Navigation Links
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 LidBac 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 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
LidBac
LidBac 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 LidBac. 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:

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 LidBac 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 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. 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 LidBac 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 LidBac 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 LidBac 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 LidBac tubes. Discussion 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 LidBac 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 LidBac 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 used. Summary 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 conditions.


'"/>

Source:


Page: All 1 2 3 4 5 6 7

Related biology technology :

1. A simple method to sequence from bacterial colonies using [a-33P] radiolabeled ddNTPs and Thermo Sequenase
Post Your Comments:
*Name:
*Comment:
*Email:
TAG: membrane lid tube for bacteria cultivation and freeze drying

(Date:4/28/2015)... PHOENIX , and ANN ARBOR, Mich. ... a small cohort published OncoTargets and Therapy ... performed better than Foundation Medicine,s FoundationOne test across a ... and clinically actionable biomarker targets. The purpose ... two commercially available NGS platforms for profiling cancer patients ...
(Date:4/28/2015)... The Pistoia Alliance, a global, not-for-profit alliance of ... to innovation in R&D, has announced the start of ... Mapping project has been set up to create better ... life sciences R&D. The project will see the Pistoia ... services to enable the universal application of ontologies. , ...
(Date:4/28/2015)... Washington, PA (PRWEB) April 28, 2015 ... will develop psychosis at some point over the course ... spectrum of symptoms that worsen as the disease progresses. ... broadcast network, announces the launch of a series dedicated ... medical experts in PD, movement disorders, and psychiatric conditions. ...
(Date:4/28/2015)... According to a 2013 report from ... medical treatment from burn injuries per year. Despite several ... formation of scars with subsequent contractures remains a major ... there is no adequate treatment. Contractures are a permanent ... can cause permanent deformity. , Synedgen has developed ...
Breaking Biology Technology:Paradigm Cancer Diagnostic Test (PCDx) Performed Better Than FoundationOne In Side by Side Clinical Study 2Paradigm Cancer Diagnostic Test (PCDx) Performed Better Than FoundationOne In Side by Side Clinical Study 3Pistoia Alliance Announces Start of Ontologies Mapping Project 2ReachMD Launches Series Focusing on Parkinson’s Disease Psychosis 2Synedgen Burn Treatment Results Presented at Premier Wound Conference 2Synedgen Burn Treatment Results Presented at Premier Wound Conference 3
... , LEXINGTON, Ky. , ... based in Lexington, KY announced today that its ... created and characterized the world,s first rat model of severe ... complete lack of the major components of the immune system. ...
... 6 Omnio Healer AB, a pioneer company,developing novel ... has entered into a partnership agreement with Eden,Biodesign. Under ... production services relating to Omnio Healer,s lead,recombinant protein candidate. ... therapeutic concept for wound healing and,anti-infection, Omnio Healer is ...
... WALTHAM, Mass., Aug. 6 Repligen Corporation (Nasdaq: ... 2010, ended June 30, 2009. Total revenue for the quarter ... quarter of fiscal year 2009. The prior year results were ... Company for royalties on the U.S. sales of Orencia(R) prior to ...
Cached Biology Technology:Transposagen Creates a Rat Model of the Human Immune System 2Transposagen Creates a Rat Model of the Human Immune System 3Omnio Healer Selects Eden Biodesign for cGMP Production of its Candidate Wound Healing and Anti-Infection Treatments 2Omnio Healer Selects Eden Biodesign for cGMP Production of its Candidate Wound Healing and Anti-Infection Treatments 3Repligen Reports First Quarter Fiscal Year 2010 Financial Results 2Repligen Reports First Quarter Fiscal Year 2010 Financial Results 3Repligen Reports First Quarter Fiscal Year 2010 Financial Results 4Repligen Reports First Quarter Fiscal Year 2010 Financial Results 5Repligen Reports First Quarter Fiscal Year 2010 Financial Results 6Repligen Reports First Quarter Fiscal Year 2010 Financial Results 7
(Date:4/13/2015)... 2015 According to a ... Market Forecast & Opportunities, 2020", the global biometrics market ... 14% till 2020. The biometrics market is garnering ... and review of biometric management systems. The constant ... with greater efficiency, are resulting in increasing installation ...
(Date:4/6/2015)... 2015 NXT-ID, Inc. (NASDAQ: NXTD ) ... focused on the growing mobile commerce market, announced ... for MINIATURE, MULTI-PURPOSE ANTENNA METHOD AND SYSTEM ... NXT-ID introduces a new revolutionary method and system ... use of miniature antenna modules for multiple purposes ...
(Date:4/1/2015)... Apr. 1, 2015  NXT-ID, Inc. (NASDAQ: NXTD ... focused on the growing mobile commerce market, announces the ... wallets is underway to early access pre-order customers. ... usage at retail outlets including Walmart, Target, AT&T, Dunkin, ... Wocket was accepted at all outlets and very easy ...
Breaking Biology News(10 mins):Global Biometrics Market to Cross US$ 21 Billion by 2020: TechSci Research 2NXT-ID Patents Miniature, Low Power Wireless Magnetic Stripe 2NXT-ID Patents Miniature, Low Power Wireless Magnetic Stripe 3NXT-ID Patents Miniature, Low Power Wireless Magnetic Stripe 4NXT-ID Reports Second Shipment of Wocket Smart Wallets Underway to Early Access Pre-order Customers and Provides User Feedback 2NXT-ID Reports Second Shipment of Wocket Smart Wallets Underway to Early Access Pre-order Customers and Provides User Feedback 3NXT-ID Reports Second Shipment of Wocket Smart Wallets Underway to Early Access Pre-order Customers and Provides User Feedback 4
... from the University of California at San Francisco (UCSF) ... dedicated to the brain that the controversy is over: ... dead. , The cerebral cortex is a sheet of ... that process particular aspects of sensation, movement and cognition. ...
... of genes has shed light on the role of gene ... in Proceedings of the National Academy of Sciences USA, was ... , Dr Robert Beiko, Professor Mark Ragan and Mr ... in an effort to map how genes are shared between ...
... used by the U.S. military to track vehicle convoys in ... little-known world of pygmy elephants in Borneo. , ... elephant's being captured and outfitted with a collar that can ... the first time, the public can track the movements of ...
Cached Biology News:MIT researcher presents new view of how the cortex forms 2Lateral thinking produces first map of gene transmission 2WWF peeks into mysterious life of Borneo's pygmy elephants 2
Human Ig Lambda-chain, clone ICO-16, Monoclonal Antibody...
Rabbit polyclonal to Warfarin ( Abpromise for all tested applications). Antigen: Chemical / Small Molecule conjugated to KLH...
WNV Core Antibody...
... Rabbit polyclonal to FANCM ( Abpromise ... Antigen: Synthetic peptide derived from ... Human FANCM. (Note: the amino acid ... available as ab27787 .) ...
Biology Products: