Name: separating bacteria by capsule amount using a discontinuous density gradient
Uploaded: 2019-01-07T14:00:00.000+00:00
Duration: 5 min 52 s
Description: Watch this Scientific Journal Video about Separating Bacteria by Capsule Amount Using a Discontinuous Density Gradient at JoVE.com

We thank Jin-Town Wang and Susannah Salter for supplying strains, and members of the Parkhill group for helpful discussions. This work was funded by the Wellcome Sanger Institute (Wellcome grant 206194), and by a Sir Henry Wellcome postdoctoral fellowship to F.L.S. (grant 106063/A/14/Z). M.J.D. is supported by a Wellcome Sanger Institute PhD Studentship.

NOTE: Ensure that any risk assessments applicable to the bacterial strains are adhered to when culturing and handling samples. Be aware that setting up too many gradients at one time can lead to musculoskeletal disorders due to the pressure on joints from the slow pipetting involved. Plan work and take precautions to avoid injury.
1. Preparation of Bacterial Strains or Mutant Libraries
<ol>
	<li>Streak out the strains to be tested on appropriate agar plates. These are stock plates for the experiment.
	<ol>
		<li>Incubate the plates overnight at the desired temperature to achieve single colonies. For this experiment, culture K. pneumoniae (NTUH-K2044 and ATCC43816 strains) on Luria broth (LB) agar at 37 &#176;C, and S. pneumoniae (23F wild type and 23F &#916;cps) on blood agar in a humidified candle jar at 37 &#176;C.</li>
	</ol>
	</li>
	<li>Pick a single colony from a stock plate (step 1.1) to inoculate 10 mL of appropriate broth using a sterile loop or cocktail stick. For screening of random mutant libraries, inoculate the broth with 10 &#181;L of the random mutant library stock (TraDIS Library).
	<ol>
		<li>Incubate K. pneumoniae strains in low salt LB media at 37 &#176;C with shaking, and S. pneumoniae strains in brain heart infusion (BHI) media, statically at 37 &#176;C.</li>
		<li>Transfer the overnight culture to a 15 mL tube and centrifuge in a bench-top centrifuge for 10 min at 3,200 x g in swing out buckets with 15 mL tube inserts and aerosol tight lids.</li>
		<li>Discard the supernatant and resuspend the pellet in 2 mL of 1x phosphate-buffered saline (PBS). 
		NOTE: Dispose of supernatant via the appropriate liquid biological waste route in the laboratory. The purpose of the centrifugation and resuspension steps (1.2.2 and 1.2.3) is to concentrate the bacteria for easy visualization on the gradient. Bacterial cultures can be loaded directly onto the gradient if preferred. Heavily capsulated strains may not form a tight pellet. If this occurs, remove as much supernatant as possible without removing any bacterial cells, add 1x PBS to a final volume of 5 mL and resuspend the pellet. Continue the protocol with step 1.2.5.</li>
		<li>For non-mucoid strains, go to step 2.</li>
		<li>Centrifuge the tubes as described in step 1.2.2.</li>
		<li>Discard the supernatant and resuspend the pellet in 2 mL of 1x PBS. Cells are now ready to use. 
		NOTE: The density of the bacterial cell suspension is not critical but needs to be sufficient for visualization in the gradient. A minimum OD600 (optical density at 600 nm) of 4 is suggested.</li>
	</ol>
	</li>
</ol>
2. Preparation of Gradient Dilutions and Mini-gradient Test
<ol>
	<li>Prepare gradient dilutions. 
	NOTE: Exact concentrations of density gradient medium (e.g., Percoll) needed in the density gradients to achieve good separation will differ depending on the bacterial strain and growth conditions used. Mini-gradient tests are performed first, to identify the concentrations that will give the best separation. These comprise 500 &#181;L of a single dilution in a 2 mL tube. If bacteria will be extracted from the gradient and subcultured for downstream applications, these steps should be performed under aseptic conditions.
	<ol>
		<li>Combine density gradient medium with 1x PBS to make the density gradient dilutions. Make dilutions of 20%, 30%, 40%, 50%, 60%, 70%, and 80% (e.g., 2 mL of density gradient medium plus 8 mL of 1x PBS = 10 mL of 20% density gradient medium).</li>
		<li>Aliquot 500 &#181;L of the 20% gradient dilution into a 2 mL tube for each strain to be tested (e.g., 4 strains = 4 tubes containing 500 &#181;L of 20% gradient dilution).</li>
		<li>Repeat step 2.1.2 for the rest of the gradient dilutions.</li>
	</ol>
	</li>
	<li>Apply bacteria to the gradient.
	<ol>
		<li>Apply 100 &#181;L of bacterial cells prepared in steps 1.2.3 and 1.2.6 to the top of each gradient dilution following the steps below.
		<ol>
			<li>Take up 100 &#181;L of cells using a 200 &#181;L pipette and place the pipette tip on the side of the tube just below the meniscus of the mini-gradient.</li>
			<li>Aspirate the bacterial cells onto the gradient extremely slowly so that they form a layer on the top of the gradient, without any mixing of the interface.</li>
			<li>Repeat steps 2.2.1.1&#8211;2.2.1.2 for all strains to be tested.</li>
		</ol>
		</li>
		<li>Using a fixed angle rotor with an aerosol tight lid, centrifuge the prepared tubes in a microcentrifuge for 10 min at 8,000 x g.</li>
		<li>After centrifugation, transfer the tubes to a rack to visualize the minimum gradient dilution required to retain cells just above the gradient layer following centrifugation.</li>
		<li>If the results are not clear, repeat the mini-gradient test with increments of 5% density gradient medium dilutions above and below the concentrations defined in step 2.1.1 (e.g., 25% and 35% dilutions should be tested if the result at 30% is ambiguous). 
		NOTE: See Figure 2A for typical results at a single density gradient medium dilution.</li>
		<li>Use the results from steps 2.2.3&#8211;2.2.4 to determine the ideal gradient dilutions to use to separate cells in larger-scale discontinuous gradients.</li>
	</ol>
	</li>
</ol>
3. Preparation of Cells for the Main Experiment
<ol>
	<li>Prepare fresh overnight cultures by inoculating 10 mL of appropriate broth as described in step 1.2.
	<ol>
		<li>Incubate the cultures overnight in appropriate conditions as described in step 1.2.1.</li>
		<li>Pellet the overnight culture as described in step 1.2.2.</li>
	</ol>
	</li>
	<li>Wash the cells as described in step 1.2.3.</li>
	<li>Discard the supernatant and resuspend the pellet in 1 mL of 1x PBS. If the strain is mucoid and does not pellet easily, resuspend the pellet in up to 2 mL of PBS or residual media. Cells are now ready to use.</li>
</ol>
4. Preparation of Discontinuous Density Gradients
NOTE: An alternative method of gradient preparation from bottom (most concentrated) to top (least concentrated) using a pipette is described in step 7.
<ol>
	<li>Pipette 1 mL of the most dilute density gradient dilution into a 5 mL polypropylene round bottom tube to form the top, most dilute, layer of the gradient. 
	NOTE: Subsequent layers of more concentrated gradient dilutions will be added below this layer using a needle, so as not to disrupt the layers. A minimum of two and maximum of three gradient layers of 1 mL each are recommended for robust separation and easy visualization of bacteria.
	<ol>
		<li>Using a 1 mL disposable syringe with a 1.5 inch needle attached, take 1 mL of the next most concentrated density gradient medium dilution into the syringe. Avoid taking up any air, as bubbles can disrupt the gradient layers.</li>
		<li>Place the needle end at the bottom of the tube containing the first layer. Aspirate the syringe contents very slowly to avoid mixing the interface. 
		NOTE: The interface of the two dilutions will rise as the more concentrated gradient dilution is added. Observe the interface by holding it up to light or an outside window. If no distinct interface is observed, discard the gradient and start again.
		<ol>
			<li>Remove the needle from the gradient very gently so as not to disturb the interface between the different gradient dilutions. Place the tube in a suitable rack to keep it upright.</li>
		</ol>
		</li>
		<li>If using three different dilutions, repeat steps 4.1.1&#8211;4.1.2.1 so that the densest dilution is at the bottom. If the gradient has been constructed successfully, there will be three distinct layers with no mixing at the interfaces.</li>
	</ol>
	</li>
</ol>
5. Adding Prepared Cells to Gradients and Separation by Centrifugation
<ol>
	<li>Add 600 &#181;L of prepared cells from step 3.3 to the top of the gradient in the tube very slowly and without mixing the interface, as described in step 2.2.</li>
	<li>Place the tubes in tube adapters and weigh the combined adapters and tubes to ensure they are balanced.
	<ol>
		<li>Place the tube adapters in a fixed angle rotor within a bench-top centrifuge. Centrifuge for 30 min at 3,000 x g.</li>
		<li>After centrifugation, carefully remove the tubes, place them in a suitable rack, and photograph the results as a record.</li>
	</ol>
	</li>
	<li>Recover the bacterial fractions for uronic acid quantification or DNA extraction by following step 6.
	<ol>
		<li>If downstream applications are not required, dispose of the samples/gradients via the appropriate liquid biological waste route in the laboratory. 
		NOTE: It is important to validate the separation for new species/strains of bacteria. Individual fractions from the gradient should be examined by microscopy, by uronic acid assay (step 8), or another suitable quantitative assay to confirm the capsule phenotype of each fraction. If the aim is to separate based on aggregation or cell size, independent appropriate assays should be used.</li>
	</ol>
	</li>
</ol>
6. Recovering Sample Fractions and Optional Outgrowth Step
<ol>
	<li>Recover the fractions from a gradient by removing and discarding any liquid from the top dilution if no bacterial fraction is present within that layer.
	<ol>
		<li>To remove the top fraction, use a P200 pipette and gently pass the pipette tip through the gradient to the fraction, and take up the fraction. Place the fraction into a 1.5 mL tube and label appropriately.</li>
		<li>To recover further fractions, still using the P200 pipette, insert the tip gently through the gradient to the fraction and recover the fraction to a fresh 1.5 mL tube. Remove excess gradient as the fractions lower down the gradient are accessed.</li>
		<li>If DNA extraction from a fraction containing very low cell numbers is required (e.g., for density-TraDISort), subculture the fraction by following the protocol from step 6.2 for optional outgrowth to obtain more cells. 
		NOTE: There will be a low level of carryover from cells at the top of the gradient into lower fractions, as fractions are removed from top to bottom. Minimize this by working carefully so as not to mix the gradient, by using a needle to remove lower fractions, and by performing re-purification of very low abundance samples where carryover may affect results.</li>
	</ol>
	</li>
	<li>Transfer cells from the low-abundance fraction recovered in steps 6.1.1&#8211;6.1.2 into 5 mL of appropriate liquid media. Place in an incubator and grow for 2 h at 37 &#176;C.
	<ol>
		<li>After 2 h of growth or when the sample has reached an OD600 of 1, transfer the culture to a 15 mL tube. Centrifuge the tube for 10 min at 3,200 x g in a centrifuge with swing out buckets and aerosol tight lids. Discard the supernatant</li>
		<li>Resuspend the cell pellet in 1 mL of 1x PBS.</li>
	</ol>
	</li>
	<li>Prepare a fresh single-concentration density gradient in a 5 mL polypropylene tube. Use the gradient concentration from just above the location of the fraction in the original gradient (e.g., for purification of the &#916;slyA mutant shown in Figure 2D, 15% density gradient medium would be used). 
	NOTE: This re-purification step is optional.
	<ol>
		<li>Apply the cells to the top of the gradient and centrifuge as described in steps 2.2 and 5.1.</li>
		<li>Remove the tubes from the centrifuge and place in an appropriate rack. Photograph the gradient and recover the fraction for DNA extraction as described in 6.1&#8211;6.1.2. 
		NOTE: The sample is now ready for DNA extraction or other downstream applications.</li>
	</ol>
	</li>
</ol>
7. Alternative Method for Gradient Preparation from Bottom (Most Concentrated) to Top (Least Concentrated) Using a Pipette
<ol>
	<li>Use the gradient dilutions determined in step 2.2.3. Using a 1,000 &#181;L pipette, add 1 mL of the densest gradient dilution to a 5 mL tube.
	<ol>
		<li>Using a 200 &#181;L pipette, add 200 &#181;L of the next dense gradient dilution very slowly to the top of the layer in the tube, so as not to mix the interface. Add another 200 &#181;L and then the final 600 &#181;L. Adding multiple smaller volumes gives more control over pipetting speed and prevents mixing of the interface. If the interface mixes, discard and start again.</li>
		<li>Repeat step 7.1.1 with the third, least dense gradient concentration if three dilutions are being used. The tube should now have gradients of either 2 mL or 3 mL depending on the results from step 2.2.3.</li>
		<li>Go to step 5 of the protocol to add cells and continue the experiment.</li>
	</ol>
	</li>
</ol>
8. Measurement of Capsule Amount by Uronic Acid Assay
<ol>
	<li>Adjust the OD600 of each recovered fraction to 4.0 by dilution with PBS. Proceed with quantification of uronic acids as previously published1.</li>
</ol>

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The authors have no financial interests to disclose.

Capsule is an important virulence factor in many bacterial species including K. pneumoniae3, Streptococcus pneumoniae9, Acinetobacter10, and Neisseria11 species. Although various methods exist for quantification and visualization of bacterial capsules, at present there is no widely used method to physically separate capsulated and non-capsulated cells. In this article, we have demonstrated a robust method for capsule-based separation of bacterial populations, with multiple potential applications in conjunction with different upstream or downstream protocols.
The presence of a surface capsule can reduce bacterial cell density, which allows separation by density gradient centrifugation (Figure 2D). We have validated this method in K. pneumoniae NTUH-K204412 and ATCC4381613 as well as in Streptococcus pneumoniae 23F14 and its &#916;cps mutant15. This method uses Percoll16 as the main constituent of the density gradient, which is a suspension of coated colloidal silica particles that has low viscosity and no toxicity towards bacteria - in principle, other substances meeting these criteria could be used to establish the density gradient.
It can be challenging to ensure that layers of different density do not mix when constructing density gradients, and if mixing does occur, the separation method will not give clean results. We have included two alternative methods for pouring the gradients, using a needle or a pipette &#8212; both are effective, and which method to use is simply a matter of preference. For all steps that involve pipetting a substance (either a bacterial suspension, or a more dilute gradient layer) above a gradient layer, pipetting multiple aliquots of smaller volumes can make it easier to achieve a sharp interface without any mixing of layers.
A limitation of this protocol is that its performance with other bacterial species cannot be guaranteed. Therefore, it is critical when examining a new bacterial species or strain to validate the density-based separation using an additional, independent capsule quantification method. Visualizing the bacteria present in each fraction by microscopy with appropriate capsule stains is a reliable method for which detailed protocols are available17. Alternatively, capsules containing uronic acids (such as those of Escherichia coli and K. pneumoniae) can be quantified by a specific assay as shown in Figure 2B1. The centrifugation-based mucoviscosity test is not suitable as an independent validation method, as this assay also depends on the density of the bacterial cells.
Another limitation of this method is that capsule production is very sensitive to culture conditions, and even small changes to growth medium, temperature, or aeration may affect the results of this assay. To minimize this issue, researchers can use a defined growth medium or a batch-consistent complex medium, keep all other growth parameters identical between experiments, and include appropriate control strains to enable the interpretation of unexpected results. Some bacterial capsules are fragile and can shear away from the cell when cultures are pipetted. To avoid the shearing of capsules, cultures should be centrifuged and resuspended no more than twice during preparation for loading on the gradient. If loss of capsule during concentration of the cultures remains problematic, bacterial cultures can be applied to a density gradient directly, with a larger volume of bacterial suspension added if necessary for visualization.
Future applications of this method are to apply it to other bacterial species, and to use this separation in conjunction with different upstream and downstream technologies. In addition to density-TraDISort8, we suggest that density gradient separation of capsulated bacteria could be used for isolation of mutants with altered capsule, for purification of capsulated cells from mixed cultures or complex samples, and for rapid profiling of capsule production in multiple strains. Finally, this technology could be used to examine other bacterial phenotypes such as aggregation.

Many bacterial species produce a polysaccharide capsule, which protects the bacterial cell from various physical stresses and from recognition and killing by the immune system. In Klebsiella pneumoniae, capsule production is an absolute requirement for infection1,2. K. pneumoniae capsule mediates resistance to antimicrobial peptides, resistance to complement-mediated killing, prevention of phagocytosis, and suppression of the innate immune response3. Excess capsule production is associated with increased virulence and community-acquired (rather than nosocomial) infections4.
A range of quantitative and qualitative tests are available to investigate capsule production. For Klebsiella species, these include the string test5, in which a toothpick touched to a colony is pulled upwards and the length of the string produced measured, and the mucoviscosity assay6, which involves the slow centrifugation of a culture followed by measuring the optical density of the supernatant. These methods are simple and quick, but lack sensitivity when used on classical Klebsiella strains rather than capsule overproducing strains. Another method of capsule quantification is the uronic acid assay, which is technically challenging and requires the use of concentrated sulfuric acid1. Finally, capsule is visible directly by microscopy (Figure 1A). Of these methods, only microscopy allows the user to observe different capsulation states within a single population, and none of these methods enables the physical separation of capsulated and non-capsulated bacteria.
Density-based separations by gradient centrifugation are routinely used in cell biology to purify different eukaryotic cell types7, but are rarely used in microbiological research. The mucoviscosity assay for Klebsiella is based on the observation that highly capsulated bacteria take more time to pellet by centrifugation, and we reasoned that this may be due to reduced overall density of capsulated cells. The method shown here was developed to separate K. pneumoniae populations physically by capsule amount, using density gradient centrifugation (Figure 1). This method was applied successfully to Streptococcus pneumoniae, indicating that it is applicable to other bacterial species. Density-gradient separation of a saturated transposon mutant library coupled with transposon-insertion sequencing (density-TraDISort) has been used to identify genes involved in the capsule production and regulation8. Similarly, this method was used in conjunction with random-prime polymerase chain reaction (PCR) of individual colonies to isolate non-capsulated K. pneumoniae mutants. This method can also be used for rapid comparisons of capsule production between different populations and conditions, or to purify capsulated bacteria from complex samples (Figure 1B). Finally, there is the option to assay other phenotypes that affect density, such as cell size or aggregation.
This manuscript demonstrates how to optimize the procedure for a new bacterial species or strain and demonstrates the construction and running of a discontinuous density gradient to separate hyper-capsulated, capsulated and non-capsulated bacteria.

<table><tbody><tr><td>Percoll</td><td>GE Healthcare</td><td>17-0891-01</td><td></td></tr><tr><td>Centrifuge 5810R with Rotor A-4-81 and 500 mL buckets</td><td>Eppendorf</td><td>5810 718.007</td><td></td></tr><tr><td>Adapters for 15 mL tubes</td><td>Eppendorf</td><td>5810 722.004</td><td></td></tr><tr><td>Fixed andgle rotor F-34-6-38</td><td>Eppendorf</td><td>5804 727.002</td><td></td></tr><tr><td>2.6 to 7 mL tube adapter</td><td>Eppendorf</td><td>5804 739.000</td><td></td></tr><tr><td>Centrifuge 5424 including Rotor FA-45-24-11</td><td>Eppendorf</td><td>5424 000.460</td><td></td></tr><tr><td>2 mL tubes</td><td>Eppendorf</td><td>0030 120.094</td><td></td></tr><tr><td>1.5 mL tubes</td><td>Eppendorf</td><td>0030 120.086</td><td></td></tr><tr><td>5 mL polypropylene round bottom tube</td><td>Falcon</td><td>352063</td><td></td></tr><tr><td>1 mL disposable syringe Luer slip</td><td>Becton Dickinson</td><td>300013</td><td></td></tr><tr><td>AGANI Needle 21G Green x 1.5&quot;</td><td>Terumo</td><td>AN 2138R1</td><td></td></tr><tr><td>P1000 pipette and tips</td><td></td><td></td><td></td></tr><tr><td>P200 pipette and tips</td><td></td><td></td><td></td></tr></tbody></table>

separating bacteria by capsule amount using a discontinuous density gradient

Capsule is a key virulence factor in many bacterial species, mediating immune evasion and resistance to various physical stresses. While many methods are available to quantify and compare capsule production between different strains or mutants, there is no widely used method for sorting bacteria based on how much capsule they produce. We have developed a method to separate bacteria by capsule amount, using a discontinuous density gradient. This method is used to compare capsule amounts semi-quantitatively between cultures, to isolate mutants with altered capsule production, and to purify capsulated bacteria from complex samples. This method can also be coupled with transposon-insertion sequencing to identify genes involved in capsule regulation. Here, the method is demonstrated in detail, including how to optimize the gradient conditions for a new bacterial species or strain, and how to construct and run the density gradient.

Representative results are shown in Figure 2. The exact result to expect will depend on the bacterial species, the set-up of the density gradients, and whether the user is examining a single strain or a pool of mutants. Most strains will migrate to a single location within a gradient, as shown in Figure&#160;2A and 2D. Applying the method to a bacterial mutant library will give rise to a major band above the gradient, a less dense band distributed through the uppermost layer of the gradient, and a minor acapsular fraction at the bottom (Figure 2B). These fractions differ in capsule amount as shown by an assay for uronic acids (Figure 2B). Transposon insertion sequencing of individual fractions results in clear localization of specific mutants within different gradient fractions, as shown for the capsule biosynthesis locus of K. pneumoniae ATCC43816 (Figure 2C). Representative results for pure cultures of K. pneumoniae NTUH-K2044 and S. pneumoniae, and different capsule biosynthesis or regulatory mutants, are shown in Figure 2D.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/58679/58679fig1.jpg" /> 
Figure 1: Schematic of the density centrifugation method to separate bacteria based on capsule, and its applications. (A) An electron microscopy image of a capsulated Klebsiella pneumoniae cell. The capsule is visible as a dense layer on the outside of the cell. (B) Applications of density centrifugation to the study of capsulated bacteria. (Bi) Density separation can be used to generate high-, low-, and no-capsule fractions of a transposon mutant library and followed by transposon insertion sequencing to define genes that influence capsule production. (Bii) Purification of capsulated bacteria from a complex sample. (Biii) Use of density-based separation for rapid comparisons of capsule amount between samples. This method also allows the visualization of heterogenous capsule production in bacterial populations, as shown in (Biii). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58679/58679fig1large.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/58679/58679fig2.jpg" /> 
Figure 2: Representative results. (A) Example of the output from mini-gradient tests. Two different K. pneumoniae strains were centrifuged on 1 mL of 15% density gradient medium. The hypermucoviscous NTUH-K2044 strain is retained above the density gradient medium layer, while ATCC43816 (which makes less capsule) migrates to the bottom of the layer. (Bi) Use of a density gradient to separate a transposon mutant library into three fractions. Note that the bottom fraction contains a low proportion of mutants and is not visible on this picture. (Bii) Validation of different capsule amounts in the top, middle, and bottom fractions using an assay for uronic acids. Cells from the top, middle, and outgrown bottom fractions were isolated and resuspended in PBS to an OD600 of 4, then capsule polysaccharides extracted and uronic acids measured1. (C) Example density-TraDISort results. Mutation locations identified are shown by blue lines above the chromosome diagram. Mutants lacking capsule can be identified as those that are present in the input library but are depleted in the top fraction while being enriched in the bottom fraction, as shown here for the capsule biosynthesis locus8. (D) An example of using density gradient centrifugation to compare capsule amount between wild type and mutant strains of K. pneumoniae NTUH-K2044 and S. pneumoniae 23F. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58679/58679fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Separating Bacteria by Capsule Amount Using a Discontinuous Density Gradient at JoVE.com

Separating Bacteria by Capsule Amount Using a Discontinuous Density Gradient

We demonstrate the use of discontinuous density gradients to separate bacterial populations based on capsule production. This method is used to compare capsule amount between cultures, isolate mutants with a specific capsule phenotype, or to identify capsule regulators. Described here is the optimization and running of the assay.

Capsule is a key virulence factor in many bacterial species, mediating immune evasion and resistance to various physical stresses. While many methods are ...

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Genetics

13.9K Views.  Wellcome Sanger Institute, Wellcome Genome Campus. Deze methode kan helpen bij het beantwoorden van belangrijke vragen in het Klebsiella-veld, zoals welke genen worden gebruikt om capsule en bijbehorende virulents te reguleren. De belangrijkste voordelen van deze techniek zijn dat het een middel biedt om gecapsuleerde en niet-gekapseizeerde stammen fysiek te scheiden, en het kan ook worden gebruikt voor snelle vergelijkingen tussen stammen van capsuleproductie. Hoewel deze methode is ontwikkeld om capsuleregulatie in Klebsiella te bestuderen,...

Video: Separating Bacteria by Capsule Amount Using a Discontinuous Density Gradient

A Microfluidic Device for Quantifying Bacterial Chemotaxis in Stable Concentration Gradients

Chemotaxis allows bacteria to approach sources of attractant chemicals or to avoid sources of repellent chemicals. Bacteria constantly monitor the concentration of specific chemoeffectors by comparing the current concentration to the concentration detected a few seconds earlier. This comparison determines the net direction of movement. Although multiple, competing gradients often coexist in nature, conventional approaches for investigating bacterial chemotaxis are suboptimal for quantifying migration in response to concentration gradients of attractants and repellents. Here, we describe the development of a microfluidic chemotaxis model for presenting precise and stable concentration gradients of chemoeffectors to bacteria and quantitatively investigating their response to the applied gradient. The device is versatile in that concentration gradients of any desired absolute concentration and gradient strength can be easily generated by diffusive mixing. The device is demonstrated using the response of Escherichia coli RP437 to gradients of amino acids and nickel ions.

This protocol describes the development of a microfluidic device for investigating bacterial chemotaxis in stable concentration gradients of chemoeffectors.

Chemotaxis allows bacteria to approach sources of attractant chemicals or to avoid sources of repellent chemicals. Bacteria constantly monitor the ...

Biology

Quantifying Bacterial Surface Swarming Motility on Inducer Gradient Plates

Bacterial swarming motility is a common microbiological phenotype that bacterial communities use to migrate over semisolid surfaces. In investigations of induced swarming motility, specific concentration&#160;of an inducer may not be able to report events occurring within the optimal concentration range to elicit the desired responses from a species. Semisolid plates containing multiple concentrations are commonly used to investigate the response within an inducer concentration range. However, separate semisolid plates increase variations in medium viscosity and moisture content within each plate due to nonuniform solidification time.
This paper describes a one-step method to simultaneously test surface swarming motility on a single gradient plate, where the isometrically arranged test wells allow the simultaneous acquisition of multiconcentration responses. In the present work, the surface swarming of Escherichia coli K12 and Pseudomonas aeruginosa PAO1 were evaluated in response to a concentration gradient of inducers such as resveratrol and arabinose. Periodically, the swarm morphologies were imaged using an imaging system to capture the entire surface swarming process.
The quantitative measurement of the swarm morphologies was acquired using ImageJ software, providing analyzable information of the swarm area. This paper presents a simple gradient swarm plate method that provides qualitative and quantitative information about the inducers&#39; effects on surface swarming, which can be extended to study the effects of other inducers on a broader range of motile bacterial species.

Here, we describe the use of inducer gradient plates to evaluate bacterial swarming motility while simultaneously obtaining multiple concentration responses.

Bacterial swarming motility is a common microbiological phenotype that bacterial communities use to migrate over semisolid surfaces. In investigations of ...

Biochemistry

Size Exclusion Chromatography to Analyze Bacterial Outer Membrane Vesicle Heterogeneity

The cell wall of Gram-negative bacteria consists of an inner (cytoplasmic) and outer membrane (OM), separated by a thin peptidoglycan layer. Throughout growth, the outer membrane can bleb to form spherical outer membrane vesicles (OMVs). These OMVs are involved in numerous cellular functions including cargo delivery to host cells and communication with bacterial cells. Recently, the therapeutic potential of OMVs has begun to be explored, including their use as vaccines and drug delivery vehicles. Although OMVs are derived from the OM, it has long been appreciated that the lipid and protein cargo of the OMV differs, often significantly, from that of the OM. More recently, evidence that bacteria can release multiple types of OMVs has been discovered, and evidence exists that size can impact the mechanism of their uptake by host cells. However, studies in this area are limited by difficulties in efficiently separating the heterogeneously sized OMVs. Density gradient centrifugation (DGC) has traditionally been used for this purpose; however, this technique is time-consuming and difficult to scale-up. Size exclusion chromatography (SEC), on the other hand, is less cumbersome and lends itself to the necessary future scale-up for therapeutic use of OMVs. Here, we describe a SEC approach that enables reproducible separation of heterogeneously sized vesicles, using as a test case, OMVs produced by Aggregatibacter actinomycetemcomitans, which range in diameter from less than 150 nm to greater than 350 nm. We demonstrate separation of &#34;large&#34; (350 nm) OMVs and &#34;small&#34; (&#60;150 nm) OMVs, verified by dynamic light scattering (DLS). We recommend SEC-based techniques over DGC-based techniques for separation of heterogeneously sized vesicles due to its ease of use, reproducibility (including user-to-user), and possibility for scale-up.

Bacterial vesicles play important roles in pathogenesis and have promising biotechnological applications. The heterogeneity of vesicles complicates analysis and use; therefore, a simple, reproducible method to separate varying sizes of vesicles is necessary. Here, we demonstrate the use of size exclusion chromatography to separate heterogeneous vesicles produced by Aggregatibacter actinomycetemcomitans.

The cell wall of Gram-negative bacteria consists of an inner (cytoplasmic) and outer membrane (OM), separated by a thin peptidoglycan layer. Throughout ...

Bioengineering

Separation of the Cell Envelope for Gram-negative Bacteria into Inner and Outer Membrane Fractions with Technical Adjustments for Acinetobacter baumannii

This method works by partitioning the envelope of Gram-negative bacteria into total, inner, and outer membrane (OM) fractions and concludes with assays to assess the purity of the bilayers. The OM has an increased overall density compared to the inner membrane, largely due to the presence of lipooligosaccharides (LOS) and lipopolysaccharides (LPS) within the outer leaflet. LOS and LPS molecules are amphipathic glycolipids that have a similar structure, which consists of a lipid-A disaccharolipid and core-oligosaccharide substituent. However, only LPS molecules are decorated with a third subunit known as the O-polysaccharide, or O-antigen. The type and amount of glycolipids present will impact an organism&#8217;s OM density. Therefore, we tested whether the membranes of bacteria with varied glycolipid content could be similarly isolated using our technique. For the LPS-producing organisms, Salmonella enterica serovar Typhimurium and Escherichia coli, the membranes were easily isolated and the LPS O-antigen moiety did not impact bilayer partitioning. Acinetobacter baumannii produces LOS molecules, which have a similar mass to O-antigen deficient LPS molecules; however, the membranes of these microbes could not initially be separated. We reasoned that the OM of A. baumannii was less dense than that of Enterobacteriaceae, so the sucrose gradient was adjusted and the membranes were isolated. The technique can therefore be adapted and modified for use with other organisms.

Separation of the Cell Envelope for Gram-negative Bacteria into Inner and Outer Membrane Fractions with Technical Adjustments for Acinetobacter baumannii

Gram-negative bacteria produce two spatially segregated membranes. The outer membrane is partitioned from the inner membrane by a periplasm and a peptidoglycan layer. The ability to isolate the dual bilayers of these microbes has been critical for understanding their physiology and pathogenesis.

This method works by partitioning the envelope of Gram-negative bacteria into total, inner, and outer membrane (OM) fractions and concludes with assays to ...

Immunology and Infection

Sperm Collection of Differential Quality Using Density Gradient Centrifugation

In sexual reproduction, a male gamete or sperm cell fuses with a female gamete to bring about fertilization. However, a large number of sperm cells with fertilizing ability are required to interact with a female gamete to ensure fertilization. As such, the fertilizing ability of individual sperm cells is critical for successful reproduction. Density gradient centrifugation has been utilized for several decades as a reproducible, fast, efficient, effective and extremely adaptable method to collect only high-quality sperm to be used in assisted reproductive technology. The protocols we described herein focus on the utilization of the discontinuous Percoll density gradient centrifugation (PDGC) technique to isolate three distinct populations of rooster sperm by their quality. We were able to collect low-, medium- and high-quality sperm. We also describe reproducible protocols that entail determining fertility potential of sperm by assessing their viability, mobility and penetrability. Collection of sperm by their quality using PDGC technique would be useful to accurately and thoroughly characterize sperm with differential fertility potential.

In this paper, we aim to describe the performance of the density gradient centrifugation technique and its application in sperm physiology research.

In sexual reproduction, a male gamete or sperm cell fuses with a female gamete to bring about fertilization. However, a large number of sperm cells with ...

Enhanced Bacterial Extracellular Vesicles Isolation for Microbiota Multi-Omics

Isolation and Purification of Bacterial Extracellular Vesicles from Human Feces Using Density Gradient Centrifugation

Bacterial extracellular vesicles (BEVs) are nanovesicles derived from bacteria that play an active role in bacteria-bacteria and bacteria-host communication, transferring bioactive molecules such as proteins, lipids, and nucleic acids inherited from the parent bacteria. BEVs derived from the gut microbiota have effects within the gastrointestinal tract and can reach distant organs, resulting in significant implications for physiology and pathology. Theoretical investigations that explore the types, quantities, and roles of BEVs derived from human feces are crucial for understanding the secretion and function of BEVs from the gut microbiota. These investigations also necessitate an improvement in the current strategy for isolating and purifying BEVs.
This study optimized the isolation and purification process of BEVs by establishing two density gradient centrifugation (DGC) modes: Top-down and Bottom-up. The enriched distribution of BEVs was determined in fractions 6 to 8 (F6-F8). The effectiveness of the approach was evaluated based on particle morphology, size, concentration, and protein content. The particle and protein recovery rates were calculated, and the presence of specific markers was analyzed to compare the recovery and purity of the two DGC modes. The results indicated that the Top-down centrifugation mode had lower contamination levels and achieved a recovery rate and purity similar to that of the Bottom-up mode. A centrifugation time of 7 h was sufficient to achieve a fecal BEV concentration of 108/mg.
Apart from feces, this method could be applied to other body fluid types with proper modification according to the differences in components and viscosity. In conclusion, this detailed and reliable protocol would facilitate the standardized isolation and purification of BEVs and thus, lay a foundation for subsequent multi-omics analysis and functional experiments.

Author Spotlight: Accelerating Research on Bacterial Extracellular Vesicles Separation and Heterogeneity 

This study describes a method to isolate and purify bacterial extracellular vesicles (BEVs) enriched from human feces via density gradient centrifugation (DGC), identifies the physical characteristics of BEVs from morphology, particle size, and concentration, and discusses the potential applications of the DGC approach in clinical and scientific research.

Bacterial extracellular vesicles (BEVs) are nanovesicles derived from bacteria that play an active role in bacteria-bacteria and bacteria-host ...

Density Gradient Ultracentrifugation

Density gradient ultracentrifugation is a common technique used to isolate and purify biomolecules and cell structures. This technique exploits the fact that, in suspension, particles that are more dense than the solvent will sediment, while those that are less dense will float. A high-speed ultracentrifuge is used to accelerate this process in order to separate biomolecules within a density gradient, which can be established by layering liquids of decreasing density in a centrifuge tube.
The video will cover the principles of density gradient ultracentrifugation, including a procedure that demonstrates sample preparation, creation of a sucrose gradient, ultracentrifugation, and collection of fractionated analytes. The applications section discusses isolation of multi-protein complexes, isolation of nucleic acid complexes, and separation using cesium chloride density gradients.
<div class="chapter_text" id="script_0">
Density gradient ultracentrifugation is a common approach to isolate and purify cell structures for biochemical experiments. The technique uses a high-speed, or ultra, centrifuge to nondestructively separate cellular components in a density gradient. This video describes the principles of density gradient ultracentrifugation, provides a general procedure using a sucrose gradient, and discusses some applications.
</div>
<div class="chapter_text" id="script_1">
Let&#39;s start by examining the principles of ultracentrifuges and density gradients. A suspension contains particles in a liquid solvent. Because of gravity, particles denser than the solvent sediment out while those less dense than the solvent float. The greater the difference in density between the particle and the solvent, the faster the separation.
An ultracentrifuge contains a unit called a rotor, which rotates at highly controlled speeds, simulating a strong gravitational field. Within this field, the differences in density between particles and the solvent are magnified.
The strength of the field depends on the speed of rotation. Even a small rotor at a relatively low rotational speed can create a force thousands of times stronger than the earth&#39;s gravitational field.
If a tube contains several liquids of different densities, centrifugation will keep them in separate layers in order of density, with the densest liquid closest to the base. Such a layering of multiple liquids is called a &#34;density gradient.&#34; There are two types. In step gradients, liquids of decreasing density are carefully layered on top one another. In continuous gradients, the liquids are mixed in varying proportions, so the density decreases smoothly from the base upwards.
Cellular organelles can be separated using a step gradient, through &#34;isopycnic density-gradient centrifugation.&#34; This is the simplest and most common centrifugation procedure.
This procedure is used to separate the cellular structures. The more dense the organelle, the further it descends-with mitochondria at the top and nucleic acids towards the bottom.
Now that you know the principles behind the technique, let&#39;s see it in the lab.
</div>
<div class="chapter_text" id="script_2">
Before the procedure is started, the manufacturer&#39;s speed and density ratings should be noted, and the ultracentrifuge checked for corrosion. This procedure uses a swinging-bucket rotor.
First, the cellular material is prepared by homogenizing the cells, which nondestructively releases their organelles. The homogenate may be fractionated through preliminary low-speed centrifugation, to remove low-density components. Next, the sucrose solutions are prepared.
Sucrose is added in increasing amounts so each solution is more concentrated, and therefore denser, than the preceding one. The exact densities of the solutions will depend on the components to be separated, which vary between organisms. The solutions should have densities between those of the components to be separated, with the last solution denser than the densest component of the analyte. Techniques for separating components denser than sucrose, like nucleic acids, are described in the applications.
The sucrose gradient is now created in a clean centrifuge tube. A pipette is used to draw up the most concentrated sucrose solution. With the tube held upright, the pipette tip is placed high against the wall, and the liquid dispensed steadily down. It&#39;s important that the working area is kept free of vibrations and other disturbances.
After replacing the tip, the remaining solutions are added in order of decreasing density. They are dispensed carefully to form distinct layers and avoid mixing. Finally, about half a milliliter of the cellular sample is added atop the gradient, and the tube is weighed. This is used to balance the weight distribution, the next step of the process.
</div>
<div class="chapter_text" id="script_3">
Centrifugation should begin as soon as possible. The tube is placed in the rotor, which is then balanced by placing blank solutions of equal weight in opposing slots. The rotor is placed in the ultracentrifuge and the system sealed. The temperature and rotation speed and time are set. Typical values are 4 &#176;C with a force of over 100,000 x g for 16 h.
After centrifugation, the tube is withdrawn from the rotor, taking care to keep it upright and undisturbed. The different cellular components have fractionated into discrete bands between the solution layers. The fractions can be collected with a syringe. Alternately, the bottom of the tube can be punctured with a fine, sterilized needle and the outflow collected in sterile tubes. The cellular components have now been isolated. They can be stored at -80 &#176;C.
</div>
<div class="chapter_text" id="script_4">
Now that we&#39;ve seen the basic procedure, let&#39;s look at some applications.
A typical application is the isolation of multi-protein complexes in plant cells. In this example, complexes responsible for cyclic electron flow are being isolated from the thylakoid, the site of the light reaction in photosynthesis. This procedure uses discrete solutions of 14 to 45% sucrose. Centrifugation occurs over 100,000 x g for 14 h at 4 &#176;C.
Because nucleic acids are denser than sucrose, isopycnic centrifugation cannot separate them from organelles nondestructively.
A different technique, known as &#34;rate-zonal centrifugation&#34; is used. It separates organelles based on their sedimentation rates, which depend not only on their densities, but also on their conformations. A continuous gradient is used to separate the components based on this property.
The procedural steps are similar to those for isopycnic cases. In this example, RNA-ribosome complexes are isolated using a continuous gradient of 5% to 20%, centrifuged at 230,000 x g. Centrifugation is interrupted after a few hours to prevent co-precipitation.
Nucleic acid strands can be separated from each other on the basis of density.
This is because strands rich in guanine and cytosine are denser than those rich in adenine and thiamine. In this case, the gradient cannot be made of sucrose, because sucrose is less dense than nucleic acids. Instead, cesium chloride gradients, typically from 1.65 to 1.75 g/mL are used, as they have sufficient density and a low viscosity.
Here we see plankton DNA being purified using a continuous cesium chloride gradient. Centrifugation occurs at over 1,000,000 x g for 18 h under vacuum.
</div>
<div class="chapter_text" id="script_5">
You&#39;ve just watched JoVE&#39;s video on ultracentrifugation with a sucrose density gradient. You should now understand how a density gradient works, how to construct a step gradient, and how to load and operate an ultracentrifuge. Thanks for watching!
</div>

Density Gradient Ultracentrifugation: Principle and Applications

Density gradient ultracentrifugation is a common technique used to isolate and purify biomolecules and cell structures. This technique exploits the fact ...

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Chemistry

Overview Of Cell Separation And Isolation

Cell separation was first achieved in 1964 by S. H. Seal, who separated large tumor cells from the smaller blood cells using filtration. Two years later, Pohl and Hawk performed experiments on how cells respond differently to a nonuniform electric field based on the cell type. Such observations were the inception of cell separation methods, which allow isolating a single cell type from a heterogeneous sample.

Commonly used cell separation techniques include filtration, density gradient centrifugation, affinity-based separation using antibodies, and flow cytometric cell sorting. Additional specialized techniques include aptamer technology, buoyancy-activated cell sorting, and laser capture microdissection. The choice of separation method is based on the characteristics of the cell type to be isolated, such as surface charge, cell size, density, morphology, physiology, and surface markers. Usually, one or more of these characteristics are employed to isolate the particular cell type efficiently.

Applications of Cell Separation and Isolation
Isolated cells can be grown in vitro to produce cell lines, which have numerous applications in areas like pharmacology, immunology, and stem cell therapy. For example, the in vitro effects of drugs can be analyzed on specific cell populations. Cell separation is also essential in screening for the appropriate B-cells for monoclonal antibody production. Single-cell analysis, such as studying gene expression patterns and epigenetic effects, also relies on the isolation of specific cells to study. Many oncological studies that contribute to our understanding of cancer cells require isolating particular tumor cells from the tissue. Thus, cell separation and isolation methods are used virtually in all major fields of modern biology.

Challenges of Cell Separation Methods
Cell separation techniques face a high noise-to-signal ratio, with a small number of specific cells (target) against a large number of varied components that form the tissue. Various factors affect the purity of the final isolate; hence, the protocols require standardization for each target cell type and application. For example, the efficiency of cells dissociating from the tissue decides the yield of isolated cells. Excess digestion increases the presence of dead cells in the isolate, whereas incomplete digestion results in cell type contamination. Cell type contamination can also occur in affinity-based cell separation. While labeling a particular cell type with specific antibodies, non-specific binding may occur, introducing different cells into&#160; the preparation. Additionally, the population size of the target cell type in the tissue is also a limiting factor &#8212; low-abundance cells often need tedious protocols to yield sufficient numbers in the isolated cell populations.

Cell separation was first achieved in 1964 by S. H. Seal, who separated large tumor cells from the smaller blood cells using filtration. Two years later, ...

JoVE Core

Cell Biology

Unit 1

Analyzing Cells and Proteins

KEY TERMS AND CONCEPTS

Centrifugation

Centrifugation is a separation technique based on differences in density or size. It is commonly used to separate solids from aqueous interferents. During centrifugation, the sample is placed in centrifugation tubes and spun at high angular velocity, which allows centrifugal force to act differentially on the different densities or masses of the components. After spinning, the supernatant liquid is decanted. Depending on the specific application, either the pellet or the supernatant is retained for further purification. Sometimes, the supernatant is subjected to additional rounds of centrifugation and downstream processing.
In principle, centrifugation separates particles based on differences in size and density. Larger particles tend to be heavier and tend to sediment first. If particles are similar in size, denser particles tend to sediment first due to higher sedimentation rates. In practice, complex mixtures such as cell lysates may not yield distinctly separated particles despite differences in density or size. To address this issue, researchers have devised a variety of centrifugation techniques.
The simplest centrifugation technique is differential centrifugation. Here, the particles to be separated have similar densities, so larger particles will sediment at lower speeds. The speed is increased stepwise until the target particles are isolated.
A more sophisticated method is equilibrium density gradient centrifugation. In this technique, the analyte is placed in a solution with a preformed density gradient or a solution that forms a density gradient during centrifugation. Here, the density of the solution increases towards the bottom of the tube, so the sedimentation rate of each analyte component decreases as it moves toward the bottom. When a component&#39;s density equals that of the solution, the centrifugal force acting on it becomes zero, and sedimentation ceases. As a result, each component is isolated in a layer equal to its density.

Centrifugation is a method of separating suspended particles from a liquid by spinning in a centrifuge at a high number of revolutions per minute.
...

Analytical Chemistry

Introduction to Separation Methods

Size Exclusion Chromatography Based Separation of Bacterial Extracellular Vesicles: A Technique to Separate Heterogenous Population of Gram-Negative Bacterial Outer Membrane Vesicles Based on Their Size

To begin, use a glass rod to mix a stock bottle of gel filtration medium, and pour the volume required to fill the column plus approximately 50% excess, into a glass bottle. Allow the beads to settle, and decant the excess liquid. Resuspend the beads in elution buffer to a final solution of approximately 70% gel and 30% buffer, and degas the solution under vacuum.
Mount the column on a ring stand in the vertical position, and fill the column with elution buffer to wet the walls, before draining the buffer until only about 1 centimeter remains. Then, carefully pipette the gel beads into the column, while simultaneously draining the excess buffer to prevent the beads from settling, until the column is packed to a height of approximately 2 centimeters below the bottom of the column reservoir.
Before loading the sample, degas the elution buffer under vacuum. Wash the column with approximately two times the column volume of elution buffer. Once the buffer reaches the top of gel layer, carefully add 2 milliliters of a 100 to 200-nanomoles per liter outer membrane vesicle sample on top of the gel layer without disturbing the surface, and let the sample enter the gel.
Slowly, add the elution buffer to the column without disturbing the gel layer, and place a 50-milliliter tube under the column. To prevent the column from drying, simultaneously add elution buffer to the top of the column.
After collecting 20 milliliters of eluate, place a rack of 1.5-milliliter tubes under the column. Collect a total of 96 1-milliliter fractions in each tube. While collecting the samples, continuously add elution buffer to the column. To clean the column, run 1 column volume of 0.1 molar sodium hydroxide through the column, followed by 2 column volumes of elution buffer.

Separating Bacteria by Capsule Amount Using a Discontinuous Density Gradient

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