.T.Esp. Mar. Bid. Ed.

,
Elsevier

279

1988, Vol. 117, pp. 279-283

JEM 01058

Short Communication

Microwave treatment for sterilization of phytoplankton

culture media
Maureen

D. Keller, Wendy K. Bellows and Robert

R.L. Guilhd

Bigelow Laboratmyfor Ocean Sciences, M&own Point, West Boothbuy Hmbor. Maine. U.S.A.
(Received 3 November 1987; revision received 25 January 1988; accepted 4 February 1988)
Abetract: A standard microwave oven for the sterilization of phytoplankton culture media and apparatus
was tested. Elimination of bacterial, algal, and fimgal contaminants is achieved in < 10 mitt for a 1.5 1 vol.
of seawater in a Teflon bottle. Empty culture tubes and vessels are sterilized in 5 min. The technique is quick,
easy, reliable, and does not contaminate with trace metals or cause precipitation. A sample protocol and
numerous precautions are discussed.
Key words: Culturing; Medium; Microwave; Phytoplankton; Sterilization

INTRODUCTION

The sterilization of culture media for the maintenance of phytoplankton stocks or

physiological experiments is often a complicating factor, for many reasons. Because of
the high temperatures occurring during sterilization by autoclaving and the rise in pH
accompanying the loss of CO,, precipitates are often formed from the combination of
inorganic constituents of seawater and components of the enrichment. These precipitates have a deleterious effect on many phytoplankters. Autoclaving can also be
time-consuming, taking several hours - especialSy if the time for cooling the liquids is
included. Further, the steam from autoclaves has been implicated as a source of trace
metal ~ont~ation
for critical analytical work, or studies of trace metal requirements
(e.g., Price et al., 1987). Alternative methods of sterilization, such as repeated pasteurization, boil-freeze-boil methods (Brand et al., 1983), or filter sterilization, are all
effective but are even more time-consuming (see Hamilton, 1973).
The use of microwaves for routine sterilization and extraction procedures has been
evaluated in many laboratory situations. Using home-type microwave ovens, protocols
for sterilizing plastic tissue culture vessels (Latimer & Matsen, 1977; Sanbom et al.,

Bigelow Contribution 87021.

Correspondence address: M.D. Keller, Bigelow Laboratory for Ocean Sciences, McKown Point, West
Boothbay Harbor, ME 04575, U.S.A.
~22-0981~88~$03.50 0 1988 Elsevier Science Publishers B.V. (Biomedical Division)

280

M.D.KELLERETAL.

1982), dental tools (Rohrer & Bulard, 1985), and agar media (Wood & Lundergan,
198 1) have been developed and were effective against a wide range of bacterial and viral
contaminants. Microwave treatment has also been used for the extraction of polar
compounds for chromatography (Ganzler et al., 1986).
Here, we evaluate the effectiveness of microwaves for the sterilization of culture
media and vessels used for growing phytoplankton cultures in experiments involving
trace elements or organic nutrition. A protocol was developed that rapidly and
repeatedly eliminated bacterial, algal, and fungal contaminants from seawater, nutrients,
and culture vessels.
METHODS

A Hot Point, 1.2~fi3, 700-W microwave oven was used, with the power setting on
High. Three 2-1 Teflon bottles, each with 1.5 1 of unfiltered Sargasso or filtered local
seawater, were prepared. To one, a culture of an unidentified marine fungus was added;
to another, a combination of several different algal cultures was added; to the third, no
addition was made (recognizing that a natural bacterial population is always present
in seawater). The bottles were left for several days and then each was microwave-treated
separately as follows: for 3 min, an additional 2 min, 3 more min, and 2 more min, for
a total exposure of 10 min; midway through the 3-min microwaving period and before
each subsampling, the liquid was agitated before proceeding. The sampling periods
chosen were 0,3,5,8, and 10 min. Triplicate l-ml samples were removed at each time.
Sterilization was tested by inoculating the samples into the following media: for algal
contaminants, f/2 (Guillard, 1975); for bacterial contaminants, f/2 p (0.1% (w/v)
bactopeptone (Difco, Detroit, Michigan) in f/2) or M (0.1% (w/v) methylamine in f/2);
and for fungal contaminants, malt extract (Difco). Each test was incubated for 3 wk
under appropriate conditions and checked several times a week.
Plastic racks (30 tubes/es) of empty polycarbonate culture tubes (30 ml) were also
treated for 0, 1, 3, and 5 min, two racks at one time. The tubes were usually wet from
the washing procedure, but if tubes were dry, a beaker of water was included in the oven
to absorb the microwaves. The manufacturer does not recommend operating the oven
empty or with dry materials only. Halfway through each microwaving period, the racks
were turned 180 for even treatment. Tubes, in triplicate, from each microwaving
treatment were tilled with f/2 medium, inoculated with algae to test for growth and later
tested for bacteria using peptone-based media.
Changes in temperature and pH following the microwave process were also recorded.
The experiment was repeated on three different occasions.
RESULTS

The effectiveness of the microwaving procedure for sterilization is illustrated in

Table I.

MICROWAVE STERILIZATION

281

OF CULTURE MEDIA

All contaminants were eliminated at 10 min for a 1.5-1 vol. of seawater. The temperature of the water immediately after the lo-min time point was 84 C. The pH of the water
before microwave treatment was 7.75, immediately afterwards, 7.55, and after cooling
7.90.
TABLE I
Time course

of microwave sterilization of 1.5 1~01s. of seawater. Algal, bacterial, and fungal contaminants
were eliminated during the time course. Presence or absence of each type of contaminant is denoted by +
(presence) or - (absence) in appropriate test media. Medium f/2 is for algae, media f/2p and M are for
bacteria, and medium Malt is for fungi.
Time (min)

f/2

f/2P

Malt

0*
3
5
8
10

+
+

+
+
+

+
+
+

+
+
+
+

* Test media performed in triplicate. Entire experiment was repeated on three different occasions with
identical results.

The microwave treated tubes were bacteria-free after 5 min. Three min or less of
treatment did not eliminate bacterial contaminants. Growth and viability of algae in
microwave treated tubes or medium were comparable to those in autoclaved media.

DISCUSSION

The mechanism of killing by microwaves is not known; no biological effects (other

than those attributable to heating) have been demonstrated. Microwaves produce heat
in two ways: by ionic polarization, in which ions accelerate in an electric field resulting
in increased collisions, or by dipole rotation, in which polar molecules attempt to align
themselves with a changing field (Decareau & Peterson, 1986). Since the final temperature of the microwave-treated solution only reached 84 C, far below that achieved by
autoclaving and for a much shorter period of time, it is unlikely that heat alone killed
the microorganisms except possibly the algae. Recently, Jeng et al. (1987) concluded
that with dry materials, heat alone is the killing mechanism in microwave sterilization;
they found that 45 min or more in either a microwave or convection dry heat oven were
necessary for the destruction of bacterial spores. It is not clear what other factors may
be operating in liquid systems. It is possible, for example, that the movement of polar
molecules within the cells results in changes to the membranes, which produces the
lethal effect. Whatever the mechanism, the ability of microwaves to render substrata free
of microbes is certain. Likewise, microwave treatment of culture vessels or media does
not produce toxic effects or otherwise reduce their usefulness for culturing. At least with
polycarbonate or Teflon ware, no degradation of the material has been observed. Even

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M.D. KELLER ETAL.

disposable (non-autoclavable) plastic ware can have its usable life extended, although
Sanbom et al. (1982) noted that after three microwavings, disposable plastic ware began
to yellow and was probably degrading.
For trace metal work, microwave treatment eliminates a significant source of metal
contamination. For our trace metal research and nutritional studies, medium is sterilized
in Teflon bottles and dispensed aseptically into polycarbonate tubes (which do not
withstand repeated sterilization when containing seawater). The protocol we describe
here was developed in the context of this experimental process, and specifically to
replace both autoclaving, which can contaminate with metals, and the time-consuming
pasteurization or boil-freeze-boil methods. Special protocols should be developed for
other purposes. In particular, protocols should be developed for microwave treatment
of racks of media dispensed into tubes prior to sterilization. Preliminary experiments
indicate that this is feasible for tubes not > l/3-1/2 full of medium, loosely capped and
in plastic racks. For tubes containing 10 ml of liquid, 2 min of exposure on high power
is the longest possible before boiling over occurs. Sterility is not achieved with this
amount of treatment, nor with five sequential treatments of 2 min each with cooling
periods between treatments. More such 2-min periods or longer treatment times at
reduced power might prove effective. More work is required on this subject.
Some ancillary observations may prove useful. No metal racks, even plastic-coated
ones, can be used in the microwave oven because arcing may result. The outer plastic
coating of such racks will also melt. For similar reasons, no aluminum foil or other
metallic objects may be used unless shielded (see Rohrer & Bulard, 1985). Vessels
containing liquid cannot be completely sealed, as they may explode from the rapid
heating, and care must be taken not to overfill tubes or vessels because liquid can be
lost by boiling. (Test ampoules containing heat-resistant bacterial spores began to
explode just seconds after treatment was started.) No completely dry materials should
be microwave treated without the inclusion in the oven of a wave absorbent, such as
a beaker of water, as recommended by the manufacturers. Cotton plugs may ignite and
some plastic caps (especially black ones) may melt, although the caps for Teflon and
polycarbonate bottles and tubes were unaffected. It is advisable to microwave-treat the
former items separately for shorter periods of time (e.g., 1 min) or to autoclave them
instead (in beakers or Petri dishes). The flasks or tubes containing media can be covered
with plastic wrap or glass beakers or caps.
Preferably, the oven should be on its own circuit to minimize voltage fluctuations,
which affect the output of the instrument. Also, since the microwave field strength is
nonuniform, leaving cold spots in the oven, it is necessary to agitate or rotate the
samples, as well as allow a sufficient period of exposure. Jeng et al. (1987) demonstrated
that the temperatures attained in vials distributed within an oven were very uneven after
a period of microwave treatment. Our own observations confirm this; temperature
differences of up to 10 C occurred within a rack of tubes containing water and exposed
for only 1.5-2 min.
Since individual microwave ovens do vary in output and in evenness of wave

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STERILIZATIONOFCULTUREMEDIA

283

distribution, it is advisable to test the protocol we describe in any oven proposed for
use.
The use of microwave ovens for sterilization of culture media and apparatus is much
more rapid and energy-efficient than more conventional methods, as well as being
equally effective if the protocols are worked out. The great advantage, of course, at least
for marine media, is that neither temperature nor pH are altered very much, precipitation
is avoided, and - presumably - viruses as well as the usual microbial contaminants are
destroyed (as demonstrated by Sanborn et al., 1982), a benefit not achievable with
standard filtration methods.

ACKNOWLEDGEMENTS

This work was supported by NSF Grant OCE-8415963. Special thanks are given to
R. Main and E. Berdalet for help with the time-course experiments.

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