Name: formulations for freeze-drying of bacteria and their influence on cell survival
Uploaded: 2013-08-03T14:00:00
Description: Watch this Scientific Journal Video about Formulations for Freeze-drying of Bacteria and Their Influence on Cell Survival at JoVE.com

Arbeidet ble støttet av den svenske Stiftelsen for strategisk miljøforskning (MISTRA) gjennom DOM programmet og Grant 211684 (BACSIN) fra EU Fellesskapet FP7 rammeprogram. Vi takker J. Engstrand for å få hjelp i å filme X-ray analyse og L. Tang for å hjelpe med det konseptuelle fortelling.

En. Dyrking og høsting av P. putida <ol><li> Forbered en forrett kultur Pseudomonas putida ved inoculating 100 ml tryptisk kjøttkraft (TSB) med en koloni av P. putida celler dyrket på agar supplert med tryptisk soyavekstmedium (TSA). Hold kultur ved 30 ° C på en ristende bord satt til 130 rpm. </li><li> Etter 7 timer overføre en alikvot fra den starterkultur ekvivalent til 1/10 av det endelige volum av hoved kultur til frisk TSB-medium. Hold cellene ved 30 ° C på en ryster ved 130 op...

<ol>
	<li>Wessman, P., Mahlin, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impact+of+matrix+properties+on+the+survival+of+freeze-dried+bacteria.">Impact of matrix properties on the survival of freeze-dried bacteria.</a> J. Sci. Food Agric. 91 (14), 2518-2528 (2011).</li><li>Ablett, S., Izzard, M. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+Scanning+Calorimetric+Study+of+Frozen+Sucrose+and+Glycerol+Solutions.">Differential Scanning Calorimetric Study of Frozen Sucrose and Glycerol Solutions.</a> Journal of the Chemical Society-Faraday Transactions. 88 (6), 789-794 (1992).</li><li>Knopp, S. A., Chongprasert, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+relationship+between+type+TMDSC+curve+of+frozen+sucrose+solutions+and+collapse+during+freeze-drying.">The relationship between type TMDSC curve of frozen sucrose solutions and collapse during freeze-drying.</a> Journal of Thermal Analysis and Calorimetry. 54 (2), 659-672 (1998).</li><li>Crowe, J. H., Leslie, S. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Is+Vitrification+Sufficient+to+Preserve+Liposomes+during+Freeze-Drying.">Is Vitrification Sufficient to Preserve Liposomes during Freeze-Drying.</a> Cryobiology. 31 (4), 355-366 (1994).</li><li>Jain, P., Sen, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+glass-forming+biopreservatives+on+head+group+rotational+dynamics+in+freeze-dried+phospholipid+bilayers:+A+P-31+NMR+study.">Effect of glass-forming biopreservatives on head group rotational dynamics in freeze-dried phospholipid bilayers: A P-31 NMR study.</a> Journal of Chemical Physics. 131 (2), (2009).</li><li>Stock, J. B., Rauch, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Periplasmic+Space+in+Salmonella-Typhimurium+and+Escherichia-Coli.">Periplasmic Space in Salmonella-Typhimurium and Escherichia-Coli.</a> Journal of Biological Chemistry. 252 (21), 7850-7861 (1977).</li><li>Crowe, J. H., Hoekstra, F. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anhydrobiosis.">Anhydrobiosis.</a> Annual Review of Physiology. 54, 579-599 (1992).</li><li>Leslie, S. B., Israeli, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Trehalose+and+Sucrose+Protect+Both+Membranes+and+Proteins+in+Intact+Bacteria+during+Drying.">Trehalose and Sucrose Protect Both Membranes and Proteins in Intact Bacteria during Drying.</a> Applied and Environmental Microbiology. 61 (10), 3592-3597 (1995).</li><li>Nesarikar, V. V., Nassar, M. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+cations+and+anions+on+glass+transition+temperatures+in+excipient+solutions.">Effect of cations and anions on glass transition temperatures in excipient solutions.</a> Pharmaceutical Development and Technology. 12 (3), 259-264 (2007).</li><li>You, Y., Ludescher, R. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effect+of+sodium+chloride+on+molecular+mobility+in+amorphous+sucrose+detected+by+phosphorescence+from+the+triplet+probe+erythrosin+B.">The effect of sodium chloride on molecular mobility in amorphous sucrose detected by phosphorescence from the triplet probe erythrosin B.</a> Carbohydr. Res. 343 (2), 350-363 (2008).</li><li>Crowe, J. H., Hoekstra, F. A., Nguyen, K. H. N., Crowe, L. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Is+vitrification+involved+in+depression+of+the+phase+transition+temperature+in+dry+phospholipids.">Is vitrification involved in depression of the phase transition temperature in dry phospholipids.</a> Biochim. Biophys. Acta Biomembr. 1280, 187-196 (1996).</li></ol>

Motivet for denne studien var å undersøke noen formulering egenskaper som kan ha betydning for celle overlevelse under frysetørking. Selv om den iboende tørking toleranse varierer mellom forskjellige arter, som illustrert i figur 1A, utviklingen av hvor godt de forskjellige formuleringer støtte celleoverlevelse er lik. Det er informativ å starte med et forhold mellom sukrose og Ficoll. Det antas at en viktig faktor for en formulering for å støtte celleoverlevelse er evnen til formuleringen ingre...

<table border="1">
	<tbody>
		<tr>
			<td>Name of the reagent</td>
			<td>Company</td>
			<td>Catalogue number</td>
			<td>Comments (optional)</td>
		</tr>
		<tr>
			<td>Ficoll PM 400</td>
			<td>GE-healthcare</td>
			<td>17-0300-10</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>HEC (Natrosol-M Pharm Grade)</td>
			<td>Ashland</td>
			<td>&nbsp;</td>
			<td>Gift from Ashland</td>
		</tr>
		<tr>
			<td>HPMC (Methocel F4M)</td>
			<td>Dow</td>
			<td>&nbsp;</td>
			<td>Gift from the Department of Pharmacy, Uppsala University</td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>Sigma-Aldrich</td>
			<td>S2395</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Sigma-Aldrich</td>
			<td>71376</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Tryptic Soy broth</td>
			<td>Merck</td>
			<td>105459</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Tryptic Soy Agar</td>
			<td>Merck</td>
			<td>105458</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Lyostar II</td>
			<td>FTS Kinetics</td>
			<td>N.A.</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Pyris Diamond DSC</td>
			<td>Perkin-Elmer</td>
			<td>N.A.</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Bruker AXS SMART CCD 1k Diffractometer</td>
			<td>Bruker</td>
			<td>N.A</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Dual-beam FEI Strata DB235 FIB/SEM</td>
			<td>FEI</td>
			<td>N.A</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Kr&uuml;ss Educational Tensiometer</td>
			<td>Kr&uuml;ss</td>
			<td>N.A.</td>
			<td>&nbsp;</td>
		</tr>
	</tbody>
</table>

formulations for freeze-drying of bacteria and their influence on cell survival

Cellular vann kan fjernes reversibelt å inaktivere mikroorganismer for å lette lagring. En slik fremgangsmåte brukes er frysetørking, som er ansett som en mild metode for dehydrering. For å legge til rette for celle overlevelse under tørking, blir cellene ofte formulert på forhånd. Formuleringen danner en matriks som bygger inn i cellene og beskytter dem fra forskjellige skadelige påkjenninger på cellene i løpet av frysing og tørking. Her presenteres en generell metode for å vurdere overlevelse av celler etter frysetørking og illustrere er det funnet ved å sammenligne resultatene oppnådd med fire forskjellige formuleringer: disakkaridet sakkarose, sukrose avledet polymer Ficoll PM400, og de respektive polysakkarider hydroksyetylcellulose (HEC ) og hydroksypropylmetylcellulose (HPMC), på to bakteriestammer, P. putida KT2440 og A. chlorophenolicus A6. I dette arbeidet illustrere hvordan vi skal forberede formuleringer for frysetørking og hvordan å undersøke mechanismer av celleoverlevelse etter rehydratisering ved karakteriserer formulering ved hjelp av differensial scanning kalorimetri (DSC), overflatespenning målinger, røntgen-analyse, og elektronmikroskopi og å relatere disse dataene til overlevelse. Polymerene som ble valgt for å få et monomert struktur av de respektive polysakkarid likner sukrose i varierende grader. Ved hjelp av denne metode oppsett vi viste at polymerene kan støtte celleoverlevelse så effektivt som disakkarider dersom visse fysikalske egenskaper av formuleringen blir styrt en.

Tabell 1 viser data for formulering sammensetning, termiske registrerte hendelser ved DSC ved oppvarming av frosne formuleringer, strukturen av de tørre prøver og overflatespenningen til formuleringen løsningene. Den T g &#39;av sukrose er bestemt til -40 ° C 2, 3 og kan være vanskelig å oppdage for sukrose konsentrasjoner under 20% vekt / vekt. Den termiske arrangement på -35 ° C har trolig sammenheng med utbruddet av is oppløsning to. Den krystallinsk...

Watch this Scientific Journal Video about Formulations for Freeze-drying of Bacteria and Their Influence on Cell Survival at JoVE.com

Formulations for Freeze-drying of Bacteria and Their Influence on Cell Survival

Frysetørking er ofte en enkel og praktisk måte å få tørre produkter av levedyktige bakterieceller. Et problem i prosessen er celle overlevelse. Vi detalj her en fremgangsmåte for å undersøke hvordan celleoverlevelse under frysetørking er påvirket av egenskapene til formuleringen benyttes.

Formuleringer for frysetørking av bakterier og deres innflytelse på Cell Survival

Cellular water can be removed to reversibly inactivate microorganisms to facilitate storage. One such method of removal is freeze-drying, which is ...

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