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Microwave sterilized media supports better microbial growth than autoclaved media

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  • The Galaxy School, Rajkot

Abstract and Figures

Microwave treated media were compared with media sterilized through conventional autoclaving in terms of their ability to support microbial growth, spore germination, and revival of lyophilized bacterial cultures. Microwave sterilized media were found to support better microbial growth. Both bacteria and yeast were able to achieve higher cell density at a faster growth rate in microwaved media. Microwave treatment was found to be suitable for media of varying compositions. Better retention of nutrient quality in microwave treated growth media due to shorter heat exposure seems to be the major reason for better microbial growth in it. Microwave sterilization can prove an attractive alternative of conventional autoclaving, especially when media are needed for immediate use, and also when high biomass yield is of particular interest.
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Research in Biotechnology, 2(5): 63-72, 2011 ISSN: 2229-791X
www.researchinbiotechnology.com
#
Present address : Intas Pharmaceuticals, Ahmedabad
Regular Article
Microwave sterilized media supports better microbial growth
than autoclaved media
Vijay Kothari*, Mohini Patadia and Neha Trivedi
#
Institute of Science, Nirma University, S-G Highway, Ahmedabad-382481, India
*Corresponding author email: vijay23112004@yahoo.co.in
; vijay.kothari@nirmauni.ac.in
Microwave treated media were compared with media sterilized through conventional
autoclaving in terms of their ability to support microbial growth, spore germination, and
revival of lyophilized bacterial cultures. Microwave sterilized media were found to support
better microbial growth. Both bacteria and yeast were able to achieve higher cell density at a
faster growth rate in microwaved media. Microwave treatment was found to be suitable for
media of varying compositions. Better retention of nutrient quality in microwave treated
growth media due to shorter heat exposure seems to be the major reason for better microbial
growth in it. Microwave sterilization can prove an attractive alternative of conventional
autoclaving, especially when media are needed for immediate use, and also when high
biomass yield is of particular interest.
Keywords: Microwave sterilization; microbial growth; generation time; autoclaving; growth
media
Autoclaving as a means of media
sterilization is a universally accepted simple
method. However this method can be
inconvenient when the plates are needed for
immediate use (Iacoviello and Rubin, 2001).
Microwave (MW) radiation has been used for
effectively disinfecting gauze pieces and
hospital white coats, aseptic packaging of
food (Bhattacharjee et al. 2009). We
performed a study to investigate whether the
sterilization of media by autoclave can be
replaced with MW treatment. Use of
microwaves for preparation of growth
medium for Aggregatibacter actinomycetem-
comitans (Bhattacharjee et al. 2009), and LB
agar plates (Iacoviello and Rubin, 2001) has
been reported. Emergency sterilization of
media using a microwave oven has been
discussed by Hengen (1997) too. But to the
best of our knowledge, there is no published
report on whether MW treatment is suitable
for media of varying compositions, and for
growth of different microorganisms. The
necessity to study the MW sterilization
conditions through the experiments for
different media and bacteria, as well as
research on sterilization mechanism
(especially non-thermal sterilization
mechanism) has been emphasized by Xi et al.
(2002). Microwaves have already been
applied for sterilization of injection ampoules
(Sasaki et al. 1995; Sasaki et al. 1998). Through
present study we show that the MW method
of sterilization is not just an alternative
method of sterilization but also promotes
faster microbial growth and higher cell
density.
Vijay kothari et al. / Research in Biotechnology, 2(5): 63-72, 2011
64
Materials and Methods
Microwave treatment of media
200 ml media (HiMedia, Mumbai)
filled in a 500 ml screw capped glass bottle
was given continuous microwave (2450
MHz) exposure at 900 W for 10 min using a
microwave oven (Electrolux EM30EC90SS).
Cap of the bottle was kept slightly loose so
that steam generated during heating can get
released. Baseplate (30 cm diameter) of the
oven rotated at a speed of 5 rpm. Volume of
medium left in the bottle after MW treatment
was ~140 ml. It took ~135 s for media to start
boiling.
Test organisms
Following microbial cultures were
procured from Microbial Type Culture
Collection (MTCC) Chandigarh: Aeromonas
hydrophila (MTCC 1739), Bacillus subtilis
(MTCC 619), Escherichia coli (MTCC 119),
Escherichia coli (MTCC 118), Pseudomonas
aeruginosa (MTCC 1688), Pseudomonas
oleovorans (MTCC 617), Salmonella paratyphi
A (MTCC 735), Shigella flexneri (MTCC 1457),
Staphylococcus aureus (MTCC 737),
Staphylococcus epidermidis (MTCC 435),
Streptococcus pyogenes (MTCC 442), Candida
albicans (MTCC 3017), Saccharomyces
cerevisiae (MTCC 170).
Growth promotion
One set of autoclaved and
microwaved medium, each containing 3 test
tubes (4 ml medium in each) was inoculated
with test organism. Inoculum standardized
to 0.5 McFarland turbidity standard was
added at 1% v/v. After proper mixing tubes
were incubated under static condition for 20
h at 35ºC for all bacteria except A. hydrophila.
Incubation temperature for latter and yeasts
was set at 30ºC. After incubation, optical
density for all the tubes was measured at 625
nm (Spectronic 20D+, Thermoscientific).
Before measuring OD a loopful from tubes
containing microwaved medium inoculated
with test organism was streaked on to a
sterile nutrient agar plate to confirm purity of
the growth (i.e. if microwaved medium was
not completely sterile, few contaminant
colonies should grow along with test
organism). Such experiments were
performed with 5 different media (Nutrient
broth, Luria Bertani broth, Mueller-Hinton
broth, Tryptone soya broth, and Tryptone
yeast extract broth) for bacteria, and 3
different media (Yeast malt broth, Potato
dextrose broth, and YPD broth) for yeasts.
All media were procured from HiMedia,
Mumbai.
Growth curve
Test organism was inoculated into
140 ml of autoclaved and microwaved
medium. Both the media were filled in 500
ml screw capped glass bottle. After
inoculation (inoculum added at 1% v/v)
contents were distributed into sterile test
tubes, and incubated under static condition
at appropriate temperature as described
above. Each tube contained 4 ml of medium.
OD was measured at regular time intervals to
prepare growth curve. Such experiments
were performed with 5 different media for
bacteria, and 3 different media for yeasts.
Spore germination
Both microwaved and autoclaved
media (140 ml medium in 500 ml bottle) were
inoculated with Bacillus coagulans spore
(SPORLAC
®
) suspension. Before inoculation
spore suspension was standardized to 0.5
McFarland turbidity standard. Contents from
bottles were then distributed into sterile test
tubes, and incubated under static condition
at 35ºC. Each tube contained 4 ml of medium.
OD
625
was measured at regular time intervals
to follow spore germination. Similar
experiment was performed with spores of
Bacillus stearothermophilus. Here one intact
spore strip (HiMedia) containing 10
5
spores
was used for inoculation, followed by
incubation at 55ºC. Spore germination with
Vijay kothari et al. / Research in Biotechnology, 2(5): 63-72, 2011
65
both organisms was checked in 5 different
nutrient media.
Rejuvenation of lyophilized cultures
Both microwaved and autoclaved
nutrient broth (140 ml medium in 500 ml
bottle) were inoculated with suspension of
test organism prepared from lyophilized
powder. Contents from bottles were then
distributed into sterile test tubes (4 ml in each
tube), and incubated under static condition at
appropriate temperature as described above.
OD
625
was measured at regular time intervals
to follow bacterial growth.
During all the above experiments
(performed in triplicate) uninoculated
autoclaved and microwaved media served as
sterility controls. The same were used as
blank while measuring OD. To confirm
sterility a loopful of content from these
controls was streaked on sterile nutrient agar
plate. Following incubation at 35ºC, absence
of any growth was taken as confirmation of
sterility.
Statistical analysis
Statistical analysis was performed using t-
test with Microsoft
®
Excel. At p<0.05 the
difference between microbial growth in
autoclaved and microwaved media was
considered significant.
Results and Discussion
Growth promotion
Results of growth promotion in
microwaved and autoclaved media are
presented in table 1-4. Cell density was
derived by plotting OD of the experimental
tube on a graph of OD
625
vs. cell no. prepared
using different McFarland turbidity
standards (Hindler and Munro, 2010).
Microwaved media supported better growth
of bacteria as well as yeast than autoclaved
media. Both amount as well as rate of growth
were higher in microwaved media. With
respect to percent increment in growth
microwaved tryptone yeast extract broth
proved most suitable medium for the growth
of 4 different bacteria- E. coli, S. aureus, S.
flexneri, and S. pyogenes. Microwaved LB
broth was most suitable medium for the
growth of S. paratyphi A and P. oleovarans. In
four out of five media, organism which
registered the highest growth increment was
a gram-positive one.
Table 1. Growth of various bacteria in nutrient broth and LB broth
Organism
Nutrient broth Luria Bertani broth
OD
625
(Mean ± SD)
Growth
increment in
microwaved
media (%)
OD
625
(Mean ± SD)
Growth
increment in
microwaved
media (%)
Autoclaved Microwaved Autoclaved Microwaved
A. hydrophila
0.47 ± 0.00
a
0.81 ± 0.01
a
71.15 0.89 ± 0.01
a
1.39 ± 0.01
a
55.82
E. coli
0.34 ± 0.04 0.43 ± 0.01 25.00 0.45 ± 0.03
b
0.64 ± 0.10
b
42.16
P. oleovarans
1.46 ± 0.08 1.58 ± 0.38 8.56
*
0.24 ± 0.00
a
0.54 ± 0.01
a
123.80
S. flexneri
0.62 ± 0.00 1.03 ± 0.00 65.75 0.68 ± 0.00
a
0.74 ± 0.00
a
8.33
S. parathyphi A
0.22 ± 0.01 0.29 ± 0.00 31.08 0.33 ± 0.00
a
0.61 ± 0.00
a
83.63
B. subtilis
0.40 ± 0.01 0.63 ± 0.00 57.03 0.51 ± 0.00
0.74 ± 0.00
45.98
S. aureus
0.32 ± 0.00 0.72 ± 0.02 124.61 0.45 ± 0.04
a
0.56 ± 0.03
a
23.95
S. epidermidis
0.48 ± 0.01
c
0.62 ± 0.00
c
29.07 0.88 ± 0.01
b
1.36 ± 0.05
b
53.67
S. pyogenes
0.42 ± 0.00 0.48 ± 0.00 13.25 0.38 ± 0.00 0.45 ± 0.01 16.53
a
2x dilution
b
3x dilution
c
4x dilution;
*
p>0.05
Vijay kothari et al. / Research in Biotechnology, 2(5): 63-72, 2011
66
Table 2. Growth of various bacteria in Mueller-Hinton broth and tryptone soya broth
Organism
Mueller-Hinton broth Tryptone soya broth
OD
625
(Mean ± SD)
Growth
increment in
microwaved
media (%)
OD
625
(Mean ± SD)
Growth
increment in
microwaved
media (%)
Autoclaved Microwaved Autoclaved Microwaved
A. hydrophila
0.84 ± 0.00 0.96 ± 0.00 13.60 0.58 ± 0.01
b
0.97 ± 0.00
b
66.09
E. coli
0.38 ± 0.00
a
0.55 ± 0.00
a
43.48 0.59 ± 0.00
b
0.82 ± 0.00
b
38.27
P. oleovarans
0.79 ± 0.20
a
1.08 ± 0.00
a
36.99 0.71 ± 0.01
b
0.82 ± 0.00
b
20.70
S. flexneri
0.30 ± 0.00 0.39 ± 0.00 27.77 0.57 ± 0.00 0.84 ± 0.00 45.92
S. parathyphi A
0.44 ± 0.00 0.58 ± 0.00 33.10 0.48 ± 0.00 0.67 ± 0.00 38.19
B. subtilis
0.26 ± 0.01
a
0.53 ± 0.02
a
105.0 0.66 ± 0.00 1.03 ± 0.00 55.87
S. aureus
0.29 ± 0.00 0.45 ± 0.01 50.50 1.13 ± 0.00 1.31 ± 0.00 16.81
S. epidermidis
0.16 ± 0.00 0.14 ± 0.00 -13.10
*
0.72 ± 0.02 1.36 ± 0.00 87.77
S. pyogenes
0.29 ± 0.00 0.32 ± 0.01 8.69 0.79 ± 0.00 0.82 ± 0.00 3.53
a
2x dilution
b
3x dilution;
*
p>0.05
Table 3. Growth of various bacteria in Tryptone yeast extract broth
Organism
Tryptone yeast extract broth
OD
625
(Mean ± SD)
Growth
increment in
microwaved
media (%)
Autoclaved Microwaved
A. hydrophila
0.53 ± 0.00 0.89 ± 0.00 53.51
E. coli
0.54 ± 0.02 0.86 ± 0.01 57.96
P. oleovarans
0.47 ± 0.02
a
0.66 ± 0.00
a
39.53
S. flexneri
0.32 ± 0.00 0.54 ± 0.01 67.69
S. parathyphi A
0.20 ± 0.00 0.31 ± 0.01 50.23
B. subtilis
0.23 ± 0.00 0.37 ± 0.00 60.85
S. aureus
0.47 ± 0.00 1.11 ± 0.00 134.17
S. epidermidis
0.18 ± 0.00 0.28 ± 0.00 56.35
S. pyogenes
0.15 ± 0.04 0.25 ± 0.02 61.14
a
2x dilution
Vijay kothari et al. / Research in Biotechnology, 2(5): 63-72, 2011
67
Table 4. Growth of yeast in different media
a
2x dilution
b
5x dilution
c
6x dilution
Table 5. Generation time of different bacteria in nutrient broth, LB broth, and Mueller-Hinton broth
Organism
Generation time (g)
(h)
Nutrient broth Luria Bertani broth Mueller-Hinton broth
Autoclaved Microwaved Autoclaved Microwaved Autoclaved Microwaved
A. hydrophila
8.36 7.71 3.19 3.14 4.86 3.19
E. coli
10.37 7.71 7.00 5.28 4.97 4.73
P. oleovarans
4.89 3.74 8.13 6.54 2.00 1.57
S. flexneri
13.08 9.70 21.50 20.06 6.71 6.66
S. parathyphi A
1.33 1.09 15.05 9.40 15.05 10.03
B. subtilis
5.89 4.09 1.89 1.24 10.03 7.50
S. aureus
13.68 8.85 15.84 12.04 6.68 6.68
S. epidermidis
6.14 4.42 3.50 3.30 9.12 15.05
S. pyogenes
6.02 5.37 10.37 5.57 21.5 10.30
Table 6. Generation time of different bacteria in tryptone soya broth and tryptone yeast extract broth
Organism
Generation time (g)
(h)
Tryptone soya broth Tryptone yeast extract broth
Autoclaved Microwaved Autoclaved Microwaved
A. hydrophila
1.00 0.59 12.75 9.83
E. coli
12.54 9.40 13.43 10.90
P. oleovarans
9.70 9.40 10.10 7.50
S. flexneri
11.14 6.02 7.50 6.02
S. parathyphi A
1.12 0.84 11.99 11.62
B. subtilis
1.06 0.59 6.89 6.84
S. aureus
3.44 1.60 1.21 0.30
S. epidermidis
1.71 0.83 3.17 0.88
S. pyogenes
5.20 5.01 60.20 25.08
Media
Organism
OD
625
(Mean ± SD)
Growth
increment in
microwaved
media (%)
Autoclaved Microwaved
Yeast malt
broth
C. albicans
0.29 ± 0.00 0.49 ± 0.00 69.65
S. cerevisiae
0.69 ± 0.07
c
1.10 ± 0.07
c
76.78
Potato
dextrose
broth
C. albicans
0.29 ± 0.00
a
0.32 ± 0.00
a
12.06
S. cerevisiae
0.69 ± 0.01
a
1.31 ± 0.00
a
88.69
YPD broth
C. albicans
0.52 ± 0.00
b
0.81 ± 0.00
b
55.53
S. cerevisiae
0.51 ± 0.01 0.66 ± 0.00 28.34
Vijay kothari et al. / Research in Biotechnology, 2(5): 63-72, 2011
68
Table 7. Generation time of yeast in in different media
Table 8. Generation time for spores in different media
Spores
Media
Generation time (g)
(h)
A MW
B. coagulans
Nutrient broth 6.27 5.18
LB broth 13.68 9.12
Mueller-Hinton broth 15.84 10.03
Tryptone soya broth 2.70 2.19
Tryptone yeast extract 8.85 6.54
B. stearothermophilus
Nutrient broth 12.54 12.18
LB broth 7.34 21.5
Mueller-Hinton broth 8.85 5.57
Tryptone soya broth 3.73 0.92
Tryptone yeast extract 16.72 15.05
Growth curve
Generation time of different
microorganisms in autoclaved and
microwaved media was estimated from
growth curve experiments (Table 5-7). All
organisms registered lesser generation time
(i.e. faster growth) and achieved higher cell
densities in microwaved media (Figure 1-2),
except S. epidermidis in Mueller-Hinton broth.
Still shorter generation time is obviously
attainable if constant shaking is provided
during incubation. Tryptone soya broth
allowed 6 of the test bacteria to achieve a
growth rate faster than in any other media.
Spore germination
Autoclaved and microwaved media
were tested for their ability to support
germination of B. coagulans and B.
stearothemophilus spores. Microwaved media
supported better germination of both the
spores. After germination organisms
registered faster growth (Table 8) and higher
cell density in microwaved media except B.
stearothermophilus in LB broth. Both
organisms registered their least generation
time in tryptone soya broth.
Media
Organism
Generation time (g)
(h)
Autoclaved Microwaved
Yeast malt
broth
C. albicans
6.54 5.28
S. cerevisiae
5.18 4.77
Potato
dextrose
broth
C. albicans
2.59 0.75
S. cerevisiae
3.71 3.42
YPD broth
C. albicans
7.71 5.57
S. cerevisiae
3.42 3.40
Vijay kothari et al. / Research in Biotechnology, 2(5): 63-72, 2011
69
Rejuvenation of lyophilized cultures
Microwaved nutrient broth supported
better rejuvenation of lyophilized bacterial
cultures. It allowed the test organisms- E. coli,
P. aeruginosa, and S. aureus to reach 93.54%,
7.01%, and 81.81% higher cell density at
faster rate (Table 9) than that in autoclaved
broth.
Table 9. Generation time of different bacteria in
nutrient broth after revival from lyophilized
form
Organism
Generation time (g)
(h)
A MW
E. coli
7.71 5.90
P. aeruginosa
6.84 5.18
S. aureus
4.77 3.62
0
5
10
15
20
25
30
0 102030405060
Time (h)
Cell no. (x 10
8
/m l)
A
MW
Figure 1. Growth curve of E. coli in
microwaved and autoclaved nutrient broth
0
10
20
30
40
50
60
70
0 102030405060
Time (h)
Cell no. (x 10
8
/ml)
A
MW
Figure 2. Growth curve of S. cerevisiae in
microwaved and autoclaved yeast malt
broth
Here we show that growth media
sterilized by microwave irradiation promotes
faster growth of various bacteria as well as
yeasts. We found that broth made by
microwaved method can be stored safely for
up to 72 h, without any indication of
contamination. Thus, the use of microwave
irradiation is an effective method of
sterilization of liquid growth media of
varying composition. Microwaves have been
known to be effective at killing
microorganisms (Najdovski et al. 1991;
Zakaira et al. 1992; Wu, 1996; Madigan et al.
2009), but a common sterilization protocol
applicable for biological media of varying
matrix is still not available.
Better retention of nutrient quality in
microwaved growth media due to shorter heat
exposure than in autoclave seems to be the
major reason for better microbial growth in
microwaved media in our experiments. Due
to longer ‘heating-holding-cooling’ cycle in
autoclave, some degree of nutrient quality
deterioration becomes unavoidable. Media
undergoes evaporation during MW
treatment, as a result of which media become
somewhat nutrient concentrated. To check
whether this factor has played any major role
behind superior performance of microwaved
media, we made E. coli grow in an nutrient
broth which was brought to initial volume by
addition of sterile distilled water after MW
treatment, thus nullifying the effect of
evaporation on nutrient composition.
Simultaneously a control was set wherein E.
coli was grown in microwaved media with no
volume adjustment. Growth of E. coli in both
the media did not differ significantly (p>0.05;
data not shown), indicating no important role
being played by evaporation during MW
treatment. Simultaneously along with this
same organism was grown in autoclaved
nutrient broth, and growth in it was lesser
than that in microwaved media prepared in
either way. Autoclaved nutrient broth with
more amount of ingredients (so as to attain
equivalence to microwaved media with no
Vijay kothari et al. / Research in Biotechnology, 2(5): 63-72, 2011
70
volume adjustment) was also inoculated with
E. coli, but it supported no higher growth
than normal autoclaved broth. Similar
results were obtained with A. hydrophila.
It is difficult to ascertain whether
better nutrient retention due to lesser heat
exposure can solely account for improved
bacterial growth in microwaved media. One
of the other possible reasons leading to better
microbial growth in microwaved media may
be the ‘MW specific athermal effects’. Despite
existence of non-thermal effects of MW being
controversial (Welt et al. 1994; Wayland et al.
1997), possibility of some role being played
by it can not be rejected fully. Non-thermal
effect was suggested to have an important
role in the inactivation of microorganisms in
suspension (Jeng et al. 1987). Microbial
destruction during microwave exposure has
also been shown to occur at lower
temperatures and shorter time periods in
comparison to conventional heating methods
(Banik et al. 2003).
As different components of
microbiological media can interact with each
other under the influence of heat, sometimes
their heat induced interactions may result in
formation of undesirable products (viz.
Amadori products), some of which may be
inhibitory to microorganisms. Inhibition of
A. actimomycetemcomitans due to Maillard
reaction products in autoclaved medium was
overcome by preparing the medium with
microwaves (Bhattacharjee et al. 2009). Due to
lesser heat exposure during MW treatment
there is a reduced probability of unwanted
interactions among different medium
components.
Bacterial species differ in their
susceptibility to MW inactivation.
Microwaves can kill bacterial spores more
effectively in presence of water than when
dry (Najdovski et al. 1991). Bacterial spores
have similar resistance to MW as to
conventional heating (Sasaki et al. 1998).
However, Celandroni et al. (2004) have
shown that the effect exerted by microwaves
on spores is different from that of autoclave.
Contrary to conventional heating,
microwaves promote formation of stable
complexes between dipicolinic acid and other
spore components, thereby preventing the
release of dipicolinic acid from spores. The
conventional heated spores exhibited a cortex
layer that was up to 10 times wider than that
of untreated spores i.e. an extremely relaxed
cortex. This relaxation of cortex was not
observed during MW treatment, cortex
maintained its original width even though
exposure time and sample temperature for
conventional heat and MW was same. For
much of the time during MW exposure,
media remain at highest reachable
temperature, strengthening the probability of
reliable sterilization despite shorter treatment
time. Superheating is a well recognized
phenomenon for homogenous suspensions
subjected to microwaves (Bond et al. 1991;
Barghust et al. 1992), which can raise the
temperature of water-based media above
normal boiling point of 100ºC, and thus can
effect microbial killing better than that
occurring under normal boiling.
Though reports of MW sterilization
for one or another particular medium have
appeared in literature in past, suitability of
MW sterilization for a wide variety of media
and growth of different microorganisms in
MW sterilized media has not been reported.
Iacoviello and Rubin (2001) reported sterile
preparation of antibiotic-selective LB agar
plates using a microwave oven. They
reported that plates prepared with a
microwave oven could be stored for short-
term (1-10 days at 4ºC) without
contamination, and showed that sterilization
of media by autoclave could be replaced with
short round of heating the media in a
microwave oven. Efficacy of the antibiotic
present in the media was not compromised
by the MW heating process. With respect to
microorganism contamination, plates
prepared with the microwave protocol were
shown to have the same shelf life as those
Vijay kothari et al. / Research in Biotechnology, 2(5): 63-72, 2011
71
prepared by the traditional autoclave
method. Liquid media that is made fresh
from powder and then boiled for several min
could be used for growing bacterial cells
without getting into too much trouble, given
that most gram-negative bacteria would not
survive the boiling treatment (Hengen, 1997).
Faster growth and greater viability of A.
actinomycetemcomitans were reported in both
liquid and solid growth media if they were
sterilized by MW radiation rather than by
autoclaving. One difference between
autoclaved and microwaved media was that
the autoclaved media were darker brown in
colour, which was suggested to be due to the
Maillard reaction products (Amadori
products). Such products are formed by
autoclaving a mixture of lysine and glucose,
and can inhibit growth of certain
microorganisms (Bhattacharjee et al. 2009).
MW sterilization thus can be recommended
for any media containing such ingredients
(e.g., lysine and glucose) whose interaction
under the influence of heat can generate
Amadori or other inhibitory products.
Bhattacharjee et al. (2009) found that broth or
plates made by MW method can be stored at
room temperature for more than a month,
indicating it to be an effective method for
sterilization of liquid or solid growth media.
Besides, it has the advantage that there can
be better control of sterilization time whereas
autoclaving time cannot be controlled easily
since it is always accompanied by extra
heating cycles during the conditioning and
exhaust cycles.
Our experiments clearly indicate
suitability of MW sterilization for liquid
nutrient media. It can be an attractive
alternative to conventional autoclaving,
especially when media are needed for
immediate use or for such organisms which
are not able to grow well in autoclaved
media, and also when high biomass yield is
of particular importance. It will be useful to
test its suitability for solid media of varying
compositions. Presence of agar in solid media
may cause bumping of contents inside vessel
during MW heating. If pressurized MW
vessels (which can be operated at pressure
equivalent to that in autoclave) of sufficiently
large volume can be made available at
reasonable cost, then MW sterilization is
likely to find wide acceptance. Such
apparatus will avoid evaporation as opposed
to open-vessel operations. If the challenges of
scale-up (in terms of volume, multiple
vessels at a time), real-time temperature
monitoring and control, and availability of a
wide range of MW-compatible materials can
be met, then MW sterilization may find
wider acceptance in microbiology
laboratories.
Acknowledgement
Authors thank Nirma Education and
Research Foundation (NERF) for financial
and infrastructural support.
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... Earlier studies revealed that the few second heat treatments are much quicker and easier methods to reduce the growth of food born microorganisms than the other conventional heating process (Cross and Fung, 1982;Rosenberg and Bogl, 1987;Kakita et al., 1995;Canumir et al., 2002). Thus, one of the common factors is still making controversy among the scientists about the thermal effects and the non-thermal effects of radiation (Dreyfuss and Chipley, 1980;Welt et al., 1994;Wayland et al., 1997;Kothari et al., 2011;Trivedi et al., 2011). Microorganisms can be killed using the thermal effect of microwave radiation while non-thermal effect has been suggested to inactivate microbial propagation (Jeng et al., 1987;Barnabas et al., 2011;Woo et al., 2000). ...
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... Such products are formed by autoclaving a mixture of lysine and glucose, and can prevent growth of microorganisms (Bhattacharjee et al., 2009). MW sterilization so can be recommended for any media containing such ingredients (e.g., lysine and glucose) whose interaction under the influence of heat can generate Amadori or other inhibitory products (Kothari et al., 2011). ...
... Such products are formed by autoclaving a mixture of lysine and glucose, and can prevent growth of microorganisms (Bhattacharjee et al., 2009). MW sterilization so can be recommended for any media containing such ingredients (e.g., lysine and glucose) whose interaction under the influence of heat can generate Amadori or other inhibitory products (Kothari et al., 2011). ...
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Different culture media in different volumes were exposed to microwave (MW) irradiation for 2, 3 and 4 minutes to investigate the ability of MW to destroy microorganisms and compared with media sterilized by conventional autoclaving method. MW sterilized media were screened for microbial growth. Exposure of different microorganisms to microwave irradiation resulted in destruction of all microorganisms within 3 minutes. Using MW for irradiation is a practical, easy, rapid and energy saving way to sterilize different types of culture media with no effects on the quality of culture media and microbial growth after sterilization. It can be used as alternative apparatus instead of autoclave in microbiology laboratories for preparing different sizes in rapid and routine experiments especially in the conditions of the weak electricity current and interruption.
... Os efeitos térmicos são causados por vibração por meio da interação entre dipolos de moléculas e a componente campo elétrico (E-Field) da onda eletromagnética e também pelo surgimento de correntes elétricas cujos portadores são íons livres, causando a transferência de energia na forma de calor. Os efeitos não térmicos, por sua vez, relacionam-se à transferência de energia, não na forma de calor, mas como efeitos eletrostáticos polares, promovendo a formação de peróxido de hidrogênio (H2O2), altamente oxidante e letal para muitos micro-organismos, além da quebra de ligações químicas [5], [6], [7], [8]. ...
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... Recent studies have demonstrated better results for food pasteurization or sterilization in terms of retention of nutrient quality [13] and microbial elimination [14], when microwave technology is used in comparison with conventional autoclaving. Specific developments have also been simulated for sterilization processes [15]. ...
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In this paper, a novel technique to achieve precise temperatures in food sterilization has been proposed. An accurate temperature profile is needed in order to reach a commitment between the total removal of pathogens inside the product and the preservation of nutritional and organoleptic characteristics. The minimal variation of the target temperature in the sample by means of a monitoring and control software platform, allowing temperature stabilization over 100 °C, is the main goal of this work. A cylindrical microwave oven, under pressure conditions and continuous control of the microwave supply power as function of the final temperature inside the sample, has been designed and developed with conditions of single-mode resonance. The uniform heating in the product is achieved by means of sample movement and the self-regulated power control using the measured temperature. Finally, for testing the sterilization of food with this technology, specific biological validation based on Bacillus cereus as a biosensor of heat inactivation has been incorporated as a distribution along the sample in the experimental process to measure the colony-forming units (CFUs) for different food samples (laboratory medium, soup, or fish-based animal by-products). The obtained results allow the validation of this new technology for food sterilization with precise control of the microwave system to ensure the uniform elimination of pathogens using high temperatures.
... Os efeitos térmicos são causados por vibração através da interação entre moléculas do tipo dipolo e a componente campo elétrico (E-Field) da onda eletromagnética e pelo surgimento de correntes elétricas cujos portadores são íons livres, o que causa a transferência de energia na forma de calor. Os efeitos não térmicos, por sua vez, são relacionados à transferência de energia, não na forma de calor, mas na forma de efeitos eletrostáticos polares, o que promove a formação de peróxido de hidrogênio (H2O2), altamente oxidante e letal para muitos micro-organismos, e mesmo a quebra de ligações químicas (CHANDRASEKARAN, RAMANATHAN e BASAK, 2013;SOLANKI, PRAJAPATI e JANI, 2011, KOTHARI, PATADIA e TRIVEDI, 2011WOO, RHEE e PARK, 2000;JENG et al., 1987). ...
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Abstract Numerous cultivation media currently exist, whether selective, non-selective, enrichment or identification. However, they all have a common goal, which is the growth of microorganisms; the constitution and quality of the culture medium must favor it. For this reason, an important factor that directly affects the quality of a culture medium is its production. Thus, this article investigated the use of a microwave oven in the production of Sabouraud dextrose agar (SDA), and the microbial inactivation compared to the autoclave in a microbiology laboratory. The quality of the medium, time exposure, and sterilization potential were performed using fungal strains of Candida spp., Cryptococcus spp., Microsporum spp., and Aspergillus spp. The results showed that the advantages of the use of a microwave oven for the preparation of SDA are practicality, speed, lower energy expense, pH, and constituents preservation of the culture medium, resulting in a richer growth compared to autoclaved SDA. The multivariate analysis of digital images allowed the detection of melanoidins (brownish tone of medium), which are responsible for the negative influence on the microorganisms growth. This research shows the use of the microwave oven as an efficient alternative for the production of the culture medium and maintaining their best quality.
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A growing body of literature has recognized the non-thermal effect of pulsed microwave radiation (PMR) on bacterial systems. However, its mode of action in deactivating bacteria has not yet been extensively investigated. Nevertheless, it is highly important to advance the applications of PMR from simple to complex biological systems. In this study, we first optimized the conditions of the PMR device and we assessed the results by simulations, using ANSYS HFSS (High Frequency Structure Simulator) and a 3D particle-in-cell code for the electron behavior, to provide a better overview of the bacterial cell exposure to microwave radiation. To determine the sensitivity of PMR, Escherichia coli and Staphylococcus aureus cultures were exposed to PMR (pulse duration: 60 ns, peak frequency: 3.5 GHz) with power density of 17 kW/cm² at the free space of sample position, which would induce electric field of 8.0 kV/cm inside the PBS solution of falcon tube in this experiment at 25 °C. At various discharges (D) of microwaves, the colony forming unit curves were analyzed. The highest ratios of viable count reductions were observed when the doses were increased from 20D to 80D, which resulted in an approximate 6 log reduction in E. coli and 4 log reduction in S. aureus. Moreover, scanning electron microscopy also revealed surface damage in both bacterial strains after PMR exposure. The bacterial inactivation was attributed to the deactivation of oxidation-regulating genes and DNA damage.
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With an automated computerized temperature control and a specialized temperature measurement system, dry spores of Bacillus subtilis subsp. niger were treated with heat simultaneously in a convection dry-heat oven and a microwave oven. The temperature of the microwave oven was monitored such that the temperature profiles of the spore samples in both heat sources were nearly identical. Under these experimental conditions, we unequivocally demonstrated that the mechanism of sporicidal action of the microwaves was caused solely by thermal effects. Nonthermal effects were not significant in a dry microwave sterilization process. Both heating systems showed that a dwelling time of more than 45 min was required to sterilize 10(5) inoculated spores in dry glass vials at 137 degrees C. The D values of both heating systems were 88, 14, and 7 min at 117, 130, and 137 degrees C, respectively. The Z value was estimated to be 18 degrees C.
Article
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Three techniques for studying effects of microwave radiation on microorganisms were introduced. Spores of Clostridium sporogenes (PA 3679) were chosen as a test organism because the kinetic parameters for thermal inactivation are well known and because of the importance of the genus Clostridium to the food industry. For the first technique, a specially designed kinetics vessel was used to compare inactivation rates of microwave-heated and conventionally heated spores at steady-state temperatures of 90, 100, and 110 degrees C. Rates were found to be similar at the 95% confidence level. The second and third techniques were designed to study the effect of relatively high power microwave exposure at sublethal temperatures. In the second approach, the suspension was continuously cooled via direct contact with a copper cooling coil in a well-mixed vessel, outside the microwave oven. The suspension was pumped through a Teflon loop in the oven, where it continuously absorbed approximately 400 W of microwave power. Inactivation occurred in both irradiated and unirradiated samples. It was suspected that copper ions entered the suspension from the copper coil and were toxic to the spores. The fact that the results were similar, however, implied the absence of nonthermal microwave effects. In the third approach, the copper coil was replaced with a silicone tubing loop in a microwave transparent vessel. The suspension was continuously irradiated at 150 W of microwave power. No detectable inactivation occurred. Results indicated that the effect of microwave energy on viability of spores was indistinguishable from the effect of conventional heating.
Article
A substantial body of information is accumulating which illustrates the beneficial effects of microwave energy, as compared with conventional heating, on a range of chemical conversions. Recently, there have been claims that rate enhancements observed are owing to superheating caused by absorption of microwaves. Recently, the authors described a simple gas thermometer suitable for temperature measurement in microwave ovens. Now, an alternative method for temperature estimation in microwave fields is outlined, based on thermochromic liquid crystals. The use of this technique is illustrated by presenting preliminary results which show conclusively that water can be readily superheated by microwaves.
Article
Methods and reagents is a unique monthly column that highlights current discussions in the newgroup bionet.molbio.methds-reagnts, available on the Internet. This month's column discusses the use of a home microwave oven for sterilizing laboratory equipment. For details on how to partake in the newsgroup, see the accompanying box.
Article
Fluoroptic temperature measurements have established that organic solvents in a microwave cavity superheat by 13–26 °C above their conventional boiling points at atmospheric pressure; this behaviour is interpreted using a model of nucleate boiling that emphasises the importance of the wetting properties of the solvents.
Article
Aggregatibacter (Actinobacillus) actinomycetemcomitans is the causative agent of localized aggressive periodontitis and endocarditis. The bacteria grow slowly even in a rich medium and rapidly lose viability after about 19 h of growth. One of the reasons for the slow growth and for the decreased viability is the conventional method of making growth media by autoclaving. Faster growth and greater viability were observed in both broth and plates if the growth media were sterilized by microwave radiation rather than by autoclaving. One difference between autoclaved and microwaved media is that the autoclaved media are darker brown in color, which is known to be due to the Maillard reaction products, also known as Amadori products. The Maillard reaction products formed by autoclaving a mixture of lysine and glucose were shown to inhibit growth of A. actinomycetemcomitans.
Article
The interdependent nature of the thermal and electromagnetic effects in the responses of biological systems to high-power-level microwave fields is investigated. This note presents an experimental approach to understanding the contribution and interaction of the thermal and electromagnetic domains. The problem of distinguishing the thermal interdependency of electromagnetic effects was addressed by inactivating spores of Bacillus subtilis in a microwave field and comparing the inactivation rate to that obtained by exposure to heat alone. Measurable differences were found in the inactivation rates.
Article
The killing activity of microwaves of 2450 MHz frequency and 325 W, 650 W and 1400 W power on some bacterial strains was investigated. Vegetative strains of Staphylococcus aureus, Streptococcus pyogenes Group A, Escherichia coli, Pseudomonas aeruginosa and Enterococcus faecalis and spores of Bacillus subtilis and Bacillis stearothermophilus in aqueous suspensions were exposed to 325 W and 650 W waves for different lengths of time. Enterococcus faecalis and spores of B. subtilis and B. stearothermophilus were exposed additionally to 1400 W waves in aqueous and 'dried' suspensions. Vegetative bacteria were promptly killed in 5 min or less, E. faecalis being slightly more resistant. Bacterial spores were only killed in aqueous suspension when a 1400 W setting was used for 10 to 20 min. Bacterial spores adhering to the tube walls after the aqueous suspension was poured out were reduced in number. We assume that the conventional microwave ovens available on the market may be used for a high level of disinfection but not for sterilization, and only then if sufficient water is present.
Article
A new microwave continuous sterilizer (MWS) for applying microwave dielectric heating as an alternative to an autoclave was developed. The developmental objectives of the MWS were: 1. Achieving sufficient sterilization for the drugs containing heat-sensitive ingredients. 2. Measuring and recording sterilization temperature of each ampoule. 3. Ensuring automatic continuous operation and linkage with the preceding and following machines in an injection ampoule production process. The temperature of the drug solution in an ampoule was heated to 140 degrees C within about 30 seconds by the MWS. Target F0 value is achieved through the maintaining heater to maintain the target temperature for 12 seconds. Ampoules are cooled with air and water after completion of heating. The MWS is capable of processing 150 ampoules per minute. The newly developed techniques which minimized temperature distribution of heated ampoule solution were: 1. Microwave irradiation in a direction opposite to the direction of ampoules transportation. 2. Microwave irradiation in the lower part of ampoule solution (i.e., heating up the drug solution by thermal convection.) 3. Microwave power control by feedback of measured temperatures. 4. Heating rate control corresponding to the dielectric property of ampoule solution. The drug stability test was performed using 3% pyridoxamine phosphate solution, and the inactivation of spores in 3% pyridoxamine phosphate solution was examined using Bacillus stearothermophilus ATCC 7953 spores. The MWS was proved to have an adequate efficiency of sterilization with less chemical degradation of the contents than an autoclave.