The work was supported by The Swedish Foundation for Strategic Environmental Research (MISTRA) through the DOM program and Grant 211684 (BACSIN) from the European Union Community FP7 Framework program. We thank J. Engstrand for assistance in filming the X-ray analysis and L. Tang for helping with the conceptual narrative.

1. Cultivation and harvest of P. putida
<ol>
	<li>Prepare a starter culture of Pseudomonas putida by inoculating 100 ml of tryptic soy broth (TSB) with a colony of P. putida cells cultured on agar supplemented with tryptic soy broth (TSA). Keep the culture at 30 &deg;C on a shaking board set at 130 rpm.</li>
	<li>After 7 hr transfer an aliquot from the starter culture equivalent to 1/10 of the final volume of the main culture to fresh TSB-medium. Keep the cells at 30 &deg;C on a shaking board at 130 rpm for 16 hr.</li>
	<li>After 16 hr of growth the cells are harvested by spinning the cell culture at 1,500 x g for 20 min at RT. Decant the supernatant and wash the cells by re-suspending the cell pellet is in a NaCl-solution that is isotonic to the growth medium. In this case a 150 mM NaCl solution was used. The cells are then centrifuged once more and the supernatant is decanted before resuspending the cells in the formulation medium, see step 3.2.</li>
</ol>
2. Cultivation of other species
<ol>
	<li>To adapt the protocol to other bacterial species the cultivation and harvest procedures should be changed according to the requirements of that species. To avoid osmotic shock when handling the cells during harvest and the washing step(s), the osmolality of the solutions needs to match that of the growth medium at harvest.</li>
</ol>
3. Formulation of cells
<ol>
	<li>Prepare the formulation solutions by weighing out the respective matrix components and dissolve them in water. To achieve isotonic conditions, either adjust the amount of excipient or add NaCl or some other cell compatible solute to reach the desired osmolality. For substances with low molecular weight such as disaccharides the amounts can easily be adjusted to reach isotonic conditions. For substances like polymers that are of high molecular weight, the tonicity has to be adjusted with cell compatible solutes, e.g. NaCl or disaccharides. Note that any addition of a substance will influence the physical properties of the formulation, which in turn may influence freeze-drying behavior and cell survival.</li>
	<li>Decant the washing solution (in this case NaCl) and re-suspend the cells in the respective formulation media.</li>
	<li>Make sure that the cells are homogeneously dispersed. Formulations of low viscosity are vortexed whereas formulations of higher viscosities are mixed using by adding, e.g., a stir bar and shaking the container until homogeneous mixing is achieved.</li>
	<li>Divide the formulations into freeze-dryer vials. The vials should be weighed empty, with sample before and after freeze-drying to calculate the amount of water removed during freeze-drying.</li>
	<li>Add rubber stoppers to the vials if the vials are to be sealed inside the freeze-dryer, see step 4.3.</li>
	<li>Enumerate the cells in each formulation before freeze-drying, see step 6.</li>
</ol>
4. Freeze-drying
<ol>
	<li>The freeze-drying conditions have to be adjusted to the physical properties of the formulation. The most important parameter is in this case the glass transition temperature, Tg, of the formulation, denoted Tg' for the freeze-concentrated sample to indicate that water is still present. The Tg' of the freeze-concentrated formulation is easily measured using differential scanning calorimetry (DSC), see step 7.</li>
	<li>Adjust the parameters of the freeze-drying process so that the temperature of the sample is always below the Tg of the sample and that the chamber pressure allows for a fast sublimation of ice, i.e. primary drying, and residual water in the formulation, i.e. secondary drying. If the sample temperature is above Tg' during the drying process there is a great risk of structural collapse of the sample which may significantly decrease the cellular survival.</li>
	<li>To protect the dry products after freeze-drying the sample atmosphere has to be controlled. If possible, seal the samples in the freeze-dryer before the vacuum is released at the end of the freeze-drying cycle. Alternatively the vials can be filled with a preferred atmosphere. It is important to avoid exposing the samples to the ambient conditions as any moisture or oxygen present in the storage atmosphere will influence the survival of the cells.</li>
	<li>Weigh the vials.</li>
</ol>
5. Rehydration
<ol>
	<li>Rehydrate the samples with deionized and sterile water. The amount of water to be added should be that same as the amount removed during freeze-drying and is calculated from the weight of the empty vial, the same vial with sample before and after freeze-drying. Vortex the samples now and again then until the solutions appear homogenous.</li>
</ol>
6. Enumeration
<ol>
	<li>Draw 100 &mu;l from each sample and make 10-fold serial dilutions. From the dilutions of interest 100 &mu;l is plated on TSA-plates. The plates are incubated at the temperature and time required.</li>
</ol>
7. Characterization of Formulation of Freezing Behavior
<ol>
	<li>Take a small amount, 5 - 10 mg, of the sample and enclose it in a DSC-aluminium pan with lid. Prepare an empty pan as a reference.</li>
	<li>Set the DSC temperature scan program. The cooling program is set to mimic the freezing step in the freeze-drying program. This is done as the cooling kinetics can influence the Tg' of the hydrated formulations. Before the heating scan begins bring down the temperature to well below the expected Tg' of the investigated formulations, normally -100 &deg;C.</li>
	<li>Choose an appropriate heating rate, normally a heating rate of 5 - 40 &deg;C/min is used. The recorded heat flow signal is expressed in watts and will be influenced by the heating rate. A higher heating rate will give a larger signal of Tg'.</li>
	<li>Set the temperature range so that it covers both Tg' and the melting of the formulation.</li>
</ol>
8. Surface Tension Measurements of the Hydrated Formulations
<ol>
	<li>The experimental setup used in this experiment is according to the du No&uuml;y method to measure surface.</li>
	<li>Clean the platinum ring and the measuring vessel carefully. The vessel is rinsed with acetone, wiped with a lint-free tissue, and then burned on the inside with a colorless flame. The ring glows red in a colorless flame.</li>
	<li>Fill the vessel with the solution and let the surface settle before measuring. For solutions where the equilibrium state is reached only after a long time, e.g. polymers, the simple setup used here will provide qualitative data.</li>
	<li>For formulations with high viscosities, e.g. solutions of 2% (w/w) HEC or HPMC, the surface tension has to be measured on solutions of lower concentrations and then extrapolated. This can be done by taking advantage of the fact that the drop in surface tension levels off at concentrations below 0.5% (w/w).</li>
</ol>
9. X-ray Analysis of the Dry Formulations
<ol>
	<li>Take a small amount of matrix/bacteria and load it onto a sample holder.</li>
	<li>Mount the sample holder into the X-ray-diffractometer.</li>
	<li>To analyze any crystalline phase present in the sample the EVA program package from Bruker can be used.</li>
</ol>
10. Electron Microscopy
<ol>
	<li>Take a small amount of matrix/bacteria out of the vials and fix it on a SEM stub.</li>
	<li>Load the SEM holder in the sputter-coater (Thermo VGScientific Polaron SC7640 used in the present work) and coat the sample with a few nm of Au/Pd or Pt. In these experiments the sputtering parameters were: 1,900 V, 20 mA, and 20 sec, for a ~ 5 - 10 nm Au/Pd coating. The coating reduces electric charging during image acquisition. It is recommended to start with a thin coating and, if charging persists and causes image artifacts, the sample should be coated again.</li>
	<li>Load the SEM holder in the SEM chamber. Select appropriate imaging parameters. For the instrument used in Figure 2 (FEI Strata DB235) we used 5 kV accelerating voltage, spotsize 3, a working distance of 5 mm and the secondary electron detector.</li>
	<li>Optionally, it is possible to use the Focused Ion Beam to cut cross-sections of the bacteria embedded in the polymer to reveal further details.</li>
</ol>

<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>

No conflicts of interest declared.

The motive for this study was to investigate some formulation properties that may be of importance for cell survival during freeze-drying. Although the intrinsic drying tolerance varies between different species, as illustrated in Figure 1A, the trend on how well the different formulations support cell survival is similar. It is informative to start with a comparison of sucrose and Ficoll. It is believed that a key factor for a formulation to support cell survival is the capability of the formulation ingredient(s) to replace water during dehydration, thus maintaining the structure of the proteins and membranes of the cell also in the dry state. Disaccharides such as sucrose but also trehalose show this property, whereas polymers such as the polysaccharide starch do not5,11. Polymers are considered too large to interact with the membrane lipids in the same fashion as disaccharides. Another property important for a successful freeze-drying of microorganisms is the ability of the supporting matrix to vitrify (i.e. become amorphous solids) during freeze-drying. The amorphous structure is beneficial for shielding and keeping cells separated. Interestingly, both disaccharides and polymers readily vitrify if the freeze-drying is properly performed. Our results show that both the sucrose and Ficoll based formulations become amorphous after freeze-drying, see Table 1, and support cell survival equally well. However, there are significant differences between the two saccharides. In contrast to sucrose, the Ficoll molecule is, with a molecular weight of 400 kDa, too large to replace water in the interactions with lipid membranes during freeze-drying 4, 5. Furthermore, as the Ficoll molecules are barred from the periplasmic space of Gram negative bacteria, again owing to their size6, cellular uptake of Ficoll is unlikely. Based on this, we suggest that the protective effect provided by the sucrose formulation is not primarily due to cellular uptake of the disaccharide, important for water replacing capacity of intracellular structures 7, 8. Rather, the capability of Ficoll to support cell survival has to do with properties common to both the Ficoll and sucrose formulation, e.g. the amorphous structure.
Focusing on the polymers, it is evident from Figure 1A that the capability of the polymer formulations to support cell survival varies. The X-ray analysis showed that NaCl crystallized during freeze-drying and coexisted with the otherwise amorphous structure of the HEC and HPMC formulations. No crystalline material was detected in the Ficoll based formulation. NaCl can form complexes with the hydroxyl moieties in carbohydrates 9, 10. In Ficoll and HEC-based formulations the NaCl to hydroxyl moiety molar ratio was 1:11 and 1:1, respectively, explaining why no crystallization was detected in the Ficoll-based formulation. The phase separation between NaCl and the cellulose polymers was also observed in the DSC-investigations. A eutectic melting was recorded during heating of the frozen HEC and HPMC-formulations indicating that the phase separation takes place in the frozen state. The morphology and microstructure of the formulation is revealed by SEM imaging (Figure 2) and corroborates the DSC and X-ray data. Bacterial cells are seen protruding from the sheets with deposited NaCl crystals clearly visible on the surface of the HEC-sheets. Based on the similarity between the survival rates for bacteria formulated in the HEC-solution and Ficoll solutions as compared to HPMC we conclude that the state of the salt, whether dispersed as crystals or dissolved in the polymer, does not correlate with cell survival. Instead cell survival shows a relationship with the surface tension of the formulations, see Figure 1B. For lower surface tensions, lower survival rates are recorded for the bacteria. Note however that the measured surface tension displays the interaction between the solutes, i.e. sucrose and the polymers, and the solvent molecule. Still it allows for an indirect speculation that surface activity of polymers can be related to their tendency to interact also with the cell surfaces in a destabilizing manner. As the concentration of the solutes increases during freezing, the negative effects may be potentiated explaining why the negative effect of the surface active polymers were not seen when only storing the bacteria in the fully hydrating formulations.

<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 water can be removed to reversibly inactivate microorganisms to facilitate storage. One such method of removal is freeze-drying, which is considered a gentle dehydration method. To facilitate cell survival during drying, the cells are often formulated beforehand. The formulation forms a matrix that embeds the cells and protects them from various harmful stresses imposed on the cells during freezing and drying. We present here a general method to evaluate the survival rate of cells after freeze-drying and we illustrate it by comparing the results obtained with four different formulations: the disaccharide sucrose, the sucrose derived polymer Ficoll PM400, and the respective polysaccharides hydroxyethyl cellulose (HEC) and hydroxypropyl methyl cellulose (HPMC), on two strains of bacteria, P. putida KT2440 and A. chlorophenolicus A6. In this work we illustrate how to prepare formulations for freeze-drying and how to investigate the mechanisms of cell survival after rehydration by characterizing the formulation using of differential scanning calorimetry (DSC), surface tension measurements, X-ray analysis, and electron microscopy and relating those data to survival rates. The polymers were chosen to get a monomeric structure of the respective polysaccharide resembling sucrose to a varying degrees. Using this method setup we showed that polymers can support cell survival as effectively as disaccharides if certain physical properties of the formulation are controlled1.

Table 1 displays data on formulation composition, thermal events recorded by DSC during heating of the frozen formulations, structure of the dry samples and the surface tension of the formulation solutions. The Tg' of sucrose has been determined to -40 &deg;C 2, 3and can be difficult to detect for sucrose concentrations below 20% w/w. The thermal event at -35 &deg;C is probably related to the onset of ice dissolution2. The crystalline structure detected by X-ray in the HEC and HPMC samples and also seen in SEM (see Figure 2b and 2c) overlap with the normal crystal form of NaCl.
Figure 1A shows survival data for the Gram-negative P. putida and the Gram-positive A. chlorophenolicus formulated in the different saccharide based formulations. Note that the trend in how well the formulations support cell survival is the same for both bacteria species. The plot shown in Figure 1B illustrates the correlation between the freeze-drying survival and the surface tensions of the formulations.
Figure 2 shows SEM-images of the dry formulations. For the four polymers shown here, the matrix formed has the appearance of "crisp paper": interconnected smooth sheets in which the bacteria are embedded and show up as corrugations. For Ficoll and Sucrose, the sheets are about 1 &mu;m thick and 10 - 20 &mu;m wide, with Ficoll having a smoother surface. HPMC and HEC form much thinner sheets, possibly due to the fact that the amount of polymer is less in these cellulose-based formulations than in the others (Table 1). Moreover, salt crystals were observed for HEC and HPMC, with the latter showing a larger amount of precipitates on the surface of the polymer sheets. Bacteria are more easily seen in the cellulose based formulations because the sheets are thinner. In Sucrose and Ficoll, they are mostly observed as corrugation of the otherwise smooth surfaces.
<table border="1">
	<tbody>
		<tr>
			<td>Formulation composition</td>
			<td>Thermal events detected in frozen formulations</td>
			<td>Structure of dry formulation</td>
			<td>Surface tension of undried formulations (mN m-1) </td>
		</tr>
		<tr>
			<td>10% Sucrose</td>
			<td>onset of ice dissolution, -35 &deg;C</td>
			<td>amorphous sucrose</td>
			<td>72 &plusmn; 0.1</td>
		</tr>
		<tr>
			<td>10% Ficoll, 150 mM NaCl</td>
			<td>Tg', -22 &deg;C</td>
			<td>amorphous Ficoll</td>
			<td>68 &plusmn; 0.3</td>
		</tr>
		<tr>
			<td>2% HEC, 150 mM NaCl</td>
			<td>eutectic melting of ice and NaCl, -28 &deg;C</td>
			<td>amorphous HEC, crystalline NaCl</td>
			<td>64 &plusmn; 0.6</td>
		</tr>
		<tr>
			<td>2% HPMC, 150 mM NaCl</td>
			<td>eutectic melting of ice and NaCl, -27 &deg;C</td>
			<td>amorphous HPMC, crystalline NaCl</td>
			<td>52 &plusmn; 0.8</td>
		</tr>
	</tbody>
</table>
Table 1. The composition of the different formulations and properties of the aqueous or dry formulations. The polymer concentrations of the HEC and HPMC were maximized to achieve a thick enough matrix cover of the bacteria but still have a workable solution as regards to viscosity.
<img alt="Figure 1" src="/files/ftp_upload/4058/4058fig1.jpg" /> 
Figure 1. A) Survival rates of freeze dried P. putida (white) and A. chlorophenolicus (grey) when formulated in solutions based on the disaccharide sucrose or the polymers Ficoll, HEC and HPMC, B) Correlation between cell survival after freeze drying and the surface tension of the un-dried formulations.
<img alt="Figure 2" src="/files/ftp_upload/4058/4058fig2.jpg" /> 
Figure 2. SEM images of P. putida in four different formulations: a) sucrose, b) Ficoll, c) HEC, d) HPMC. The bacteria are difficult to see in a) and b) because the polymer sheet are quite thick and can envelop the bacteria completely; in c) and d) the bacteria show up more prominently on the surface. In d), they can be distinguished from the large amount of salt precipitates because of their shape and contrast, as they appear as oblong, dark corpuscles.

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

Freeze-drying is often an easy and convenient way to obtain dry products of viable bacterial cells. An issue of the process is cell survival. We detail here a procedure to investigate how cell survival during freeze-drying is influenced by the properties of the formulation used.

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

formulations-for-freeze-drying-bacteria-their-influence-on-cell

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23.2K Views.  Swedish University of Agricultural Sciences. Freeze-drying is often an easy and convenient way to obtain dry products of viable bacterial cells. An issue of the process is cell survival. We detail here a procedure to investigate how cell survival during freeze-drying is influenced by the properties of the formulation used.

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

Obtaining Hemocytes from the Hawaiian Bobtail Squid Euprymna scolopes and Observing their Adherence to Symbiotic and Non-Symbiotic Bacteria

Studies concerning the role of the immune system in mediating molecular signaling between beneficial bacteria and their hosts have, in recent years, made significant contributions to our understanding of the co-evolution of eukaryotes with their microbiota. The symbiotic association between the Hawaiian bobtail squid, Euprymna scolopes and the bioluminescent bacterium Vibrio fischeri has been utilized as a model system for understanding the effects of beneficial bacteria on animal development. Recent studies have shown that macrophage-like hemocytes, the sole cellular component of the squid host's innate immune system, likely play an important role in mediating the establishment and maintenance of this association. This protocol will demonstrate how to obtain hemocytes from E. scolopes and then use these cells in bacterial binding assays. Adult squid are first anesthetized before hemolymph is collected by syringe from the main cephalic blood vessel. The host hemocytes, contained in the extracted hemolymph, are adhered to chambered glass coverslips and then exposed to green fluorescent protein-labeled symbiotic Vibrio fischeri and non-symbiotic Vibrio harveyi. The hemocytes are counterstained with a fluorescent dye (Cell Tracker Orange, Invitrogen) and then visualized using fluorescent microscopy.

Obtaining Hemocytes from the Hawaiian Bobtail Squid Euprymna scolopes and Observing their Adherence to Symbiotic and Non-Symbiotic Bacteria

This video will demonstrate how to obtain hemocytes (blood cells) from the Hawaiian bobtail squid, Euprymna scolopes for use in cell biological and bacterial adhesion assays. Hemocytes will be stained with a fluorescent dye and exposed to GFP-labeled bacteria.

Studies concerning the role of the immune system in mediating molecular signaling between beneficial bacteria and their hosts have, in recent years, made ...

Analyzing the Function of Small GTPases by Microinjection of Plasmids into Polarized Epithelial Cells

Epithelial cells polarize their plasma membrane into biochemically and functionally distinct apical and basolateral domains where the apical domain faces the 'free' surfaces and the basolateral membrane is in contact with the substrate and neighboring cells. Both membrane domains are separated by tight junctions, which form a diffusion barrier. Apical-basolateral polarization can be recapitulated successfully in culture when epithelial cells such as Madin-Darby Canine Kidney (MDCK) cells are seeded at high density on polycarbonate filters and cultured for several days 1 2. Establishment and maintenance of cell polarity is regulated by an array of small GTPases of the Ras superfamily such as RalA, Cdc42, Rab8, Rab10 and Rab13 3 4 5 6 7. Like all GTPases these proteins cycle between an inactive GDP-bound state and an active GTP-bound state. Specific mutations in the nucleotide binding regions interfere with this cycling 8. For example, Rab13T22N is permanently locked in the GDP-form and thus dubbed 'dominant negative', whereas Rab13Q67L can no longer hydrolyze GTP and is thus locked in a 'dominant active' state 7. To analyze their function in cells both dominant negative and dominant active alleles of GTPases are typically expressed at high levels to interfere with the function of the endogenous proteins 9. An elegant way to achieve high levels of overexpression in a short amount of time is to introduce the plasmids encoding the relevant proteins directly into the nuclei of polarized cells grown on filter supports using microinjection technique. This is often combined with the co-injection of reporter plasmids that encode plasma membrane receptors that are specifically sorted to the apical or basolateral domain. A cargo frequently used to analyze cargo sorting to the basolateral domain is a temperature sensitive allele of the vesicular stomatitis virus glycoprotein (VSVGts045) 10. This protein cannot fold properly at 39&deg;C and will thus be retained in the endoplasmic reticulum (ER) while the regulatory protein of interest is assembled in the cytosol. A shift to 31&deg;C will then allow VSVGts045 to fold properly, leave the ER and travel to the plasma membrane 11. This chase is typically performed in the presence of cycloheximide to prevent further protein synthesis leading to cleaner results. Here we describe in detail the procedure of microinjecting plasmids into polarized cells and subsequent incubations including temperature shifts that allow a comprehensive analysis of regulatory proteins involved in basolateral sorting.

This article details the procedures involved in overexpression and analysis of small GTPases in polarized epithelial cells using microinjection technique.

Epithelial cells polarize their plasma membrane into biochemically and functionally distinct apical and basolateral domains where the apical domain faces ...

Single Cell Transcriptional Profiling of Adult Mouse Cardiomyocytes

While numerous studies have examined gene expression changes from homogenates of heart tissue, this prevents studying the inherent stochastic variation between cells within a tissue. Isolation of pure cardiomyocyte populations through a collagenase perfusion of mouse hearts facilitates the generation of single cell microarrays for whole transcriptome gene expression, or qPCR of specific targets using nanofluidic arrays. We describe here a procedure to examine single cell gene expression profiles of cardiomyocytes isolated from the heart. This paradigm allows for the evaluation of metrics of interest which are not reliant on the mean (for example variance between cells within a tissue) which is not possible when using conventional whole tissue workflows for the evaluation of gene expression (Figure 1). We have achieved robust amplification of the single cell transcriptome yielding micrograms of double stranded cDNA that facilitates the use of microarrays on individual cells. In the procedure we describe the use of NimbleGen arrays which were selected for their ease of use and ability to customize their design. Alternatively, a reverse transcriptase - specific target amplification (RT-STA) reaction, allows for qPCR of hundreds of targets by nanofluidic PCR. Using either of these approaches, it is possible to examine the variability of expression between cells, as well as examining expression profiles of rare cell types from within a tissue. Overall, the single cell gene expression approach allows for the generation of data that can potentially identify idiosyncratic expression profiles that are typically averaged out when examining expression of millions of cells from typical homogenates generated from whole tissues.

Single cell expression profiling allows the detailed gene expression analysis of individual cells. We describe methods for the isolation of cardiomyocytes, and preparing the resulting lysates for either whole transcriptome microarray or qPCR of specific targets.

While numerous studies have examined gene expression changes from homogenates of heart tissue, this prevents studying the inherent stochastic variation ...

Isolation and Culture of Individual Myofibers and their Satellite Cells from Adult Skeletal Muscle

Muscle regeneration in the adult is performed by resident stem cells called satellite cells. Satellite cells are defined by their position between the basal lamina and the sarcolemma of each myofiber. Current knowledge of their behavior heavily relies on the use of the single myofiber isolation protocol. In 1985, Bischoff described a protocol to isolate single live fibers from the Flexor Digitorum Brevis (FDB) of adult rats with the goal to create an in vitro system in which the physical association between the myofiber and its stem cells is preserved 1. In 1995, Rosenblattmodified the Bischoff protocol such that myofibers are singly picked and handled separately after collagenase digestion instead of being isolated by gravity sedimentation 2, 3. The Rosenblatt or Bischoff protocol has since been adapted to different muscles, age or conditions 3-6. The single myofiber isolation technique is an indispensable tool due its unique advantages. First, in the single myofiber protocol, satellite cells are maintained beneath the basal lamina. This is a unique feature of the protocol as other techniques such as Fluorescence Activated Cell Sorting require chemical and mechanical tissue dissociation 7. Although the myofiber culture system cannot substitute for in vivo studies, it does offer an excellent platform to address relevant biological properties of muscle stem cells. Single myofibers can be cultured in standard plating conditions or in floating conditions. Satellite cells on floating myofibers are subjected to virtually no other influence than the myofiber environment. Substrate stiffness and coating have been shown to influence satellite cells' ability to regenerate muscles 8, 9 so being able to control each of these factors independently allows discrimination between niche-dependent and -independent responses. Different concentrations of serum have also been shown to have an effect on the transition from quiescence to activation. To preserve the quiescence state of its associated satellite cells, fibers should be kept in low serum medium 1-3. This is particularly useful when studying genes involved in the quiescence state. In serum rich medium, satellite cells quickly activate, proliferate, migrate and differentiate, thus mimicking the in vivo regenerative process 1-3. The system can be used to perform a variety of assays such as the testing of chemical inhibitors; ectopic expression of genes by virus delivery; oligonucleotide based gene knock-down or live imaging. This video article describes the protocol currently used in our laboratory to isolate single myofibers from the Extensor Digitorum Longus (EDL) muscle of adult mice (6-8 weeks old).

Isolation and culture of myofibers is the gold standard in vitro system to study the transition of satellite cells through quiescence, activation and differentiation. Importantly, the single myofiber culture system preserves the myofiber/stem cell association, which is an essential component of the muscle stem cell niche.

Muscle  regeneration in the adult is performed by resident stem cells called satellite  cells. Satellite cells are defined by  their position between the ...

Strategies for Tracking Anastasis, A Cell Survival Phenomenon that Reverses Apoptosis

Anastasis (Greek for &#8220;rising to life&#8221;) refers to the recovery of dying cells. Before these cells recover, they have passed through important checkpoints of apoptosis, including mitochondrial fragmentation, release of mitochondrial cytochrome c into the cytosol, activation of caspases, chromatin condensation, DNA damage, nuclear fragmentation, plasma membrane blebbing, cell shrinkage, cell surface exposure of phosphatidylserine, and formation of apoptotic bodies. Anastasis can occur when apoptotic stimuli are removed prior to death, thereby allowing dying cells to reverse apoptosis and potentially other death mechanisms. Therefore, anastasis appears to involve physiological healing processes that could also sustain damaged cells inappropriately. The functions and mechanisms of anastasis are still unclear, hampered in part by the limited tools for detecting past events after the recovery of apparently healthy cells. Strategies to detect anastasis will enable studies of the physiological mechanisms, the hazards of undead cells in disease pathology, and potential therapeutics to modulate anastasis. Here, we describe effective strategies using live cell microscopy and a mammalian caspase biosensor&#160;for identifying and tracking anastasis in mammalian cells.

The term anastasis refers to the phenomenon in which dying cells reverse a cell suicide process at a late stage, repair themselves, and ultimately survive. Here we demonstrate protocols for detecting and tracking cells that undergo anastasis.

Anastasis (Greek for &#8220;rising to life&#8221;) refers to the recovery of dying cells. Before these cells recover, they have passed through important ...

A Standardized Method for the Analysis of Liver Sinusoidal Endothelial Cells and Their Fenestrations by Scanning Electron Microscopy

Liver sinusoidal endothelial cells are the gateway to the liver, their transcellular fenestrations allow the unimpeded transfer of small and dissolved substances from the blood into the liver parenchyma for metabolism and processing. Fenestrations are dynamic structures - both their size and/or number can be altered in response to various physiological states, drugs, and disease, making them an important target for modulation. An understanding of how LSEC morphology is influenced by various disease, toxic, and physiological states and how these changes impact on liver function requires accurate measurement of the size and number of fenestrations. In this paper, we describe scanning electron microscopy fixation and processing techniques used in our laboratory to ensure reproducible specimen preparation and accurate interpretation. The methods include perfusion fixation, secondary fixation and dehydration, preparation for the scanning electron microscope and analysis. Finally, we provide a step by step method for standardized image analysis which will benefit all researchers in the field.

The fenestrated liver sinusoidal endothelial cell is a biologically important filter system that is highly influenced by various diseases, toxins, and physiological states. These changes significantly impact on liver function. We describe methods for the standardisation of the measurement of the size and number of fenestrations in these cells.

Liver sinusoidal endothelial cells are the gateway to the liver, their transcellular fenestrations allow the unimpeded transfer of small and dissolved ...

Measurement and Analysis of Extracellular Acid Production to Determine Glycolytic Rate

Extracellular measurement of oxygen consumption and acid production is a simple and powerful way to monitor rates of respiration and glycolysis1. Both mitochondrial (respiration) and non-mitochondrial (other redox) reactions consume oxygen, but these reactions can be easily distinguished by chemical inhibition of mitochondrial respiration. However, while mitochondrial oxygen consumption is an unambiguous and direct measurement of respiration rate2, the same is not true for extracellular acid production and its relationship to glycolytic rate 3-6. Extracellular acid produced by cells is derived from both lactate, produced by anaerobic glycolysis, and CO2, produced in the citric acid cycle during respiration. For glycolysis, the conversion of glucose to lactate- + H+ and the export of products into the assay medium is the source of glycolytic acidification. For respiration, the export of CO2, hydration to H2CO3 and dissociation to HCO3- + H+ is the source of respiratory acidification. The proportions of glycolytic and respiratory acidification depend on the experimental conditions, including cell type and substrate(s) provided, and can range from nearly 100% glycolytic acidification to nearly 100% respiratory acidification 6. Here, we demonstrate the data collection and calculation methods needed to determine respiratory and glycolytic contributions to total extracellular acidification by whole cells in culture using C2C12 myoblast cells as a model.

Glycolysis is a defining metabolic marker in multiple biological systems. Monitoring glycolysis by measuring the extracellular flux of H+ is common, but requires correction to be quantitative and unambiguous. Here, we demonstrate how to gather and correct extracellular flux data to distinguish between respiratory and glycolytic sources of extracellular acidification.

Extracellular measurement of oxygen consumption and acid production is a simple and powerful way to monitor rates of respiration and glycolysis1. Both ...

Isolation of Specific Genomic Regions and Identification of Associated Molecules by enChIP

The identification of molecules associated with specific genomic regions of interest is required to understand the mechanisms of regulation of the functions of these regions. To enable the non-biased identification of molecules interacting with a specific genomic region of interest, we recently developed the engineered DNA-binding molecule-mediated chromatin immunoprecipitation (enChIP) technique. Here, we describe how to use enChIP to isolate specific genomic regions and identify the associated proteins and RNAs. First, a genomic region of interest is tagged with a transcription activator-like (TAL) protein or a clustered regularly interspaced short palindromic repeats (CRISPR) complex consisting of a catalytically inactive form of Cas9 and a guide RNA. Subsequently, the chromatin is crosslinked and fragmented by sonication. The tagged locus is then immunoprecipitated and the crosslinking is reversed. Finally, the proteins or RNAs that are associated with the isolated chromatin are subjected to mass spectrometric or RNA sequencing analyses, respectively. This approach allows the successful identification of proteins and RNAs associated with a genomic region of interest.

The identification of molecules associated with specific genomic regions of interest is required to understand the mechanisms of regulation of the functions of these regions. This protocol describes procedures to perform engineered DNA-binding molecule-mediated chromatin imunoprecipitation (enChIP) for identification of proteins and RNAs associated with a specific genomic region.

The identification of molecules associated with specific genomic regions of interest is required to understand the mechanisms of regulation of the ...

Measurement of Survival Time in Brachionus Rotifers: Synchronization of Maternal Conditions

Rotifers are microscopic cosmopolitan zooplankton used as models in ecotoxicological and aging studies due to their several advantages such as short lifespan, ease of culture, and parthenogenesis that enables clonal culture. However, caution is required when measuring their survival time as it is affected by maternal age and maternal feeding conditions. Here we provide a protocol for powerful and reproducible measurement of the survival time in Brachionus rotifers following a careful synchronization of culture conditions over several generations. Empirically, poor synchronization results in early mortality and a gradual decrease in survival rate, thus resulting in weak statistical power. Indeed, under such conditions, calorie restriction (CR) failed to significantly extend the lifespan of B. plicatilis although CR-induced longevity has been demonstrated with well-synchronized rotifer samples in past and present studies. This protocol is probably useful for other invertebrate models, including the fruitfly Drosophila melanogaster and the nematode Caenorhabditis elegans, because maternal age effects have also been reported in these species.

Measurement of Survival Time in Brachionus Rotifers: Synchronization of Maternal Conditions

Rotifers are microscopic zooplankton used as models in ecotoxicological and aging studies. Here we provide a protocol for powerful and reproducible measurement of survival time in Brachionus rotifers. Synchronization of culture conditions over several generations is of particular importance because maternal condition affects life history of offspring.

Rotifers are microscopic cosmopolitan zooplankton used as models in ecotoxicological and aging studies due to their several advantages such as short ...

Investigating the Detrimental Effects of Low Pressure Plasma Sterilization on the Survival of Bacillus subtilis Spores Using Live Cell Microscopy

Plasma sterilization is a promising alternative to conventional sterilization methods for industrial, clinical, and spaceflight purposes. Low pressure plasma (LPP) discharges contain a broad spectrum of active species, which lead to rapid microbial inactivation. To study the efficiency and mechanisms of sterilization by LPP, we use spores of the test organism Bacillus subtilis because of their extraordinary resistance against conventional sterilization procedures. We describe the production of B. subtilis spore monolayers, the sterilization process by low pressure plasma in a double inductively coupled plasma reactor, the characterization of spore morphology using scanning electron microscopy (SEM), and the analysis of germination and outgrowth of spores by live cell microscopy. A major target of plasma species is genomic material (DNA) and repair of plasma-induced DNA lesions upon spore revival is crucial for survival of the organism. Here, we study the germination capacity of spores and the role of DNA repair during spore germination and outgrowth after treatment with LPP by tracking fluorescently-labelled DNA repair proteins (RecA) with time-resolved confocal fluorescence microscopy. Treated and untreated spore monolayers are activated for germination and visualized with an inverted confocal live cell microscope over time to follow the reaction of individual spores. Our observations reveal that the fraction of germinating and outgrowing spores is dependent on the duration of LPP-treatment reaching a minimum after 120 s. RecA-YFP (yellow fluorescence protein) fluorescence was detected only in few spores and developed in all outgrowing cells with a slight elevation in LPP-treated spores. Moreover, some of the vegetative bacteria derived from LPP-treated spores showed an increase in cytoplasm and tended to lyse. The described methods for analysis of individual spores could be exemplary for the study of other aspects of spore germination and outgrowth.

Investigating the Detrimental Effects of Low Pressure Plasma Sterilization on the Survival of Bacillus subtilis Spores Using Live Cell Microscopy

This protocol illustrates the important consecutive steps required to assess the relevance of monitoring vitality parameter and DNA repair processes in reviving Bacillus subtilis spores after treatment with low pressure plasma by tracking fluorescence-labelled DNA repair proteins via time-resolved confocal microscopy and scanning electron microscopy.

Plasma sterilization is a promising alternative to conventional sterilization methods for industrial, clinical, and spaceflight purposes. Low pressure ...