We thank Dr. Carmen R. Beuz&#243;n and Roc&#237;o Carvajal-Holguera for helpful discussions and suggestions. This work was supported by the grant PID2020-116995RB-I00 funded by MICIU/AEI/ 10.13039/5011100011033 and the VI Plan Propio de Investigaci&#243;n y Transferencia from the Universidad de Sevilla.

NOTE: Prior to performing the procedure of elimination of phages, we describe the preparation of a Salmonella culture infected with 9NA bacteriophages. In Figure 1, a general representation of the complete procedure for bacteriophage removal in bacterial cultures is illustrated.
1. Preparation of Salmonella cultures infected with bacteriophages
<ol>
	<li>Bacterial culture preparation 
	NOTE: In this study, Salmonella enterica subsp. enterica serovar Typhimurium strain ATCC 14028 opvAB::lacZ (SV8011)13 was used.
	<ol>
		<li>Slowly thaw the vial retrieved from -80 &#176;C that contains the S. enterica strain of interest by placing it on ice.</li>
		<li>Streak out the S. enterica strain from the thawed dimethyl sulfoxide stock onto a Lennox broth (LB) plate (10 g/L tryptone, 5 g/L NaCl, 5 g/L yeast extract, and 15 g/L agar, see Table of Materials). Incubate the plate at 37 &#176;C for 24 h.</li>
		<li>Using an inoculation needle, transfer a colony from the S. enterica plate and inoculate it into 5 mL of liquid LB in a glass tube. Incubate at 37 &#176;C for 24 h with aeration by shaking at 200 rpm.</li>
	</ol>
	</li>
	<li>Bacteriophage preparation 
	NOTE: Bacteriophage 9NA, which belongs to the family Siphoviridae (order Caudovirales)14, was used in this protocol. Notably, other Salmonella enterica bacteriophages that use the O-antigen of the lipopolysaccharide as a receptor can be used.
	<ol>
		<li>Prepare the bacteriophage broth by adding 0.1 mL of concentrated bacteriophage lysate to a mixture of 100 mL of nutrient broth (NB; 5 g/L peptone and 3 g/L yeast extract), 2 mL of 50x E salts (38.78 mM MgSO47 H2O, 520.5 mM Citric acidH2O, 2.87 M K2HPO4 anhydrous and 829 mM NaNH4HPO44 H2O) and 0.4 mL of 50% glucose. Mix thoroughly by inversion.
		<ol>
			<li>A representative example of bacteriophage concentration could be 109 PFU/mL (Plaque Forming Units/mL), but lower concentrations can be used with similar results (see Table of Materials). Estimate bacteriophage concentration following step 1.2.3.</li>
		</ol>
		</li>
		<li>Prepare lysate by following the steps described below.
		<ol>
			<li>Prepare the S. enterica inoculum in 5 mL of LB (10 g/L tryptone, 5 g/L NaCl, and 5 g/L yeast extract). Incubate at 37 &#176;C and 200 rpm for 24 h.</li>
			<li>Mix 4 mL of bacteriophage broth with 1 mL of the overnight culture of S. enterica. Incubate the mix at 37 &#176;C and 200 rpm for 8 h.</li>
			<li>To discriminate between bacteria and bacteriophages, centrifuge the mix at 2,377 x g for 20 min. Recover the supernatant in a glass tube, add 800 &#181;L of chloroform (see Table of Materials), and vortex it. Make sure to only transfer the upper phase when a mixture with chloroform is used. 
			NOTE: It is important to prevent the transfer of residual chloroform, which could affect the efficiency of the procedure. The chloroform step is common in both isolation and propagation protocols as a way of maximizing released phages. However, chloroform may inactivate some phages or destroy their infectivity15. Therefore, this step is only applicable to phages resistant to chloroform.</li>
			<li>Maintain the lysates at room temperature for 2 h and then store them at 4 &#176;C.</li>
		</ol>
		</li>
		<li>Titrate the lysate to quantify the number of bacteriophages/number of PFU per mL present in the prepared lysate. To do this, perform the overlay technique described below.
		<ol>
			<li>Prepare 5 mL of LB of inoculum for S. enterica. Incubate at 37 &#176;C for 24 h with aeration by shaking at 200 rpm.</li>
			<li>Perform serial dilutions of the lysate into LB to reach a countable number of PFU/mL. To determine the necessary dilution factors, identify the initial bacteriophage lysate concentrated. For a representative example of bacteriophage concentration at around 109 PFU/mL, the dilution factors that result in a countable number of plaques are between 106-1010.</li>
			<li>Add 100 &#181;L of the appropriate lysate dilution and 60 &#181;L of the overnight culture of S. enterica prepared the day before to 5 mL of LB soft agar, prepared by mixing liquid LB and LB-agar in a proportion 1:1 and maintaining at 56 &#176;C to avoid solidification. Mix carefully, avoiding bubbles.</li>
			<li>Pour each mix into the top of an LB plate and keep on the bench until the medium solidifies.</li>
			<li>Incubate plates for 24 h at 37 &#176;C. Count the number of plaques on each plate. To determine the number of PFU/mL, multiply the number of plaques of each plate by the dilution factor (FD). To ensure efficient phage infection, 109-1012 PFU/mL are necessary. 
			PFU/mL= No. plaques in the plate x FD</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Infection of Salmonella cultures with bacteriophages
	<ol>
		<li>Dilute an overnight culture of S. enterica to 1:100 in a final volume of 5 mL of LB and add 0.1 mL of lysate (concentrated at 109-1012 PFU/mL). Incubate at 37 &#176;C and 200 rpm for 24 h.</li>
	</ol>
	</li>
	<li>Determination of phage titer as PFU in plate assay
	<ol>
		<li>To quantify the number of bacteriophages in the infected culture, perform plate assays to determine the phage titer as PFU/mL as described below.
		<ol>
			<li>Transfer 1 mL of the culture to a tube. Centrifuge at 17,115 x g for 2 min and pour the supernatant into a new labeled vial.</li>
			<li>Add 100 &#181;L of chloroform to the vial and vortex to remove bacterial debris from the solution. Follow step 1.2.3.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>
2. Bacteriophage removal from infected Salmonella enterica cultures (see Figure 2)
NOTE: To monitor the bacteriophage removal process, different aliquots are taken throughout the cleaning protocol for titration. In total, there are eight aliquots to confirm that the number of PFU/mL is decreasing throughout the cleaning process until complete elimination.
<ol>
	<li>Removal of bacteriophage in suspension
	<ol>
		<li>Dilute the bacterial culture containing phages to 1:100 in 5 mL of 0.22 &#181;m-filtered LB in a 50 mL conical centrifuge tube. Titration point 1: This is the first aliquot for titration (the starting infected Salmonella culture).</li>
		<li>Centrifuge the suspension for 10 min at 2,377 x g. Carefully remove the supernatant. Do not use a pipette; pour the tube. Try to leave some volume at the bottom of the conical centrifuge tube to ensure the presence of cells.</li>
		<li>Wash bacterial cells with 10 mL of 0.22 &#181;m-filtered LB medium. Repeat centrifugation and washing steps 3x. Resuspend the pellet in 5 mL of 0.22 &#181;m-filtered LB. Titration point 2: This is the second aliquot for titration.</li>
		<li>Transfer 50 &#181;L of the above bacterial suspension through a sterile 0.45 &#181;m filter (see Table of Materials) using a vacuum filtration system. Rinse the filter with 100 mL of 0.22 &#181;m-filtered LB. 
		NOTE: The head size of bacteriophage 9NA is 0.067 &#181;m. In contrast, Salmonella is 2-5 &#181;m long and 0.5-1.5 &#181;m wide. Thus, if 0.45 &#181;m membranes are used, most bacteria will not pass because they are larger than 0.45 &#181;m, while most phages will pass through the 0.45 &#181;m filter pores.</li>
	</ol>
	</li>
	<li>Removal of bacteriophage contained inside Salmonella cells
	<ol>
		<li>To facilitate phage release from bacteria cells, incubate the 0.45 &#181;m filter containing cells for 2 h at 37 &#176;C inside the incubator without disassembling the system. 
		NOTE: This step of incubation is required to promote the lysis of the infected bacteria and the release of intracellular phages into the filter.</li>
		<li>Wash the 0.45 &#181;m filter using a vacuum filtration system, rinsing the filter with 100 mL of 0.22 &#181;m-filtered LB.</li>
		<li>Recover bacterial cells from the filter in 2 mL of 2x LB (20 g/L tryptone, 10 g/L NaCl, 10 g/L yeast extract; see Table of Materials). To do this, transfer the 0.45 &#181;m filter to a flask using sterile forceps. Then, add 2 mL of 2x LB over the filter and pipette several times to release cells from the filter.</li>
		<li>Centrifuge 1 mL of this suspension for 2 min at 10,354 x g. Discard the supernatant and wash bacterial cells 3x with 1 mL of filtered LB medium. Resuspend cells in 1 mL of 2x LB. Titration point 3: This is the third aliquot for titration. 
		NOTE: The use of a more concentrated LB (2x LB) is highly recommended because, as described below, commercial lipopolysaccharide (LPS) will be added, and this LPS dissolves in water, thus diluting the medium.</li>
	</ol>
	</li>
	<li>Avoid reinfection by deceiving the bacteriophages released from bacterial cells with commercial lipopolysaccharide as described below.
	<ol>
		<li>Incubate 20 &#181;L of the bacterial suspension in the presence of a commercial S. enterica lipopolysaccharide (LPS; see Table of Materials) to a final concentration of 3.75 mg/mL in 1 mL of 0.22 &#181;m-filtered LB for 2 h at 37 &#176;C with shaking at 200 rpm. 
		NOTE: Phages released from cells into the medium may bind to the commercial S. enterica LPS instead of bacterial LPS.</li>
		<li>After 2 h of incubation, transfer 1 mL of culture to a microcentrifuge tube and centrifuge the suspension for 2 min at 10,354 x g. Titration point 4: Use the supernatant as the fourth aliquot for titration.</li>
		<li>Wash pellet 3x with 1 mL of filtered LB medium.</li>
		<li>After washing, resuspend the pellet in 1 mL of 2x LB. Incubate 20 &#181;L of this mix in 1 mL of 0.22 &#181;m-filtered LB with 0.8 mg/mL of commercial LPS for 2 h at 37 &#176;C with shaking at 200 rpm. Titration point 5: Use the rest of the bacterial suspension for the fifth titration.</li>
		<li>After 2 h of incubation, transfer 1 mL of culture to a microcentrifuge tube and pellet by centrifugation for 2 min at 10,354 x g. Titration point 6: Use the supernatant as the sixth aliquot for titration by pouring the supernatant into a new vial.</li>
		<li>Wash bacterial cells 3x with filtered LB. After washing, resuspend collected cells in 1 mL of 0.22 &#181;m-filtered LB.</li>
		<li>Pass 1 mL of bacterial suspension through a sterile 0.45 &#181;m filter using a vacuum filtration system. Rinse the filter using 100 mL of 0.22 &#181;m-filtered LB.</li>
		<li>Recover cells from the 0.45 &#181;m filter in 2 mL of 2x LB medium. To that end, transfer the 0.45 &#181;m filter to a flask using sterile forceps. Then, add 2 mL of 2x LB over the filter and pipette several times to try to release cells from the filter.</li>
		<li>Pellet 1 mL of cells by centrifugation for 2 min at 10,354 x g. Wash 3x with 0.22 &#181;m-filtered LB. After washing, resuspend collected cells in 1 mL of 2x LB. Titration point 7: This is the seventh aliquot for titration. This aliquot will inform about the efficiency of removal of phages in bacterial cultures.</li>
	</ol>
	</li>
</ol>
3. Preparation of bacteriophage-free Salmonella culture after bacteriophage removal
<ol>
	<li>Prepare the phage-free inoculum. Add 100 &#181;L of cells from the bacterial suspension to 900 &#181;L of LB containing 0.45 mg/mL LPS. Incubate at 37 &#176;C for 24 h with shaking at 200 rpm.</li>
	<li>Titration point 8: Prepare the eighth aliquot for titration (the final potential un-infected culture). This point is to confirm the absence of phage re-infection.</li>
</ol>

Using Bacterial LPS to Eliminate Bacteriophages from Salmonella Cultures

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The authors have nothing to disclose.

Diverse strategies are employed by bacteriophages to recognize and infect bacterial hosts. Different molecular structures on the surface of bacteria can act as phage receptors: protein, polysaccharide, lipopolysaccharides (LPS), and carbohydrate moieties20. In Gram-negative bacteria, LPS is a common receptor for phages. In addition, other receptors are outer membrane proteins, pili, and flagella21.
The specific interaction between bacteriophages and bacteria based on the recognition of LPS8 has been exploited in this work for the development of a highly efficient protocol for the elimination of bacteriophages in infected bacterial cultures (Figure 1 and Figure 2). Our protocol does not favor the selection of phage-resistant cells; it only eliminates bacteriophages (Figure 6). Both phage-susceptible and phage-resistant cells remain in bacterial culture after performing this protocol of phage removal.
The traditional standard practice when a phage infection occurs is to attempt to eliminate all the contaminated material, followed by cleaning and sterilization22. The decontamination procedure involves exposing the bacterial culture to stressful conditions, such as high temperatures, in order to partially or completely eliminate the bacterial cells. As described in representative results, the crucial step in this protocol is the incubation of phages-infected bacterial cultures with commercial LPS, a non-harmful substance for bacterial cultures. This helps to preserve the viability of bacterial cultures and offers significant advantages for industrial applications in fermenters and bioreactors.
The incubation time in this protocol is 2 h to ensure sufficient time for phage lysis of bacterial cells. If different bacterial strains and bacteriophages are to be used, this parameter should be considered and defined by the user. In this case, an assay similar to that described in Figure 4 should be performed prior to the experiment.
Interestingly, the efficacy of this cleaning protocol could also be analyzed by employing an assay that monitors the phage content of a given sample. In this sense, epigenetic biosensors are a novel tool for bacteriophage detection23. A well-known phage biosensor able to detect coliphages, which use LPS as a receptor, is the opvAB::gfp system13,18,23,24. This phage biosensor detects an increase in the OpvABON subpopulation in the presence of phages that use O-antigen as a receptor. In this sense, we could use an opvAB::gfp fusion to monitor LPS-binding phages in various steps of this protocol and/or diverse media and conditions. These approaches could be valuable in determining the timing and locations at which an effective protocol may be necessary.
While LPS recognition is common, phages can also utilize a variety of other surface receptors on bacterial cells for attachment and infection. Here, we have used the Gram-negative Salmonella as a representative enterobacteria and the bacteriophage 9NA that uses LPS as a receptor and genome ejection trigger. Other enterobacteria phages (e.g., Escherichia coli T5) bind loosely to LPS and require an outer membrane protein for genome injection. The protocol described is applicable for bacteriophages that recognize and need the O-antigen of LPS for successful infection, such as 9NA, Det7, and P2213,25,26,27. Accordingly, the successful implementation of this protocol for the phage decontamination of bacterial cultures involves determining whether the source of the phage infection requires recognizing LPS in the host.
In conclusion, and despite the potential limitations of the protocol, our representative results clearly demonstrate that this method is a powerful tool to clean bacterial Salmonella cultures of bacteriophages that use LPS as a receptor and genome ejection trigger.

Microbial populations are faced with multiple challenges in natural environments, and an especially severe threat is the potential of infection by bacteriophages, the viruses that infect bacteria1. These viruses are widespread on the planet, exhibiting great diversity and abundance2,3,4,5. Although bacteriophages are diverse in size, morphology, and genomic organization, all share the same structure: a DNA or RNA genome enveloped by a capsid formed by phage-encoded proteins6. Bacteria have evolved a diverse array of defense mechanisms against them7. A key aspect of bacteriophage infection, which is relevant for characterization as well as for detection, is the receptor binding domains present on tail fibers. Bacteriophages have proteins on their surface called receptor-binding proteins or tail fibers for recognizing and binding to specific receptor sites on the surface of the bacterial cell. In the case of Gram-negative bacteria, the recognition of surface structures, such as lipopolysaccharides (LPS), outer membrane proteins, pili, and/or flagella, are involved in phage-bacteria interaction8. This interaction between bacteriophages and bacteria is highly specific and depends mainly on their ability to attach to host surfaces. The O-antigen of lipopolysaccharide is a commonly used receptor9.
The investigation of bacteriophage-bacteria interactions is not only fascinating from a biological standpoint but also has practical applications in areas such as phage therapy and biotechnology. While bacteriophages have valuable roles in various contexts, for example, altering microbial populations10, their presence can be undesirable in certain industrial processes. In pharmaceuticals, biotechnology, and food production, the presence of bacteriophages can impact the quality and safety of the final products, making their removal essential to meet quality standards. In bioprocessing and biomanufacturing, where bacterial cultures are used to produce various compounds (e.g., proteins, enzymes, or antibiotics), the presence of bacteriophages can lead to the disruption of production processes due to their ability to balance the bacterial population in every shared environment. Phages can occasionally turn the industrial microbiologist&#39;s professional life into a nightmare11. The design of effective procedures to remove phages is critical to ensure consistent and reliable production, enhancing process efficiency. Apart from these industrial aspects, in a research laboratory setting, where precision and reproducibility are crucial, the elimination of bacteriophages is essential for obtaining accurate and reliable results. Furthermore, the removal of phages can also be used to simulate varying environments to test different hypotheses12. Removing phages can also be very useful in the research environment since many phage-based studies, such as the enumeration of bacteria following phage application, would benefit from a step to remove phages in order to produce much more reliable viable counts.
Phage removal based on colony isolation would take several days to ensure that colonies are phage-free, whereas the procedure described here allows the generation of phage-free cultures in hours. This protocol permits us to follow the evolution of bacterial cultures without stopping them from isolating colonies. In this sense, it is possible to simulate fluctuating environments (presence and/or absence of phages) to test different hypotheses. Furthermore, this protocol permits the qualitative and quantitative analysis of the presence of phages in a bacterial culture.
In summary, designing cost-effective procedures for the removal of bacteriophages is crucial for maintaining product quality, safety, and process efficiency in various industries and for advances in basic and applied research. Here, we describe a highly effective protocol based on the use of LPS for the removal of bacteriophages, which use LPS as a receptor, from infected Salmonella cultures, which is both time-efficient and requires minimal equipment.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>20 mL syringe</td>
			<td>BD Discardit II</td>
			<td>300296</td>
			<td>No special requirements</td>
		</tr>
		<tr>
			<td>50 mL conical tubes</td>
			<td>Avantor</td>
			<td>525-0610</td>
			<td>No special requirements</td>
		</tr>
		<tr>
			<td>90 x 14 mm Petri dishes</td>
			<td>Deltalab</td>
			<td>200209</td>
			<td>No special requirements</td>
		</tr>
		<tr>
			<td>Agar</td>
			<td>Sigma-Aldrich</td>
			<td>A1296</td>
			<td>No special requirements</td>
		</tr>
		<tr>
			<td>Bacteriophage lysate</td>
			<td></td>
			<td></td>
			<td>Minimal concentration: 109 PFU/mL</td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>Eppendorf</td>
			<td></td>
			<td>No special requirements</td>
		</tr>
		<tr>
			<td>Chloroform</td>
			<td>Panreac</td>
			<td>131252</td>
			<td>No special requirements</td>
		</tr>
		<tr>
			<td>Citric acid &#183; H2O</td>
			<td>Merck</td>
			<td>1.00247</td>
			<td></td>
		</tr>
		<tr>
			<td>Colony counter</td>
			<td></td>
			<td></td>
			<td>No special requirements</td>
		</tr>
		<tr>
			<td>Evans Blue&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>E-2129</td>
			<td></td>
		</tr>
		<tr>
			<td>Flasks</td>
			<td></td>
			<td></td>
			<td>No special requirements</td>
		</tr>
		<tr>
			<td>Fluorescein sodium salt</td>
			<td>Sigma-Aldrich</td>
			<td>F-6377</td>
			<td></td>
		</tr>
		<tr>
			<td>Forceps</td>
			<td></td>
			<td></td>
			<td>No special requirements</td>
		</tr>
		<tr>
			<td>Glass tubes</td>
			<td></td>
			<td></td>
			<td>No special requirements</td>
		</tr>
		<tr>
			<td>Glass tubes for lysate&#160;</td>
			<td></td>
			<td></td>
			<td>No special requirements</td>
		</tr>
		<tr>
			<td>Glucose&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>G7021</td>
			<td></td>
		</tr>
		<tr>
			<td>K2HPO4</td>
			<td>Merck</td>
			<td>1.05104.1000</td>
			<td></td>
		</tr>
		<tr>
			<td>K2HPO4 anhydrous&#160;</td>
			<td>Merck</td>
			<td>1.05104</td>
			<td></td>
		</tr>
		<tr>
			<td>Lipopolysaccharide from Salmonella enterica serotype Typhimurium</td>
			<td>Sigma-Aldrich</td>
			<td>L6511-25 mg</td>
			<td>Dissolved in sterile water</td>
		</tr>
		<tr>
			<td>Membrane 0.45 &#181;m&#160;</td>
			<td>MF-Millipore</td>
			<td>HAWP02500</td>
			<td>No special requirements</td>
		</tr>
		<tr>
			<td>MgSO4 &#183; 7 H2O</td>
			<td>Merck</td>
			<td>1.05886</td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Sigma-Aldrich</td>
			<td>S9888</td>
			<td>No special requirements</td>
		</tr>
		<tr>
			<td>NaNH4HPO4 &#183; 4 H2O</td>
			<td>Sigma-Aldrich</td>
			<td>S9506</td>
			<td></td>
		</tr>
		<tr>
			<td>Peptone</td>
			<td>iNtRON</td>
			<td>Ba2001</td>
			<td>No special requirements</td>
		</tr>
		<tr>
			<td>Syringe Filter 0.22 &#181;m</td>
			<td>Millex</td>
			<td>SLGSR33SB</td>
			<td>No special requirements</td>
		</tr>
		<tr>
			<td>Toothpicks</td>
			<td></td>
			<td></td>
			<td>No special requirements</td>
		</tr>
		<tr>
			<td>Tryptone</td>
			<td>Panreac</td>
			<td>403682.1210</td>
			<td>No special requirements</td>
		</tr>
		<tr>
			<td>Vacuum pump</td>
			<td>Thermo Scientific</td>
			<td></td>
			<td>No special requirements</td>
		</tr>
		<tr>
			<td>Yeast extract</td>
			<td>iNtRON</td>
			<td>48045</td>
			<td>No special requirements</td>
		</tr>
	</tbody>
</table>

bacteriophage removal from infected salmonella cultures

Bacteriophages, or simply phages, play a vital role in microbial environments, impacting bacterial populations and shaping their evolution and interactions. These organisms are viruses that infect and replicate within bacterial hosts. Phages are ubiquitous on Earth, highly diverse, and very abundant. While bacteriophages have valuable roles in different environments and are a key area of research in microbiology and ecology, their presence can be undesirable in certain industrial processes or products. Considering the abundance and ubiquity of bacteriophages on Earth, the design of procedures for the removal of bacteriophages from bacterial cultures is crucial in diverse laboratory and industrial applications to preserve the integrity of the cultures and ensure accurate experimental results or product quality. Here, we have fine-tuned a protocol to eliminate the bacteriophages from infected Salmonella enterica cultures, using a strategy based on the use of lipopolysaccharides (LPS) located in the outer membrane of Gram-negative bacteria. Bacterial LPS plays an important role in host recognition by phages, and we make use of this property to design an effective procedure for the removal of phages, which use LPS as a receptor, in Salmonella bacterial cultures.

Salmonella enterica and other Gram-negative bacteria have an outer membrane containing LPS. The O-antigen of LPS is a commonly used receptor by bacteriophages 9NA to infect Salmonella cultures16,17.
Given the specific affinity of bacteriophages for the O-antigen or core polysaccharide regions of LPS, we wanted to examine whether Salmonella enterica commercial&#160;LPS could be used as a decoy to exclude 9NA bacteriophages. To do it, we mixed known concentrations of the commercial LPS and 9NA lysate, followed by a titration. Commercial LPS and 9NA bacteriophages were mixed in a total volume of 200 &#181;L and incubated for 2 h at 37 &#176;C without shaking. For titration, 100 &#181;L of the LPS-9NA mixture and 60 &#181;L of an overnight culture were added to 5 mL of LB soft agar and poured onto the top of an LB plate. Plates were incubated for 24 h at 37 &#176;C. As displayed in Figure 3, the lysate titer decreases proportionally when the concentration of S. enterica commercial LPS increases. These results indicate that the commercial LPS functions correctly as a decoy for 9NA bacteriophages.
Interestingly, the time required for phage 9NA to lyse Salmonella cells and release phages is facilitated by the number of bacteria and phages (Figure 4). In order to study how this aspect could affect the phage removal protocol, we tested different bacteria: phage ratios (1:1, 1:10, 1:100, and 1:1000). As seen in Figure 4, there is a drop in OD600 nm of the culture at 1.5-2.5 h after the addition of 9NA phage. OD600 nm values nearing zero are indicative that the bacterial culture is being lysed18. For this reason, incubation times in this protocol were defined as 2 h in order to ensure sufficient time for phage contained inside Salmonella cells to lyse bacteria and be released. This time should be estimated for each host-phage system prior to performing this protocol.
Once we determined that commercial LPS works as a decoy and the lysis time for 9NA infected bacteria, we performed the protocol described for cleaning infected Salmonella cultures of the bacteriophages (Figure 1 and Figure 2). In order to monitor the presence of bacteriophages along each step of the protocol, we did plaque assays to calculate the infectivity of the resulting cultures in 100 &#181;L of the resulting mixture at different points (Figure 5). We can observe that repeated washing and filtering are not sufficient for the elimination of bacteriophages from cultures (titration points 1-3); however, the number of phages decreases as soon as we employ a step of incubation with commercial LPS (titration point 4). The crucial step for the complete removal of bacteriophages in a bacterial culture is the second incubation with commercial LPS (titration point 6). This step is essential for successful removal of 9NA bacteriophage in Salmonella cultures.
An interesting aspect of this protocol would be to know the level of phage resistance after phage removal steps. A procedure that separates phages from phage-resistant bacteria has no utility. For this reason, it is crucial to reveal that the bacteria remaining in the culture is phage-susceptible. To demonstrate that susceptible-phage cells remain in the cultures after the procedure, we used Evans Blue Uranine (EBU) plate assay to screen for phage contamination19. EBU plates were made of LB medium supplemented with 10 mL/L K2HPO4 25%, 5 mL/L glucose 50%, 2.5 mL/L fluorescein 1%, 1.25 mL/L Evans Blue 1%, and 15 g/L agar. Cross-streaking on EBU plates with 9NA phage was used to discriminate phage-resistant and phage-sensitive isolates (Figure 6). The bacterial cultures obtained at the end of the cleaning protocol were used to get isolated colonies, which were checked for phage contamination (Figure 6B). We can observe the existence of both resistant and sensitive cells. This protocol does not favor the selection of phage-resistant cells; it only eliminates bacteriophages.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/66855/66855fig01.jpg" /> 
Figure 1: Brief outline of the procedure for elimination of bacteriophages in Salmonella cultures. The workflow is divided into different stages: preparation of bacterial culture and lysate, infection of the bacterial culture with bacteriophages, removal of bacteriophages from infected bacterial cultures, and preparation of a phage-free bacterial inoculum. <a href="https://www.jove.com/files/ftp_upload/66855/66855fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/66855/66855fig02.jpg" /> 
Figure 2: Procedure for bacteriophage removal from infected Salmonella enterica cultures. The process consists of three phases: 1) Removal of bacteriophages in suspension, 2) Removal of phages contained inside bacterial cells, and 3) Avoidance of reinfection. <a href="https://www.jove.com/files/ftp_upload/66855/66855fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/66855/66855fig03.jpg" /> 
Figure 3: LPS-decoy assay to measure the efficiency of Salmonella enterica commercial LPS to bind bacteriophages. Titration of a 9NA lysate (PFU/mL) at increasing concentrations of Salmonella enterica commercial LPS. The experiment was carried out in triplicate. Mean and standard deviation are presented. <a href="https://www.jove.com/files/ftp_upload/66855/66855fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/66855/66855fig04.jpg" /> 
Figure 4: Bacteriophage 9NA lysis time in Salmonella cultures. Growth curves of the Salmonella enterica cultures in the presence of bacteriophage 9NA at bacteria:phage ratios of 1:1, 1:10, 1:100, and 1:1000. The experiment was carried out in triplicate. Mean and standard deviation are presented. <a href="https://www.jove.com/files/ftp_upload/66855/66855fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/66855/66855fig05.jpg" /> 
Figure 5: Plaque assay for infectivity testing during bacteriophage removal in Salmonella cultures. (A) Titration of eight aliquots was taken at different points of the protocol (titration points 1-7 are marked in Figure 2). For this experiment, Salmonella enterica serovar&#160;Typhimurium strain ATCC 14028 opvAB::lacZ (SV8011) and bacteriophage 9NA were used. Experiments were performed in triplicate, and the mean and standard deviations are shown.&#160;(B)&#160;Soft agar plates with Salmonella enterica were obtained following the overlay technique using aliquots from eight titration points. The plates correspond from the left to the right with titration points 1-8. (C) Optical density at 600 nm of bacterial culture at different times of the phage-remove protocol. <a href="https://www.jove.com/files/ftp_upload/66855/66855fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/66855/66855fig06.jpg" /> 
Figure 6: Testing for phage-resistant bacteria after bacteriophage removal procedure. (A) Schematic diagram of a typical EBU agar plate used for the cross-streak agar assay: the vertical dark region at the center represents the zone of 9NA lysate. The dot represents the location where the tested cells are inoculated at a safe distance from the lysate zone, and the horizontal solid lines represent either the phage-resistant cells that are growing across the lysate zone or the phage-sensitive cells that are not growing beyond the lysate zone. (B) EBU plate assays for testing 11 colonies were obtained at the end of the removal protocol. Control R and S are examples of phage-resistant and phage-sensitive isolates, respectively. <a href="https://www.jove.com/files/ftp_upload/66855/66855fig06large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Author Spotlight: Efficiently Eliminating Bacteriophages from Infected Salmonella Cultures Using Lipopolysaccharides

Bacteriophages, ubiquitous and diverse on Earth, infect and replicate within bacterial hosts, playing a crucial role in microbial ecosystems. Despite their importance, their presence may disrupt industrial processes. We have developed a method using bacterial lipopolysaccharides to eliminate bacteriophages from Salmonella cultures.

Bacteriophage Removal from Infected Salmonella Cultures

Bacteriophages, or simply phages, play a vital role in microbial environments, impacting bacterial populations and shaping their evolution and ...

bacteriophage-removal-from-infected-salmonella-cultures

Research

JoVE Journal

Biology

723 Views.  Universidad de Sevilla. Bacteriophages, ubiquitous and diverse on Earth, infect and replicate within bacterial hosts, playing a crucial role in microbial ecosystems. Despite their importance, their presence may disrupt industrial processes. We have developed a method using bacterial lipopolysaccharides to eliminate bacteriophages from Salmonella cultures.

Video: Author Spotlight: Efficiently Eliminating Bacteriophages from Infected Salmonella Cultures Using Lipopolysaccharides

Primary Neuronal Cultures from the Brains of Late Stage Drosophila Pupae

In this video, we demonstrate the preparation of primary neuronal cultures from the brains of late stage Drosophila pupae. The procedure begins with the removal of brains from animals at 70-78 hrs after puparium formation. The isolated brains are shown after brief incubation in papain followed by several washes in serum-free growth medium. The process of mechanical dissociation of each brain in a 5 ul drop of media on a coverslip is illustrated. The axons and dendrites of the post-mitotic neurons are sheered off near the soma during dissociation but the neurons begin to regenerate processes within a few hours of plating. Images show live cultures at 2 days. Neurons continue to elaborate processes during the first week in culture. Specific neuronal populations can be identified in culture using GAL4 lines to drive tissue specific expression of fluorescent markers such as  GFP or RFP. Whole cell recordings have demonstrated the cultured neurons form functional, spontaneously active cholinergic and GABAergic synapses. A short video segment illustrates calcium dynamics in the cultured neurons using Fura-2 as a calcium indicator dye to monitor spontaneous calcium transients and nicotine evoked calcium responses in a dish of cultured neurons. These pupal brain cultures are a useful model system in which genetic and pharmacological tools can be used to identify intrinsic and extrinsic factors that influence formation and function of central synapses.

This video demonstrates the preparation of primary neuronal cultures from the brains of late stage Drosophila pupae. Views of live cultures show neurite outgrowth and imaging of calcium levels using Fura-2.

In this video, we demonstrate the preparation of primary neuronal cultures from the brains of late stage Drosophila pupae. The procedure begins with the ...

Preparation of Neuronal Cultures from Midgastrula Stage Drosophila Embryos

This video illustrates the procedure for making primary neuronal cultures from midgastrula stage Drosophila embryos. The methods for collecting embryos and their dechorionation using bleach are demonstrated. Using a glass pipet attached to a mouth suction tube, we illustrate the removal of all cells from single embryos. The method for dispersing cells from each embyro into a small (5 l) drop of medium on an uncoated glass coverslip is demonstrated. A view through the microscope at 1 hour after plating illustrates the preferred cell density. Most of the cells that survive when grown in defined medium are neuroblasts that divide one or more times in culture before extending neuritic processes by 12-24 hours. A view through the microscope illustrates the level of neurite outgrowth and branching expected in a healthy culture at 2 days in vitro. The cultures are grown in a simple bicarbonate based defined medium, in a 5% CO2 incubator at 22-24&deg;C. Neuritic processes continue to elaborate over the first week in culture and when they make contact with neurites from neighboring cells they often form functional synaptic connections. Neurons in these cultures express voltage-gated sodium, calcium, and potassium channels and are electrically excitable. This culture system is useful for studying molecular genetic and environmental factors that regulate neuronal differentiation, excitability, and synapse formation/function.

This video demonstrates the preparation of primary neuronal cultures from midgastrula stage Drosophila embryos. Views of live cultures show cells 1 hour after plating and differentiated neurons after 2 days of growth in a bicarbonate-based defined medium. The neurons are electrically excitable and form synaptic connections.

This video illustrates the procedure for making primary neuronal cultures from midgastrula stage Drosophila embryos. The methods for collecting embryos ...

Primary Dissociated Midbrain Dopamine Cell Cultures from Rodent Neonates

The ability to create primary cell cultures of dopamine neurons allows for the study of the presynaptic characteristics of dopamine neurons in isolation from systemic input from elsewhere in the brain. In our lab, we use these neurons to assess dopamine release kinetics using carbon fiber amperometry, as well as expression levels of dopamine related genes and proteins using quantitative PCR and immunocytochemistry. In this video, we show you how we generate these cultures from rodent neonates.
The process involves several steps, including the plating of cortical glial astrocytes, the conditioning of neuronal cell culture media by the glial substrate, the dissection of the midbrain in neonates, the digestion, extraction and plating of dopamine neurons and the addition of neurotrophic factors to ensure cell survival.
The applications suitable for such a preparation include electrophysiology, immunocytochemistry, quantitative PCR, video microscopy (i.e., of real-time vesicular fusion with the plasma membrane), cell viability assays and other toxicological screens.

Primary dissociated midbrain dopamine cell cultures allow for the study of presynaptic characteristics of dopamine neurons. They can be used to monitor real-time dopamine release kinetics and protein/mRNA levels of regulators of dopamine exocytosis. Here, we show you how to generate these cultures from rodent neonates.

The ability to create primary cell cultures of dopamine neurons allows for the study of the presynaptic characteristics of dopamine neurons in isolation ...

Toxin Induction and Protein Extraction from Fusarium spp. Cultures for Proteomic Studies

Fusaria are filamentous fungi able to produce different toxins. Fusarium mycotoxins such as deoxynivalenol, nivalenol, T2, zearelenone, fusaric acid, moniliformin, etc... have adverse effects on both human and animal health and some are considered as pathogenicity factors. Proteomic studies showed to be effective for deciphering toxin production mechanisms (Taylor et al., 2008) as well as for identifying potential pathogenic factors (Paper et al., 2007, Houterman et al., 2007) in Fusaria. It becomes therefore fundamental to establish reliable methods for comparing between proteomic studies in order to rely on true differences found in protein expression among experiments, strains and laboratories. The procedure that will be described should contribute to an increased level of standardization of proteomic procedures by two ways. The filmed protocol is used to increase the level of details that can be described precisely. Moreover, the availability of standardized procedures to process biological replicates should guarantee a higher robustness of data, taking into account also the human factor within the technical reproducibility of the extraction procedure.
The protocol described requires 16 days for its completion: fourteen days for cultures and two days for protein extraction (figure 1). 
Briefly, Fusarium strains are grown on solid media for 4 days; they are then manually fragmented and transferred into a modified toxin inducing media (Jiao et al., 2008) for 10 days. Mycelium is collected by filtration through a Miracloth layer. Grinding is performed in a cold chamber. Different operators performed extraction replicates (n=3) in order to take into account the bias due to technical variations (figure 2). Extraction was based on a SDS/DTT buffer as described in Taylor et al. (2008) with slight modifications. Total protein extraction required a precipitation process of the proteins using Aceton/TCA/DTT buffer overnight and Acetone /DTT washing (figure 3a,3b). Proteins were finally resolubilized in the protein-labelling buffer and quantified. Results of the extraction were visualized on a 1D gel (Figure 4, SDS-PAGE), before proceeding to 2D gels (IEF/SDS-PAGE). The same procedure can be applied for proteomic analyses on other growing media and other filamentous fungi (Miles et al., 2007).

Toxin Induction and Protein Extraction from Fusarium spp. Cultures for Proteomic Studies

Protein extraction for proteomic analyses in fungal species requires high levels of standardization to be accomplished according with the minimum information about a proteomic experiment (MIAPE) guidelines. We present a video-protocol that includes a procedure for minimizing experimental bias during toxin induction and protein extraction from Fusarium spp.

Fusaria are filamentous fungi able to produce different toxins. Fusarium mycotoxins such as deoxynivalenol, nivalenol, T2, zearelenone, fusaric acid, ...

Chronic Salmonella Infected Mouse Model

The bacterial infected mouse model is a powerful model system for studying areas such as infection, inflammation, immunology, signal transduction, and tumorigenesis. Many researchers have taken advantage of the colitis induced by Salmonella typhimurium for the studies on the early phase of inflammation and infection. However, only few reports are on the chronic infection in vivo. Mice with Salmonella persistent existence in the gastrointestinal tract allow us to explore the long-term host-bacterial interaction, signal transduction, and tumorigenesis. We have established a chronic bacterial infected mouse model with Salmonella typhimurium colonization in the mouse intestine over 6 months. To use this system, it is necessary for the researcher to learn how to prepare the bacterial culture and gavage the animals. We detail a methodology for prepare bacterial culture and gavage mice. We also show how to detect the Salmonella persistence in the gastrointestinal tract. Overall, this protocol will aid researchers using the bacterial infected mouse model to address fundamentally important biological and microbiological questions.

Chronic Salmonella Infected Mouse Model

Establish a chronic bacterial infected mouse model with persistent Salmonella typhimurium colonization in intestine for 27 weeks.

The bacterial infected mouse model is a powerful model system for studying areas such as infection, inflammation, immunology, signal transduction, and ...

Establishing Primary Adult Fibroblast Cultures From Rodents

The importance of using primary cells, rather than cancer cell lines, for biological studies is becoming widely recognized. Primary cells are preferred in studies of cell cycle control, apoptosis, and DNA repair, as cancer cells carry mutations in genes involved in these processes. Primary cells cannot be cultured indefinitely due to the onset of replicative senescence or aneuploidization. Hence, new cultures need to be established regularly. The procedure for isolation of rodent embryonic fibroblasts is well established, but isolating adult fibroblast cultures often presents a challenge. Adult rodent fibroblasts isolated from mouse models of human disease may be a preferred control when comparing them to fibroblasts from human patients. Furthermore, adult fibroblasts are the only available material when working with wild rodents where pregnant females cannot be easily obtained. Here we provide a protocol for isolation and culture of adult fibroblasts from rodent skin and lungs. We used this procedure successfully to isolate fibroblasts from over twenty rodent species from laboratory mice and rats to wild rodents such as beaver, porcupine, and squirrel.

This article describes a protocol for isolation and maintenance of primary fibroblast cultures from skin and lung tissue of wild rodents.

The importance of using primary cells, rather than cancer cell lines, for biological studies is becoming widely recognized.  Primary cells are preferred ...

Primary Cell Cultures from Drosophila Gastrula Embryos

Here we describe a method for preparing and culturing primary cells dissociated from Drosophila gastrula embryos. In brief, a large amount of staged embryos from young and healthy flies are collected, sterilized, and then physically dissociated into a single cell suspension using a glass homogenizer. After being plated on culture plates or chamber slides at an appropriate density in culture medium, these cells can further differentiate into several morphologically-distinct cell types, which can be identified by their specific cell markers. Furthermore, we present conditions for treating these cells with double stranded (ds) RNAs to elicit gene knockdown. Efficient RNAi in Drosophila primary cells is accomplished by simply bathing the cells in dsRNA-containing culture medium. The ability to carry out effective RNAi perturbation, together with other molecular, biochemical, cell imaging analyses, will allow a variety of questions to be answered in Drosophila primary cells, especially those related to differentiated muscle and neuronal cells.

Primary Cell Cultures from Drosophila Gastrula Embryos

We provide a detailed protocol for preparing primary cells dissociated from Drosophila embryos. The ability to carry out the effective RNAi perturbation, together with other molecular, biochemical and cell imaging methods will allow a variety of questions to be addressed in Drosophila primary cells.

Here we describe a method for preparing and culturing primary cells dissociated from Drosophila gastrula embryos.   In brief, a large amount of staged ...

Streamlined Purification of Plasmid DNA From Prokaryotic Cultures

We describe the complete process of AcroPrep Advance Filter Plates for 96 plasmid preparations, starting from prokaryotic culture and ending with high purity DNA. Based on multi-well filtration for bacterial lysate clearance and DNA purification, this method creates a streamlined process for plasmid preparation. Filter plates containing silica-based media can easily be processed by vacuum filtration or centrifuge to yield appreciable quantities of plasmid DNA. Quantitative analyses determine the purified plasmid DNA is consistently of high quality with average OD260/280 ratios of 1.97. Overall, plasmid yields offer more pure DNA for downstream applications, such as sequencing and cloning. This streamlined method of using AcroPrep Advance Filter Plates allows for manual, semi-automated or fully-automated processing.

This protocol is a cost effective alternative for efficient parallel clarification and plasmid DNA purification from E. coli cultures. The AcroPrep Advance process starts with an optimized lysate clarification filter plate followed by purification on a high binding capacity DNA binding filter plate.

We describe the complete process of AcroPrep Advance Filter Plates for 96 plasmid preparations, starting from prokaryotic culture and ending with high ...

Rapid Fibroblast Removal from High Density Human Embryonic Stem Cell Cultures

Mouse embryonic fibroblasts (MEFs) were used to establish human embryonic stem cells (hESCs) cultures after blastocyst isolation1. This feeder system maintains hESCs from undergoing spontaneous differentiation during cell expansion. However, this co-culture method is labor intensive, requires highly trained personnel, and yields low hESC purity4. Many laboratories have attempted to minimize the number of feeder cells in hESC cultures (i.e. incorporating matrix-coated dishes or other feeder cell types5-8). These modified culture systems have shown some promise, but have not supplanted the standard method for culturing hESCs with mitomycin C-treated mouse embyronic fibroblasts in order to retard unwanted spontaneous differentiation of the hESC cultures. Therefore, the feeder cells used in hESC expansion should be removed during differentiation experiments. Although several techniques are available for purifying the hESC colonies (FACS, MACS, or use of drug resistant vectors) from feeders, these techniques are labor intensive, costly and/or destructive to the hESC. The aim of this project was to invent a method of purification that enables the harvesting of a purer population of hESCs. We have observed that in a confluent hESC culture, the MEF population can be removed using a simple and rapid aspiration of the MEF sheet. This removal is dependent on several factors, including lateral cell-to-cell binding of MEFs that have a lower binding affinity to the styrene culture dish, and the ability of the stem cell colonies to push the fibroblasts outward during the generation of their own "niche". The hESC were then examined for SSEA-4, Oct3/4 and Tra 1-81 expression up to 10 days after MEF removal to ensure maintenance of pluripotency. Moreover, hESC colonies were able to continue growing from into larger formations after MEF removal, providing an additional level of hESC expansion.

Despite ongoing efforts to transition cultures to feeder-free conditions, the derivation and culture of human embryonic stem cells (hESC) remain largely dependent on co-cultures with mouse embryonic feeders (MEFs). Here, we show a novel methodology for rapidly removing feeders from hESC cultures prior to experimentation.

Mouse embryonic fibroblasts (MEFs) were used to establish human embryonic stem cells (hESCs) cultures after blastocyst isolation1. This feeder system ...

Na&iuml;ve Adult Stem Cells Isolation from Primary Human Fibroblast Cultures

Over the last decade, several adult stem cell populations have been identified in human skin 1-4. The isolation of multipotent adult dermal precursors was first reported by Miller F. D laboratory 5, 6. These early studies described a multipotent precursor cell population from adult mammalian dermis 5. These cells--termed SKPs, for skin-derived precursors-- were isolated and expanded from rodent and human skin and differentiated into both neural and mesodermal progeny, including cell types never found in skin, such as neurons 5. Immunocytochemical studies on cultured SKPs revealed that cells expressed vimentin and nestin, an intermediate filament protein expressed in neural and skeletal muscle precursors, in addition to fibronectin and multipotent stem cell markers 6. Until now, the adult stem cells population SKPs have been isolated from freshly collected mammalian skin biopsies.
Recently, we have established and reported that a population of skin derived precursor cells could remain present in primary fibroblast cultures established from skin biopsies 7. The assumption that a few somatic stem cells might reside in primary fibroblast cultures at early population doublings was based upon the following observations: (1) SKPs and primary fibroblast cultures are derived from the dermis, and therefore a small number of SKP cells could remain present in primary dermal fibroblast cultures and (2) primary fibroblast cultures grown from frozen aliquots that have been subjected to unfavorable temperature during storage or transfer contained a small number of cells that remained viable 7. These rare cells were able to expand and could be passaged several times. This observation suggested that a small number of cells with high proliferation potency and resistance to stress were present in human fibroblast cultures 7.
We took advantage of these findings to establish a protocol for rapid isolation of adult stem cells from primary fibroblast cultures that are readily available from tissue banks around the world (Figure 1). This method has important significance as it allows the isolation of precursor cells when skin samples are not accessible while fibroblast cultures may be available from tissue banks, thus, opening new opportunities to dissect the molecular mechanisms underlying rare genetic diseases as well as modeling diseases in a dish.

Na&#239;ve Adult Stem Cells Isolation from Primary Human Fibroblast Cultures

We report a method to isolate na&#239;ve multipotent skin-derived precursor (SKP) cells from primary human fibroblast cultures. We show that these SKPs derived from fibroblast cultures share similar stem cell properties to the ones derived directly from human skin biopsies. These cells express the neural crest marker, nestin, in addition to the multipotent markers such as OCT4 and Nanog.

Over the last decade, several adult stem cell  populations have been identified in human skin 1-4.  The isolation of multipotent adult dermal precursors ...

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