1. Bacterial Contaminants of Plant
Tissue Culture
Plant Tissue Culture
MBT 722
2011
Prof. Naim Iraki
Amer Wazwaz
1000316

2. Importance of Controlling Contamination
The medium contains many different bacterial
nutrients, both original constituents of the medium
and exudates from the plant cells.((Bradbury 1988
When microbe(s( come in contact with plant tissue or
.medium then we will have a contamination
Plant ]tissues/ cells[ growing in vitroare considered
to be under some stress conditions and may be
predisposed to direct infection, even by bacteria
not normally pathogenic to them.((Bradbury 1970

3. Contaminating Bacteria Can Be Divided into
Epiphytes
Common
Disinfection can be enough
Endophytes
More problematic
Disinfection is not enough
Antibiotics are needed
Pathogenic bacteria can be a contaminant

4. Contaminating Bacteria May Originatefrom
Explants
Lab environment

5. Contaminating Bacteria May Originatefrom
Operators
Mites and Thrips

6. Contaminating Bacteria May Originatefrom
Ineffective sterilization techniques
Can contaminate cultures at any procedural step if we don‘t take strict standards

7. Procedures for producing aseptic cultures
require attention to
Indexing explants and cultures for contaminants
Identifying the source of those contaminants
Identifying and characterizing the contaminants
Eliminating the contaminants with
improved cultural practices ,antibiotics orother chemical
agents

8. Indexing Cultures
Serial stem slices inoculated intoliquid and agar-solidified
different bacterial media 3
Incubated for three weeks at30°C, detected most contaminants
from more than60 aquatic ,marsh , andornamental woody
plant species ((Kane. 1995
Usually, a contaminant would grow on two of the three media
((Kane. 1995
Contaminated cultures are sometimes rooted and transferred
to the greenhouse instead of being discarded((Kane. 1995

9. Cultures and Medium Indexing
a. Healthy bacteria-index-negative (left( b. Medium-indexed plate
and index-positive (right( cultures of showing different bacterial types
triploid watermelon .one week post-indexing
Pious Thomas , CURRENT SCIENCE, VOL. 87, NO. 1, 10 JULY2004

10. Characterization and Identification
Purification by standard bacteriological methods
Characterization by biochemical tests, Gram staining
Identification by traditional tests which are
Labor-intensive and time consuming
Can be performed in any laboratory with common chemicals
Through comparing with the standard strains ofBergey's Manual
(Krieg and Holt 1984(

11. Modern Identification Techniques
Biolog system
Detectscarbon source utilization with
the reduction oftetrazolium dye in
response to cellular respiration
Can identify yeasts and fungi
Through comparing the results with
a database of responses

12. Modern Identification Techniques
Analytical Profile Index or API system
Carbon source utilization test
Visual detection of the test
(Leifert et at. 1989; Vemiere et al., 1993(
Enzymatic oxidation/reduction interactions
Allows the identification ofa limited number of Bacteria

13. Modern Identification Techniques
Fatty Acid Analysis Profiles FAP
Uses gas chromatography to identify
over 140 separate fatty acids
Fatty acid profile is matched to a library
of 700+ bacterial species representing
over 180 genera
Can identify yeasts and fungi also
Match fatty acid methyl esters with those
of known organisms (Buckley et al.. 1995; Chase
(et al., 1992; Stead et at, 1992

14. Modern Identification Techniques
16S rRNA
PCR amplification/ probes for known sequence
Using this system depends uponthe number and diversity of
bacteria in the databases
Many soil and plant bacteria have not been not characterized
((Buckley et al. 1995
For a more accurate identification the use of more than one
test isRecommended ( (Jones et al. 1993( and ( Verniere et al. 1993

15. Rate of occurrence of microbial
contaminant in plant tissue culture
Odutayo et al. Afr. J. Agric. Res. Vol. 2)3(, pp. 067-072, March2007

16. The occurrence of bacteria isolates in
plant tissue culture
Odutayo et al. Afr. J. Agric. Res. Vol. 2)3(, pp. 067-072, March2007

17. Antibiotic Treatments
Choosing an antibiotic depends on the type of bacteria
Gram negative or Gram positive
Ideal antibiotics should be
soluble, stable, unaffected by pH, unaffected by media,
lacking side effects, broadly active, bactericidal,
suitable incombination, non-resistance inducing,
.inexpensive and nontoxic to human health
Many antibiotics exist that have not yet been evaluated on
plants or their bacterial contaminants(Falkiner, 1990; Seckinger,
.(1995

18. Antibiotic Treatments
Antibiotics may be inactivated by environmental conditions
heat/ light
Antibiotic sensitivity is reduced in plant tissue culture media
due todifferent favorable pH degrees
Antibiotic concentration ] MBC [ for a particular bacteria
should be determined
Phytotoxicity varies greatly among plant species and explant
types, so preliminary testing with plant cultures is important

19. Effects ofdifferent concentrations of antibiotics at
different durations of time to ensure
contamination free cultures
Habiba et al. Plant Tissue Cult. 12)2( : 117-124,2002

20. Antibiotic Effects on Shoots Number
and Multiplication Rate
Multiplication rate of
Pelargoniumshoots
before (week
0(,during andafter
treatment with
carbenicillin or
cefotaxime
end of cefotaxime treatment*
A. Wojtania et al.J. Fruit Ornam. end of carbenicillin treatment**
Plant Res. 104 vol. 13,2005

21. Antibiotic Effects on Shoots Number
and Multiplication Rate
Pelargonium shoots after 3 weeks of the growth on the medium containing
(cefotaxime (A( and carbenicillin (B
A. Wojtania et al. J. Fruit Ornam. Plant Res. 104 vol. 13,2005

22. Conclusion
Several steps can reduce bacterial contaminants
Properly training the operators
Indexing cultures at initiation stage/culture cycle
Identifying contaminants and testing to
determine the proper antibiotic

23. Thank You
Questions