Wilhelm Bockelmann, Institut fr Mikrobiologie, Bundesanstalt fr
Milchforschung; Kiel, Germany
Susanne Wagner, Eppendorf AG
Miniaturization plays an ever increasing role in everyday lab routines.
Ongoing developments of tubes, pipettes, and pipette tips enable drastic
reductions in the amount of material required. More economical use of reagents,
the opportunity of using smaller sample quantities, and the elimination
of bottlenecks thanks to reduced space requirements, save time and money
and thus form the ideal prerequisites for increasing sample throughput.
The membrane-lid tube LidBac
is a significant contributory factor in the miniaturization process to the
microbiological laboratory. For the first time ever, it is now possible
to cultivate miniprep bacteria cultures in exactly the format required for
the preparation. Following cultivation, the bacteria no longer have to be
pipetted into a new tube since the culture is grown in the tube of the subsequent
preparation. The automated production process enables the membrane-lid tube
to be used directly, without need for prior sterilization. The cleaning
process before and after cultivation, as required when conventional glass
tubes are used, is no longer necessary.
The experiments described in this article demonstrate the suitability
of the membrane-lid tube LidBac
for bacteria cultivation on a
miniprep scale (1 to 2 ml). A comparison is made between the growth behavior
of bacteria in LidBac
and in conventional Erlenmeyer flasks.
The membrane-lid tube is suitable for virtually all stages of the experiment
whenever ventilation, drying, or a similar step is necessary. This is underlined
by a comparison of the results obtained by freeze-drying in LidBac
with those produced in a standard tube.
Material and methods
consists of a polypropylene (PP) membrane-lid with an
integrated PP hydrophobic membrane and a Safe-Lock microcentrifuge tube.
The membrane is gas-permeable, hydrophobic,and impermeable to bacteria (0.2
m pore size). The membrane lid is compatible with all 1.5 ml and
2 ml Eppendorf Tubes. After the tube has been filled, the membrane-lid
is simply pressed into the tube. As there is no hinged connection to the
tube, the lid can be removed easily at any time to enable the tube to be
used for follow-up applications.
Four parallel experiments were carried out in order to investigate the growth
behavior of Escherichia coli HB101 and Bacillus subtilis 168 when cultivated
. A positive control was carried out in parallel in
the form of bacteria cultivation in Erlenmeyer flasks. To ensure an identical
initial bacteria concentration in the different test tubes, LB (Luria Bertani)
medium was given a 1% inoculation of overnight culture. After being mixed
thoroughly, 1 ml bacteria culture each was transferred to 1.5 ml membrane-lid
tubes (Eppendorf LidBac
)and 5 ml each was transferred to a 50
ml Erlenmeyer flask with cellulose stopper.
The membrane-lid tubes were then incubated in the Eppendorf Thermomixer
R at 1,400 rpm and 37C. The Erlenmeyer flasks were sealed with cellulose
stoppers and incubated in a linear oscillating water bath (Kttermann,
approx. 200 strokes/min, at 37 C). The growth kinetics of the bacteria
were determined by means of absorption measurements at 620 nm.
Evaporation experiments were carried out with water since no significant
differences were expected from experiments with culture medium containing
1 ml water was transferred into each 1.5 ml LidBac
tubes were sealed with their membrane-lid and then incubated in the Thermomixer
R at 37 C and 1,400 rpm (room temperature 22-23 C, relative humidity
45-50 %). As a positive control, 5 ml water was transferred into each 50
ml Erlenmeyer flask, which was then sealed with a cellulose stopper and
incubated in a linear oscillating water bath at 37 C.
The weight of the tubes was determined in five parallel measurements,
carried out after 3, 6, 9,18, and 24 hours.
Since the cell morphology of bacteria often changes when insufficient amounts
of oxygen are supplied for example, under sub-optimal air conditions,
long, thin nutrient-deprived phenotypes are formed in bacteria that normally
grow in short, rodlike shapes an analysis of cell morphology of cultivated
bacteria enables the tubes to be assessed in regard to their suitability
for bacteria cultivation.
Following overnight cultivation, the bacteria strains E. coli HB101 (facultatively
anaerobic) and B. subtilis 168 (strictly aerobic) were inoculated at a concentration
of 1% into the LB medium. 1 ml of this medium was then transferred into
each 1.5 ml LidBac
tube and 5 ml of the medium was placed into
each 50 ml Erlenmeyer flask. The vessels were then sealed (LidBac
with its membrane-lid, the Erlenmeyer flasks with cellulose stoppers) and
incubated under the same conditions as for bacteria cultivation. After 3,
6,16, and 24 hours, samples were removed from both vessel types and examined
under a microscope. To prevent any effect on the morphology of the bacteria
caused by the increase in the oxygen supply as a result of the tubes being
opened, a new tube was used for each measurement.
In five parallel preparations, a comparison was made between freeze-drying
(Beta 2-16, Christ, Osterode, Germany) E. coli HB101 in membrane-lid tubes
and in standard tubes.
The medium used for lyophilization was 5 % lactose (in distilled water).
1 ml of each bacteria culture, grown in both LidBac
tubes, was pelleted in the early stationary phase (in the Eppendorf Centrifuge
5402 at 4 C and 14,000 x g), resuspended in 1 ml of 5 % lactose, centrifuged,
and then diluted in 500 l /200 l medium.The LidBac
then sealed with their membrane-lid and placed into a custom-made aluminum
block with the corresponding bores. The standard tubes were transferred,
unsealed, to the same block. For a eutectic point of the cultures in 5 %
lactose of approx. 13 C, a vacuum of 1.03 mbar was set. To dry the samples,
the heating temperature of the sample area was raised gradually from 5C
to +5 C over a period of 12 hours. The sample temperature was monitored
continuously using a Pt100 sensor. After 4, 8,12, and 24 hours, the samples
were removed from the drying process and the residual volume in the tubes
was determined. The drying process had to be interrupted briefly each time
the samples were removed.
Results of bacteria cultivation
The time required for the cultivation of E. coli HB101 / B. subtilis 168
corresponds to the usual times for 5 ml cultures in
Erlenmeyer flasks (see Figs. 1 and 2). Undelayed growth was possible with
both tube types and bacteria species up to an absorption of 1.8.
Sealed Safe-Lock microcentrifuge tubes (1.5 ml filled with 1ml culture)
were selected as a negative control. The cell concentrations obtained in
these tubes were much lower.
The assumption that evaporation phenomena are independent of the medium
used (LB or water) was confirmed by the results of the growth of E. coli
HB101 (results not shown).
Whereas there was virtually no evaporation with Erlenmeyer flasks that had
been sealed with cellulose stoppers, the membrane-lid tubes displayed negligible
evaporation values (< 7 % in 6 hours; approx. 20 % in 16 hours) (results
During both cultivation processes, no differences in cell morphology were
noted with E. coli HB101 or with B. subtilis 168.
Results of freeze-drying
The speed at which solutions are freezedried in membrane-lid tubes corresponds
to the requirements of standard protocols. It is possible to freeze-dry
500 l medium completely within 12 -13 hours and 200 l within 8 hours (see
Fig. 3). Drying in the open standard tubes was quicker than in LidBac
The experiments mentioned here were carried out to test the suitability
of the membrane-lid tube LidBac
both for bacteria cultivation
as well as for freeze-drying.
The results show virtually identical growth behavior of the bacteria cultivated
in comparison to conventional cultivation methods in
Erlenmeyer flasks. The same bacterial densities were achieved within the
usual times. The slight concentration of the culture during incubation,
caused by the vapor permeability of the membrane, had no adverse effect
on the growth behavior of the bacteria.
The experiments showed that LidBac
is also suitable for lyophilization.
Although freeze-drying in the membrane-lid tube is slower than in open tubes,
it still meets the requirements of standard protocols. It is possible to
dry biological samples within one working day or overnight. The main advantage
is the sterile condition achieved by the bacteria-proof membranelid when
is used. Conversely, when open tubes are used, the chemical
sterilization of the drying chamber of the freeze-dryer requires a lot of
time and effort. For this reason, sterile conditions are rarely achieved
in laboratory freezedrying units, and are not necessary when LidBac
is a quick and easy way to eliminate several work steps, reduce the quantities of
media required, save space in the lab, and
simplify drying processes under sterile
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