( ...)A membrane-lid tube for bacteria cultivation and freeze-drying

Wilhelm Bockelmann, Institut fr Mikrobiologie, Bundesanstalt fr Milchforschung; Kiel, Germany
Susanne Wagner, Eppendorf AG Introduction 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 Lid_Bac from Eppendorf 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. Target The experiments described in this article demonstrate the suitability of the membrane-lid tube Lid_Bac for bacteria cultivation on a miniprep scale (1 to 2 ml). A comparison is made between the growth behavior of bacteria in Lid_Bac 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 Lid_Bac with those produced in a standard tube. Material and methods
Lid_Bac Lid_Bac 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. Bacteria cultivation Growth kinetics:
Four parallel experiments were carried out in order to investigate the growth behavior of Escherichia coli HB101 and Bacillus subtilis 168 when cultivated in Lid_Bac. 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 Lid_Bac)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:

Evaporation experiments were carried out with water since no significant differences were expected from experiments with culture medium containing growth.

1 ml water was transferred into each 1.5 ml Lid_Bac tube. The 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. Cell morphology:

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 Lid_Bac tube and 5 ml of the medium was placed into each 50 ml Erlenmeyer flask. The vessels were then sealed (Lid_Bac 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. Freeze-drying 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 Lid_Bac and standard 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 Lid_Bac tubes were 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 Growth kinetics:
The time required for the cultivation of E. coli HB101 / B. subtilis 168 in Lid_Bac 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. Evaporation:
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 not shown).

Cell morphology:
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 Lid_Bac tubes. Discussion The experiments mentioned here were carried out to test the suitability of the membrane-lid tube Lid_Bac both for bacteria cultivation as well as for freeze-drying.

The results show virtually identical growth behavior of the bacteria cultivated in Lid_Bac 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 Lid_Bac 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 Lid_Bac 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 Lid_Bac is used. Summary Lid_Bac 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 conditions.


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