이 작품은 빌과 멜린다 게이츠 재단 (PneuCarriage 프로젝트 그랜트 52099)와 빅토리아 정부의 운영 인프라 지원 프로그램에 의해 지원되었다. 아일린 던 자넷 스트라 칸, 그리고 원고의 중요한 읽기 김 토끼 감사합니다.

1. 타이핑에 대한 세포 현탁액의 준비 <ol><li> 멸균 일회용 1 μL 루프를 사용하여 고체 비 선택적 말 혈액 한천에 성장 폐렴 구균의 신선한 하룻밤 순수 배양의 작은 스윕을 100 ㎕의 심장 주입 (HI) 배양액을 접종. 서스펜션은 눈에 띄게 혼탁 한 것으로 나타납니다. 부드럽게 유화 튜브를 교반. 참고 : 뇌 심장 주입 국물 같은 다른 미디어도 적합 할 수있다. 혈청 (10 % 예를 들면 최종 농도 (v / v) 말 혈청) 및 사용 전 약 1 시간 동안 37 ° C에서 배양을 첨가하여 반응을 향상시키기 위해 사용될 수있다. </li></ol> 2. 세포 현탁액의 농도를 확인 단계 1.1에서 제조 한 세포 현탁액은 음성 대조군으로 사용되며, 입력 혈청을 추가하기 전에 셀 밀도에 대해 검사되어야한다. <ol><li> 1 μL 루프를 사용 GLAS 상 단계 1.1에서 제조 한 균액의 저하를 전송할의 현미경 슬라이드. </li><li> 포셉 한 켤레를 사용하여 서스펜션에 작은 원형의 커버 슬립을 배치합니다. </li><li> 위상차 설정을 사용하여 현탁액의 농도 (도 1a)을 확인하기 위해 현미경으로 400 배의 배율 하에서 제제를 관찰한다. <ol><li> 접종은 (그림 2A) 너무 무거운 경우, 희석하는 단계 1.1에서 만든 세포 현탁액에 더 많은 국물을 추가합니다. 조심스럽게 다시 섞는다. </li><li> 접종이 너무 가벼운 경우에, 예를 들어, 50 세포 단계 1.1에서 만든 세포 현탁액 준비에 더 많은 박테리아를 추가하고 부드럽게 섞는다, 미세한 필드 (그림 2B)에서 볼 수 &lt;있습니다. 참고 : 경험을 바탕으로, 세포 현탁액의 밀도가 떨고 그냥 눈에 띄게 혼탁이 의심되는 빛에 들고으로 판단 할 수있다. </li></ol></li><li> 세포의 적절한 숫자가 미세한 필드에 표시 될 때까지 반복 2.1-2.3 단계를 반복합니다. 참고 : 유리 슬라이드 및 coverslIPS는 날카로운 위험을 제시한다. 사용 후, 슬라이드 커버 슬립은 전염성 물질로 오염되어 생물의 날카로운 물건 용기에 처리해야합니다. </li></ol> 3. 타자 <ol><li> 유리 현미경 슬라이드에 (예를 들어, 1 μL 루프를 사용하여) 준비된 접종 ~ 1 μl를 전송합니다. 생물의 날카로운 물건 용기에 루프를 폐기하십시오. </li><li> 슬라이드에 상온 항혈청의 동량 (1 μL의 백금이)를 첨가하고 모두 루프 잘 방울 섞는다. </li><li> 포셉 한 쌍을 사용하여, 드롭를 만지지 않도록주의하면서, 서스펜션에 작은 원형의 커버 슬립을 배치합니다. 마르지 슬라이드의 유체를 방지하기 위해 즉시 항혈청을 추가 한 후 정지에 커버 슬립을 놓습니다. </li><li> 현미경을 사용하여, 400 배의 배율 위상차에 따라 서스펜션을 관찰합니다. 폐렴 구균 세포의 &#39;팽창&#39;거의 또는 전혀 (40)에서 볼 수 없습니다 때 부정적인 Quellung 반응은 관찰더 항혈청과 폐렴 구균 세포 현탁액에서 관찰되는 것과 유사한 0X 배율 (음성 대조군)을 추가했다. 대조군의 세포에 비해 세포가 확대 또는 &#39;팽윤&#39;더 볼 때 나타나는 Quellung 양성 반응이 관찰된다. 참고 : 모호한 반응을 실온에서 50-10 분간 배양 한 후, 이상 항혈청과 반응을 반복하여 반응을 조사함으로써, 예를 들어 더 조사한다. </li><li> 긍정적 인 반응이 관찰 될 때까지 각각의 항혈청 풀의 연속적인 테스트와 폐렴 구균 혈청 형을 실시한다. 또한, 풀은 혈청 형의 지역 빈도에 따라 &#39;라운드&#39;에 적용 할 수 있습니다. 참고 : &#39;라운드&#39;의 테스트는 가장 흔한 혈청​​ 형에 대한 항체를 포함하는 풀을 먼저 테스트되도록, 노력과 비용을 최소화. 여러 풀의 동시 테스트는 interp에 유용 할 수있는 추가 부정적인 컨트롤을 제공합니다retation. 특히 &#39;라운드&#39;의 모든 풀을 부의 경우, 풀의 다음 라운드로 분리를 테스트합니다. </li><li> 반응성 풀에 포함 된 적절한 유형 또는 그룹 항혈청을 사용하여 분리를 테스트합니다. 적절한 경우, 추가 혈청 형을 구별하는 요소 항혈청을 사용합니다. 참고 : 제조업체에서 제공하는 폐렴 구균 항혈청에 키가 적절한 풀, 유형, 그룹 또는 인자 항혈청 (있는 교차 반응을 포함하여) 결과의 해석의 선택에 사용됩니다. </li><li> 부정적인 결과가 모든 풀을 얻을 경우, (91 혈청 형 (21)에 대한 항체를 포함) Omniserum 시약 분리를 테스트합니다. Omniserum 시약 분리 폐렴을 테스트 할 때 긍정적 Quellung 반응이 여전히 발견되지 않는 경우 분리가 거의 없거나 전혀없는 캡슐을 발현 또는 혈청 형에 대한 항체가 Omniserum에 표시되지 않도록되어 있으므로,이 일 수있다. 후자는 새로운 혈청 형을 포함 할 수있다. </li> &lt;/ OL&gt;

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Infect. 17, 1-13 (2011).</li><li>Casewell, M. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Experiences+in+the+use+of+commercial+antisera+for+the+capsular+typing+of+klebsiella+species.">Experiences in the use of commercial antisera for the capsular typing of klebsiella species.</a> J. Clin. Pathol. 25, 734-737 (1972).</li></ol>

저자가 공개하는 게 없다.

이 기술에서 중요한 단계는 순수 배양 브로스에 균체의 적절한 수를 더한 함께 현탁액을 제조하는 데 사용되는 약속된다. 그것은 한천 접시에 식민지의 성장과 형태가 다른 폐렴 구균 균주 사이에서 변화 주​​목해야한다. 일부는 루프를 잡을하고 국물에 잘 유화 없습니다 어려울 수 있습니다 건조하고 거친 식민지를 생산하고 있습니다. 세포 현탁액을 준비 할 때 그 때문에, 박테리아의 큰 청소가 필요할 수있다. 어떤 경우에는, 자신의 상대적인 크기는 항혈청으로 세포에 비교 될 수 있도록 개별 셀, 그리고 골재, 음성 대조군에 첨가 명백한 것이 필수적이다. 또한, 너무 많은 박테리아 세포의 캡슐 항원의 과잉 Quellung 반응 (10)을 억제 할 수 위음성 반응이 발생할 수 있습니다. 이러한 이유로, 최적 셀 밀도 세균 현탁액 (도 1)를 생성하는 것이 중요하다. 지속연금 농축 포르말린 몇 방울을 첨가하여 몇 달 동안 유지 될 수있다. 이것은 쉽게 배치 테스트를 가능하게하고 또한 실험실 취득한 감염의 위험을 감소 국물을 살균, 이것은 독성, 부식성, 발암 성, 민감하고, 가연성 처리 포르말린의 안전 위험, 비교 검토해야하지만. 또, 몇 가지 균주는 포르말린 (데이터는 도시되지 않음)의 존재 하에서 약한 반응을 보여줄 것을 관찰했다. Quellung 반응은 현미경을 사용하여 검사합니다. 위상차의 사용은 캡슐의 시인성을 향상시킨다. 일부 실험실에서, 반응은 1,000 X 배율 (14, 16)에 기름 침수를 사용하여 볼 수 있습니다. 또한, 일부 실험실은 더 긍정적 인 반응을 10, 16, 22의 가시성을 향상시키기 위해 카운터 얼룩 (메틸렌 블루)를 사용합니다. 여기에 제시된 방법은 석유 나없이 볼mmersion 및 400X 배율 15, 17 세 미만. 이 방법은 빠르고, 간단하고 23 비교적 읽기 쉽다. 그것은 항혈청 및 테스트 절차를 순서에 보장하기 위해 좋은 내부 품질 관리 절차를 유지하는 것이 중요합니다. 외부 품질 보증 프로그램에 참여하는 것은 종종 유용합니다. 폐렴 구균의 혈청 형에 대한 Quellung 반응의 주요 제한은 필요 항혈청의 고비용이다. 그러나이 때문에 각 반응에 사용되는 항혈청의 소량으로 상기 프로토콜에서 최소화된다. 몇 가지 다른 방법은 폐렴 구균 23, 24 혈청 형에 대해 설명 하였다. 그러나 Quellung는 황금 표준 남아 새로운 방법의 개발은 Quellung 반응 (23)에 대한 표준화가 필요합니다. 해당 항혈청을 사용 Quellung 반응은 또한 다른 캡을 식별하고 입력하는 데에 이용 될 수있다ULE 박테리아 (25)을 생산. Quellung 반응이 현재 및 신흥 폐렴 구균 혈청 형의 특성뿐만 아니라, 백신의 효능 및 포가 및 질환에 대한 백신의 장기적인 영향에 대한 정보를 제공하는 수단이다.

연쇄상 구균 폐렴 (폐렴 구균)는 질병의 부담이 자원이 부족한 국가의 1에 주로 떨어지는, 매년 5 세 미만 약 80 만 어린이의 죽음에 대한 책임이 있습니다. 폐렴 구균 질환은 패혈증, 수막염과 폐렴이 심한 생명을 위협하는 감염으로 급성 중이염과 같은 지역화 감염 정도에 이르기까지 다양합니다. 폐렴 구균의 90 캡슐 혈청 형 3, 4를 설명 하였다. 현재 폐렴 구균 백신은 침습성 질환 (5, 6)의 대부분을 담당하는 혈청 형에 대한 보호 기능을 제공합니다. 그러나, 백신의 광범위한 사용은 비 인두 운송 및 침입 질병 7, 8 두, nonvaccine 혈청 형에 의해 백신에 포함 된 혈청 형의 교체와 관련이있다. 이 역학에 대한 혈청 형의 중요성을 강조감시 및 장기 백신 영향 9 연구. 폐렴 구균 입력을위한 황금 표준 메서드는 먼저 1902 년에 네우 펠드에 의해 기술 된 Quellung 반응으로 알려진 캡슐 반응 시험 (오스트리아 10 참조). 일치의 높은 수준의 실험실 사이, 그리고 Quellung 반응 및 다른 혈청 형의 방법으로 11 ~ 13 사이에서 발견되었습니다. Quellung 반응 10, 14 ~ 16의 몇 가지 변형이 있습니다. 여기서 설명한 방법은 카운터 얼룩이나 오일 침지 렌즈의 사용을 필요로하지 않으며, 오히려, 준비된 시편의 방울​​ 타이핑 혈청과 혼합하고 400X 배율 하에서 즉시 조사했다. Quellung 반응은 일반적으로 시판되는 항혈청을 사용하여 수행된다. 여기에 설명 된 방법보다 비용 효과적인 만드는 다른 방법 (16, 17)에 비해 적게 항혈청을 이용한다. 일단 ISOL식사는 긍정적 인 반응이 관찰 될 때까지 순차적으로 항혈청 풀 테스트되어, 폐렴으로 확인됩니다. 각 풀 항혈청 91 폐렴 구균 혈청 형 (18)에 대해 제기 항혈청의 다른 혼합물을 포함합니다. 풀이 구축되면, 반응성 풀에 포함 된 개별 타입 및 그룹 항혈청은 순서대로 개별적으로 테스트된다. 그룹 항혈청 (예 : 그룹 22 항혈청은 혈청 형 22F 및 22A와 반응) 특정 그룹의 모든 혈청과 반응하는 반면 형 항혈청은 일반적으로, 하나의 혈청 (예, 타입 1 항혈청은 혈청 형 1과 반응)과 반응. 그룹 내의 일부 혈청 형은 추가 인자 항혈청 (예를 들어 혈청 형 22F는 요소 (22B)와 반응 아니지만 22C)를 사용하여 구별된다. 혈청 형이 (19, 20)을 판정 할 때까지이 경우 테스트는 모든 관련 인자 항혈청으로 계속된다. Quellung 반응은 간단하고 신속 14이며 모든 L로 수행 될 수있다aboratory이는 좋은 품질의 현미경 및 항혈청의 적절한 세트가 장착되어 있습니다.

<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

폐렴 구균의 90 가지 캡슐 혈청 형 (폐렴 구균)가있다. 뿐만 아니라 폐렴 역학을 이해하기위한 도구 것으로, 캡슐 혈청 형 백신의 효능과 효과 연구를위한 유용한 정보를 제공 할 수 있습니다. Quellung 반응은 폐렴 구균 캡슐 혈청 형에 대한 황금 표준 방법입니다. 방법은 캡슐 형 다당류에 대한 감독 모으고 특정 항혈청과 폐렴 구균 세포 현탁액을 테스트하는 것을 포함한다. 항원 항체 반응은 미시적으로 관찰된다. 세균 세포 현탁액 1) 준비, 유리 슬라이드에 세포 및 항혈청의 2) 혼합, 3) 현미경을 사용 Quellung 반응을 읽기 : 프로토콜은 세 가지 주요 단계가 있습니다. Quellung 반응을 수행 할 합리적으로 간단하고 적당한 현미경 및 항혈청을 사용할 수 있습니다 어디든지 적용 할 수있다.

<html> 형식 별 항체 굴절률 (10)의 변화로 이어지는, 폐렴 구균의 캡슐에 바인딩 할 때 긍정적 인 Quellung 반응이 발생합니다. 현미경으로 볼 때, 박테리아는 &#39;부어&#39;와 14 개의 표시가 나타납니다. 그림 1A는 HI 국물에 폐렴 구균 세포를 보여줍니다. 형식 별 항혈청이 세균 현탁액에 첨가 될 때, 폐렴 구균 (그림 1B), &#39;부어&#39;굴절 더 둥근 나타납니다. 국물에 추가 너무 많은 박테리아 세포는 세포의 응집 또는 위음성 반응이 발생할 수 있습니다. (그림 2A) 또는 &#39;너무 빛&#39;(그림 2B) &#39;너무 무거운&#39;이다 HI 국물에 폐렴 구균 세포의 접종 2는 그림. 08fig1highres.jpg &quot;SRC =&quot;/ files/ftp_upload/51208/51208fig1.jpg &quot;/&gt; 그림 1. 부정과 긍정적 인 Quellung 반응. 혈청 형 9V의 폐렴 구균의 준비가 더 항혈청 (1A)로 및 그룹 9 항혈청으로 분리 (1B), 400 배 배율에서 조회되었습니다. 후자는 &#39;팽윤&#39;전형적 Quellung 양성 반응을 보이는 세균 세포의 라운딩 나타낸다. <img alt="그림 2" fo:content-width="5in" fo:src="/files/ftp_upload/51208/51208fig2highres.jpg" src="/files/ftp_upload/51208/51208fig2.jpg" /> 그림 2. 너무 무겁고 너무 가벼운 접종. 400 배 배율에서 볼 (2A) 너무 가벼운 (2B) 너무 무거운 폐렴 구균 세포 현탁액의 준비.

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

Quellung 반응은 폐렴 구균 혈청 형을위한 황금 표준 기술이다.이 기술은 현미경 특정 폐렴 구균 항혈청을 이용하여 일반적으로 세계 기준과 연구 실험실에서 사용된다.

의 캡슐 혈청 형<em&gt; 폐렴 구균</em&gt; Quellung 반응을 이용하여

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

Research

JoVE Journal

Immunology and Infection

Capsular Serotyping of Streptococcus pneumoniae Using the Quellung Reaction

45.2K 조회수.  Murdoch Childrens Research Institute.  Quellung 반응은 폐렴 구균 혈청 형을위한 황금 표준 기술이다.이 기술은 현미경 특정 폐렴 구균 항혈청을 이용하여 일반적으로 세계 기준과 연구 실험실에서 사용된다. 

비디오: 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.

사이 Synergism을 맡은 이후로 발광 생물 이미징을 사용하여 연쇄상 구균 pneumoniae</em유아 마우스에서>와 인플루엔자 바이러스

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

연구

면역학 및 감염병학

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.

라이브 셀 이미징 바실러스 subtilis과 연쇄상 구균 pneumoniae 자동 시간 저속 현미경을 사용하여

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.

연쇄상 구균 폐렴으로 비강 내 식민지 화 중 염증 반응의 특성화

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.

막 극성, 멤브레인 무결성 및 세포 내 이온 농도의 모니터링 변경<em&gt; 폐렴 구균</em&gt; 형광 염료를 사용

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.

의 캡슐 혈청 형<em&gt; 폐렴 구균</em&gt; 라텍스 응집에 의해

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.

의 Zebrafish의 애벌레 모델의 선천성 면역 반응의 비 침습적 영상<em&gt; 연쇄상 구균 iniae</em&gt; 감염

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.

그룹 B의 쥐 모델<em&gt; 연쇄상 구균</em&gt; 질 식민

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.

면역 적격 설치류에서 강력한 폐렴 모델에 대해 항균 효능을 평가하기<i&gt; S. 폐렴</i&gt;<i&gt; H. 인플루엔자</i&gt;<i&gt; K. 폐렴</i&gt;<em&gt; P. 녹농균</em&gt; 또는<em&gt;를했다. baumannii가</em

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.

마우스 감염 모델에서 뇌 막 염에 구 이렇게 뵙 Biofilms의 조사 역할에 대 한 감염 intracranial Subarachnoidal 경로

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.