United States

Department of

Agriculture

Forest Service

Southern Forest

Experiment Station

Research Note

SO-369

Julv 1992

A Method to Preclude Moisture Condensation

in Plated Tissue Cultures

Alex M. Diner

SUMMARY

Excessive condensate normally accumulates in

in vitro-illuminated petri dishes containing plant tissue

cultures, causing avariety of problems. Adark-colored

rubber net-mesh placed over the petri dishes prevented such condensation, even when charcoal-supplemented media are used under high light intensity

in a growth chamber.

INTRODUCTION

Petri dishes containing agar-solidified media are

commonly used in microbiology as well as in plant and

animal tissue culture. However, the illumination employed to satisfy the photosynthetic light requirements

of higher plant tissues frequently leads to the accumulation of condensate .under the petri dish lids. This

problem is compounded by the need to seal the rim of

covered dishes during the standard several-week tissue incubation periods to prevent desiccation of the

medium and to maintain asepsis. During these periods, condensate accumulates and eventually drips

onto the medium. The resultant pools of moisture not

only alter the environment of plant tissues in situ, but

may cause the spread of any contaminant microorganisms from small, isolated colonies otherwise available for selective removal. Free moisture has also

been implicated in the development of vitreous tissues

(von Arnold and Eriksson 1984). Moreover, this condensate restricts both the visual examination of cultures and their photography through the covered petri

dish. Stacking one or two empty petri dishes atop the

dish containing the tissues is common practice and

sometimes helps reduce the formation of condensate.

However, the use of charcoal-supplemented media

(Amerson and others 1988) nullifies any benefit this

practice might have; evaporation of water from the

illuminated (and therefore slightly warmed) black substrate presumably accounts for the unusually large

amounts of condensate that accumulate.

The inexpensive and simple method described in

this article completely precludes the accumulation of

condensate even in charcoal-supplemented media

and under conditions of high light intensity.

MATERIALS AND METHODS

We observed that condensate will characteristically

not accumulate contiguous to any black (magic

marker) lettering used for tissue treatment identification on the petri dish lid surface. This led to the suspicion that light absorption by the dark lettering, and the

resultant local and slight warming of the petri dish

plastic lid 2 to 3 mm on either side of the ink%ne,

precluded the accumulation of condensate. Therefore, 115 mm* sheets of a black, soft-rubber mesh

(Softliner, Nalle Plastics, Austin, TX) were placed directly upon Parafilm-sealed plastic petri dishes in a

20 “C growth chamber under 100,150, and 250 microEinsteins meter -* second -’ @Em -* s-l) cool-white

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1
fluorescent illumination. The diamond-shaped mesh

network consisted of 6 x 10 mm openings bordered

by a 2 mm-thick mesh. Petri dishes contained loblolly

pine (Pinus faeda L.) adventitious micropropagules on

nutrient media with and without 1 percent (w/v) activated charcoal. A second group of plates containing

similar propagules was similarly illuminated, either directly or by having two empty petri dishes stacked on

top of each other.

All plates were examined for the presence of condensate or free water following a 2 week incubation.

Light intensities incident upon the petri dish lid surfaces were measured with and without the intervening

Softliner.

RESULTS AND DISCUSSION

Plates containing medium with or without activated

charcoal and illuminated (all intensities) with no intervening empty petri dishes showed a large amount of

condensate on both the inner surface of the lid and

the medium surface, the latter having dripped from the

lid. Plant tissues in plates containing the charcoal-supplemented medium were thus obscured from view.

Plated media stacked with empty dishes were less wet

than were the directly illuminated plates. All plates

covered with the Softliner mesh were free of water on

all inner surfaces. The mesh reduced light intensity on

the petri dish lids 40 percent; light intensities effective

for in vitro micropropagation of Pinus (Amerson and

others 1966) and other genera (Diner 1990) could

therefore be maintained employing incident illumination of at least 150 FE m -* s-l. Pine propagules in

plates covered by the Softliner grew and developed

normally.

The use of Softliner or a similar product should

benefit research in plant tissue culture employing tissue containers that otherwise would accumulate unwanted condensate from the agar-solidified, aqueous

medium. A mesh color other than black may prove

advantageous under circumstances of extreme tissue

temperature-sensitivity where conversion of radiant to

thermal energy by the Softliner would be less efficient.

LITERATURE CITED

Amerson, Henry V; Frampton, L. John, Jr; Mott, Robert

L.; Spaine, Pauline C. 1988. Tissue culture of conifers

using loblolly pine as a model. In: Hanover, James

W.; Keathley, Daniel E., eds. Genetic manipulation of

woody plants. New York: Plenum Press: 117-l 37.

Diner, Alex M. 1990. Clonal micropropagation of mature Lark New Forests. 4: 63-66.

von Arnold, Sara; Eriksson, Tag. 1984. Effect of agar

concentration on growth and anatomy of adventitious shoots of Picea abies (L.) Karst. Plant Cell,

Tissue and Organ Culture 3: 257-264.

Alex M. Diner is a plant physiologist at Biotechnology of the Southern Pines and Hardwoods Unit, U.S. Department of Agriculture, Forest

Service, Southern Forest Experiment Station, Alabama A&M University, Huntsville, AL 35762.

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