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White Paper 6/2023: BACT-R
Obtaining an anaerobic atmosphere
for growth of stricly anaerobic
bacteria in medical bacteriology
Evaluation of automated evacuation-replacement system
by AC. Tessedre (*), A-J. Waligora-Dupriet (*), F. Doucet-Populaire (*,**), M-J. Butel (*)
Abstract
The performance of an automatic evacuation-replacement system – BACT-R (Sobioda,
Montbonnot Saint Martin, France) – to create an anaerobic atmosphere in jars was
evaluated for approximately fifty anaerobic strains. This system was compared to the
growth of 51 strains in anaerobic cabinets and jars using anaerobic condition generation
bags. The study showed that the anaerobic condition created by the evacuation-
replacement system allows for the growth of isolated strictly anaerobic bacteria in
medical bacteriology identical to that of the two compared systems. The jar opening
tests showed the absence of the toxic effect of oxygen on 9 strains with the exception
of one strain. On the other hand, the growth was slowed for six of the strains.
Keywords: Anaerobic condition, technique.
Introduction species can tolerate contact with oxygen in the air for
While strictly anaerobic micro-organisms constitute a many hours (Bacteroides fragilis for example); others
significant part of the commensal flora, they are implicated are killed after contact lasting only minutes (Clostridium
in a large number of generalised or localised infections, butyricum for example). Different systems can be used
or of endogenous or exogeneous origin. Their sensitivity to obtain an anaerobic atmosphere.
to oxygen and their often slow growth make it difficult Anaerobic cabinets, biological safety cabinets
to bring them to light. In addition, anaerobic infections blanketed with an oxygen-free gas, allow work to be
are frequently mixed infections, associating aerobic and performed under continuous anaerobic condition and is
strict anaerobic bacterial, requiring the use of selective the ideal equipment for isolating anaerobic bacteria in
environments. So, it is very important to optimise the biological samples. It is the only devices that allows the
culture growth conditions in order to obtain cultures as growth of micro-organisms that are extremely sensitive
quickly and as reliably as possible. to oxygen. Nevertheless, these anaerobic cabinets are
The degree of sensitivity to oxygen of anaerobic highly expensive to purchase and very cumbersome.
bacteria varies from once species to another. Certain Most frequently, discontinuous anaerobic condition
Σελίδα 2
White Paper 6/2023: BACT-R
systems are used in medical biological laboratories. I. Materials and methods
The most common systems are jars or individual
bags. Anaerobic condition is created thanks to the
use of mixtures of reagents that trap the oxygen A) Bacterial strains
and produce carbon dioxide; for example GENbox Fifty-one species from the laboratory collection were
anaer® or GENbag anaer® (bioMérieux, Marcy l’Étoile, included. These species originated from clinical samples
France), Anaerogen® (Oxoïd, Dardilly, France) or or collections. They were divided as follows: 24 belonging
Anaerocult® (VWR, Fontenay-sous-Bois, France). These to the Clostridium genus, 17 were Gram negative bascilli
reagents are a good alternative, allowing good strict (Bacteroides, Prevotella, and Fusobacterium genus) and
anaerobic grown isolated in medical bacteriology (1), 10 des cocci or non–spore-forming Gram positive bascilli
(2) and are less expensive in terms of initial investment. (Table I).
Anaerobic condition can also be obtained with the
evacuation-replacement technique. This technique B) Anaerobic Cabinet
requires the use of jars that have an evacuation system, The anaerobic cabinet used was a biological safety
a vacuum source and an oxygen-free gas mixture. A cabinet with rigid plates, temperature regulated to 37 °C
number of automatic systems are available on the market: (Don Whitley Scientific, AES Laboratoire, Bruz, France).
BACT-R (Sobioda, Montbonnot Saint-Martin, France), The gas used was a blend of H2 :CO2 :N2 = 10 :10 :80 v/v.
Anoxomat (Mart, Lichtenvoorde, Hollande), MACS-mics The anaerobic conditions were verified continuously due
(AES Laboratoire, Bruz, France). Anoxomat was recently to the presence of a resazurin solution in the presence
quoted, proving the interest in these types of systems of cysteine. This solution, colourless in the absence of
(3). BACT-R, tested in our study, is a microprocessor- oxygen, turns pink if the anaerobic condition fails.
controlled programmable system that creates a vacuum
of about 0.6 bar in a jar and then fills it with the blend C) Anaerobic condition generation bag
of gases chosen. This operation is repeated six times to The anaerobe generation systems used were GENbags
obtain an anaerobic condition quickly. (bioMérieux, Marcy-1’Étoile, France). These bags allow
The study’s objective was to validate the use of a the anaerobic condition to be generated quickly, without
BACT-R system by testing growth on about 50 strict the addition of water or a catalyser. Their chemical
anaerobic species that belong to the main types that can composition (activated carbon, sodium ascorbate, and
be responsible for anaerobic infections. The growth of other organic and mineral components) absorb oxygen
these species was compared using four different types and liberate carbon dioxide.
of anaerobic condition generation: the BACT-R system
with two different gases, the anaerobic cabinet, and a D) BACT-R
chemical anaerobe generation system. The BACT-R system allows for an ana
Σελίδα 2
es was compared using four different types and liberate carbon dioxide.
of anaerobic condition generation: the BACT-R system
with two different gases, the anaerobic cabinet, and a D) BACT-R
chemical anaerobe generation system. The BACT-R system allows for an anaerobic condition
The rapid creation of an anaerobic atmosphere does to be established after six evacuation-replacement
not prevent the bacteria from being killed if the contact cycles. The manufacturer’s recommended gas mixture
time with air is too long. For this reason, jar opening is H2 :CO2 :N2 = 5 : 5 :90 v/v. The 5 per cent hydrogen
tests were included in this study in order to provide content gas blanket to be maintained in the laboratory.
recommendations for the use of this system. For reasons of the study, the equipment was adapted to
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White Paper 6/2023: BACT-R
Table I. Bacterial strains used to evaluate the BACT-R system deliver both this gas mixture and the gas used for the
Genera and bacterial species Number of anaerobic cabinet (10 per cent H2). The jars were equipped
isolates tested with a palladium catalyst. The catalyst was regenerated
Spore-forming Gram positive bacilli after each use (1 h 30 at 180 °C) to eliminate the water of
Clostridium perfringens 3 condensation. Note that the catalyst regeneration can be
Clostridium butyricum 5 performed at 37 °C, also, but for a period of 12 hours (i.e.
Clostridium difficile 10 overnight).
Clostridium sporogenes 2
Clostridium bifermentans 1 E) Environment
Clostridium ramosum 1 The bacterial strains, stored at -80 °C in brain heart
Clostridium paraputrificum 2
infusion broth additivated with 15 per cent glycerol, were
Gram negative bacilli
inoculated on freshly prepared Columbia blood agar (5
per cent). For the bacterial enumeration, the inoculation
Bacteroides fragilis 4
was performed using TGYH (tryptone, glucose, hemin
Bacteroides ovatus 1
and yeast extract) brain heart infusion broth.
Bacteroides vulgatus 1
Bacteroides caccae 1
F) Method
Bacteroides thetaiotaomicron 1
1) Validation of bacterial strain growth using the
Bacteroides distasonis 1
BACT-R system
Bacteroides stercoris 1
After verifying the purity of the microbial cultures, a
Prevotella bivia 1
colony of each strain was inoculated in TGYH broth and
Prevotella melaninogenica 1
incubated for 24 to 48 hours according to the bacterial
Prevotella intermedia 1
species. Dilutions of 100 in 100 were made and each
Fusobacterium varium 1
dilution was inoculated on four Columbia blood agar using
Fusobacterium nucleatum 1
the WASP spiral automatic plater (Don Whitley Scientific,
Fusobacterium necrophorum
Σελίδα 3
dilution was inoculated on four Columbia blood agar using
Fusobacterium nucleatum 1
the WASP spiral automatic plater (Don Whitley Scientific,
Fusobacterium necrophorum 2
AES Laboratoire, Bruz, France). The blood agar plates
Gram positive cocci
were then placed as quickly as possible under anaerobic
Peptostreptococcus anaerobias 1
condition using the four different systems
Finegoldia magna 2
∙ the anaerobic cabinet,
Peptostreptococcus asaccharolyticus 1 ∙ the GENbag system,
Micromonas micros 1 ∙ the BACT-R system using the manufacturer‘s
Non-spore-forming Gram positive bacilli recomended gas for laboratories (5 per cent H2),
Propionibacterium acnés 2 ∙ the BACT-R system using the anaerobic cabinet
Eubacterium lentum 1 gas (10 per cent H2).
Bifidobacterium longum 1
Bifidobacterium bifidum 1 The bacterial enumeration was performed after 24 and 48
hours of incubation and expressed in log10 UFC/mL and
the sizes of the colonies were compared.
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White Paper 6/2023: BACT-R
2) Jar opening tests These tests were performed on the bacterial stains most
In order to optimise the use of the BACT-R system, sensitive to the toxicity of oxygen: Clostridium butyricum
comparative bacterial enumeration was performed (2 strains), Clostridium difficile (3 strains), Fusobacterium
after anaerobic incubation (1) without opening the necrophorum (2 strains), Fusobacterium nucleatum
jar, and voluntarily opening the jar in two steps (after (1 strains), Micromonas micros (1 strains).
1 hour and after 5 hours of incubation) (2) for 5
minutes each time, and (3) for 30 minutes each time.
This was done to mimic the conditions encountered
in a medical bacteriology laboratory. The gas used
was the manufacturer’s recommended gas. The
bacterial enumeration and observation of the colonies
was performed after 24 and 48 hours of incubation
and compared to the growth of the same strains
incubated for 24 and 48 hours in the anaerobic cabinet.
Table II. Bacterial enumeration as a function of the systems used for the anaerobic condition,
expressed in log10 UFC/mL (mean ± standard deviation) after 48 hours incubation
Bacterial strains Anaerobic Cabinet GENbag BACT-R
H2 :CO2 :N2 10 :10 :80 H2 :CO2 :N2 5 :5 :90 H2 :CO2 :N2 10 :10 :80
Clostridium 7.3 ± 1.0 7.2 ± 1.2 7.3 ± 1.0 7.4 ± 1.2
C perfringens 8.2 ± 0.2 8.2 ± 0.3 8.0 ± 0.1 8.6 ± 0.9
C difficile 7.2 ± 0.6 6.9 ± 0.6 7.2 ± 0.5 7.1 ± 0.6
C butyricum 6.2 ± 0.6 6.0 ± 0.7 6.2 ± 0.6 6.1 ± 0.4
Autres Clostridium a
7.6 ± 1.1 7.7 ± 1.3 7.5 ± 1.1 7.6 ± 1.4
Gram negative bacilli 8.8 ± 1.4 8.8 ± 1.3 8.8 ± 1.5 8.8 ± 1.3
Bacteroides fragilis 8.8 ± 0.7 8.7 ± 0.7 8.8 ± 0.7 8.8 ± 0.7
Autres Bacteroides a
9.5 ± 0.3 9.5 ± 0.3 9.6 ± 0.3 9.5 ± 0.3
Prevotella sp a
7.8 ± 2.8 8.0 ± 2.3 7.7 ± 2.3 8.0 ± 2.4
Fusobacterium sp a
8.1 ± 1.9 8.1 ± 2.0 8.1 ± 1.9 8.2 ± 1.9
Gram positive cocci a
7.5 ± 0.8 7.4 ± 0.9 7.4 ± 0.9 7.4 ± 0.9
Non-spore-forming Gram positive 8.1 ± 0.3 8.0 ± 0.3 8.1 ± 0.4 8.1 ± 0.4
bacillia
a
see the list of bacteria specials (Table I).
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Σελίδα 4
7.4 ± 0.9 7.4 ± 0.9 7.4 ± 0.9
Non-spore-forming Gram positive 8.1 ± 0.3 8.0 ± 0.3 8.1 ± 0.4 8.1 ± 0.4
bacillia
a
see the list of bacteria specials (Table I).
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Σελίδα 5
White Paper 6/2023: BACT-R
Table III. Biological enumeration of the species without and with jar opening during incubation, expressed in log10 UFC/mL
24 hours incubation 48 hours incubation
Strains Anaerobic BACT-R Anaerobic BACT-R
bacterial Cabinet Without Opening Opening Cabinet Without Opening Opening
opening 2 x 5 mina 2 x 30 mina opening 2 x 5 mina 2 x 30 mina
8.4 8.3 n/eb n/e 8.4 8.3 8.3 8.3
6.8 n/e n/e n/e 6.8 6.8 6.8 6.7
8.9 8.7 n/e n/e 8.9 8.7 8.7 8.8
8.2 8.2 8.1 n/e 8.2 8.2 8.2 8.1
6.5 6.1 n/e n/e 6.5 6.2 6.2 5.5
8.0 7.8 7.9 n/e 8.0 7.8 7.9 7.8
6.3 6.2 6.3 6.3 6.4 6.4 6.4 6.3
5.0 5.0 4.9 4.9 5.0 5.0 4.9 4.9
No culture present 5.4 5.5 5.6 5.6
a
1st opening at H +1 after starting incubation, 2nd opening at H +5 after starting incubation.
b
n/e : not enumerable, punctiform colonies.
Results and discussion
The comparison of the biological enumeration of the 51 recommended by the manufacturer yields results similar
species in the different anaerobic atmospheres did not to the gas traditionally used in anaerobic cabinets (richer
show any significant variation after 48 hours of incubation in hydrogen and CO2) (Table II). Only two strains showed
(Table II). Therefore it can be said that the BACT-R system greater growth by a half log in the presence of more
offers a similar growth to the other anaerobe generation hydrogen-rich gas (one strain of C. perfringens, and one
systems, be the continuous (anaerobic cabinet) or strain of C. sporogenesi.
discontinuous (GENbag) (average variation of 0.1 to 0.2 The discontinuous anaerobic condition generation
log10 UFC/mL). Only the C. difficile species cultivated a method requires the laboratories to leave jars open
little less well with the GENbag (6.9 ± 0.6 compared to in time to observe the isolation. In order to measure
7.2 or 7.1 ± 0.6 with the other techniques). the consequence of opening the jars on the anaerobic
The growth of species highly sensitive to oxygen, such bacteria growth, bacterial enumera-tion tests wer
Σελίδα 5
ime to observe the isolation. In order to measure
7.2 or 7.1 ± 0.6 with the other techniques). the consequence of opening the jars on the anaerobic
The growth of species highly sensitive to oxygen, such bacteria growth, bacterial enumera-tion tests were
as Fusobacterium sp or C. butyricum showed extremely performed after opening the jars in two occa-sions
similar results between the BACT-R system and the and during the incubation time. The opening time was
other anaerobic condition generation techniques. For the 5 minutes (minimum time) and 30 minutes.
species currently isolated in medical bacteriology, the gas
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Σελίδα 6
White Paper 6/2023: BACT-R
These tests were performed on species that are highly associated with the evacuation-replacement cycles that
sensitive to oxygen (2 species of F. necrophorum, allow, from when the atmosphere is created, to be in
1 species of F. nucleatum, 2 species of C. butyricum‘), control of the culture conditions. Finally, this system, with
on 3 species of C. difficile, micro-organism used regularly the same gas, allows establishment of a microaerophilic
for research in medical bacteriology laboratories, non- atmosphere with only three evacuation-replacement
sporing in the selective isolation environment used and cycles and a capnophile atmosphere with a blanket of
therefore more sensitive to the toxic action of oxygen and carbon dioxide.
on a slow-growth species Micromonas micros (Table III).
The strains of C. butyricum and M. micros have a similar
growth regardless of the jar opening. Only the species of Bibliography
Fusobacterium and C. difficile showed growth retardation
compared to the anaerobic cabinet or unopened jar. This (1) Delaney ML, Onderdonk AB. Evaluation of the AnaeroPack
growth retardation was important both due to the opening system for growth of clinically significant anaerobes. J Clin
of the jar and the period it was opened. Nevertheless, the Microbiol, 1997, 35, 558-562.
biological enumeration at 48 hours was identical with the (2) Van Horn KG, Warren K, Baccaglini EJ. Evaluation of the An-
exception of one species of C. difficile where the growth aeroPack system for growth of anaerobic bacteria. J Clin
was less than 0.7 log10 UFC/mL.
Microbiol, 1997, 35, 2170-2173.
(3) Shahin M, Jamal W, Verghese T, Rotimi VO. Comparative
Conclusion
evaluation of anoxomat and conventional anaerobic GasPak
This study showed that the BACT-R system allows creating
jar systems for the isolation of anaerobic bacteria. Med Prine
an anaerobic condition where the quality is identical to
Pract. 2003,12. 81-86.
the two atmospheres compared, the anaerobic cabinet
and the anaerobic condition generation bags.
This automated evacuation-replacement procedure,
much less expensive than an anaerobic cabinet, with
the advantage of being very easy to use. This system
remains, however, a discontinuous anaerobe generation
system, but the anaerobic condition is created faster than
when using bags (3).
The jar opening tests show that if the anaerobic
condition is quickly recreated, its effect on bacterial
growth is inconsequential, causing only a delay in growth.
Nonetheless, it must be kept in consideration that the
oxygen in the air is toxic for the anaerobic bacteria and
therefore if the jar may be opened on multiple occasions
for adding plates or observing the cultures, the opening
time must be kept as short as possible.
In the BACT-R system, there are integral controls
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Σελίδα 6
for the anaerobic bacteria and
therefore if the jar may be opened on multiple occasions
for adding plates or observing the cultures, the opening
time must be kept as short as possible.
In the BACT-R system, there are integral controls
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