J. Exp. Mar. Biol. Ecol., 1988, Vol. 117, pp.
279-283
the standing crop of benthic and
Fish. Res. Board. Can., Vol. 25,
re der Kieler Bucht im J ahresgang.
~weier Waldbãche des Naturparks
)fi of some intertidal invertebrates
,p.411-419.
of marine benthos. IBP Handbook.
, U.K., pp. 284-372.
stigations in ecological energetics.
ld biochemical composition of the
eth.J. SeaRes., Vol. 17,pp. 84-95.
'i Luleâ skãrgãrd sommaren 1976.
)f Macoma balthica (L.). Biol. BulI.
Anim. Ecol., Vol. 46, pp. 593-605.
nem Okosystem "Zostera-Wiese"
11., No 18.
and assimilation of a Mysis relicta
'p. 129-137.
e bivalve Scrobicularia plana (Da
'oductive output and recruitment
íO.
N eanthes ( = N ereis) virens (S ars ).
Energieãquivalenten aquatischer
Vniv. Rostock Math. Naturwiss.
nd planktonic invertebrates of
rganismgroups. Ophelia, Vol. 23,
lhia, Pennsylvania, third edition.
>branch tissues. Ecology, Vol. 45,
ophistobranch Navanax inermis.
:etical investigations. Pol. Arch.
)Qnal and long-term population
}erman Bight. Neth. J. Sea Res.,
conversion factors for benthic
York, fifth edition, 552 pp.
Jn of energy and organic content
'ature (London), Vol. 191, p. 299.
Iimpet, Acmaea scabra (Gould).
. Caloric measurements of some
(ar. Biol., Vol. 19, pp. 258-261.
nvertebrates from the Canadian
279
Elsevier
JEM 01058
Short Communication
Microwave treatment for sterilization of phytoplankton
culture media
Maureen D. Keller, Wendy K. Bellows and Robert R. L. Guillard
Bigelow Laboratory for Ocean Sciences. McKown Point. West Boothbay Harbor. Maine. V.S.A.
(Received 3 November 1987; revision received 25 January 1988; accepted 4 February 1988)
Abstract: A standard microwave oven for the sterilization of phytoplankton culture media and apparatus
was tested. Elimination ofbacterial, algal, and fungaI contaminants is achieved in < 10 min for a 1.5 I vol.
of seawater in a Teflon bottle. Empty culture tubes and vessels are sterilized in 5 mino The technique is quick,
easy, reliable, and does not contaminate with trace metais 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 2 , precipitates are often formed from the combination of
inorganic constituents of seawater and components of the enrichment. These pre-
cipitates have a deleterious effect on many phytoplankters. Autoclaving can also be
time-consuming, taking several hours - especially if the time for cooling the liquids is
included. Further, the steam from autoclaves has been implicated as a source of trace
metal contamination for criticaI analytical work, or studies of trace metal requirements
(e.g., Price et aI., 1987). Alternative methods of sterilization, such as repeated pasteuri-
zation, boil-freeze-boil methods (Brand et aI., 1983), or filter sterilization, are alI
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. U sing home-type microwave ovens, protocols
for sterilizing plastic tissue culture vessels (Latimer & Matsen, 1977; Sanborn et aI.,
Bigelow Contribution 87021.
Correspondence address: M. D. Keller, Bigelow Laboratory for Ocean Sciences, McKown Point, West
Boothbay Harbor, ME 04575, U.SA
0022-0981/88/$03.50 © 1988 Elsevier Science Publishers B.V. (Biomedical Division)
280 M.D. KELLER ETAL. MICROWAV

1982), dental tools (Rohrer & Bulard, 1985), and agar media (Wood & Lundergan, All contaminants were elimi:
1981) have been developed and were effective against a wide range of bacterial and viral ture ofthe water immediately a
contaminants. Microwave treatment has also been used for the extraction of polar before microwave treatment v
compounds for chromatography (Ganzler et ai., 1986). 7.90.
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 Time course of microwave sterilizati<
were eliminated during the time cour
repeatedly eliminated bacterial, algal, and fungal contaminants from seawater, nutrients, (presence) or - (absence) in appro]
and culture vessels. bac

Time (min)
METHODS
O'
A Hot Point, 1.2-ft3, 700-W microwave oven was used, with the power setting on 3
5
High. Three 2-1 Teflon bottles, each with 1.51 of unfiltered Sargasso or filtered local 8
seawater, were prepared. To one, a culture of an unidentified marine fungus was added; 10
to another, a combination of several different algal cultures was added; to the third, no
• Test media performed in triplicat<
addition was made (recognizing that a natural bacterial population is always present identical results.
in seawater). The botdes 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
The microwave treated tu'
a total exposure of 10 min; midway through the 3-min microwaving period and before
treatment did not eliminate 1
each subsampling, the liquid was agitated before proceeding. The sampling periods
microwave treated tubes or I
chosen were 0,3,5,8, and 10 mino Triplicate 1-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 The mechanism of killing 1
under appropriate conditions and checked several times a week. than those attributable to he2
Plastic racks (30 tubes/ea) of empty polycarbonate culture tubes (30 ml) were also in two ways: by ionic polariz;
treated for O, 1, 3, and 5 min, two racks at one time. The tubes were usually wet from in increased collisions, or by
the washing procedure, but iftubes were dry, a beaker ofwater was included in the oven themselves with a changing fi
to absorb the microwaves. The manufacturer does not recommend operating the oven ture of the microwave-treate<
empty or with dry materials only. Halfway through each microwaving period, the racks autoclaving and for a much :
0
were turned 180 for even treatment. Tubes, in triplicate, from each microwaving the microorganisms except ç
treatment were filled with f/2 medium, inoculated with algae to test for growth and later that with dry materials, heat
tested for bacteria using peptone-based media. they found that 45 min or mo
Changes in temperature and pH following the microwave process were also recorded. neceSSarY for the destruction
The experiment was repeated on three different occasions. be operating in liquid systern
molecules within the cells n
RESULTS lethal effect. Whatever the m~
of microbes is certain. Likew
The effectiveness of the microwaving procedure for sterilization is illustrated in not produce toxic effects or o
Table I. polycarbonate or Teflon war

edia (Wood & Lundergan,
: range ofbacterial and viral
for the extraction of polar
the sterilization of culture
:s in experiments involving
~veloped that rapidly and
tts from seawater, nutrients,
with the power setting on
i Sargasso or filtered local
-marine fungus was added;
was added; to the third, no
Ipulation is always present
ach was microwave-treated
e min, and 2 more min, for
owaving period and before
lng. The sampling periods
vere removed at each time.
following media: for algal
inants, f/2p (0.1% (w/v)
(w/v) methylamine in f/2);
st was incubated for 3 wk
week.
re tubes (30 ml) were also
bes were usually wet from
~r was included in the oven
nmend operating the oven
rowaving period, the racks
, from each microwaving
to test for growth and later
'fOcess were also recorded.
:rilization IS illustrated
---------------III!II!
MICROWAVE STERILIZATION OF CULTURE MEDIA 281
All contaminants were eliminated at 10 min for a 1.5-1 voI. ofseawater. The tempera-
ture ofthe water immediately after the lO-min time point was 84 o C. The pH ofthe 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 vols. of seawater. Algal, bacterial, and fungai 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 M Malt
0* + + + +
3 + + + +
5 + + +
8 +
10
* Test media performed in triplicate. Entire experiment was repeated on three difTerent occasions with
identical results.
The microwave treated tubes were bacteria-free after 5 mino 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 efTects (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 tempera-
ture ofthe 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 ai. (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 efTect. Whatever the mechanism, the ability of microwaves to render substrata free
of microbes is certain. Likewise, microwave treatment of culture vessels or media does
In not produce toxic efTects or otherwise reduce their usefulness for culturing. At least with
polycarbonate or Teflon ware, no degradation ofthe material has been observed. Even
282 M. D. KELLER ET AL. MICROWAVI

disposable (non-autoclavable) plastic ware can have its usable life extended, although distribution, it is advisable to
Sanborn et aI. (1982) noted that after three microwavings, disposable plastic ware began use.
to yellow and was probably degrading. The use of microwave oven:
F or trace metal work, microwave treatment eliminates a significant source of metal more rapid and energy-efficil
contamination. For our trace metal research and nutritional studies, medium is sterilized equally efTective ifthe protocol
in Teflon bottles and dispensed aseptically into polycarbonate tubes (which do not for marine media, is that neithf
withstand repeated sterilization when containing seawater). The protocol we describe is avoided, and - presumably
here was developed in the context of this experimental process, and specifically to destroyed (as demonstrated .
replace both autoclaving, which can contaminate with metals, and the time-consuming standard filtration methods.
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 > 1/3-1/2 full ofmedium, loosely capped and
This work was supported b
in plastic racks. For tubes containing 10 ml of liquid, 2 min of exposure on high power
R. Main and E. Berdalet for
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 BRAND, L. E., W. G. SUNDA & R R
rates by zinc, manganese, and ir<
ones, can be used in the microwave oven because arcing may resulto The outer plastic DECAREAu,R.V.& R.A.PETERSON
coating of such racks will also melt. For similar reasons, no aluminum foil or other U.K., 224 pp.
GANZLER, K., A. SALGO & K. VAL
metallic objects may be used unless shielded (see Rohrer & Bulard, 1985). Vessels
for chromatography. J. Chromato,
containing liquid cannot be completely sealed, as they may explode from the rapid GUILLARD, RRL., 1975. Culture o
heating, and care must be taken not to overfill tubes or vessels because liquid can be invertebrate animais, edited by W
lost by boiling. (Test ampoules containing heat-resistant bacterial spores began to pp.29-66.
HAMILTON, RD., 1973. Sterilizati<
explode just seconds after treatment was started.) No completely dry materials should measurements, edited by J.R Ste
be microwave treated without the inclusion in the oven of a wave absorbent, such as JENG, D.K.H., K.A. KACZMAREK,
sterilization in the dry state. App
a beaker ofwater, as recommended by the manufacturers. Cotton plugs may ignite and
LATlMER,J.M. & J.M. MATSEN, 19'
some plastic caps (especially black ones) may melt, although the caps for Teflon and tion in a clinicaI microbiology la1
polycarbonate bottles and tubes were unaffected. It is advisable to microwave-treat the PRICE, N,M., P.A. THOMPSON & F
former items separately for shorter periods of time (e.g., 1 min) or to autoclave them coas tal marine diatom Tha/assim
ROHRER, M.D. & RA. BULARD, .
instead (in beakers or Petri dishes). The flasks or tubes containing media can be covered SANBORN, M.R, S.K. WAN & RI
with plastic wrap or glass beakers or caps. for reuse. App/. Environ. Microbic
Preferably, the oven should be on its own circuit to minimize voltage fluctuations, WOOD, N.J. & c.A. LUNDERGAN
Vol. 16, p. 417.
which affect the output of the instrument. AIso, 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. J eng et aI. (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 o C occurred within a rack of tubes containing water and exposed
for only 1.5-2 mino
Since individual microwave ovens do vary in output and in evenness of wave

able life extended, although
sposable plastic ware began
. significant source of metal
studies, medium is sterilized
onate tubes (which do not
I. The protocol we describe
Irocess, and specifically to
~s, and the time-consuming
oIs should be developed for
:d for microwave treatment
n. Preliminary experiments
tedium, loosely capped and
of exposure on high power
I is not achieved with this
)f 2 min each with cooling
longer treatment times at
~d on this subject.
racks, even plastic-coated
ay result. The outer plastic
no aluminum foil or other
& Bulard, 1985). Vessels
ly explode from the rapid
.sels because liquid can be
bacterial spores began to
Iletely dry materiaIs should
a wave absorbent, such as
otton plugs may ignite and
~h the caps for Teflon and
lble to microwave-treat the
min) or to auto clave them
ining media can be covered
imize voltage fluctuations,
nicrowave field strength is
rry to agitate or rotate the
et ai. (1987) demonstrated
ven were very uneven after
confirm this; tem per ature
ttaining water and exposed
and in evenness of wave
MICROWAVE STERILlZATION OF CULTURE MEDIA 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 efTective ifthe 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 ai., 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.
REFERENCES
BRAND, L. E., W. G. SUNDA & R. R. L. GUILLARD, 1983. Limitation of marine phytoplankton reproduction
rates by zinc, manganese, and iron. Limnol. Oceanogr., Vol. 28, pp. 1182-1198.
DECAREA U, R. V. & R. A. PETERSON, 1986. Microwave processing and engineering. Ellis Harwood, Chichester,
U.K., 224 pp.
GANZLER, K., A. SALGO & K. VALKO, 1976. Microwave extraction. A novel sample preparation method
for chromatography. J. Chromatogr., Vol. 371, pp. 299-306.
GUILLARD, R. R. L., 1975. Culture of phytoplankton for feeding marine invertebrates. In, Culture of marine
invertebrate animais, edited by W.L. Smith & M.H. Chanley, Plenum Press, New York, New York,
pp.29-66.
HAMILTON, R.D., 1973. Sterilization. In, Handbook ofphycologieal methods. Culture methods and growth
measurements, edited by J.R. Stein, Cambridge University Press, Cambridge, U.K., pp. 181-194.
JENG, D.K.H., K.A. KACZMAREK, A.G. WOODWORTH & G. BALASKY, 1987. Mechanism ofmicrowave
sterilization in the dry state. Appl. Environ. Microbiol., Vol. 53, pp. 2133-2137.
LATIMER, J. M. & J. M. MATSEN, 1977. Microwave oven irradiation as a method for bacterial decontamina-
tion in a clinicai microbiology laboratory. J. Clin. Mierobiol., Vol. 6, pp. 340-342.
PRICE, N.M., P.A. THOMPSON & P.J. HARRISON, 1987. Selenium: an essential element for growth ofthe
coastal marine diatom Thalassiosira pseudonana (Bacillariophyeeae). J. Phyeol., Vol. 23, pp. 1-9.
ROHRER, M.D. & R.A. BULARD, 1985. Microwave sterilization. J. Am. Dent. Assoe., Vol. 110, pp. 194.
SANBORN, M. R., S. K. WAN & R. BULARD, 1982. Microwave sterilization of plastic tissue culture vessels
for reuse. Appl. Environ. Microbiol., Vol. 44, pp. 960-964.
WOOD, N.J. & c.A. LUNDERGAN, 1981. Microwave sterilization of tis sue culture media. Hort Scienee,
Vol. 16, p. 417.