Antibiotics in Plant Tissue Culture Protocol

• Preparation of Selected Antibiotics
  
• Antibiotics Reference Guide
  
• Antimycotics Reference Guide

In general, antibiotics require storage in a refrigerator or freezer. Aminoglycosides (e.g. kanamycin) are hygroscopic and should be stored in a desiccator. Storage of many powdered antibiotics at -20 °C is not recommended and increases the risk of water condensation. All antibiotics should be protected from direct sunlight. Rifampicin and amphotericin B are very sensitive to light and should be stored in the dark.

The labels of antibiotic containers usually state the activity (potency, active matter per amount of powder) of the product in µg/mg or units/mg of powder. The amount of powder to use for a given antibiotic will change according to the activity of the product. For example, if a product has an activity of 1000 units/mg, twice as much powder will be required than for the same product with an activity of 2000 units/mg.

The relationship between the weight (mg) of antibiotic to use, the activity of the powder (µg/mg or units/mg), the volume of solution to prepare (ml) and the concentration (µg/ml) of antibiotic desired in the solution is:

Weight = (Volume x Concentration) / Activity

Most antibiotic solutions will remain stable stored at -0 °C for up to 3 months. However, Rifampicin should be freshly prepared for each use. Most antibiotics are heat labile and should be filter sterilized using membranes that minimize adsorption (e.g. cellulose acetate).

Preparation of Selected Antibiotics

Nystatin Preparation (Product No. N9767)

Nystatin is an antifungal agent produced from Streptomyces spp. It exhibits fungicidal and fungistatic activity when prepared as an aqueous suspension. Nystatin is only slightly soluble in water and alcohol. Aseptic suspensions can be prepared by surface sterilizing the powder in a small amount of dimethyl sulfoxide (DMSO) (to wet the powder) and then suspending the powder in sterile water. This procedure should be performed under aseptic conditions with sterile equipment (glassware, etc.)

Nystatin preparations are heat labile. Aqueous suspensions should be stored at -0 °C and protected from light. Nystatin suspensions should have a pH of approximately 6.5-8.0 and will decompose at a pH below 2.0 or above 9.0.

Carbenicillin Preparation (Product No. C3416)

Carbenicillin is a white to off-white, hygroscopic powder which is soluble in either water or alcohol. Carbenicillin is most effective against gram-negative bacteria but may also have some effect against gram-positive bacteria. Aqueous solutions of carbenicillin are reported to be stable for up to 24 hours at room temperature, and for up to 72 hours when stored at 0-5 °C.

Cefotaxime Preparation (Product No. C7039)

Cefotaxime is a white to off-white powder which is freely soluble in water. Variations in color of the freshly prepared solutions do not necessarily indicate changes in potency. Store this product in an airtight container protected from light. Aqueous solutions of cefotaxime at a pH of 4.3-6.2 are stable for 14-21 days when stored at 0-5 °C. Cefotaxime is most effective against gram-negative bacteria.

G418 Disulfate Salt (Product No. G1279)

Although it is related to Gentamicin, G418 Disulfate Salt is not normally used as a standard antibiotic. Its most common application is in molecular biology as a selection agent. G418 Disulfate Salt is toxic to bacteria, yeast, protozoa, helminths, and mammalian cells. Resistance is conferred by one of two dominant genes of bacterial origin which can be expressed in eukaryotic cells.

G418 Disulfate Salt is water soluble and can be stored at room temperature for as long as 1 year. Aqueous solutions should be stored frozen. The amount of G418 Disulfate Salt required for selection will vary with each cell type and growth cycle. Although cells that are multiplying will be affected sooner than those that are not, cells that are in log phase will still require 3 to 7 days for selection.

Hygromycin B (Product No. H9773)

Hygromycin B is an aminoglycoside antibiotic which is effective against prokaryotic and eukaryotic micro-organisms and cells. Similar to G418 Disulfate Salt, its most common application is in molecular biology as a selection agent. Insect and mammalian cells transformed with the hph gene, which encodes for hygromycin-B-phosphotransferase, are resistant to hygromycin B. Hygromycin B is provided as a powder. The recommended concentration range for use as a selection agent is 10 - 400 µg/ml.

Prokaryotes - 100 µg/ml
Lower eukaryotes - 200 µg/ml
Higher eukaryotes - 150-400 µg/ml

Refer to pertinent scientific references for more specific application information.

 

Antibiotics Reference Guide

Product	Prod. No.	Mol. Wt.	Gram (+) bacteria	Gram (-) bacteria	Myco- bacteria	Myco- plasma	Toxicity to microbes (µg/mL)	Toxicity to plant tissue (µg/mL)	Working conc. (µg/mL) and applications1	Product Solubility	Liquid Storage
Carbenicillin	C3416	422.4	X	XX			500	>1000	up to 500 (CC,PR,EC)	Water	-0 °C 2-8 °C 3 days
Cefotaxime	C7039	477.4	X	XX			90	>100	up to 500 (CC,PR,EC)	Water	-0 °C 2-8 °C 22 days
Chloramphenicol	C1919	323.1	XX	XX	X	X	128	>1	10-35 (TS)	EtOH	2-8 °C 30 days
G418 Disulfate Salt	G1279	692.7							10-500	Water	-20 °C 6 mo.
Gentamicin sulfate	G1264		X	XX		XX	50	>50	up to 250 (CC)	Water	2-8 °C 12 mo.
Hygromycin B	H9773	527.5							100-200	Water	2-8 °C 6 mo.
Kanamycin	K1377	582.6	XX	XX		XX	100	>2	16 (SSC); up to 40 (CC,PR,EC)	Water	2-8 °C 12 mo.
Paromycin	P8692	615.6	XX	X						Water	2-8 °C 21 days
Penicillin G, potassium	P7794	372.5	XX	X			1005		up to 100	Water	2-8 °C 7 days
Penicillin G, sodium	P3032	356.4	XX	X			1005		up to 100	Water	2-8 °C 7 days
Rifampicin	R7382	823.0	XX	XX	XX		15	>25	50 (TS); up to 25 (PC,TEC)	DMSO	2-8 °C 1 day
Vancomycin	V1130	1485.7	XX				5	>100	up to 10 (PC); up to 40 (cc,EC,STM)	Water	2-8 °C 7 days

XX = Effective against most species
X = Effective against certain species

¹Application key: CC = callus culture; PR = plant regeneration; EC = cotyledon, hypocotyl, or leaf disc culture;

TS = transformant selection; SSC = stem section culture; PC = protoplast culture; TEC = tuber explant culture;

STM = shoot tip micropropagation

Antimycotics Reference Guide

Product	Prod. No.	Mol. Wt.	Yeasts	Molds	Toxicity to microbes (µg/mL)	Toxicity to plant tissue (µg/mL)	Working conc. (µg/mL) and applications1	Product Solubility	Liquid Storage
Cycloheximide	C7698	281.4						EtOH	2-8 °C 30 days
Pentachloronitrobenzene	P8556	295.3		XX				EtOH DMSO
Thiabendazole	T5535	201.2		XX				1 N HCl DMSO

XX = Effective against most species
X = Effective against certain species

¹Application key: CC = callus culture; PR = plant regeneration; EC = cotyledon, hypocotyl, or leaf disc culture;

TS = transformant selection; SSC = stem section culture; PC = protoplast culture; TEC = tuber explant culture;

STM = shoot tip micropropagation