder; 2) pH change; 3) inability to promote growth when properly used.

 


 

Luria Broth Base, Miller, LB (Product No. L 1900)
Luria Agar Base, Miller, LB (Product No. L 2025)

Luria Broth and Agar are media used for culturing bacteria used in molecular biology studies. The basic steps for preparing these products are listed below:

  1. Suspend the appropriate amount of powder in 1 liter of distilled/deionized water. Dissolve the agar medium by heating to a boil.
  2. Sterilize the medium in a validated autoclave at 1 kg/cm2 (15 psi), 121 °C. The medium should attain a temperature of 121 °C for at least 15 minutes. Refer to the Sterilization of Media Protocol.
  3. Dispense the medium as desired (before or after autoclaving).

STORAGE

All medium preparations should be stored at 0-5 °C. Store dry medium at room temperature (below 30 °C). The dehydrated granules should be light tan, homogeneous and free-flowing. Deterioration of powdered medium may be recognized by: 1) granulation, clumping, or particulate matter throughout the powder; 2) pH change; 3) inability to promote growth when properly used.

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Media Composition Table

   
B 1662
C 1176
C 1551
L 1900
L 2025
M 6409
M 6907
N 4019
N 7519
O 3506
P 2182
P 6685
 
Bacteria screening medium 523
Corn meal agar
Czapek-Dox broth
Luria broth base, Miller
Luria agar base, Miller
Malt extract broth
Malt extract agar
Nutrient agar 1.5%
Nutrient broth
Oatmeal agar
Potato dextrose agar
Potato dextrose broth
 
Component
(g/L)
B1662 C1176 C1551 L1900 L2025 M6409 M6907 N4019 N7519 O3506 P2182 P6685
Agar 8.0 15.0 15.0   15.0   15.0 15.0   12.5 15.0  
Beef extract               3.0 3.0      
Casein hydrolysate 8.0                      
Corn meal, infusion from   2.0                    
Dextrin             12.75          
Ferrous sulfate     0.01                  
Glucose (dextrose)           6.0         20.0 20.0
Glycerol             2.35          
Malt extract           6.0            
Maltose           1.8 2.75          
Magnesium sulfate     0.5                  
Magnesium sulfate • 7H2O 0.15                      
Oatmeal                   60.0    
Peptone             0.78 5.0 5.0      
Potassium chloride     0.5                  
Potassium phosphate monobasic 2.0                      
Phosphate, dipotassium     1.0                  
Potatoes, infusion from                     4.0 4.0
Sodium chloride       0.5 0.5     8.0        
Sodium nitrate     3.0                  
Sucrose (saccharose) 10.0   30.0                  
Tryptone       10.0 10.0              
Yeast extract 4.0     5.0 5.0 1.2            
  B1662 C1176 C1551 L1900 L2025 M6409 M6907 N4019 N7519 O3506 P2182 P6685
Grams to prepare 1L 32.15 17.0 35.0 15.5 30.5 15.0 33.6 31.0 8.0 72.5 39.0 24.0

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