This work was supported by the Bill and Melinda Gates Foundation (PneuCarriage project, Grant 52099) and the Victorian Government&#8217;s Operational Infrastructure Support Program.&#160;Thanks to Eileen Dunne, Janet Strachan, and Kim Hare for critical reading of the manuscript.

1. Preparation of Cell Suspension for Typing
<ol>
	<li>Using a sterile disposable 1 &#181;l loop, take a small sweep of fresh overnight pure culture of pneumococci grown on solid nonselective horse blood agar and inoculate 100 &#181;l Heart Infusion (HI) broth. The suspension should appear to be just visibly turbid. Gently agitate the tube to emulsify. 
	Note: Other media such as Brain Heart Infusion broth may also be suitable. Addition of serum (e.g. a final concentration of 10% (v/v) horse serum) and incubation at 37 &#176;C for approximately 1 hr before use may be used to enhance the reaction.</li>
</ol>
2. Checking Density of Cell Suspension
The cell suspension prepared in step 1.1 will also be used as the negative control, and should be checked for cell density before adding the typing sera.
<ol>
	<li>Using a 1 &#181;l loop, transfer a drop of the inoculum prepared in step 1.1 onto a glass microscope slide.</li>
	<li>Using a pair of forceps, place a small circular coverslip on the suspension.</li>
	<li>Using phase-contrast setting, observe the preparation microscopically under 400X magnification, to check the density of the suspension (Figure 1A).
	<ol>
		<li>If the inoculum is too heavy&#160;(Figure 2A), add more broth to the cell suspension made in step 1.1 to dilute it. Gently mix again.</li>
		<li>If the inoculum is too light, for example, there are &#60;50 cells visible in the microscopic field&#160;(Figure 2B), add more bacteria to the cell suspension preparation made in step 1.1 and gently mix. 
		Note: With experience, the density of the cell suspension can be judged by shaking and holding up to the light, where it should appear to be just visibly turbid.</li>
	</ol>
	</li>
	<li>Repeat steps 2.1-2.3 until a suitable number of cells are visible in the microscopic field. 
	Note: Glass slides and coverslips present a sharps hazard. After use, slides and coverslips are contaminated with infectious material and must be disposed into a biohazard sharps container.</li>
</ol>
3. Typing
<ol>
	<li>Transfer ~1 &#181;l of the prepared inoculum (e.g. using a 1 &#181;l loop) onto the glass microscope slide. Discard the loop into the biohazard sharps container.</li>
	<li>Add an equal volume (1 &#181;l loopful) of room-temperature antiserum onto the slide and mix both drops well with the loop.</li>
	<li>Using a pair of forceps, place a small circular coverslip on the suspension, taking care not to touch the drop. Place the coverslip on the suspension immediately after the addition of antisera to prevent the fluid on the slide from drying out.</li>
	<li>Using a microscope, observe the suspension under phase-contrast with 400X magnification. A negative Quellung reaction is observed when little or no &#8216;swelling&#8217; of the pneumococcal cells can be seen at 400X magnification, similar to what is observed in the pneumococcal cell suspension with no antisera added (negative control). A positive Quellung reaction is observed when the cells appear enlarged or &#39;swollen&#39; and more visible when compared to the cells in the negative control. 
	Note: Equivocal reactions should be investigated further, for example by examining the reaction after 5-10 min incubation at room temperature, or by repeating the reaction with more antiserum.</li>
	<li>Conduct pneumococcal serotyping with successive testing of individual antisera pools until a positive reaction is observed. Alternatively, pools can be applied in &#8216;rounds&#8217; depending upon local incidence of serotypes. 
	Note: Testing in &#8216;rounds&#8217; ensures that the pools containing antibodies to the most common serotypes are tested first, minimizing effort and cost. Concurrent testing of several pools also provides additional negative controls, which can be useful in interpretation. If all the pools in a particular &#8216;round&#8217; are negative, test the isolate with subsequent rounds of pools.</li>
	<li>Test the isolate using the appropriate type or group antiserum contained within the reactive pool. Where appropriate, use factor antisera to further differentiate the serotype. 
	Note: Keys to pneumococcal antisera supplied by the manufacturer are used in the selection of appropriate pool, type, group or factor antisera and interpretation of results (including any cross-reactions).</li>
	<li>If negative results are obtained with all pools, test the isolate with Omniserum reagent (contains antibodies against 91 serotypes21). If a positive Quellung reaction is still not observed when testing a pneumococcal isolate with the Omniserum reagent, this may be because the isolate is expressing little or no capsule, or that antibodies against the serotype are not represented in the Omniserum. The latter category may include new serotypes.</li>
</ol>

<ol>
	<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>Bogaert, D., De Groot, R., Hermans, P. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Streptococcus+pneumoniae+colonisation:+the+key+to+pneumococcal+disease.">Streptococcus pneumoniae colonisation: the key to pneumococcal disease.</a> Lancet Infect. Dis. 4, 144-154 (2004).</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>Calix, J. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biochemical,+genetic,+and+serological+characterization+of+two+capsule+subtypes+among+Streptococcus+pneumoniae+Serotype+20+strains:+discovery+of+a+new+pneumococcal+serotype.">Biochemical, genetic, and serological characterization of two capsule subtypes among Streptococcus pneumoniae Serotype 20 strains: discovery of a new pneumococcal serotype.</a> J. Biol. Chem. 287, 27885-27894 (2012).</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>Johnson, H. 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=Systematic+evaluation+of+serotypes+causing+invasive+pneumococcal+disease+among+children+under+five:+the+pneumococcal+global+serotype+project.">Systematic evaluation of serotypes causing invasive pneumococcal disease among children under five: the pneumococcal global serotype project.</a> PLoS Med. 8, (2010).</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>Obaro, S. K., Adegbola, R. A., Banya, W. A., Greenwood, B. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Carriage+of+pneumococci+after+pneumococcal+vaccination.">Carriage of pneumococci after pneumococcal vaccination.</a> Lancet. 348, 271-272 (1996).</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>Austrian, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+quellung+reaction,+a+neglected+microbiologic+technique.">The quellung reaction, a neglected microbiologic technique.</a> Mt. Sinai. J. Med. 43, 699-709 (1976).</li><li>Konradsen, H. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Validation+of+serotyping+of+Streptococcus+pneumoniae+in+Europe.">Validation of serotyping of Streptococcus pneumoniae in Europe.</a> Vaccine. 23, 1368-1373 (2005).</li><li>Lovgren, M., 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=Evolution+of+an+international+external+quality+assurance+model+to+support+laboratory+investigation+of+Streptococcus+pneumoniae,+developed+for+the+SIREVA+project+in+Latin+America,+from.">Evolution of an international external quality assurance model to support laboratory investigation of Streptococcus pneumoniae, developed for the SIREVA project in Latin America, from.</a> J. Clin. Microbiol. 45, 3184-3190 (2007).</li><li>Reasonover, 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=The+International+Circumpolar+Surveillance+interlaboratory+quality+control+program+for+Streptococcus+pneumoniae.">The International Circumpolar Surveillance interlaboratory quality control program for Streptococcus pneumoniae.</a> J. Clin. Microbiol. 49, 138-143 (2011).</li><li>Hare, K. M., 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=#34;Dodgy+6As&amp;#34;:+differentiating+pneumococcal+serotype+6C+from+6A+by+use+of+the+Quellung+reaction.">#34;Dodgy 6As&amp;#34;: differentiating pneumococcal serotype 6C from 6A by use of the Quellung reaction.</a> J. Clin. Microbiol. 47, 1981-1982 (2009).</li><li>Kong, F., Gilbert, G. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Using+cpsA-cpsB+sequence+polymorphisms+and+serotype-/group-specific+PCR+to+predict+51+Streptococcus+pneumoniae+capsular+serotypes.">Using cpsA-cpsB sequence polymorphisms and serotype-/group-specific PCR to predict 51 Streptococcus pneumoniae capsular serotypes.</a> J. Med. Microbiol. 52, 1047-1058 (2003).</li><li>Castillo, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+and+characterization+of+Streptococcus+pneumoniae.">Identification and characterization of Streptococcus pneumoniae.</a> Laboratory Methods for the Diagnosis of Meningitis caused by Neisseria meningitidis, Streptococcus pneumoniae, and Haemophilus influenzae. 2nd ed. , 73-86 (2011).</li><li>Kaijalainen, T., Rintamaki, S., Herva, E., Leinonen, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+gene-technological+and+conventional+methods+in+the+identification+of+Streptococcus+pneumoniae.">Evaluation of gene-technological and conventional methods in the identification of Streptococcus pneumoniae.</a> J. Microbiol. Methods. 51, 111-118 (2002).</li><li>Sorensen, U. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Typing+of+pneumococci+by+using+12+pooled+antisera.">Typing of pneumococci by using 12 pooled antisera.</a> J. Clin. Microbiol. 31, 2097-2100 (1993).</li><li>Diagnostica SSI, <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Key+to+pneumococcal+factor+antisera.">Key to pneumococcal factor antisera.</a> , (2013).</li><li>Diagnostica SSI, <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pneumococcal+antisera+for+in+vitro+diagnostic+use.">Pneumococcal antisera for in vitro diagnostic use.</a> , (2011).</li><li>Lund, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Laboratory+diagnosis+of+Pneumococcus+infections.">Laboratory diagnosis of Pneumococcus infections.</a> Bull. 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The authors have nothing to disclose.

A critical step in this technique is ensuring a pure culture is used to prepare the suspension along with adding a suitable number of bacterial cells to the broth. It should be noted that colony growth and morphology on agar plates varies between different pneumococcal isolates. Some produce dry and rough colonies, which can be difficult to pick off with a loop and do not emulsify well in the broth. For this reason, a larger sweep of bacteria may be required when preparing the cell suspension. In any case, it is imperative that individual cells, and not aggregates, are obvious in the negative control so their relative size can be compared to the cells with antisera added. Additionally, too many bacterial cells may result in a false negative reaction, where an excess of capsular antigen may inhibit the Quellung reaction10. For this reason, it is important to create a bacterial suspension with a suitable cell density (Figure 1). The suspension may be preserved for several months by the addition of several drops of concentrated formalin. This enables easier batch testing and also sterilizes the broth reducing the risk of laboratory-acquired infections; although this must be weighed against the safety risk of handling formalin, which is toxic, corrosive, carcinogenic, sensitizing, and flammable. In addition, we have observed that some isolates show weaker reactions in the presence of formalin (data not shown).
Quellung reactions are examined using a microscope. The use of phase contrast enhances the visibility of the capsule. In some laboratories, the reaction is viewed using oil-immersion at 1,000X magnification14,16.&#160; In addition, some laboratories use a counter stain (methylene blue) to further enhance the visibility of a positive reaction10,16,22. The method presented here is viewed without oil-immersion and under 400X magnification15,17. This method is quick, simple and relatively easy to read23.
It is important to maintain good internal quality control procedures to ensure the antisera and testing procedures are in order. Participation in an external quality assurance program is often beneficial.
A major limitation of the Quellung reaction for serotyping of pneumococci is the high cost of the antisera required. However, this is minimized in the above protocol due to the small amount of antiserum used for each reaction. Several other methods have been described for serotyping pneumococci23,24. However, Quellung remains the gold standard and the development of new methods requires standardization against the Quellung reaction23.&#160; Using appropriate antisera, the Quellung reaction can also be utilized to identify and type other capsule producing bacteria25. The Quellung reaction is instrumental in the characterization of current and emerging pneumococcal serotypes, as well as providing information on vaccine efficacy and the long-term impact of vaccination on carriage and disease.

Streptococcus pneumoniae (the pneumococcus) is responsible for the death of an estimated 800,000 children under the age of five annually, with the burden of disease falling predominantly in resource-poor countries1. Pneumococcal diseases range in severity from localized infections such as acute otitis media to severe life threatening infections such as sepsis, meningitis and pneumonia2. Over 90 capsular serotypes of pneumococci have been described3,4. Current pneumococcal vaccines provide protection against the serotypes that are responsible for the majority of invasive disease5,6. However, widespread use of vaccines has been associated with replacement of serotypes included in the vaccine by nonvaccine serotypes, both in nasopharyngeal carriage and invasive disease7,8. This highlights the importance of serotyping for epidemiological surveillance and long-term vaccine impact studies9.
The gold standard method for pneumococcal typing is the capsular reaction test, known as the Quellung reaction, first described by Neufeld in 1902 (see Austrian10). A high degree of concordance has been found between laboratories, and between the Quellung reaction and other serotyping methods11-13. There are several variations of the Quellung reaction10,14-16. &#160;The method described here does not require the use of a counter stain or the oil immersion lens; rather, a drop of prepared specimen is mixed with the typing serum and examined immediately under 400X magnification.
The Quellung reaction is usually performed using commercially available antisera. The method described here uses less antisera compared with some other methods16,17, making it more cost effective. Once an isolate is identified as a pneumococcus, it is sequentially tested with antisera pools until a positive reaction is observed. Each pool antiserum contains different mixtures of antisera raised against 91 pneumococcal serotypes18. Once the pool is established, the individual type and group antisera that are included in the reactive pool are tested individually in sequence. Type antisera generally react with a single serotype (e.g. Type 1 antiserum reacts with serotype 1), whereas group antisera react with all the serotypes in a particular group (e.g. Group 22 antiserum reacts with serotypes 22F and 22A). Some serotypes within groups are further differentiated using factor antisera (e.g. Serotype 22F reacts with factor 22b, but not 22c). In this case testing is continued with all relevant factor antisera until serotype is determined19,20. The Quellung reaction is simple and quick14 and can be performed in any laboratory that is equipped with a good quality microscope and appropriate set of antisera.

<table border="0" cellpadding="0" cellspacing="0" width="1305">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;">Pneumococcus (Neufeld) antisera</td>
			<td>SSI Diagnostica</td>
			<td></td>
			<td><a href="http://www.ssi.dk/ssidiagnostica">http://www.ssi.dk/ssidiagnostica</a> 
			Bring all antisera to room temperature before use</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Pneumococcus (Omni) antiserum</td>
			<td>SSI Diagnostica</td>
			<td>2438</td>
			<td>Bring all antisera to room temperature before use</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Optical Microscope</td>
			<td>Leica Microsystems</td>
			<td>Leica DML</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Calibrated Disposable Inoculating Loops 1 &#181;l</td>
			<td>Copan</td>
			<td>CD175S01</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Horse Blood Agar (HBA) plates</td>
			<td>Thermo Fisher Scientific</td>
			<td>PP2001</td>
			<td><a href="http://www.thermofisher.com.au/">http://www.thermofisher.com.au/</a> 
			Bring to room temperature before use</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Heart Infusion (HI) broth</td>
			<td>Media Preparation Unit (MPU), The University of Melbourne</td>
			<td>40HIN</td>
			<td>Bring to room temperature before use</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Glass microscope slides 76 mm x 26 mm</td>
			<td>Lomb Scientific</td>
			<td>7101</td>
			<td>Sharps hazard: Dispose in a biohazard sharps container</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Circular glass coverslips</td>
			<td>NeuVitro&#160;</td>
			<td>GG-5</td>
			<td></td>
		</tr>
		<tr height="20">
			<td colspan="2" height="20" style="height:20px;">*Excluding the antisera, all other materials can be purchased from alternative suppliers</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td colspan="4" height="20" style="height:20px;">*Plates prepared from defibranated sheep blood are suitable but blood plates prepared from citrated blood or human blood are not</td>
		</tr>
	</tbody>
</table>

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.

A positive Quellung reaction occurs when type-specific antibody binds to the capsule of the pneumococcus, leading to a change in its refractive index10. When viewed under a microscope, the bacteria appear &#8216;swollen&#8217; and more visible14. Figure 1A shows pneumococcal cells in HI broth. When type-specific anti-serum is added to the bacterial suspension, pneumococci appear &#8216;swollen&#8217;, refractive and more rounded (Figure 1B).
Too many bacterial cells added to the broth may result in aggregation of cells or a false negative reaction. Figure 2 shows inocula of pneumococcal cells in HI broth that are &#8216;too heavy&#8217; (Figure 2A) or &#8216;too light&#8217; (Figure 2B).
<img alt="Figure 1" fo:content-width="5in" fo:src="/files/ftp_upload/51208/51208fig1highres.jpg" src="/files/ftp_upload/51208/51208fig1.jpg" /> 
Figure 1.&#160;Negative and positive Quellung reaction. Preparations of serotype 9V pneumococcal isolate with no antiserum (1A) and with group 9 antiserum (1B), were viewed under 400X magnification. The latter shows the &#8216;swelling&#8217; and rounding of bacterial cells typically seen in a positive Quellung reaction.
<img alt="Figure 2" fo:content-width="5in" fo:src="/files/ftp_upload/51208/51208fig2highres.jpg" src="/files/ftp_upload/51208/51208fig2.jpg" /> 
Figure 2.&#160;Too heavy and too light inocula. Preparations of pneumococcal cell suspensions that are too heavy (2A) or too light (2B), when viewed under 400X magnification.

Watch this Scientific Journal Video about Capsular Serotyping of Streptococcus pneumoniae Using the Quellung Reaction at JoVE.com

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.

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 ...

capsular-serotyping-streptococcus-pneumoniae-using-quellung

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Immunology and Infection

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Video: Capsular Serotyping of Streptococcus pneumoniae Using the Quellung Reaction

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 ...

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 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.

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.

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

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, ...

Intracranial Subarachnoidal Route of Infection for Investigating Roles of Streptococcus suis Biofilms in Meningitis in a Mouse Infection Model

Streptococcus suis is not only a major bacterial pathogen of pigs worldwide but also an emerging zoonotic agent. In humans and pigs, meningitis is a major manifestation of S. suis infections. A suitable infection model is an essential tool to understand the mechanisms of diseases caused by pathogens. Several routes of infection in mice have been developed to study the pathogenesis of S. suis infection. However, the intraperitoneal, intranasal, and intravenous routes of infection are not suitable for studying the roles of S. suis surface components in meningitis directly in the brain, such as the extracellular matrix from biofilms. Although intracisternal inoculation has been used for S. suis infection, the precise injection site has not been described. Here, the intracranial subarachnoidal route of infection was described in a mouse model to investigate the roles of biofilms in S. suis meningitis. S. suis planktonic cells or biofilm state cells were directly injected into the subarachnoid space of mice through the injection site located 3.5 mm rostral from the bregma. Histopathological analysis and increased mRNA expression of TLR2 and cytokines of the brain tissue from mice injected with biofilm state cells clearly indicated that S. suis biofilm plays definitive roles in S. suis meningitis. This route of infection has obvious advantages over other routes of infection, allowing the study of the host-bacterium interaction. Furthermore, it permits the effect of bacterial components on host immune responses directly in the brain to be assessed, and mimics bacterial entrance into the central nervous system. This route of infection can be extended for investigating the mechanisms of meningitis caused by other bacteria. In addition, it can also be used to test the efficacy of drugs against bacterial meningitis.

Intracranial Subarachnoidal Route of Infection for Investigating Roles of Streptococcus suis Biofilms in Meningitis in a Mouse Infection Model

Here, we describe the intracranial subarachnoidal route of infection in mice to study roles of biofilms in Streptococcus suis meningitis. This infection model is also suitable for studying the pathogenesis of other bacterial meningitis and the efficacy of new drugs against bacterial meningitis.

Streptococcus suis is not only a major bacterial pathogen of pigs worldwide but also an emerging zoonotic agent. In humans and pigs, meningitis is a major ...

Measuring Dengue Virus RNA in the Culture Supernatant of Infected Cells by Real-time Quantitative Polymerase Chain Reaction

At present, real-time polymerase chain reaction (PCR) technology is an indispensable tool for the detection and quantification of viral genomes in research laboratories, as well as for molecular diagnosis, because of its sensitivity, specificity, and convenience. However, in most cases, the quantitative PCR (qPCR) assay generally used to detect virus infection has relied on the purification of viral nucleic acid prior to the PCR step. In this study, the fluorescence-based reverse transcription qPCR (RT-qPCR) assay is developed through the combination of a processing buffer and a one-step RT-PCR reagent so that the whole process, from the harvest of the culture supernatant of virus-infected cells until real-time detection, can be performed without viral RNA purification. The established protocol enables the quantification of a wide range of RNA concentrations of dengue virus (DENV) within 90 min. In addition, the adaptability of the direct RT-qPCR assay to the evaluation of an antiviral agent is demonstrated by an in vitro experiment using a previously reported DENV inhibitor, mycophenolic acid (MPA). Moreover, other RNA viruses, including yellow fever virus (YFV), Chikungunya virus (CHIKV), and measles virus (MeV), can be quantified by direct RT-qPCR with the same protocol. Therefore, the direct RT-qPCR assay described in this report is useful for monitoring RNA virus replication in a simple and rapid manner, which will be further developed into a promising platform for a high-throughput screening study and clinical diagnosis.

Real-time quantitative polymerase chain reaction analysis combined with reverse transcription (RT-qPCR) has been widely used to measure the level of RNA virus infections. Here we present a direct RT-qPCR assay, which does not require an RNA purification step, developed for the quantification of several RNA viruses, including dengue virus.