Physical & Chemical Means of Controlling Contamination in Plant Tissue

Physical & Chemical Means of
Controlling Contamination in
Plant Tissue Culture
Dr. Gary Seckinger

Dr. Kenneth C. Torres
PhytoTechnology Laboratories
“Like death and taxes, contamination or the
threat of contamination is always with us and
we need all the weapons to combat them.”
L.L. Coriell
National Cancer Institute
Monog. 7:33, 1962
24 May 2004 2004 WORLD CONGRESS on In Vitro Biology

Physical Means of Controlling
Contamination
Eliminate vector transmission of contaminants:

• Seal Culture Vessels
– Parafilm, Nescofilm
– PVC Film (Austratec)
• Seal Cultures in Plastic Bags:

– e.g., Zip-lock Bags,
Star*Pac Bags

Chemical Control of Contamination
The Use of Antimicrobials in

Plant Tissue Culture
24 May 2004 2004 WORLD CONGRESS on In

Vitro Biology
Antibiotics (Antimicrobials)
Bacteristatic – Inhibit bacterial growth; bacteria viable

but “dormant”. Growth resumes in antibiotic-free
medium.
Bactericidal – Lethal to bacteria; no subsequent
growth in antibiotic-free medium
Antifungal (Antimycotics) – Fungistatic & Fungicidal
Broad-spectrum Antimicrobials – Inhibit or eliminate
bacteria and fungi.
Antiviral – Inactivate virus

Ideally, Antibiotics in Plant Tissue Culture are:
Soluble Systemic in plant tissues

Stable Broad spectrum of activity
Unaffected by media Low chance of bacterial
pH or components resistance
Inexpensive Lethal to microbe
Minimal side effects Usable in combinations
(no phytotoxicity)
Gilbert, et al., Ann Appl Biol 119:113, 1991
Falkiner, Acta Hort 225:53, 1988 & Int Assoc Plant Tiss Cult 60:137, 1990

Theory vs. Reality
Theoretically…
All (microbial) contaminants can be eliminated from
plant tissue cultures by one or more antibiotics.
In reality…
This is seldom possible.
No substitute for careful, aseptic work.
Last resort when conventional techniques fail.

So Many Antibiotics Available… Which to Use?
Ideally:
• Isolate contaminant
• Streak it on bacterial medium plate
• Apply antibiotic discs
• Determine which antibiotic(s) are most effective
Alternate approach:
• Streak contaminant through selective media
• Determine partial characterization or identity
• Hit the library… Which antibiotics are effective?
Any phytotoxicity reported?
Antibiotic Effectiveness… More Reality
Antibiotic effectiveness can be affected by:

• pH of medium – e.g., some are more effective at alkaline
pH’s
• Ion concentration of medium – high cation concentration
can reduce effectiveness of some antibiotics
Many antibiotics have short solution stability:
• e.g., the Penicillin group → 24-48 hr at room temp.
Phytotoxicity varies:
• Between plant species
• Between genotypes within a species
• Between plant parts – organs vs. intact plant
How Much Do I Use?
MIC vs. MBC

MIC – Minimum Inhibitory Concentration
• Inoculate bacteria on medium w/ increasing concentrations
of antibiotic(s)
• Lowest concentration of antibiotic to prevent bacterial
growth = MIC
MBC – Minimum Bactericidal Concentration
• Subculture from medium at & above MIC conc.
• Lowest antibiotic conc. in MIC test at which no bacteria
grow on bacterial medium w/out antibiotic = MBC
Target: Phytotoxic Concentration =2
MBC
If One is Good… Are 2+ Better?
Most antibiotics have narrow spectrum of activity

e.g., Gram +, Gram -, or Antimycotic
Plant cultures may contain more than one type of
contaminant… may contain numerous types
Therefore,
Antibiotic combinations may be necessary
Worse yet…
Some microbial species difficult to eliminate w/ one
antibiotic, e.g., Hypomicrobium contamination
Neither 500 µg/ml Streptomycin or Carbenicillin effective.
Combined 100 µg/ml of both was effective.
Horsch & King, Plant Cell Tiss Oran Cult 2:21-28, 1983

Antibiotic Effects on Growth & Development
Negative:
• Detrimental to plastids & mitochondria
• Impair chlorophyll formation
• Inhibit amino acid incorporation
• Inadvertent development of resistant cell lines by
mutation or selection.
• Antibiotic combinations may have synergistic
phytotoxicity (i.e., 2 antibiotics show little/no toxicity
when used individually; significant toxicity when
combined).

Effect on Multiplication
Grading antibiotics based on their inhibitory effect on

plant growth (reduced multiplication rate in vitro):
Most Inhibitory
Streptomycin ⇒ Polymyxin-B ⇒
Rifampicin ⇒ Carbenicillin
Least Inhibitory
Leifert, et al.
Plant Cell Tiss Org Cult 29:153, 1992

Antibiotic Effects on Growth & Development
Positive:
• Promote growth of cell cultures
• Enhance morphogenesis while inhibiting callus
growth
• Stimulate root development in cuttings.

Do I Have to Add Antibiotics to the Medium?
Not necessarily…
Stock Plant Treatment – Reduced phytotoxicity
• Hevea shoots-tips ⇒ inhibited growth w/ antibiotics
in medium.
• No inhibition if stock plant sprayed every 2 days for
2 weeks before culture.
• Contamination controlled
Enjalric, et al., Acta Hort 225: 57, 1988
Other studies have shown reduced bacterial growth
in shoot-tips & buds w/ regular antibiotic treatments
to stock pants.
Alternatives to Antibiotics in Medium (cont.)
Explant Treatment
• Shoot-tips sprayed before excision from stock plant
• Shoot tips/ buds dipped or soaked after excision
– e.g., Prunus shoot tips treated 1 min w/ Rifampicin before
culture gave better control than Rifampicin in medium.
• Tuber/ corm treatments
– Cyclamen tubers soaked 1-4 days before culture
produced non-contaminated cultures w/out phytotoxicity.
– Antibiotics in medium were phytotoxic.
Combining stock plant or explant treatments w/ in vitro
treatment may be necessary.
Antibacterial Antibiotics
Bacteristatic Bactericidal
Reversible inhibition of Lethal to Bacteria.
bacterial growth.
May have bactericidal activity Bacteristatic at low
at high concentrations. concentrations.
Includes: Includes:
• Choramphenicols • Aminoglycosides
• Tetracyclines • Cephalosporins
• Macrolides (Erythromycin) • Penicillins
• Polymyxins (Polymyxin-B)
• Rifamycins (Rifampicin)
The Chloramphenicols
• First broad-spectrum antibiotics discovered.

• First isolated from Streptomyces venezuelae.
• Bacteristatic activity by interfering with bacterial
protein synthesis.
• Activity spectrum similar to Tetracyclines includes
Gram +, Gram -, and anaerobic bacteria.

Chloramphenicol
• Solubility:
– 2.5 mg/ml in water (increased with urea).
– 50 mg/ml in ethanol.
• Solution Stability:
– 50% lost in 290 days at 20° C.
– 10% lost on heating to 115° C for 29 minutes.
– Protect from light, stable over pH 2.0-7.0.
• Incompatible with gentamicin.
• Application: [5 µg/ml for selection]
– Safe: up to 300 µg/ml stimulated corn protoplast.
Agricell Rep 9:38, 1987
– Toxic: 5-50 µg/ml Tobacco, beet, carrot, sunflower
George, Plant Prop by Tiss Cult, 1991

The Tetracyclines (Quinones)
• Semi-synthetic antibiotics originally derived from

Streptomyces spp.
• Bacteristatic activity by interfering with bacterial
protein synthesis.
• Broad-spectrum activity including Gram +, Gram -,
and mycoplasmas.
• Includes: Tetracycline (only widely used antibiotic
in this group).

Tetracycline
• Solubility:
– Free base – alcohol.
– HCl salt – water.
– Hydrolyzes in water (tetracycline precipitates out).
– 10% lost in 24 hours at room temperature or 48 hours at 5° C.
• Incompatible with Penicillins, Polymyxin-B, and
B-complex vitamins.
• Applications:
– Safe: 25-100 µg/ml, Woody plants
Young, et al., Plant Sci Lett 34:203-209, 1984
– Toxic: Cherry, beet, carrot, tobacco
George, Plant Prop by Tiss Cult, 1993

The Macrolides
• Derived from Streptomyces spp.

• Bacteriostatic/bactericidal activity is dependent on
dose and specific microbe.
• Antibiotic activity due to interference with bacterial
protein biosynthesis.
• Effective against Gram (+) bacteria.
• Erythromycin – only widely used antibiotic in this
group.

Erythromycin
• Hygroscopic powder, mixture of Macrolide antibiotics.

• Solubility:
– 2 mg/ml in water (Merck Index).
– 5 days or longer at 5° C.
– Protect from light.
• Resistance shown in many organisms.
• Antagonism & synergism with Ampicillin and Gentamicin;
incompatible with Penicillin-G.
• Application:
– 32 µg/ml (comb.). Plant Cell Rep 7:622, 1989
– Toxic: Tobacco

The Aminoglycosides
• Derived from Actinomycetales (bacterial order).

• Bind to 30S ribosomal subunit in bacterial cells and
inhibit protein synthesis.
• Most active against Gram -.
• Gram + and anaerobic bacteria are naturally
resistant.
• Includes:
– Kanamycin
– Neomycins
– Streptomycin
– Gentamicin

Kanamycin
• Water Soluble.
– Stable at room temperature.
– Recommend 5° storage long term.
– Solutions darken without affecting potency.
• Resistance reported:
– Cross resistance with neomycin.
– Streptomycin.
• Effectiveness reduced with increasing Calcium Ions.
• Application:
– Safe: 1.5-4.5 µg/ml enhanced shoot differentiation of tobacco and
carrot calli. Owens, Plant Sci Lett 16:225-230, 1979
– Toxic: Beet, carrot, radish, Brassica spp, Tobacco

Neomycin Sulfate (Fradiomycin Sulphate)
• Mixture of sulfates of Streptomyces fradiae.

• Water Soluble.
– 5° C for long term storage, yields some color change. Maximum of
two years.
• Used in conjunction with other antibiotics (e.g., Polymyxin
B) to prevent development of resistant strains.
• Application:
– 1000 µg/ml shoot tip pretreatment. Agricell Rep 20:22, 1993
– Toxic: Cherry, tobacco

Streptomycin Sulfate
• Water Soluble.
– 1 month at room temperature.
– A few months at 5° C.
– 18 months at -20° C.
• Resistance reported in most sensitive organisms.
• Cross-resistance with neomycin and kanamycin.

Streptomycin Sulfate (Cont.)
• Applications:
– 16 µg/ml (comb). Plant Cell Rept 20:22, 1993
– 25 µg/ml (comb). Ann Appl Biol 119:113, 1991
– 50-100 µg/ml (comb). J Appl Biol 119:113, 1991
– 72 µg/ml enhanced morphogenesis in tobacco and
carrot calli. Plant Sci Lett 16:225, 1979
– 1000 µg/ml (comb). Agricell Rep 20:22, 1993
– 100-200 µg/ml Syngonium & Philodendron
Acta Hort 212:87, 1987
– Toxic: Tobacco, sunflower, cherry, beet, carrot

Gentamicin Sulfate
• Mixture of Gentamicins C1, C1A, C2, C2A, C2B.

• Water Soluble.
– Recommend 5° for long-term.
• Incompatible with Penicillins, Erythromycin, and
Chloramphenicol.
• Resistance appearing in many Gram - bacteria.

Gentamicin Sulfate (Cont.)
• Resistance appearing in many Gram (-) bacteria.

• Application:
– 50 µg/ml (comb).
Agricell Rept 20:22, 1993.
– 50 µg/ml no toxicity on Cattleya seedlings grown In Vitro.
Amer J Bot 66:825, 1979.
– 250 µg/ml reduced explant contamination in Piper sp.
S Afr J Bot 58:500, 1992.
– Toxic: Datura, Cymbidium, Vanda

The Cephalosporins
• Semi-synthetic antibiotics derived from Cephalosporin C.

– Natural antibiotic produced by Cephalosporium acremonium
mold.
• Inhibit Bacterial cell wall synthesis.
• Classified by “generations” based on antibacterial activity
and introduction date.
• Third Generation:
– Have wider spectrum and greater potency against Gram -
bacteria.
– Activity against Gram + bacterium is less than earlier
generations.
• Includes:
– Cefotaxime

Cefotaxime
• Water Soluble. Protect from light.

– 24 hr at room temp.
– 22 days at 5° C.
– 112 days at -10° C.
– Optimum pH 4.3-6.2.
• Activity enhanced by:
– Aminoglycosides (e.g., Gentamicin).
• Incompatible with alkaline solutions.
Cefotaxime (Cont.)
• Applications:
– 25-100 µg/ml (comb), woody plants.
Plant Sci Lett 34:203,1984
– 50-100 µg/ml enhanced wheat regeneration.
J Plant Physiol 140:372,1992
– 100 µg/ml enhanced walnut shoot multiplication.
J Exp Bot 44:1211, 1993
– 200 µg/ml enhanced pear regeneration.
J Hort Sci 64:553, 1989
– 250 µg/ml enhanced apple regeneration.
Vitis 33:117, 1994
– 200 µg/ml increased apple shoot growth.
Plant Growth Reg 15:55, 1991
– 500 µg/ml induced Dianthus embryogenesis.
J Plant Physiol 141:721, 1993

The Penicillins
• First antibiotics for therapeutic use.

• Bactericidal activity by inhibiting biosynthesis of
bacterial cell wall.
• Inactivated by penicillinase-producing bacteria.
• Includes:
– Penicillin G (Benzylpenicillin) – Gram (+).
– Ampicillin – broader spectrum than Penicillin G.
– Carbenicillin – narrower spectrum than Ampicillin.
– Ticarcillin – similar spectrum to Penicillin G.

Penicillin G (Benzylpenicillin)
• Off-white powder produced by Pennicillium notatum.

• Water Soluble.
• Stability:
– 7 days at 5° C.
– 18 months at -20° C.
– Optimum pH 5.5-7.5
• pH above or below the accepted range accelerates hydrolysis of
B-Lactam Ring.
– Dilute Solutions more stable than concentrated solutions.
• Incompatible with metal ions, rubber, vitamin C,
Amphotericin, and Tetracycline.

Penicillin G (Cont.)
• Applications: Approximately 1650 U/mg

– 25-50 U/ml (comb).
Ann Appl Biol 119:113, 1991.
– Stimulated embryogenesis in Dianthus.
Agicell Rep 22:3, 1994.
– 10-100 µg/ml Cattleya seedling in vitro.
Amer J Bot 66:825, 1979.
– Prevent seed germination & inhibit organogenesis in
several species.

Ampicillin
• Water Soluble.
– As with penicillin 24 hours at room temp.
– Maximum stability at pH 5.5-7.5.
• Incompatibility with aminoglycides
• Applications:
– 160 µg/ml reduced contamination in Piper sp
explants.
S Afr J Bot 58:500, 1992.

Carbenicillin (α-Carboxybenzylpenicillin)
• Water Soluble
• Hygroscopic
– 72 hours at 5° C.
– Gentamicin, Ticarcillin, and Clavulanic acid.
• Incompatible with:
– Aminoglycosides, Tetracyclines, Amphotericin.
Carbenicillin (Cont.)
• Applications:
– 250 µg/ml Cattleya seedlings in vitro.
Amer J Bot 66:845, 1979.
– 500 µg/ml induced rapid callus in apple leaf explants;
inhibited regeneration.
Plant Cell Tiss Org Cult 37:257, 1994.
– Stimulated embryogenesis in Dianthus.
J Plant Physiol 141:721, 1993.
– Breakdown produces phenylacetic acid (auxin).
Plant Cell Rep 11:93, 1992.
– Toxic: Beet, carrot

Ticarcillin
• Water Soluble.
– As with Penicillin (24 hr at room temp).
• Activity is enhanced by clavulanic acid (Timentin)
which is a B-lactamase inhibitor.
– 15-30 parts Ticarcillin: 1 part Clavulanic acid.
• Synergism reported with Aminoglycosides.
• Cross resistance between Ticarcillin and
Carbenicillin.
• Applications primarily in transformation systems

Polymyxin-B
• Bonds to bacterial membranes and alter their permeability.

• Bactericidal activity against Gram -.
• Water Soluble, Hygroscopic Powder.
– 1 yr at 5° C.
• Activity enhanced with Rifampicin.
• Incompatible with Ampicillin, Tetracycline, Amphotericin.
– Rapidly inactivated by acids and bases.
• Applications:
– 6-24 µg/ml (comb) Woody plants
Young, Plant Sci Lett 34:203, 1984.

Rifampicin (Rifamycin, Rifampin)
• Rifamycin group; interferes with bacterial nucleic

acid synthesis.
• Spectrum: Gram +, few Gram -, Anaerobic
bacteria, Mycobacteria, (some viruses at high
concentrations).
• Solubility:
– DMSO [Dimethylsulfoxide] (per DBR),
– Slightly soluble in water and ethanol.

Rifampicin (Continued)

– Erythromycin, Gentamicin, and Vancomycin.
• Incompatible with Penicillins.
• Applications:
– 6-24 µg/ml (comb) Wood plants
Young, Plant Sci Lett 34:203, 1984
– 10 µg/ml (comb) Camellia
Haldeman, et al., HorSci 22:306, 1987
– 75 µg/ml (comb) Hevea
Enjalric, et al., Acta Hort 225:87, 1988
Antifungals (Antimycotics)
Amphotericin B
Cycloheximide
Nystatin
Griseofulvin
Petachloronitrobenzene (PCNB)
Thiabendazole

Amphotericin B (“Amphoterrible”)
• Interferes with membrane permeability by binding

to sterols.
• Soluble in DMSO.
– Water-soluble preparations available (Deoxycholate-
sodium phosphate complex = Fungizone)
– 5 days at room temp.
– 1 month at 5° C.
– 1+ Yr at -20° C.
– Inactivated by low pH.

Amphotericin B (Cont)
• Incompatible with Penicillins, Gentamicin,

Kanamycin, Polymyxin-B, and Streptomycin.
• Applications:
– Up to 10 µg/ml on Cattleya seedlings in vitro.
Amer J Bot 66:825, 1991.
– 62-125 µg/ml (comb).
Ann Appl Biol 199:113, 1991.

Cycloheximide
• Antifungal and antiviral activity.

• Solubility:
– 5 mg/ml in water (may require sonication).
– 1 to 3 months at 5° C (aqueous pH 3.0-5.0)
– 1 to 3 years at -20° C.
• Autoclavable

Nysatin (Fungicidin)
• Polyene antibiotic with fungistatic and fungicidal

activity by interferring with cell membrane
permeability.
• Solubility:
– Soluble in DMF (dimethylformamide).
– Very slightly soluble in water; used as active
suspension.

Nysatin (Cont.)
• Suspension Stability:
– 5 days at room temp.
– 1 month at 5° C.
– 2 yr at -20° C.
• Applications:
– 16 µg/ml (comb).
Plant Cell Rep 7:622,1989.

Wide-Spectrum Antimicrobials
PPM™ (Plant Preservative Mixture)

8-Hydroxyquinoline

PPM™ (Plant Preservative Mixture)

• Patented product of Plant Cell Technologies
• Active ingredients
5-Chloro-2-methyl-3(2H)-isothiazolone
2-Methyl-3(2H)-isothiazolone
• Broad spectrum biocide (bacteria & fungi)
• Inhibits enzymatic activity in citric acid cycle and
electron transport chain
• May inhibit translocation of sugars & amino acids
• Translocation across plant cell wall impeded
(Freshly isolated protoplasts are very sensitive to PPM)
PPM™ (Cont.)
• Heat stability:
– Typically loses ~10% potency when autoclaved in
standard plant TC media (MS, B5, WPM, etc.).
– Binds to polypeptides when autoclaved so potency
reduced in protein-rich media (e.g., w/ 1 g/L Casein
Hydrolysate conc. of ~2X required).
• Recommended Concentrations:
– Seeds – 20-30+ ml/L for 8-12 hr.
– Shoot-tips (1+ cm) – 40-50 ml/L for 4-12 hr.
– Tubers – 40-50 ml/L for 12-24 hr.
– General TC media – 0.5-2 ml/L
– Callus, organogenesis, embryogenesis – 0.5-0.75 ml/L

8-Hydroxyquinoline
• Antibacterial and antifungal activity by intereferring
with microbial nucleic acid synthesis.
• Water Soluble.
– No references.
– Stable in practical uses.
• Applications:
– Up to 1 µg/ml on Sedum callus with little toxicity.
Protoplasma 158:19, 1990.

Antiviral
Ribavirin (Virazole™)
Solubility: Water
Solution Stability: 24 hr at 2-6° C (prepare fresh)
Mode of action not fully understood; may be due to
competition w/ guanosine in formation of viral mRNA cap
structure or enzymes involved in production of structural
viral proteins. (www.rxmed.com)
Virus elimination in shoot-tips & buds augmented by heat
(thermo) therapy or electric shock treatment.
Some reports indicate mutation rate may be increased.
Concentration range: 20-100 µg/ml
Repeated subculture required: 7-10 for sugarcane; 6.5 months
for bamboo
Toxicity above 30 µg/ml reported.
Concluding Comments
• Since most microbial contaminants are unknowns,

and,
• Since microorganisms have different sensitivities to
antibiotics it is difficult to specifically target
microbial contaminants.
• Unfortunately, it is common practice to use the
“spray and pray” technique for antibiotic
application… Best to check the literature first.
• Broad-spectrum biocides frequently have
bacteristatic/ fungistatic activity… continuous use
may be required to control contaminants.

References
• O’Neil, MJ (ed). 2001. The Merck Index 13th

Edition. Merck & Co., Inc. Whitehouse Station, NJ.
• United States Pharmacopeia (USP XXIV). 2000.
• George, EF. 1993. Plant Propagation by Tissue
Culture, Part 1. The Technology, 2nd Ed.,
Exegetics Ltd.
• Reynolds, JEF (ed). 1989. Martindale The Extra
Pharmacopoeia. The Pharmaceutical Press.
London.
• Dawson, RMC. 1986. Data for Biochemical
Research. Clarendon Press. Oxford.
• TCA Manual. Procedure #15143. 1976.

Physical &amp; Chemical Means of Controlling Contamination in Plant Tissue

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Physical & Chemical Means of

Dr. Gary Seckinger

24 May 2004 2004 WORLD CONGRESS on In Vitro Biology

Eliminate vector transmission of contaminants:

• Seal Cultures in Plastic Bags:

24 May 2004 2004 WORLD CONGRESS on In Vitro Biology

The Use of Antimicrobials in

24 May 2004 2004 WORLD CONGRESS on In

Bacteristatic – Inhibit bacterial growth; bacteria viable

24 May 2004 2004 WORLD CONGRESS on In Vitro Biology

Soluble Systemic in plant tissues

24 May 2004 2004 WORLD CONGRESS on In Vitro Biology

24 May 2004 2004 WORLD CONGRESS on In Vitro Biology

Antibiotic effectiveness can be affected by:

MIC vs. MBC

Most antibiotics have narrow spectrum of activity

24 May 2004 2004 WORLD CONGRESS on In Vitro Biology

24 May 2004 2004 WORLD CONGRESS on In Vitro Biology

Grading antibiotics based on their inhibitory effect on

24 May 2004 2004 WORLD CONGRESS on In Vitro Biology

24 May 2004 2004 WORLD CONGRESS on In Vitro Biology

• First broad-spectrum antibiotics discovered.

24 May 2004 2004 WORLD CONGRESS on In Vitro Biology

24 May 2004 2004 WORLD CONGRESS on In Vitro Biology

• Semi-synthetic antibiotics originally derived from

24 May 2004 2004 WORLD CONGRESS on In Vitro Biology

24 May 2004 2004 WORLD CONGRESS on In Vitro Biology

• Derived from Streptomyces spp.

24 May 2004 2004 WORLD CONGRESS on In Vitro Biology

• Hygroscopic powder, mixture of Macrolide antibiotics.

24 May 2004 2004 WORLD CONGRESS on In Vitro Biology

• Derived from Actinomycetales (bacterial order).

24 May 2004 2004 WORLD CONGRESS on In Vitro Biology

24 May 2004 2004 WORLD CONGRESS on In Vitro Biology

• Mixture of sulfates of Streptomyces fradiae.

24 May 2004 2004 WORLD CONGRESS on In Vitro Biology

24 May 2004 2004 WORLD CONGRESS on In Vitro Biology

24 May 2004 2004 WORLD CONGRESS on In Vitro Biology

• Mixture of Gentamicins C1, C1A, C2, C2A, C2B.

24 May 2004 2004 WORLD CONGRESS on In Vitro Biology

• Resistance appearing in many Gram (-) bacteria.

24 May 2004 2004 WORLD CONGRESS on In Vitro Biology

• Semi-synthetic antibiotics derived from Cephalosporin C.

24 May 2004 2004 WORLD CONGRESS on In Vitro Biology

• Water Soluble. Protect from light.

24 May 2004 2004 WORLD CONGRESS on In Vitro Biology

• First antibiotics for therapeutic use.

24 May 2004 2004 WORLD CONGRESS on In Vitro Biology

• Off-white powder produced by Pennicillium notatum.

24 May 2004 2004 WORLD CONGRESS on In Vitro Biology

• Applications: Approximately 1650 U/mg

24 May 2004 2004 WORLD CONGRESS on In Vitro Biology

24 May 2004 2004 WORLD CONGRESS on In Vitro Biology

24 May 2004 2004 WORLD CONGRESS on In Vitro Biology

24 May 2004 2004 WORLD CONGRESS on In Vitro Biology

• Bonds to bacterial membranes and alter their permeability.

24 May 2004 2004 WORLD CONGRESS on In Vitro Biology

• Rifamycin group; interferes with bacterial nucleic

24 May 2004 2004 WORLD CONGRESS on In Vitro Biology

• Activity enhanced by:

24 May 2004 2004 WORLD CONGRESS on In Vitro Biology

• Interferes with membrane permeability by binding

24 May 2004 2004 WORLD CONGRESS on In Vitro Biology

Physical & Chemical Means of Controlling Contamination in Plant Tissue

Physical & Chemical Means of Controlling Contamination in Plant Tissue