Letters in Applied Microbiology 1997, 24, 265268

Comparison of aerobic and anaerobic methods for the

microbiological monitoring of chilled packaged meat
during storage
R.G.Bell, N. Penney and S.M. Moorhead
Meat Industry Research Institute of New Zealand (Inc.), Hamilton, New Zealand
1202/96: received 26 August 1996 and accepted 27 August 1996

Aerobic and anaerobic plate counts were

compared for routine monitoring of the microflora, dominated by lactic acid bacteria,
developing on vacuum- and carbon dioxide-packaged raw meat during chilled storage.
No statistical differences were observed between aerobic and anaerobic enumerations,
made on plate count and blood agar plates, of the microflora developing on beef striploins
packaged under vacuum or carbon dioxide during 14 weeks storage at 0C. With both
techniques the spoilage microflora development differed between the two packaging
regimes. The results indicate that there is no necessity for aerobic plate counts to be replaced
by anaerobic plate counts in the routine microbiological examination of the spoilage
microflora developing on chilled meats packaged under anoxic modified atmospheres.
R .G . B E LL , N . PE NN E Y A ND S .M . M O OR HE A D. 1997.

INTRODUCTION

Vacuum or modified atmosphere packaging extends the storage life of chilled meat by restricting the growth of spoilage
micro-organisms. In vacuum packs the oxygen-deficient
environment surrounding the product precludes the growth
of obligate aerobes and slows the growth of facultative
bacteria, which must rely on fermentative metabolism rather
than the more energy-efficient oxidative metabolism. In oxygen-free saturated carbon dioxide packs, microbial growth is
further restricted by a high partial pressure of carbon dioxide
(Gill 1989). Consequently, the spoilage microflora developing
on chilled meats in such packs will be limited to facultative
and anaerobic bacteria, especially those that can tolerate high
concentrations of carbon dioxide.
Aerobic plate counts are widely used to monitor spoilage
microflora development on products after storage in vacuum
or carbon dioxide packs (Newton and Gill 1978 ; Enfors et al.
1979 ; Grau et al. 1985 ; Penney et al. 1994). However, it
seems a little incongruous to use aerobic plate counts to
enumerate a microflora developing within an anaerobic package. The present authors, as many before them, have justified
this apparently illogical practice on the grounds that the
Correspondence to : Dr R. G. Bell, Meat Industry Research Institute of New
Zealand (Inc.), PO Box 617, Hamilton, New Zealand.
1997 The Society for Applied Bacteriology

developing microflora are composed of facultative and aerotolerant anaerobes that can grow, and therefore be enumerated, using either aerobic or anaerobic incubation. The
present study was undertaken to verify this assertion by
comparing the use of aerobic and anaerobic plate counts for
monitoring spoilage microflora development on vacuum- and
carbon dioxide-packaged meat during chilled storage.
MATERIALS AND METHODS
Meat

Eight beef striploins were obtained from a commercial hot

deboning plant and transported, in an insulated container, to
the laboratory within 1 h of slaughter. On arrival at the
laboratory each striploin was cut into four pieces of approximately 800 g each.
Packaging

Half of the quarter-striploins (16) were conventionally

vacuum packed in Cryovac BB4L (W.R. Grace, Porirua, New
Zealand) pouches with a stated oxygen transmission rate of
50 ml/m2/atm/24 h at 23C. The remaining 16 quarterstriploins were packed under carbon dioxide, 15 l/kg meat,

266 R .G . B E LL ET A L.

in aluminium foil laminate pouches (Securefresh Pacific,

Auckland, New Zealand) with an immeasurably low oxygen
transmission rate.
Storage

Both vacuum- and carbon dioxide-packed hot deboned

quarter-striploins were placed into a chiller operating at
5C and held for 24 h before being transferred to a storage chiller operating at 0C. After 6, 8, 10, 12 and 14 weeks
storage, three vacuum and three carbon dioxide packs were
removed and subjected to microbiological examination.
Microbiological examination

On arrival at the laboratory, a 5 cm2 area on each of five whole

striploins, i.e. before quartering, were swab-sampled using
the wet and dry swab technique (Cook 1991). After various
periods of chilled storage, groups of three vacuum- and three
carbon dioxide-packed quarter-striploins were similarly
swab-sampled to monitor spoilage microflora development.
Initial homogenates were prepared in sterile dilution fluid
(01% peptone, 085% NaCl), and 01 ml volumes of appropriate dilutions thereof spread in duplicate onto the surface
of two sets of Plate Count Agar (PCA ; Difco, Detroit, MI,
USA) and single sets of deMan Rogosa Sharp Agar (MRS ;
Oxoid, Basingstoke, UK) and Blood Agar (BA ; made from
Columbia Base, Oxoid, plus 5% defibrinated sheep blood).
One set of PCA plates and the MRS plates were incubated
aerobically at 25C and the colonies that developed after 72
h were enumerated. The other set of PCA plates and the BA
plates were pre-reduced in an anaerobic chamber for 24 h at
15C prior to use and then incubated at 25C in Oxoid HP11
anaerobic jars with Difco Anaerobic System pouches. The
colonies that developed after 72 h were enumerated. All
numerical data were collated and compared statistically using
the Sigmastat for Windows software package ( Jandel Scientific Software, San Rafael, CA, USA).
RESULTS AND D ISCUSSION

The development of spoilage microflora on vacuum- and

carbon dioxide-packed striploins during chilled storage as
influenced by enumeration medium and incubation environment is summarized in Table 1. With vacuum, and especially
with carbon dioxide, packaging the growth rate advantage
afforded to the lactic acid bacteria by the prevailing conditions
in the packs during chilled storage is clearly demonstrated by
the increasing similarity between aerobic counts obtained on
non-selective PCA and MRS, which selects for lactic acid
bacteria. These results show lactic acid bacteria proliferating
from numerically insignificance in the initial microflora to
dominance in the spoilage microflora (Table 1).

Irrespective of incubation regime or growth medium, the

lag phase associated with the development of the spoilage
microflora on carbon dioxide-packed product was significantly longer than that for product under vacuum. This
observation supports the well-established principle that lag
phase extension plays an important role in storage life extension of chilled beef achieved under carbon dioxide over that
attained under vacuum (Gill and Penney 1988). A statistical
consequence of this effect is that the microbial population on
vacuum-packed meat from weeks 6 to 14 shows a log normal
distribution whereas populations on meat packed under carbon dioxide do not. In practical terms maximum numbers
were reached much earlier during chilled storage in vacuum
packs than in packs containing carbon dioxide. Consequently,
vacuum and carbon dioxide packs must be considered separately with respect to the effects of incubation environment
and growth medium.
With vacuum-packed product, aerobic and anaerobic incubation on PCA did not produce significantly different results
(P  0767, one-way analysis of variance). With anaerobic
incubation, counts obtained for PCA and BA plates were
not significantly different (P  0929, one-way analysis of
variance). Comparison of all four incubation growth medium
combinations by a four way comparison showed no significant
differences between medians (P  0943, Kruskal Wallis oneway analysis of variance of ranks).
With product packed under carbon dioxide the Kruskal
Wallis one-way analysis of variance of ranks was used for all
comparisons because the log distribution of the counts was
not normal. Anaerobic and aerobic counts on PCA were
not significantly different (P  0319). Counts obtained with
anaerobic incubation of PCA and BA were not significantly
different (P  0693) and a four way comparison also showed
no significant differences (P  0625).
These results show quite clearly that the use of aerobic
incubation conditions for enumerating micro-organisms
developing on chilled meat packaged under vacuum or anoxic
modified atmospheres is not inappropriate for the routine
microbiological examination of such products. It should be
noted that in routine examinations, as was the case in this
study, no special precautions would be taken with respect
to oxygen toxicity during sampling, dilution and plating
procedures.
Product spoilage occurs as a direct consequence of the
development of a climax microbial community under the
selective influence of the conditions found, in the case of
whole tissue, on the meat surface. Generally such climax
populations, the spoilage microflora, lack species diversity.
Lactic acid bacteria are the dominant physiological group in
the microflora developing on vacuum- and carbon dioxidepackaged chilled meats. Although lactic acid bacteria is not
an official taxonomic classification, speciation into Lactobacillus, Carnobacterium, Streptococcus, Leuconostoc and Pedi-

1997 The Society for Applied Bacteriology, Letters in Applied Microbiology 24, 265268

A ER OB I C V S A N AE RO B IC PL A TE CO U NT S 267

Table 1 Influence of aerobic or

anaerobic incubation and growth medium

on the enumeration at 25C of the
spoilage microflora
developing on vacuum- and carbon
dioxide-packed beef quarter-striploins
during storage at 0C

Spoilage microflora (cfu log10 cm2)

Aerobic
Anaerobic

Storage time (weeks)

PCA
MRS
PCA
BA

Vacuum-packed
0
2852011
ND
2162030
2272024
6
4242063
4172065
4262098
4112043
8
6182041
5832006
5792010
5862011
10
6712050
6212057
6412048
6322058
12
6662089
7262041
6612093
6692098
14
7752093
7412038
7762095
7632099
Carbon dioxide-packed
0
2852011
ND
2162030
2272024
6
2242016
1512052
1752030
1822034
8
2662033
2102066
2032123
2172043
10
2752012
2402028
2502022
2502021
12
3702131
3562168
3452178
3542159
14
4732089
5152126
5082124
5122114

PCA, Plate count agar ; MRS, de Man Rogosa Sharp agar ; BA, blood agar medium ; ND,
not detected (limit of detection log10 10 cfu cm2).

ococcus is of questionable utility in routine monitoring of

spoilage development.
In contrast, the spoilage microflora other than the lactic
acid bacteria on vacuum- or carbon dioxide-packaged chilled
meats may be of more consequence for assessing product
life than its magnitude would suggest. For example, 10%
Enterobacteriaceae, in a population of 107 cells cm2, may
cause spoilage whereas 10% Brochothrix thermosphacta might
not if oxygen were effectively excluded from the pack (Davidson et al. 1968). Furthermore, with routine examination of
vacuum- or carbon dioxide-packaged meat, the microflora
enumerated may not be consistent with the spoilage symptoms, e.g. clostridial blown pack spoilage (Broda et al. 1996).
In such cases routine examination using aerobic incubation
would fail to isolate to causative bacteria, and the use of
special investigatory anaerobic procedures would be both
justified and required. However, as a first action for routine
examination of vacuum- or carbon dioxide-packaged meat
showing no abnormal or unexpected spoilage symptoms, the
use of anaerobic procedures would provide no information
not more conveniently acquired using aerobic procedures.
Therefore, there is no justification for advocating the replacement of aerobic plate counts by anaerobic plate counts in the

routine microbiological examination of vacuum- or carbon

dioxide-packaged chilled meats.

ACKNOWLEDGEMENT

The financial support of the New Zealand Meat Research

and Development Council is gratefully acknowledged.

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1997 The Society for Applied Bacteriology, Letters in Applied Microbiology 24, 265268

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1997 The Society for Applied Bacteriology, Letters in Applied Microbiology 24, 265268