A RESEARCH NOTE

COMPARISON OF MIKROCLAVETMSTERILIZATION SYSTEM

AND CONVENTIONAL AUTOCLAVING FOR THE
STERILIZATION OF MICROBIOLOGICAL MEDIA'

K E N N Y F. CHUANG and DANIEL Y . C . FUNG

Department of Animal Sciences and Industry
Kansus State University
Manhattan, KS 66506-1600

Received for Publication March 24, 1994

ABSTRACT

The viable cell count performance of nutrient agar prepared by the

MikroClaveTMsterilization system MSS-SO0 was evaluated using Escherichia coli
0157:H7,Salmonella typhimurium, a d Yersinia enterocolitica with conventionully
autoclaved media as a comparison. N o signijkant dijjerence wus found between
agar prepared by the two methodd. 7he MikroClave' was able to sterilize corn-
mon bacteriological media such as E o ~ i nMethylene Blue (EMB) a p r , nutrimr
agar, Yeust und Mold (YM) agar, lactose broth, nutrient broth, und Yeust und
Mold (YM) broth.

INTRODUCTION

Conventional methods for sterilization of media usually use moist heat and steam
sterilization techniques. The autoclave is the most common piece of' laboratory
equipment using steam to sterilize bacteriological media (121C, 15 psi for 15
min or longer). However, a complete sterilization cycle (come up time, steriliza-
tion time, and exhaust) for liquid media requires 45-60 min, which is very time
consuming.

'Contribution No. 944255, Kansas Agricultural Experiment Sfittion, Manhattan, Kansas. This marerial
is based upon work supported by the Cooperative State Research Service, U . S . Department o f
Agriculture, under Agreement No. 89-341887-451 1.
Journal of Rapid Methods and Automation in Microbiology 3 (1994) 77-8 I . All Ri,yht.s Re.wrvuJ
% Copyright 1994 by Food & Nutrition Pre.s.\, lnc. , 7rumbull, Connrrticut 71
7x K E N N Y F . C H U A N G and DANIEL Y C FUNG

Microwave ovens have been used in the food processing industry for many
years (Decareau 1985). The effects of microwave radiation on many micro-
organisms in different food systems were reviewed by Fung and Cunningham
(1980). The conventional microwave oven is a convenient and efficient way of
preparing agars for autoclaving or melting previously sterilized agar for plating
and cultivation o f bacteria (Fung and Lin 1984; Liang and Fung 1988).
Recently, a new instrument called the MikroClavelM(Fig. I ; CEM Corp., Mat-
thews, NC). has been developed combining microwave energy and pressurized
vessels for rapid sterilization of bacteriological media. Ranxiley and Meehan (1991)
found n o significant differences in thermal death times ( D and Z values) ofBuci1lu.s
s r r c i r ~ ) t h ~ ~ t - t ~ i ~ ) ~using the
~1iiIu . s MikroClave' sterilization system and a conven-
tional autoclave sterilization system. The unit can simultaneously sterilize 12,
100-ml vessels of media each. One vessel is connected to the built-in pressure
controller. The MikroClave'" heats the media to 70 psi in 70 s per vessel and
will automatically terminate the cycle. Sterilization o f 1200 ml of microbiological
media can he completed in approximately 7 niin. The sterilized media can be
ready t o use after an additional 5-7 min of cooling time. The purpose o f this
investigation was to evaluate the performance of nutrient agar prepared by the
MikroClavel " MSS-500 sterilization system and the conventional autoclave
system in the enumeration of selected bacteria. Also the sterility of other com-
nion laboratory media prepared by MikroClave'M was evaluated.

MATERIALS AND METHODS

Pure Cultures
t'schcrichiu coli 01 57:H7 Eh7-2, Sulniotiell~i/vphitmriutn NR-CDC, and Yor-
.siniti pntproc.o/itrc.tiATCC
27729 were obtained from the Kansas State University
food microbiology culture collection and used as test organisms. Cultures were
grown in Brain Heart Infusion Broth (Dit'co, Detroit, MI) at 37C ior 24 h before
use as inocula to test the performance of nutrient afar on viable cell counts.

Media Tested
F.MB agar (Difco, Detroit. M l ) , nutrient agar (Difco, Detroit, MI), Y M agar
(Difco, Detroit. M I ) , nutrient broth (Diico, Detroit. M I ) , lactose broth tDit'co,
Detroit. M I ) . and Y M broth IDifco, Detroit, MI) were used tor testing the steriliza-
tion ability ofthe MikroClave'" steriliAon 5ystem. Nutrient agar (Difco, Detroit,
M I ) was uhed l o r viable cell count\ o f the pure culture\.
 STERILIZATION OF MICROBIOLOGICAL MEDIA 79

FIG. I . DIAGRAM OF THE MIKROCLAVETMMSS-500 STERILIZATION SYSTEM

(CEM CORPORATION, MATTHEW, NC)

Microwave Oven
A “MikroClavelM sterilization System” model MSS-500 (CEM Corp., Mat-
thews, NC) was used in this study. This system is operated at 2450 MHz, and
has a power output of 1800 Watts. Up to 12 sterilization vessels with I 0 0 ml
capacity each can be sterilized simultaneously in the system. The system was set
at “flashing” mode for operation. The average time needed for sterilizing one
vessel with media (100 ml) is 70 s.

Media Prepared by Conventional Method

An appropriate amount of dehydrated nutrient agar (DIFCO) was distributed
in 600 nil of distilled water in a 1000-ml beaker. The beaker was placed in the
center of a conventional microwave oven and heated until the agar melted com-
pletely (Fung and Lin 1984). The melted medium (100 ml) was then transferred
to Wheton bottles (250 ml). All bottles were sterilized in the autoclave for 15
min at 15 psi (121C). After a complete sterilization cycle, all bottles were tempered
in a 48 2C water bath and used to compare agar prepared by MikroClaveTM
sterilization system.
80 KENNY F . CHUANG and DANIEL Y . C . FUNG

Media Prepared by MikroClaveTh’Sterilization System

Nutrient agar (DIFCO) was prepared as previously discussed. The melted
medium (100 ml) was then transferred to MikroClaveTh’ sterilization vessels and
sterilized using the “flashing sterilization” mode. After completing the steriliza-
tion cycle, all vessels were tempered in 48 f 2C water bath and used for viable
cell counts.

Performance of Media
Performance of media prepared by the conventional method and MikroClaveTM
sterilization method was evaluated by performing viable cell counts of pure cultures
of Escherichia coli 0157:H7. Salmonella Vphimurium. and Yersinia enterocolitica
using standard plate count methods (Marshall 1993).
Sterility of EMB agar, nutrient agar, YM agar, lactose broth, nutrient broth,
and Y M broth prepared by the MikroClaveTh’ checked by observing turbidity
development of broth or growth of colonies in sterile agar after 24 h incubation
at 37C incubator.

RESULTS AND DISCUSSION

Results of viable cell counts (log,, CFU/ml) and statistical analysis of target
bacteria populations using nutrient agar prepared by the conventional method and
the MikroClaveT” sterilization system are recorded in Table 1. No significant
difference was observed in agar prepared by those two methods for enumerating
selected bacteria. MikroClaveTMwas more convenient to use and less time con-
suming especially for small batches of media.
During preliminary experiments, contamination of broth and agar was observed
from MikroClaveTMsterilization system. The source of the contamination was
determined to be from the lip of the sterilization vessel. Apparently, these vessels
were used previously to study bacterial spores and residual spores at the edge
lip were not destroyed by microwave treatment. By wiping with alcohol and fur-
ther UV light treatment (30 min), the contamination was eliminated. After solv-
ing the problem, all subsequent agar and broth prepared in the MikroClaveTM
were found to be sterile.
In conclusion, agar and broth can be sterilized by the MikroClaveTMeffective-
ly. There is no difference in performance of nutrient agar prepared by conven-
tional autoclaving and the MikroClave’M sterilization system for viable cell counts
of target bacteria. We recommend the use of the MikroClaveTMsterilization system
for rapid sterilization of microbiological media. Since the unit cannot sterilize
glassware or media in tubes and can only prepare 1200 ml of media at one time,
 STERILIZATION OF MICROBIOLOGICAL MEDIA 81

TABLE 1 .
PERFORMANCE OF NUTRIENT AGAR PREPARED BY MIKROCLAVETM AND
AUTOCLAVE METHODS IN VIABLE CELL COUNTS OF ESCHENCHL4 COL10157:H7.
AND SALMONELLA TYPHIMURIUM, YERSINIA ENTEROCOLITICA
tog CEUjmla

E . c o l i 0157:H7 S. typhimurium Y . enterocolitica

MikroClaveTM
sterilization 9.27 8.74 8.80
system

Conventional 9.26 8.75 8.83

method

Pierson
correlation 0.999 0.999 0.999
coefficient

a A l l data are t h e average of five trails.

it will not replace the conventional autoclave in a microbiology laboratory.

However, it is useful for making small batches of media rapidly.

REFERENCES

DECAREAU, R.V. 1985. Microwaves in the Food Processing Industry, Academic

Press, New York.
FUNG, D.Y.C. and CUNNINGHAM, F.E. 1980. Effects of microwave cook-
ing on microorganisms in foods. J . Food Prot. 43, 641-650.
FUNG, D.Y.C. and LIN, C.C.S. 1984. Melting agar by microwave energy. J .
Food Prot. 43, 770-772.
LIANG, C. and FUNG, D.Y.C. 1988. Performance of some heat-sensitive dif-
ferential agars prepared and melted by microwave energy. J. Food Prot. 51,
577-578.
MARSHALL, R.T. (ed.) 1993. Standard Methods for the Examination of Dairy
Products, 16th Ed., American Public Health Assoc., Washington, DC.
RAMALEY, R. and MEEHAN, Z. 1991. Microwave sterilization of micro-
biological media. CEM Co., Matthew, NC.