Pathology Media – Plant Tissue Culture Protocol

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

Fungal and Bacterial Detection Media

Prod. No.	Name	Product Use	Preparation
B1662	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
C1176	Corn meal agar	Cultivation of phytopathological fungi. To encourage the production of chlamydospores by Candida albicans, add 1% Tween 80 (Product No. P4780).	17.0 g/L; boil, autoclave 15 min. at 121 °C
C1551	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
L1900	Luria broth base	Cultivation of bacteria used in molecular biology studies.	15.5 g/L; boil, autoclave 15 min. at 121 °C
L2025	Luria agar base		30.5 g/L; boil, autoclave 15 min. at 121 °C
M6409	Malt extract broth	Isolation and detection of yeasts and molds.	15.0 g/L; boil, autoclave 15 min. at 121 °C
M6907	Malt extract Agar		33.6 g/L; boil, autoclave 15 min. at 121 °C
N4019	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
N7519	Nutrient broth		8.0 g/L; boil, autoclave 15 min. at 121 °C
O3506	Oatmeal agar	Cultivation of fungi, particularly macrospore formation.	72.5 g/L; boil, autoclave 15 min. at 121 °C
P2182	Potato dextrose agar	Cultivation of bacteria, fungi, yeast and molds.	39.0 g/L; boil, autoclave 15 min. at 121 °C
P6685	Potato dextrose broth		24.0 g/L; boil, autoclave 15 min. at 121 °C

Media Preparation and Storage

C1176 - Corn meal agar
C1551 - Czapek-Dox broth
L1900 - Luria broth base, Miller
L2025 - Luria agar base, Miller
M6409 - Malt extract broth
M6907 - Malt extract agar

N4019 - Nutrient agar 1.5%
N7519 - Nutrient broth
O3506 - Oatmeal agar
P2182 - Potato dextrose agar
P6685 - Potato dextrose broth

Corn Meal Agar (C1176)

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 (W3500) and heat to boiling or until the medium is completely dissolved. Add 1% polyoxyethylenesorbitan monooleate (TWEEN^® 80) (P4780) 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 (C1551)

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.

Luria Broth Base, Miller, LB (L1900)
Luria Agar Base, Miller, LB (L2025)

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.

Malt Extract Broth (M6409)
Malt Extract Agar (M6907)

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 (N7519)
Nutrient Agar 1.5% (N4019)

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 (O3506)

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 (P6685)
Potato Dextrose Agar (P2182)

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.

Media Composition Table

B1662 - Bacteria screening medium 523
C1176 - Corn meal agar
C1551 - Czapek-Dox broth
L1900 - Luria broth base, Miller
L2025 - Luria agar base, Miller
M6409 - Malt extract broth
M6907 - Malt extract agar

N4019 - Nutrient agar 1.5%
N7519 - Nutrient broth
O3506 - Oatmeal agar
P2182 - Potato dextrose agar
P6685 - 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