This work was part of the PneuCarriage project, funded by the Bill and Melinda Gates Foundation with contribution by the Victorian Government&#8217;s Operational Infrastructure Support Program.

1. Preparation of in-house Latex Reagents
<ol>
	<li>Preparation of glycine buffered saline (GBS)
	<ol>
		<li>Add 1.5 g glycine, 11.7 g sodium chloride and 100 ml of distilled water to a 200 ml glass beaker.</li>
		<li>Mix to dissolve using a magnetic stirrer.</li>
		<li>Transfer mixture to a 500 ml measuring cylinder and add distilled water to make a total of 200 ml.</li>
		<li>Transfer the solution back to the 200 ml beaker, check the pH and adjust to 8.2 with 5 M sodium hydroxide. NOTE: Sodium hydroxide is corrosive, wear personal protective equipment.</li>
		<li>Filter sterilize the solution using 0.22 &#181;M filters and several 60 ml syringes into a 250 ml preautoclaved screw capped glass bottle.</li>
		<li>Store at RT.</li>
	</ol>
	</li>
	<li>Preparation of GBS with 0.2% bovine serum albumin
	<ol>
		<li>Add 0.2 g of bovine serum albumin (BSA) powder and 100 ml of GBS to a 200 ml glass beaker and mix to dissolve using a magnetic stirrer.</li>
		<li>Filter sterilize the solution through 0.22 &#181;M filters using several 60 ml syringes into a 250 ml preautoclaved screw capped glass bottle.</li>
		<li>Store at 4 &#176;C.</li>
	</ol>
	</li>
	<li>Latex reagent preparation
	<ol>
		<li>Label a 2 ml round bottom microfuge tube. 
		NOTE: round bottom tubes are used to minimize particle settling which may affect antibody attachment.</li>
		<li>Using a P1000 pipette with sterile tips add 975 &#181;l of GBS into the microfuge tube, then add 25 &#181;l of appropriate pneumococcal antiserum for the latex reagent being prepared (i.e., pool, group, type or factor). Invert five times to mix.</li>
		<li>Dilute polystyrene latex particles 1:10 by adding 120 &#181;l of latex particles to 1,080 &#181;l of sterile physiological saline. 
		NOTE: This can be made in larger volumes, but should be made fresh each time the latex reagents are produced.</li>
		<li>Add 1,000 &#181;l of the 1:10 latex suspension (prepared in step 1.3.3) to the 1,000 &#181;l 1:40 antiserum suspension (prepared in step 1.3.2). Invert five times to mix.</li>
		<li>Incubate at 37 &#176;C on a slowly rotating wheel (e.g., 4 rpm for a wheel of 38.5 cm diameter) for 2 hr.</li>
		<li>Centrifuge for 15 min at 1,100 x g. Discard the supernatant and gently resuspend the pellet in 2 ml of sterile saline.</li>
		<li>Centrifuge for 15 min at 1,100 x g. Discard the supernatant and add 1 ml of GBS and gently resuspend the pellet.</li>
		<li>Pipette the latex suspension into a labeled 5 ml screw capped tube. Add another 1 ml of GBS.</li>
		<li>Add 2 ml of GBS containing 0.2% BSA (w/v) (pH 8.2) (prepared in step 1.2). Add 40 &#181;l of 10% (w/v) solution of sodium azide as a preservative. 
		NOTE: Sodium azide is hazardous if inhaled, and is a skin and eye irritant. It is desirable to purchase a prepared 10% solution rather than the powdered form, as this minimizes the risk of inhalation exposure. Personal protective equipment (including gloves and splash goggles) should be worn when handling this reagent. Refer to the Material safety data sheet for details.</li>
		<li>Store latex reagents at 4 &#176;C.</li>
	</ol>
	</li>
	<li>Preparation of negative control latex reagent
	<ol>
		<li>Label a 2 ml round bottom microfuge tube. 
		NOTE: round bottom tubes are used to minimize particle settling which may affect antibody attachment.</li>
		<li>Using a P1000 pipette with sterile tips add 975 &#181;l of GBS into the microfuge tube, then add 25 &#181;l normal rabbit antiserum. Invert five times to mix.</li>
		<li>Proceed as in steps 1.3.3 to 1.3.10 above</li>
	</ol>
	</li>
</ol>
2. Quality Control (QC) of Latex Reagents
<ol>
	<li>Bring latex reagent to RT. Immediately before use, gently mix the latex reagent by inverting the tube several times.</li>
	<li>Check reagent for autoagglutination by pipetting 15 &#181;l onto a glass microscope slide and proceed as in steps 4.7 and 4.8 below. There should be no agglutination of the latex particles. The reagent should appear white and smooth.</li>
	<li>Test the reagent against a panel of low passage pneumococcal isolates. Use freshly grown cultures prepared as in step 3. The panel of isolates should include isolates of the &#8216;target&#8217; serotype (the specific serotype for the reagent being tested) and &#8216;non-target&#8217; serotypes (isolates of other serotypes that should not normally cross react with the reagent). Include at least one isolate of each target and non-target serotype for each latex reagent undergoing QC. If there is only one target or non-target serotype, test two different isolates of the particular serotype. 
	NOTE: The testing panel should include several isolates of each serotype, some of which are uncommon in invasive disease and/or type culture collections. It is preferable to include some carriage isolates, as they are often more demanding to serotype (data not shown), thereby further testing the capacity of the reagents and ensuring that the reagents are likely capable of serotyping both invasive and carriage isolates.</li>
	<li>Proceed with testing of latex reagents with target and non-target pneumococcal isolates as per the method described in steps 3 and 4 below.</li>
	<li>Quality control reagents at time of production and then at least annually thereafter. 
	NOTE: If a reagent does not pass QC, the batch must be discarded and a new preparation made. In the event of repeat QC failure we recommend the reagents be produced using the alternate methods described in Ortika et al., 20138 and in the discussion below.</li>
</ol>
3. Preparation of Pneumococcal Cultures for Serotyping
<ol>
	<li>Using a sterile loop select a single colony of the pneumococcal isolate and streak this out onto a solid nonselective blood agar so that the primary inoculum covers approximately one third of the plate surface. Streak for single colonies. 
	NOTE: Plates prepared from defibrinated horse or sheep blood are suitable; but blood plates prepared with human blood or citrated animal blood are not.</li>
	<li>Incubate plate O/N&#160;at 37 &#176;C in an atmosphere of 5% CO2.</li>
	<li>Observe the growth on the plate for purity and assess that there is sufficient growth for serotyping. 
	NOTE: A pure culture of the pneumococcal isolate is required for serotyping. In our experience, confluent, or near confluent growth covering approximately one third of the plate surface is usually sufficient to complete serotyping. As pneumococcal cultures may be prone to autolysis, cultures prepared for serotyping should be tested within 24 hr of subculture. Do not take the culture plate out of the incubator more than 15 to 20 min before serotyping commences.</li>
</ol>
4. Conducting Latex Agglutination Serotyping
<ol>
	<li>Remove all latex reagents from refrigerator and allow them to warm to RT for approximately 30 min. Immediately before use, gently invert the tubes to mix the reagents.</li>
	<li>Label a microscope slide.</li>
	<li>Place 15 &#181;l of negative control latex reagent onto the microscope slide.</li>
	<li>Using a sterile disposable 1 &#181;l loop take a sweep of the pneumococcal culture so that the loop is approximately half full.</li>
	<li>Gently smear the inoculum onto the surface of the slide close to the drop of negative control latex reagent. The inoculum should be visible on the glass slide.</li>
	<li>Quickly and thoroughly mix the inoculum into the negative control latex reagent using the loop. Ensure that the drop does not spread further than its initial size.</li>
	<li>Pick up the slide, holding it at both ends. Rock the slide back and forth for 1 min. Take care to keep the liquid moving slowly, and ensure that the size of the drop does not increase. 
	NOTE: Alternatively use a plate rocker.</li>
	<li>Observe for macroscopic agglutination (i.e., by naked eye) and clearing of the suspension against a black background.</li>
	<li>If the negative control latex reagent shows no agglutination or clearing continue testing the culture, replacing the test latex reagents for the negative control latex reagent in steps 4.1 to 4.8 above. A fresh loop must be used each time. Proceed with testing of the latex reagents starting with the pools.</li>
	<li>Test each of the &#39;pool&#39; latex reagents in succession until a positive test is obtained. 
	NOTE: The order of testing of the pools may be done in &#8216;rounds&#8217; to reflect the local incidence of particular serotypes (e.g.,&#160;by testing for the three most likely pools on the first slide. If a positive reaction is not obtained, then test the next three most likely of the remaining pools on the second slide; and so on until a positive reaction is obtained). This ensures that the most common serotypes are tested first, minimizing the time and reagents needed.</li>
	<li>Using the antisera manufacturer&#8217;s key determine which individual antisera are represented in the positive pool.</li>
	<li>Test each of the groups or types contained in the positive pool individually to determine the &#8216;group&#8217; or &#8216;type&#8217; as in steps 4.1 to 4.8 above.</li>
	<li>Determine the result by reference to the antisera manufacturer&#8217;s key. If a &#8216;type&#8217; is determined (e.g., serotype 5), then no further testing is needed and this result is recorded.</li>
	<li>If a &#8216;group&#8217; is determined (e.g., group 19), then refer to the manufacturers key to determine the factors to be tested. Continue testing with the individual &#8216;factor&#8217; latex reagents and determine the final serotype (e.g., serotype 19B).</li>
</ol>

<ol>
	<li>Rudan, I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epidemiology+and+etiology+of+childhood+pneumonia+in+2010:+estimates+of+incidence,+severe+morbidity,+mortality,+underlying+risk+factors+and+causative+pathogens+for+192+countries.">Epidemiology and etiology of childhood pneumonia in 2010: estimates of incidence, severe morbidity, mortality, underlying risk factors and causative pathogens for 192 countries.</a> J Glob Health. 3 (10401), (2013).</li><li>O'Brien, K. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Burden+of+disease+caused+by+Streptococcus+pneumoniae+in+children+younger+than+5+years:+global+estimates.">Burden of disease caused by Streptococcus pneumoniae in children younger than 5 years: global estimates.</a> Lancet. 374, 893-902 (2009).</li><li>Henrichsen, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Six+newly+recognized+types+of+Streptococcus+pneumoniae.">Six newly recognized types of Streptococcus pneumoniae.</a> J Clin Microbiol. 33, 2759-2762 (1995).</li><li>Hausdorff, W. P., Bryant, J., Paradiso, P. R., Siber, G. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Which+pneumococcal+serogroups+cause+the+most+invasive+disease:+implications+for+conjugate+vaccine+formulation+and+use,+part+I.">Which pneumococcal serogroups cause the most invasive disease: implications for conjugate vaccine formulation and use, part I.</a> Clin Infect Dis. 30, 100-121 (2000).</li><li>Mulholland, K., Satzke, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Serotype+replacement+after+pneumococcal+vaccination.">Serotype replacement after pneumococcal vaccination.</a> Lancet. 379, 1388-1389 (2012).</li><li>Weinberger, D. M., Malley, R., Lipsitch, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Serotype+replacement+in+disease+after+pneumococcal+vaccination.">Serotype replacement in disease after pneumococcal vaccination.</a> Lancet. 378, 1962-1973 (2011).</li><li>Satzke, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Standard+method+for+detecting+upper+respiratory+carriage+of+Streptococcus+pneumoniae:+Updated+recommendations+from+the+World+Health+Organization+Pneumococcal+Carriage+Working+Group.">Standard method for detecting upper respiratory carriage of Streptococcus pneumoniae: Updated recommendations from the World Health Organization Pneumococcal Carriage Working Group.</a> Vaccine. , (2013).</li><li>Ortika, B. D., Habib, M., Dunne, E. M., Porter, B. D., Satzke, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Production+of+latex+agglutination+reagents+for+pneumococcal+serotyping.">Production of latex agglutination reagents for pneumococcal serotyping.</a> BMC Res Notes. 6 (49), (2013).</li><li>Adegbola, R. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Serotype+and+antimicrobial+susceptibility+patterns+of+isolates+of+Streptococcus+pneumoniae+causing+invasive+disease+in+The+Gambia+1996-2003.">Serotype and antimicrobial susceptibility patterns of isolates of Streptococcus pneumoniae causing invasive disease in The Gambia 1996-2003.</a> Trop Med Int Health. 11, 1128-1135 (2006).</li><li>Slotved, H. C., Kaltoft, M., Skovsted, I. C., Kerrn, M. 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D., Satzke, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Capsular+serotyping+of+Streptococcus+pneumoniae+using+the+Quellung+reaction.">Capsular serotyping of Streptococcus pneumoniae using the Quellung reaction.</a> J. Vis. Exp. (84), (2014).</li><li>Arai, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+antiserum-coated+latex+particles+for+serotyping+Streptococcus+pneumoniae.">Use of antiserum-coated latex particles for serotyping Streptococcus pneumoniae.</a> Microbiol Immunol. 45, 159-162 (2001).</li><li>Lafong, A. C., Crothers, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simple+latex+agglutination+method+for+typing+pneumococci.">Simple latex agglutination method for typing pneumococci.</a> J Clin Pathol. 41, 230-231 (1988).</li><li>Ortega-Vinuesa, J. L., Bastos-Gonzalez, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+review+of+factors+affecting+the+performances+of+latex+agglutination+tests.">A review of factors affecting the performances of latex agglutination tests.</a> J Biomater Sci Polym Ed. 12, 379-408 (2001).</li><li>Yap, K. 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The authors have nothing to disclose.

Latex agglutination is a simple, quick and inexpensive method for pneumococcal serotyping. Commercial pneumococcal latex agglutination reagents are available, but they currently do not differentiate all known pneumococcal serotypes10,12. However, latex agglutination reagents can be easily produced in-house using purchased antisera raised against specific pneumococcal capsular antigens. In the method described here, antibodies are passively attached to latex particles to make a set of latex agglutination reagents.
A positive latex reaction occurs when specific antibodies bound to the latex particles attach to antigens on the polysaccharide capsule of the pneumococcal isolate 18,19. The latex particles attached to the antibodies allow the specific antibody-antigen reaction to be visualized without magnification.
Latex agglutination is a suitable method for high throughput laboratories and also for resource-poor settings. Key advantages of the latex agglutination method are that no specialized equipment is required to perform the test, reagents have a shelf life of at least 2 years when stored at 4 &#176;C8, and that it is inexpensive, simple and quick to perform. Serotyping a pneumococcal isolate by latex agglutination takes approximately 10 min, and up to four latex reagents can be tested in parallel on the one slide. Furthermore, if the prevalence of serotypes is known for the relevant epidemiological setting, testing can be performed in rounds starting with the pools containing the most common serotypes similarly to the Quellung test17. This approach minimizes the number of tests needed to determine the serotype. The method is also highly reproducible between different operators (data not shown). A disadvantage of latex agglutination serotyping is that producing the reagents is time consuming, taking approximately 4 hr; although multiple reagents can easily be produced in parallel. Furthermore, some antigens are common across different pneumococcal serotypes, resulting in cross-reactions with some antisera (e.g., serotype 29, 42 and Group 35). However, these cross reactions are well characterized when using commercial antisera (which is usually preabsorbed to remove the more problematic cross-reacting antibodies) and additional reaction tables are provided by the manufacturer in order to distinguish which serotype is present. Latex reagents may also cross react if the antibodies are not aligned properly on the latex particles; these are detected as QC failures. In our experience, rigorous QC is central to the success of this method, even when commercially available antisera are used for production. QC takes approximately 15 min per reagent and relies on a set of appropriate QC isolates as described above.
The method described here results in reagents that give a positive reaction well within the test period. In our experience, false positives are rare in practice (data not shown). Occasional false negative reactions are observed; usually these relate to known issues with a particular antiserum (e.g., serogroup 6 isolates may not react with pool B antiserum), or down regulation of capsule expression by the isolate. Using the serotyping algrorithm provided by the manufacturer is also important, as in most cases a false positive or negative reaction will lead to a &#8216;blind end&#8217; result. In these cases, and when reactions are difficult to interpret, we recommend repeat-testing and/or testing by an alternative method such as the Quellung reaction.
Some troubleshooting is occasionally required to make satisfactory latex reagents. In the method described here, the antibody is attached to latex particles via passive adsorption15,19, so a critical variable is the concentration of antibody used, which determines how well the surface of the latex particles is covered and the subsequent alignment of the antibody active sites15,20. Consequently, if insufficient or excess antibody is attached to the latex particles, the antibody-antigen reactions will not be optimal, and unsatisfactory reagents may be produced15,20,21. As antibody titers vary between different lots of antisera, a standard set of dilutions may not produce reagents that perform well8. A useful starting point is to use a 1:40 dilution of antiserum, and if this is not successful, dilute the antiserum further. In our experience this usually resolves the problem8. In a small number of cases reagents may show weak agglutination that is not accompanied by clearing of the suspension when tested with target isolates. In these cases diluting the antiserum does not improve the quality of the reagent, but satisfactory reagents can usually be produced by omitting the centrifugation and wash steps8,22.
Antibody coated latex particles are used commonly in diagnostic microbiology for detection, identification or serotyping of many different microbes15,20. As such, the method described here may be suitable for preparing latex reagents for other purposes, provided suitable antisera is available and adequate QC procedures are adopted.

Streptococcus pneumoniae (the pneumococcus) is a major cause of morbidity and mortality in children under the age of five years old world-wide, particularly in resource-poor settings1,2. Pneumococcal disease ranges from localized infections to life threatening conditions such as pneumonia, sepsis and meningitis1,2.
More than 90 serotypes of pneumococci have been identified based on differences in their capsular polysaccharide3. Current vaccines target the capsular polysaccharide and provide protection from the major serotypes causing invasive disease4. Serotyping pneumococcal isolates is important for assessing the impact of vaccination on carriage and disease as well as providing broader epidemiological information5,6. The current &#8216;gold standard&#8217; method for pneumococcal serotyping is the Quellung test, but it is time consuming and requires some skill to perform7. Latex agglutination is an alternative serotyping method recommended by the World Health Organization7. Latex agglutination is quick and simple to perform, and is employed in many laboratories worldwide8-11. Importantly, latex agglutination has shown comparable accuracy to the Quellung test for pneumococcal serotyping10,12-14. Overall, this method is ideal for resource-poor settings as well as high throughput laboratories.
Latex agglutination reagents (&#8216;latex reagents&#8217;) are created by the attachment of antibodies to latex particles15. In a positive reaction, these labeled particles agglutinate in the presence of specific antigen. Commercial latex reagents are available for a restricted range of serotypes. Latex reagents can also be prepared in-house using commercially available antisera10. Mixtures of antisera (&#8216;pools&#8217;) as well as specific antisera to pneumococcal serogroups (e.g., group 19), individual serotypes (e.g., serotype 5), and to specific antigens (&#8216;factors&#8217;, e.g., factor 19c recognizing serotype 19A) for further defining serotypes within groups are available16.
This manuscript outlines the major steps in the production of latex reagents using passive attachment of commercially available antisera to latex particles. The quality control (QC) aspects of this method are described, and a protocol for the use of latex reagents for pneumococcal serotyping is included.

<table border="0" cellpadding="0" cellspacing="0" width="1085">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">Name of Material/ Equipment</td>
			<td style="width:180px;">Company</td>
			<td style="width:127px;">Catalog Number</td>
			<td style="width:263px;">Comments/Description</td>
		</tr>
		<tr height="27">
			<td height="27" style="height:27px;width:299px;">Microparticles based on polystyrene, 800 nm</td>
			<td style="width:180px;">Sigma</td>
			<td style="width:127px;">65984-10 ml-F</td>
			<td style="width:263px;">Or other volumes as required</td>
		</tr>
		<tr height="47">
			<td height="47" style="height:47px;width:299px;">Normal rabbit serum</td>
			<td style="width:180px;">Antibodies Australia&#160;</td>
			<td style="width:127px;">NRS-1ml</td>
			<td style="width:263px;">Or other volumes as required, bring to room temperature before use</td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;width:299px;">Pneumococcus (Neufeld) antisera</td>
			<td style="width:180px;">SSI Diagnostica</td>
			<td style="width:127px;">Various</td>
			<td style="width:263px;">Bring to room temperature before use 
			<a href="http://www.ssi.dk/ssidiagnostica">http://www.ssi.dk/ssidiagnostica</a></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:299px;">Sterile Bovine Albumin&#160;</td>
			<td style="width:180px;">Sigma</td>
			<td style="width:127px;">A-4503</td>
			<td style="width:263px;">Bring to room temperature before use</td>
		</tr>
		<tr height="47">
			<td height="47" style="height:47px;width:299px;">Sodium azide 10% (v/v) solution&#160;</td>
			<td style="width:180px;">VWR International</td>
			<td style="width:127px;">ROAC3902/100ml</td>
			<td style="width:263px;">Caution: hazardous if inhaled; skin and eye irritant</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">Eppendorf Safe-Lock tubes, 2 ml</td>
			<td style="width:180px;">Eppendorf</td>
			<td style="width:127px;">0030 120.094</td>
			<td style="width:263px;">Round bottom</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">Sodium chloride</td>
			<td style="width:180px;">Merck</td>
			<td style="width:127px;">10241.AP</td>
			<td style="width:263px;"></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:299px;">Calibrated disposable inoculating loops 1 &#181;l</td>
			<td style="width:180px;">Copan</td>
			<td style="width:127px;">CD175SO1</td>
			<td style="width:263px;"></td>
		</tr>
		<tr height="28">
			<td height="28" style="height:28px;width:299px;">Glass microscope slides 76 mm x 26 mm</td>
			<td style="width:180px;">Thermo Fisher Scientific</td>
			<td style="width:127px;">LBS 2950RC</td>
			<td style="width:263px;"></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:299px;">5 M NaOH</td>
			<td style="width:180px;">BDH</td>
			<td style="width:127px;">10252</td>
			<td style="width:263px;">Caution: highly corrosive</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">Glycine</td>
			<td style="width:180px;">Merck</td>
			<td style="width:127px;">10119.05</td>
			<td style="width:263px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">Horse Blood Agar (HBA) plates</td>
			<td style="width:180px;">Thermo Fisher Scientific</td>
			<td style="width:127px;">PP2001</td>
			<td>Bring to room temperature before use 
			<a href="http://www.thermofisher.com.au/">http://www.thermofisher.com.au</a></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:299px;">Rotating wheel</td>
			<td style="width:180px;">Wyble Engineering Development Corporation</td>
			<td style="width:127px;">Not available</td>
			<td></td>
		</tr>
		<tr height="29">
			<td height="29" style="height:29px;width:299px;">Polystyrene flat bottom screw cap tube, 5 ml</td>
			<td style="width:180px;">Technoplas</td>
			<td style="width:127px;">S5016SU</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">60 ml syringes without needles</td>
			<td style="width:180px;">Terumo</td>
			<td style="width:127px;">SS-60L</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">Millex-GP syringe filter unit, 0.22 &#181;m</td>
			<td style="width:180px;">Merck Millipore</td>
			<td style="width:127px;">SLGP033RS</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">Brochure on Neufeld antisera</td>
			<td style="width:180px;">Statens Serum Institut</td>
			<td style="width:127px;"></td>
			<td>Key to determining serotypes 
			<a href="http://www.ssi.dk/ssidiagnostica">http://www.ssi.dk/ssidiagnostica</a></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:299px;">Key to pneumococcal factor serum</td>
			<td style="width:180px;">Statens Serum Institut</td>
			<td align="right" style="width:127px;">18058</td>
			<td style="width:263px;">For determining serotype within Groups 
			<a href="http://www.ssi.dk/ssidiagnostica">http://www.ssi.dk/ssidiagnostica</a></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;"></td>
			<td style="width:180px;"></td>
			<td style="width:127px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;"></td>
			<td style="width:180px;"></td>
			<td style="width:127px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td colspan="2" height="21" style="height:21px;">*alternative sources are available for most materials</td>
			<td style="width:127px;"></td>
			<td></td>
		</tr>
	</tbody>
</table>

capsular serotyping of streptococcus pneumoniae by latex agglutination

Latex agglutination reagents are widely used in microbial diagnosis, identification and serotyping. Streptococcus pneumoniae (the pneumococcus) is a major cause of morbidity and mortality world-wide. Current vaccines target the pneumococcal capsule, and there are over 90 capsular serotypes. Serotyping pneumococcal isolates is therefore important for assessing the impact of vaccination programs and for epidemiological purposes. The World Health Organization has recommended latex agglutination as an alternative method to the &#8216;gold standard&#8217; Quellung test for serotyping pneumococci. Latex agglutination is a relatively simple, quick and inexpensive method; and is therefore suitable for resource-poor settings as well as laboratories with high-volume workloads. Latex agglutination reagents can be prepared in-house utilizing commercially-sourced antibodies that are passively attached to latex particles. This manuscript describes a method of production and quality control of latex agglutination reagents, and details a sequential testing approach which is time- and cost-effective. 
This method of production and quality control may also be suitable for other testing purposes.

A positive latex reaction occurs when type-specific antibody attached to the latex particle binds to the capsule of the pneumococcus, and agglutination of the antibody labeled particles ensues15. Figure 1A shows a positive reaction which is characterized by visible agglutination and clearing of the background suspension. A negative reaction is characterized by the latex agglutination reagent suspension remaining smooth and white (Figure 1B). The reagents are optimized so that a positive reaction should be observed before the end of the one min time interval (usually detectable in approximately 20 sec). We do not recommend reading reactions after the 1 min&#160;time interval. Very occasionally, reactions display weak agglutination around the edge of the drop that is not accompanied by clearing of the background suspension, or appear &#8216;stringy&#8217; with no background clearing (data not shown). These are most likely negative reactions. In such cases, we recommend retesting and/or further investigation using another serotyping method (for example the Quellung reaction17).
<img alt="Figure 1" fo:content-width="5in" src="/files/ftp_upload/51747/51747fig1highres.jpg" width="500" /> 
Figure 1.&#160;Positive and negative latex agglutination reactions. Preparations of a pneumococcal isolate mixed with latex agglutination reagent showing a positive reaction (A) or a negative reaction (B). Agglutination accompanied by clearing of the background is seen in the positive reaction (A).There is no visible agglutination with the negative control latex reagent or in a negative test reaction (B). Photographs8 used with permission. <a href="https://www.jove.com/files/ftp_upload/51747/51747fig1highres.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Capsular Serotyping of Streptococcus pneumoniae by Latex Agglutination at JoVE.com

Capsular Serotyping of Streptococcus pneumoniae by Latex Agglutination

Latex agglutination testing is a simple, rapid and inexpensive method for serotyping Streptococcus pneumoniae, and has also been widely applied in diagnostic microbiology. This manuscript describes the in-house production of latex agglutination reagents, quality control procedures and the application of this technique to pneumococcal serotyping.

Capsular Serotyping of Streptococcus pneumoniae by Latex Agglutination

Latex agglutination reagents are widely used in microbial diagnosis, identification and serotyping. Streptococcus pneumoniae (the pneumococcus) is a major ...

capsular-serotyping-of-streptococcus-pneumoniae-by-latex-agglutination

Research

JoVE Journal

Immunology and Infection

Using Bioluminescent Imaging to Investigate Synergism Between Streptococcus pneumoniae and Influenza A Virus in Infant Mice

During the 1918 influenza virus pandemic, which killed approximately 50 million people worldwide, the majority of fatalities were not the result of infection with influenza virus alone. Instead, most individuals are thought to have succumbed to a secondary bacterial infection, predominately caused by the bacterium Streptococcus pneumoniae (the pneumococcus). The synergistic relationship between infections caused by influenza virus and the pneumococcus has subsequently been observed during the 1957 Asian influenza virus pandemic, as well as during seasonal outbreaks of the virus (reviewed in 1, 2). Here, we describe a protocol used to investigate the mechanism(s) that may be involved in increased morbidity as a result of concurrent influenza A virus and S. pneumoniae infection. We have developed an infant murine model to reliably and reproducibly demonstrate the effects of influenza virus infection of mice colonised with S. pneumoniae. Using this protocol, we have provided the first insight into the kinetics of pneumococcal transmission between co-housed, neonatal mice using in vivo imaging 3.

Using Bioluminescent Imaging to Investigate Synergism Between Streptococcus pneumoniae and Influenza A Virus in Infant Mice

A concurrent infection with influenza A virus is one of the factors implicated in the induction of invasive pneumococcal disease during asymptomatic Streptococcus pneumoniae carriage. Here we describe a mixed infection method using infant mice to investigate the synergism between these two respiratory pathogens.

During the 1918 influenza virus pandemic, which killed approximately 50 million people worldwide, the majority of fatalities were not the result of ...

Particle Agglutination Method for Poliovirus Identification

In the Global Polio Eradication Initiative, laboratory diagnosis plays a critical role by isolating and identifying PV from the stool samples of acute flaccid paralysis (AFP) cases. In the World Health Organization (WHO) Global Polio Laboratory Network, PV isolation and identification are currently being performed by using cell culture system and real-time RT-PCR, respectively. In the post-eradication era of PV, simple and rapid identification procedures would be helpful for rapid confirmation of polio cases at the national laboratories. In the present study, we will show the procedure of novel PA assay developed for PV identification. This PA assay utilizes interaction of PV receptor (PVR) molecule and virion that is specific and uniform affinity to all the serotypes of PV. The procedure is simple (one step procedure in reaction plates) and rapid (results can be obtained within 2 h of reaction), and the result is visually observed (observation of agglutination of gelatin particles).

A recently developed novel particle agglutination (PA) assay utilizing virus receptor molecule allowed a rapid and easy identification of poliovirus (PV). In this article, we will show the procedure for the PA assay for PV identification.

In the Global Polio Eradication Initiative, laboratory diagnosis plays a critical role by isolating and identifying PV from the stool samples of acute ...

Live Cell Imaging of Bacillus subtilis and Streptococcus pneumoniae using Automated Time-lapse Microscopy

During the last few years scientists became increasingly aware that average data obtained from microbial population based experiments are not representative of the behavior, status or phenotype of single cells. Due to this new insight the number of single cell studies rises continuously (for recent reviews see 1,2,3). However, many of the single cell techniques applied do not allow monitoring the development and behavior of one specific single cell in time (e.g. flow cytometry or standard microscopy).
Here, we provide a detailed description of a microscopy method used in several recent studies 4, 5, 6, 7, which allows following and recording (fluorescence of) individual bacterial cells of Bacillus subtilis and Streptococcus pneumoniae through growth and division for many generations. The resulting movies can be used to construct phylogenetic lineage trees by tracing back the history of a single cell within a population that originated from one common ancestor. This time-lapse fluorescence microscopy method cannot only be used to investigate growth, division and differentiation of individual cells, but also to analyze the effect of cell history and ancestry on specific cellular behavior. Furthermore, time-lapse microscopy is ideally suited to examine gene expression dynamics and protein localization during the bacterial cell cycle. The method explains how to prepare the bacterial cells and construct the microscope slide to enable the outgrowth of single cells into a microcolony. In short, single cells are spotted on a semi-solid surface consisting of growth medium supplemented with agarose on which they grow and divide under a fluorescence microscope within a temperature controlled environmental chamber. Images are captured at specific intervals and are later analyzed using the open source software ImageJ.

Live Cell Imaging of Bacillus subtilis and Streptococcus pneumoniae using Automated Time-lapse Microscopy

This protocol provides a step-by-step procedure to monitor single cell behavior of different bacteria in time using automated fluorescence time-lapse microscopy. Furthermore, we provide guidelines how to analyze the microscopy images.

During the last few years scientists became increasingly aware that average data obtained from microbial population based experiments are not ...

Characterization of Inflammatory Responses During Intranasal Colonization with Streptococcus pneumoniae

Nasopharyngeal colonization by Streptococcus pneumoniae is a prerequisite to invasion to the lungs or bloodstream1. This organism is capable of colonizing the mucosal surface of the nasopharynx, where it can reside, multiply and eventually overcome host defences to invade to other tissues of the host. Establishment of an infection in the normally lower respiratory tract results in pneumonia. Alternatively, the bacteria can disseminate into the bloodstream causing bacteraemia, which is associated with high mortality rates2, or else lead directly to the development of pneumococcal meningitis. Understanding the kinetics of, and immune responses to, nasopharyngeal colonization is an important aspect of S. pneumoniae infection models.
Our mouse model of intranasal colonization is adapted from human models3 and has been used by multiple research groups in the study of host-pathogen responses in the nasopharynx4-7. In the first part of the model, we use a clinical isolate of S. pneumoniae to establish a self-limiting bacterial colonization that is similar to carriage events in human adults. The procedure detailed herein involves preparation of a bacterial inoculum, followed by the establishment of a colonization event through delivery of the inoculum via an intranasal route of administration. Resident macrophages are the predominant cell type in the nasopharynx during the steady state. Typically, there are few lymphocytes present in uninfected mice8, however mucosal colonization will lead to low- to high-grade inflammation (depending on the virulence of the bacterial species and strain) that will result in an immune response and the subsequent recruitment of host immune cells. These cells can be isolated by a lavage of the tracheal contents through the nares, and correlated to the density of colonization bacteria to better understand the kinetics of the infection.

Characterization of Inflammatory Responses During Intranasal Colonization with Streptococcus pneumoniae

Colonization of the murine nasopharynx with Streptococcus pneumoniae and the subsequent extraction of adherent or recruited cells is described. This technique involves flushing the nasopharynx and collection of the fluid through the nares and is adaptable for various readouts, including differential cell quantification and analysis of mRNA expression in situ.

Nasopharyngeal colonization by Streptococcus pneumoniae is a prerequisite to invasion to the lungs or bloodstream1. This organism is capable of colonizing ...

Monitoring Changes in Membrane Polarity, Membrane Integrity, and Intracellular Ion Concentrations in Streptococcus pneumoniae Using Fluorescent Dyes

Membrane depolarization and ion fluxes are events that have been studied extensively in biological systems due to their ability to profoundly impact cellular functions, including energetics and signal transductions. While both fluorescent and electrophysiological methods, including electrode usage and patch-clamping, have been well developed for measuring these events in eukaryotic cells, methodology for measuring similar events in microorganisms have proven more challenging to develop given their small size in combination with the more complex outer surface of bacteria shielding the membrane. During our studies of death-initiation in Streptococcus pneumoniae (pneumococcus), we wanted to elucidate the role of membrane events, including changes in polarity, integrity, and intracellular ion concentrations. Searching the literature, we found that very few studies exist. Other investigators had monitored radioisotope uptake or equilibrium to measure ion fluxes and membrane potential and a limited number of studies, mostly in Gram-negative organisms, had seen some success using carbocyanine or oxonol fluorescent dyes to measure membrane potential, or loading bacteria with cell-permeant acetoxymethyl (AM) ester versions of ion-sensitive fluorescent indicator dyes. We therefore established and optimized protocols for measuring membrane potential, rupture, and ion-transport in the Gram-positive organism S. pneumoniae. We developed protocols using the bis-oxonol dye DiBAC4(3) and the cell-impermeant dye propidium iodide to measure membrane depolarization and rupture, respectively, as well as methods to optimally load the pneumococci with the AM esters of the ratiometric dyes Fura-2, PBFI, and BCECF to detect changes in intracellular concentrations of Ca2+, K+, and H+, respectively, using a fluorescence-detection plate reader. These protocols are the first of their kind for the pneumococcus and the majority of these dyes have not been used in any other bacterial species. Though our protocols have been optimized for S. pneumoniae, we believe these approaches should form an excellent starting-point for similar studies in other bacterial species.

Monitoring Changes in Membrane Polarity, Membrane Integrity, and Intracellular Ion Concentrations in Streptococcus pneumoniae Using Fluorescent Dyes

Unlike that seen for eukaryotes, there is a paucity of studies that detail membrane depolarization and ion concentration changes in bacteria, primarily as their small size makes conventional methods of measurement difficult. Here, we detail protocols for monitoring such events in the significant Gram-positive pathogen Streptococcus pneumoniae utilizing fluorescence techniques.

Membrane depolarization and ion fluxes are events that have been studied extensively in biological systems due to their ability to profoundly impact ...

Capsular Serotyping of Streptococcus pneumoniae Using the Quellung Reaction

There are over 90 different capsular serotypes of Streptococcus pneumoniae (the pneumococcus). As well as being a tool for understanding pneumococcal epidemiology, capsular serotyping can provide useful information for vaccine efficacy and impact studies.&#160;The Quellung reaction is the gold standard method for pneumococcal capsular serotyping. The method involves testing a pneumococcal cell suspension with pooled and specific antisera directed against the capsular polysaccharide. The antigen-antibody reactions are observed microscopically. The protocol has three main steps: 1) preparation of a bacterial cell suspension, 2) mixing of cells and antisera on a glass slide, and 3) reading the Quellung reaction using a microscope. The Quellung reaction is reasonably simple to perform and can be applied wherever a suitable microscope and antisera are available.

Capsular Serotyping of Streptococcus pneumoniae Using the Quellung Reaction

The Quellung reaction is the gold standard technique for serotyping Streptococcus pneumoniae. This technique utilizes a microscope and specific pneumococcal antisera and is commonly used in reference and research laboratories worldwide.

There are over 90 different capsular serotypes of Streptococcus pneumoniae (the pneumococcus). As well as being a tool for understanding pneumococcal ...

Proteomic Profiling of Macrophages by 2D Electrophoresis

The goal of the two-dimensional (2D) electrophoresis protocol described here is to show how to analyse the phenotype of human cultured macrophages. The key role of macrophages has been shown in various pathological disorders such as inflammatory, immunological, and infectious diseases. In this protocol, we use primary cultures of human monocyte-derived macrophages that can be differentiated into the M1 (pro-inflammatory) or the M2 (anti-inflammatory) phenotype. This in vitro model is reliable for studying the biological activities of M1 and M2 macrophages and also for a proteomic approach. Proteomic techniques are useful for comparing the phenotype and behaviour of M1 and M2 macrophages during host pathogenicity. 2D gel electrophoresis is a powerful proteomic technique for mapping large numbers of proteins or polypeptides simultaneously. We describe the protocol of 2D electrophoresis using fluorescent dyes, named 2D Differential Gel Electrophoresis (DIGE). The M1 and M2 macrophages proteins are labelled with cyanine dyes before separation by isoelectric focusing, according to their isoelectric point in the first dimension, and their molecular mass, in the second dimension. Separated protein or polypeptidic spots are then used to detect differences in protein or polypeptide expression levels. The proteomic approaches described here allows the investigation of the macrophage protein changes associated with various disorders like host pathogenicity or microbial toxins.

Macrophages are the key cells involved in host pathogenicity. Macrophages display phenotypic and functional diversity that can be analysed and detected by proteomic analysis. This article describes how to perform 2D electrophoresis of primary cultures of human macrophages differentiated into M1 or M2 phenotype.

The goal of the two-dimensional (2D) electrophoresis protocol described here is to show how to analyse the phenotype of human cultured macrophages. The ...

Non-invasive Imaging of the Innate Immune Response in a Zebrafish Larval Model of Streptococcus iniae Infection

The aquatic pathogen, Streptococcus iniae, is responsible for over 100 million dollars in annual losses for the aquaculture industry and is capable of causing systemic disease in both fish and humans. A better understanding of S. iniae disease pathogenesis requires an appropriate model system. The genetic tractability and the optical transparency of the early developmental stages of zebrafish allow for the generation and non-invasive imaging of transgenic lines with fluorescently tagged immune cells. The adaptive immune system is not fully functional until several weeks post fertilization, but zebrafish larvae have a conserved vertebrate innate immune system with both neutrophils and macrophages. Thus, the generation of a larval infection model allows the study of the specific contribution of innate immunity in controlling S. iniae infection.
The site of microinjection will determine whether an infection is systemic or initially localized. Here, we present our protocols for otic vesicle injection of zebrafish aged 2-3 days post fertilization as well as our techniques for fluorescent confocal imaging of infection. A localized infection site allows observation of initial microbe invasion, recruitment of host cells and dissemination of infection. Our findings using the zebrafish larval model of S. iniae infection indicate that zebrafish can be used to examine the differing contributions of host neutrophils and macrophages in localized bacterial infections. In addition, we describe how photolabeling of immune cells can be used to track individual host cell fate during the course of infection.

Non-invasive Imaging of the Innate Immune Response in a Zebrafish Larval Model of Streptococcus iniae Infection

Here, we present a protocol for the generation and imaging of a localized bacterial infection in the zebrafish otic vesicle.

The aquatic pathogen, Streptococcus iniae, is responsible for over 100 million dollars in annual losses for the aquaculture industry and is capable of ...

A Murine Model of Group B Streptococcus Vaginal Colonization

Streptococcus agalactiae (group B Streptococcus, GBS), is a Gram-positive, asymptomatic colonizer of the human gastrointestinal tract and vaginal tract of 10 - 30% of adults. In immune-compromised individuals, including neonates, pregnant women, and the elderly, GBS may switch to an invasive pathogen causing sepsis, arthritis, pneumonia, and meningitis. Because GBS is a leading bacterial pathogen of neonates, current prophylaxis is comprised of late gestation screening for GBS vaginal colonization and subsequent peripartum antibiotic treatment of GBS-positive mothers. Heavy GBS vaginal burden is a risk factor for both neonatal disease and colonization. Unfortunately, little is known about the host and bacterial factors that promote or permit GBS vaginal colonization. This protocol describes a technique for establishing persistent GBS vaginal colonization using a single &#946;-estradiol pre-treatment and daily sampling to determine bacterial load. It further details methods to administer additional therapies or reagents of interest and to collect vaginal lavage fluid and reproductive tract tissues. This mouse model will further the understanding of the GBS-host interaction within the vaginal environment, which will lead to potential therapeutic targets to control maternal vaginal colonization during pregnancy and to prevent transmission to the vulnerable newborn. It will also be of interest to increase our understanding of general bacterial-host interactions in the female vaginal tract.

A Murine Model of Group B Streptococcus Vaginal Colonization

The purpose of this protocol is to imitate human group B Streptococcus (GBS) vaginal colonization in a murine model. This method may be used to investigate host immune responses and bacterial factors contributing to GBS vaginal persistence, as well as to test therapeutic strategies.

Streptococcus agalactiae (group B Streptococcus, GBS), is a Gram-positive, asymptomatic colonizer of the human gastrointestinal tract and vaginal tract of ...

A Robust Pneumonia Model in Immunocompetent Rodents to Evaluate Antibacterial Efficacy against S. pneumoniae, H. influenzae, K. pneumoniae, P. aeruginosa or A. baumannii

Efficacy of candidate antibacterial treatments must be demonstrated in animal models of infection as part of the discovery and development process, preferably in models which mimic the intended clinical indication. A method for inducing robust lung infections in immunocompetent rats and mice is described which allows for the assessment of treatments in a model of serious pneumonia caused by S. pneumoniae, H. influenzae, P. aeruginosa, K. pneumoniae or A. baumannii. Animals are anesthetized, and an agar-based inoculum is deposited deep into the lung via nonsurgical intratracheal intubation. The resulting infection is consistent, reproducible, and stable for at least 48 h and up to 96 h for most isolates. Studies with marketed antibacterials have demonstrated good correlation between in vivo efficacy and in vitro susceptibility, and concordance between pharmacokinetic/pharmacodynamic targets determined in this model and clinically accepted targets has been observed. Although there is an initial time investment when learning the technique, it can be performed quickly and efficiently once proficiency is achieved. Benefits of the model include elimination of the neutropenic requirement, increased robustness and reproducibility, ability to study more pathogens and isolates, improved flexibility in study design and establishment of a challenging infection in an immunocompetent host.

A Robust Pneumonia Model in Immunocompetent Rodents to Evaluate Antibacterial Efficacy against S. pneumoniae, H. influenzae, K. pneumoniae, P. aeruginosa or  A. baumannii

A method for establishing lung infections in immunocompetent rodents is described. With proficiency, this method can be performed quickly and easily to induce stable infection with many isolates of S. pneumoniae, H. influenzae, P. aeruginosa, K. pneumoniae and A. baumannii.

Efficacy of candidate antibacterial treatments must be demonstrated in animal models of infection as part of the discovery and development process, ...