BACTERIAL AND FUNGAL INDEXING OF TISSUE CULTURES

Dr. Mike Kane

Department of Environmental Horticulture

University of Florida

INTRODUCTION

Rapid production of pathogen eradicated plants is a fundamental goal of the micropropagation process. Since the surfaces of plants are normally covered with a diverse bacterial and fungal microflora, the primary objective of Stage I is the complete elimination of contaminating organisms and the subsequent establishment of the sterile explant in vitro. This is usually accomplished through surface sterilization of excised shoot tips (or other explants) with sterilants such as alcohol and/or sodium hypochlorite. Once the explants have been established in vitro, it is essential that cultures be indexed (screened) for the presence of microbial contaminants before being multiplied in Stage II. Besides increased culture mortality, the presence of latent infections can result in variable growth, tissue necrosis, reduced shoot proliferation and reduced rooting. Indexing is accomplished by inoculating tissue sections onto enriched selective microbiological media that will promote the growth of bacterial, fungal or other contaminants. Since secondary contamination can occur as a result of poor aseptic technique or contaminant vectors such as mites, it is important that Stage II cultures also be routinely indexed.

In reality, many laboratories use only visual methods to screen culture contamination (the so- called eyeball determination). There are several important reasons why visual screening for microbial contamination is not adequate. First, visible growth of specific contaminants may be reduced in plant culture media (6). Certain contaminants such as Methylobacteria have evolved close biochemical relationships with the plants and do not grow independently. Finally, bacteria, fungi and other contaminants often persist as endophytes within plants cultures that appear visually contaminant-free (3,4). Numerous procedures have been developed to screen for cultivable contaminants in culture (1,2,6,7,8). Knauss (4) has developed procedures which screen for the presence of systemic bacteria and fungi by culturing 0.5-1.0 mm thick internodal and nodal stem cross sections from Stage I plantlets in enriched broth media that promote microbial growth (Figure 1). This procedure has been effective in establishing cultures of foliage plants such as Dieffenbachia that are free of pathogens such as Xanthomonas dieffenbachiae and Erwinia chrysanthemi (3,4). We use a modified procedure, as detailed by Knauss (4), to routinely screen for cultivable bacteria and fungi in our Stage I and Stage II cultures. These procedures are outlined below.


PROCEDURES

Indexing Media & Preparation

Difficulties often exist in detecting contamination without knowledge of appropriate culture media for the contaminants present. Therefore, we routinely use three enriched media (Table 1). Sabouraud dextrose medium is formulated for the culture and growth of fungi including yeast, mold and aciduric microorganisms. Yeast extract dextrose broth medium is used to stimulate bacterial growth. AC broth is a sterility test medium for a wide variety of microorganisms. These media are manufactured by Difco Laboratories (Detroit, MI) and distributed through Fisher Scientific Supply (Norcross, GA). Both solid and liquid broth indexing media are used. The solid indexing media are prepared by the addition of 10 g/liter agar. Media are prepared without pH adjustment, and dispensed as 10 ml volumes into 20 ml glass scintillation vials covered with autoclavable screw caps. After autoclaving, the agar-supplemented medium is cooled and solidified as 45° slants.

Culture Indexing Procedure: Stage I Cultures

Culture exhibiting visible contamination are discarded. Shoots are aseptically removed from the culture vessel and placed on a sterile petri dish half. Roots are excised and thin stem tissue cross sectional disks are cut. Each of the three liquid media are inoculated with a single stem disk from the same culture. The tissue is partially crushed before being inoculated into the liquid media. Agar-solidified slants of each medium are inoculated by first gently stabbing the medium at the base of the slant and then dragging the tissue up along the surface, leaving it at the top of the slant. The remaining shoot culture is transferred onto fresh medium. For reference, the scintillation vials are labelled with the same culture number as the transferred shoot culture. Uninoculated vials serve as controls. Inoculated indexing media and controls are maintained in the dark at 22 - 30ºC for three weeks. Shoot cultures that screen negative for cultivable contaminants are indexed again after 4 weeks (Fig. 1).

Culture Indexing Procedure: Stage II Cultures

Cultures that initially screen negative for contamination can become contaminated as the result of poor aseptic technique or the result of mite contamination. All cultures within a transfer block are numbers. Fifteen percent (15%) of the cultures are randomly selected and indexed using the aforementioned procedures.

Expected Results

Rapid clouding of the inoculated liquid media is a positive indication of the presence of cultivable contaminants in the tissue sample. The presence of contaminants on the agar-solidified media is confirmed by development of colonies on the surface of the medium and\or development of a halo in the medium where the tissue sample was stabbed. The uninoculated controls should not display microbial growth. When contamination is detected in one medium, it is detected in the other two media more than 95% of the time. Detection of slower growing contaminants is usually facilitated on agar-solidified media. Although these media will promote the growth of diverse microorganisms, it is important to note that growth of all bacteria and fungi will not be supported on these medium. Other media such as Leifert and Waites (6,7) detect a wide range of contaminants with the exception of some Xanthosomonas, Methylobacterium and Hyphomycrobium species and all mycoplasma-like organisms. Likewise, George and Falkinham Mycrobacterium Detection Medium TT (2) has been developed to detect bacteria of the Mycobacterium avium-intracellulare and M. scrofulaceum (MAIS) group.


LITERATURE CITED

  1. Debergh, P.C. and A.M. Vanderschaeghe. 1988. Some symptoms indicating the presence of bacterial contaminants in plant tissue cultures. Acta Hort. 225:77-82.
  2. George, K.L. and J.O. Falkinham. 1986. Selective medium for the isolation and enumeration of Mycobacterium avium-intracellulare and M. scrofulaceum. Can. J.Microbiology 32:10-14.
  3. Holland, M.A. and J.C. Polacco. 1994. PPFMs and other covert contaminants: is there more to plant physiology that just plant? Annu. Rev. Plant Physiol. Mol. Biol. 45:197-09.
  4. Knauss, J.F. 1976. A tissue culture method for producing Dieffenbachia picta cv. `Perfection' free of fungi and bacteria. Proc. Fla. State Hort. Soc. 89:293-296.
  5. Knauss, J.F. and M.E. Knauss. 1979. Contamination of plant tissue cultures. Proc. Fla. State Hort. Soc. 92:341-343.
  6. Liefert, C., W.M. Waites, and J.R. Nicholas. 1989. Bacterial contaminants of micropropagagated plant cultures. J. Applied Bacteriology 67:353-361.
  7. Leifert, C. and W.M. Waites. 1992. Bacterial growth in plant tissue culture media. J. Applied Bacteriology 72:460-466.
  8. Viss. P.R., E.M. Brooks and J.A. Driver. 1991. A simplified method for the control of bacterial contamination in woody plant tissue culture. In Vitro Cell Dev. Biol. 27P:42.

Indexing Procedure

Figure 1. Indexing Procedure (modified from Knauss [4]).


TABLE 1. FUNGAL AND BACTERIAL INDEXING MEDIA1

SABOURAUD DEXTROSE BROTH2

YEAST EXTRACT DEXTROSE BROTH

AC BROTH

10 Grams Neopeptone

10 Grams Yeast Extract

20 Grams Proteose Peptone No. 3

20 Grams Bacto Dextrose

10 Grams Dextrose

3 Grams Bacto Beef Extract

3 Grams Bacto Malt Extract

3 Grams Bacto Yeast Extract

5 Grams Bacto Dextrose

0.2 Grams Ascorbic Acid

1Media available through DIFCO Laboratories, Detroit, Michigan.

2Media also solidified by addition of 10 g/liter agar