Plant Tissue Culture Protocols

Antibiotics

 
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.

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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 G6896   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 K4378 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 P8306 372.5 XX X     1005   up to 100 Water 2-8°C
4 days
Penicillin G, sodium P8431 356.4 XX X     1005   up to 100 Water 2-8°C
4 days
Rifampicin R7382 823.0 XX XX XX   15 >25 50 (TS);
up to 25
(PC,TEC)
DMSO 2-8°C
1 day
Streptomycin sulfate S0774 1457.4 X XX X   100 >16   Water 2-8°C
30 days
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
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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
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