This project was supported by Midwestern University, IL Office of Research and Sponsored Programs (ORSP) and Midwestern University College of Dental Medicine-Illinois (CDMI).

1. HSV 균주 및 취급 참고 : 재조합 비 확산 HSV-1 (KOS) gL86과 HSV-2 (KOS) 333gJ을 - V. Twiari (36, 37)에 의해 제공되었다 사용되는 베타 - 갈 락토시다 아제 리포터 활동. <ol><li> 1에서 -80 ° C에서 단일 많이 매장에서 바이러스를 사용하여 둘 베코의 변형 이글의 중간 20 % 소 태아 혈청 (FBS)와 (DMEM)의 1의 비율로 사용 할 때까지 우유를 탈지. 바이러스 많이 저장하기 전에 입출력 -nitrophenyl-β-D-갈 락토 (ONPG) 및 5- 브로 모 -4- 클로로 -3- 인돌 릴 β-D-갈 락토 (X-GAL) 분석으로 바이러스의 농도를 결정한다. </li><li> 이전에 (그림 1) 14 설명한 바와 같이, 기자 바이러스 항목 분석을 사용하여 각 실험 분석 실행을 위해 X-걸 염색에 의한 바이러스의 생존과 감염 (MOI)의 다양성을 결정합니다. </li><li> 원하는 MOI로 바이러스 (수신 거부 MEM)을 희석. 단일 층 (파라 포름 알데히드를 0.5 ㎖ / 웰) 수정 염색 전합니다. 별도의 microti에서 바이러스의 생존 컨트롤을 배치polymicrobial 분석 플레이트와 병렬 터 플레이트. </li></ol> 2. 헬라 299 셀 처리 <ol><li> 4.5 g / L 글루코스, 10 % 열 - 불 활성화 된 소 태아 혈청 (FBS), 겐타 마이신 (50 μg의 / ml) 및 L- 글루타민으로 DMEM에서 37 ° C, 5 % CO 2에서 성장한다. 트립신 용액과 80 %의 합류 통로 세포 (0.05 % 트립신, 에틸렌 디아민 테트라 아세트산, 0.53 M EDTA 5 ㎖ / 플라스크). </li></ol> 3. C. 알비 칸스 처리 참고 : C를 임상 실험에서 얻어진 소스 알비 매체 2X 탈지 우유 Remmel에서 -80 ° C에 저장된다. <ol><li> 문화 사부로 포도당 중간 (37 ° C)에 주식을 동결. 24 시간 배양 C. 후 Fungisel 매체에 알비 칸스 (37 ° C, 48 시간) 사용. </li><li> 세균 튜브 (GT) 형태의 생성 (3 ML의 FBS를, 대표적인 식민지를 선택 3 시간 37 ° C, 복근 600, 0.3). 배양 후, 세척 GT (HBSS를, 배, XG 4000 참조). (37 예열 HBSS로 세척 GT 추가# 176; C; 0.32 복근 600). GT 양식은 혈구 수에 의해 결정되는 관찰 세포의 99 %이어야한다. </li><li> 대표 Fungisel 식민지를 선택하여 효모 형태 (YF) 주식 현탁액을 (HBSS, 3 ㎖를, 0.32 복근 600). YF 형태의 수를 카운트 / 혈구 현미경을 사용 ㎖로. YF 양식은 혈구 수에 의해 결정되는 관찰 세포의 99 %이어야한다. </li><li> 곰팡이 주식을 작동합니다 (25 ml의 HBSS에 GT 또는 YF 주식의 250 μl를 37 ° C, 10 5 CFU / ㎖) </li></ol> 4. S. 구균 처리 <ol><li> 스토어 S. 구균 ATCC 25923 (-80 ° C; Remmel 우유의 2 배를 탈지). 양 혈액 아가 상 배양 (5 %, 37 ℃, 24 시간). 대표적인 식민지를 선택하고 재고 2 일 이내에 매체 염을 만니톨로 전송 (37 ° C, 18 시간을). </li><li> S.합니다 구균 재고 서스펜션 (3 ml의 HBSS, 1.32 복근 600 10 8 CFU / ㎖) <ol><li> S. 작업 확인 구균 재고 (100 μL25 ml의 HBSS의 재고; 105 CFU / ㎖). </li></ol></li></ol> 5. 칸디다 및 S. 구균 현탁액 <ol><li> 혼합 C. 확인 알비 칸스 및 S. 구균 현탁액 (250 μL의 YF 또는 GT 재고 및 25 ml의 HBSS 100 μL S. 구균 주). </li></ol> 6. Polymicrobial 바이오 필름 분석 <ol><li> 2 × 105 HeLa 세포 / ㎖의 200 μL (85 % 합류 레벨)와 종자를 96 웰 플레이트. 락 플레이트 (30-45 분 37 ° C) 배양 전 (37 ° C, 5 % CO 2 배양기 18 시간). 워시 단일 층 (1 배, 수신 거부 MEM) 다음 HSV와 시드 (HSV-1 (KOS) gL86 또는 HSV-2 (KOS) 33 GJ - 100 μL에서 탈퇴 MEM, MOI 50, 10). 판을 품어 (37 ° C, 3 시간 5 % CO 2). 하루에 단 하나의 바이러스 균주를 사용합니다. </li><li> 감염 단일 층을 씻으 (1 배, 인산염 완충 생리 식염수 마그네슘이와 칼슘이와 (PBS) 100 μL). 25 &amp; # 떠나는 따뜻한 HBSS와 PBS를 교체(181) 각각 잘 리터. <ol><li> YF, GT 및 / 또는 S. 추가 (, 셀 비율 = 5 100 μL 대상 : 1; N = 16) 작동 구균 현탁액. 표 1에 나타낸 바와 같이 판 부화 (정적를 30 분, 37 ° C, 5 % CO 2). </li></ol></li><li> 배양 후, 한 번에 흡인 한 열은 즉시 마그네슘이와 칼슘이 300 ㎕의 PBS로 보충. (시 50 분 희석 200 μL; 필터 살균 RIPA)는 무선 면역 분석 용해 버퍼를 추가 한 다음 두 번이 단계를 반복합니다. </li><li> 빠르게 헬라 세포 용 해물 후 만니톨 염 (MS) 및 / 또는 Fungisel (F) 미디어 (그림 2)에 50 μl를 배치 ​​씹다. 90 ° 각도로 유리로드 절곡하여 분해물을 확산. (37 ° C에서 18 시간) 번호판을 품어. 수동 접시 당 콜로니의 수를 계산합니다. 컨트롤 S. 구성 구균 및 / 또는 C. HSV-감염되지 않은 HeLa 세포에 albicans에 부착. </li></ol> 7. 이미징 연구 &lt;/ P&gt; <ol><li> 각 라운드 유리 커버 슬립을 씻으 (12mm, 50 ml의 아세톤 비이커 100). 건조하고 소독 된 커버 (매직 잉크를 킴 와이프, 유리 페트리 접시). 알코올 불꽃 소독 집게로 멸균 24 웰 플레이트의 우물에 건조 멸균 된 커버를 놓습니다. </li><li> 세척 멸균 라운드 커버 글라스를 포함하는 24 웰 플레이트의 웰에, HeLa 세포 (96 웰 플레이트에 사용되는 5 배 볼륨 1 ml)에 추가합니다. 위의 단계 6.4 6.2.1에 설명 된대로 다음 부화 및 프로세스, 5 배 96 웰 플레이트에 사용되는 볼륨의 템플릿 (표 2)에 따라 바이러스, 세균 및 곰팡이를 추가합니다. </li><li> 최종 세척 한 후, 메탄올 슬라이드를 범람하고 증발 할 수 있도록하여 현미경 세포를 고정합니다. 염색 할 때까지 실온에서 접시를 저장합니다. </li><li> 밝은 필드 현미경 (1000 배 최종 원래 배율)의 경우 탈 이온수로 메탄올 고정 된 커버를 포함하는 우물을 입력합니다. 즉시 물을 대기음. 그램 크리스탈 바이올렛 각 커버 슬립 커버. 워시가 아닌 바운드 얼룩 (탈 이온수)의 무료 된 커버. 현장에서 건조 된 커버는 레이블 슬라이드 매체를 장착 하드 세트 (그림 3)로를 준수합니다. </li><li> 형광 현미경 (100 배 대물, 1000 배 최종 원래 배율)의 경우 단계 7.4에 설명 된대로 세척은 본질적으로 메탄올 무료로 된 커버. 우물에서 커버 슬립을 건조. <ol><li> 건조 후, 커버 전표를 제거하고 표시된 슬라이드에 배치합니다. (- 5 μL 1) 다음 형광 이소 티오 시아 네이트 (FITC) 공역 계 단순 포진 바이러스 유형의 1시 20분 희석의 커버 슬립을 커버하는 1 + 2 GD 항체를 충분한 양을 추가합니다. </li><li> 30 분 동안 37 ℃에서 습도 챔버에 슬라이드를 부화. 부화 후, PBS의 4 변화에 커버 슬립을 씻는다. </li><li> 최종 세척 후, 표시된 슬라이드에 커버 슬립을 반환합니다. 4 &#39;, 6-diamidino -2- 페닐 인돌 (; 수분 챔버에서 30 분 동안 37 ° C DAPI)로 염색. 부화 후 4 찬으로 된 커버를 세척PBS의 GES 후 하드 세트 장착 매체와 표지 슬라이드에 부착합니다. </li><li> 설치 매체가 어둠 속에서 실온에서 24 시간 동안 치료하도록 허용합니다. 밝은 필드 현미경 또는 FITC와 DAPI 차단 필터 형광 현미경 중 하나에 기름 목표 아래 커버 슬립을 검사합니다. 공동 현지화 (그림 4) 적어도 50 분야 (생물 최소 100 세포) 사진을 검사합니다. </li></ol></li></ol>

The authors have nothing to disclose.

현재 어떤 정보는 원핵 생물 진핵 세포 및 바이러스 성 즉, 다수의 분류 학적 영역을, 교차 호스트 마이크로 바이 옴의 준 상임 이사국에 영구적 인 사이의 복잡한 상호 작용에 사용할 수 없습니다. 그러므로 우리는 S.으로 생물막 개시를 연구하기 위해 체외 모델 시스템에서 새로운 개발 구균 및 C. HSV-1 또는 HSV-2에 알비 칸스는 헬라 229 (헬라) 세포 <su...

<html> 인간 마이크로 바이 옴 몸에 지리적 영역을 공유하는 여러 분류 학적 왕국에서 다양한 생물이 포함되어 있습니다. 세포 표면에 준수는 마이크로 바이 옴의 식민지화 과정의 일부입니다 생물막 형성에 필수적인 첫 번째 단계입니다. 마이크로 바이 옴에 포함 된 만성 및 지속적인 감염의 원인 바이러스가 될 수 있습니다. 이 바이러스에 의한 만성 세포 감염이 추정되는 수용체의 가용성에 변화를 일으킬 수 있습니다. 또한 1, 2, 세포 내 병원균에 의한 세포 항목도 차례로 박테리아와 곰팡이를 포함한 다른 마이크로 바이 옴 회원의 첨부 파일을 변경할 수 있습니다 호스트 막 유동성 / 소수성에 영향을 미칠 수 . 인간 숙주의 동일한 지리적 영역에서 - 지역화 공동이 여러 병원체 사이에서 발생할 수있는 상호 작용을 이해하기 위해, 우리는 점막 표면에 존재하는 분류학 왕국의 스펙트럼을 나타내는 병원체의 상호 작용을 연구 할 수 있어야한다. t &quot;&gt;는 헤르페스는 마이크로 바이 옴의 상임 이사국으로 인간의 100 %에 존재하는 미생물의 가족은 3,4. 또한 그들은 또한 지속적으로 증상의 유무에 모두 흘려 될 수있다. 특히, 단순 포진 바이러스 1 및 단순 헤르페스 바이러스 -2 (HSV-1, HSV-2, 각각)는 oronasopharynx 및 생식기의 마이크로 바이 영구 부재이다. 면역 능력이 개인, 두 HSV-1, HSV-2 원인 gingivostomatitis은 물론 음부 포진 5-8.이 사이트에서 HSV는 만성 지속적 nectins, 헤파 란 황산, 지질 뗏목 및 헤르페스 바이러스 항목 중재자의 표면 발현의 변화 세포 결과로 HSV 9. 항목 흘리기 바이러스 증상 / 종양 괴사 특징으로하는 잠재 감염의 원인 인자 수용체 (HVEM / TNFR) 10-25. 일부 박테리아와 곰팡이, 예를 들면 황색 포도상 구균과 C. albicans에 대한이 잠재적으로 공유 나타냅니다 수용체있는 기회 병원균 동안,또한 oronasopharynx (26, 27)의 점막 마이크로 바이 옴의 구성원으로 상주 할 수 있습니다. oronasopharynx S. 내 구균 및 C. 알비 칸스는 식민지의 두 가지 위치를 차지한다. 자연 치아와 호스트에서 구강 점막은 HSV-1과 C.에서 공유 알비 칸스, 전방 코 콧 구멍은 S.에 의해 점유하는 동안 구균 28. 그러나에도 불구하고 체외 조사 결과 그 S. 구균 입 상피 세포에 부착, 29, 30 S. 정상 조직은 29, 30 존재할 때 구균은 드물게 구강 검체에서 분리된다. 리틀는 임상 연구 결과 그 이상 생식기 공동 식민지의 틈새 시장에 관한 알려져있다 S. 구균은 C.하면서, 생식기 염증, 방전 및 성교통 특징, 에어로빅 질염과 연관된 알비 구강 31-35에서 관찰 된 것과 유사한 점막 병변을 생성한다. 따라서, 구강과 생식기 microbi 이러한 회원하지만의 상호 작용에 관한 알려진 작은 오메 크로스 분류 학적 왕국 숙주 세포 표면 5 준수를 통해 biofilm 형성을 시작하기에 영향을 미치기 능력이있다. 이 프로토콜은 효과적으로 공동 정착 / 감염의 기능적 결과를 결정하는데 적용되었다.

<table border="0" cellpadding="0" cellspacing="0" width="1185">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:391px;">C.albicans</td>
			<td style="width:195px;"></td>
			<td style="width:119px;"></td>
			<td style="width:481px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:391px;">BBL Sabouraud Dextrose</td>
			<td style="width:195px;">BD</td>
			<td style="width:119px;">211584</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:391px;">Fungisel Agar</td>
			<td style="width:195px;">Dot Scientific</td>
			<td style="width:119px;">7205A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:391px;">S.aureus</td>
			<td style="width:195px;"></td>
			<td style="width:119px;"></td>
			<td style="width:481px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:391px;">Mannitol Salt Agar</td>
			<td style="width:195px;">Troy Biologicals</td>
			<td style="width:119px;">7143B</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:391px;">Sheep blood agar</td>
			<td style="width:195px;">Troy Biologicals</td>
			<td style="width:119px;">221239</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:391px;">Hela cells</td>
			<td style="width:195px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:391px;">1xDMEM (Dubelcco&#39;s Modified Eagle Medium, with 4.5 g/L glucose and L-glutamine, without sodium pyruvate</td>
			<td style="width:195px;">Corning</td>
			<td style="width:119px;">10-017-CM</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:391px;">Gentamicin 50mg/ml</td>
			<td style="width:195px;">Sigma</td>
			<td style="width:119px;">1397</td>
			<td>50&#181;g/ml final concentration in the complete DMEM</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:391px;">Trypsin EDTA (0.05% Trypsin, 0.53M EDTA)Solution 1X</td>
			<td style="width:195px;">Corning</td>
			<td>25-052-CI</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:391px;">Fetal Bovine Serum</td>
			<td style="width:195px;">Atlanta Biologicals</td>
			<td style="width:119px;">S11150</td>
			<td>10% final concentration in the complete DMEM</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:391px;">Other medium and reagents</td>
			<td style="width:195px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:391px;">ONPG</td>
			<td style="width:195px;">Thermo Scientific</td>
			<td style="width:119px;">34055</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:391px;">Ultra-Pure X gal</td>
			<td style="width:195px;">Invitrogen</td>
			<td style="width:119px;">15520-018</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:391px;">1x HBSS (Hanks&#39; Balanced Salt Solution)</td>
			<td style="width:195px;">Corning</td>
			<td style="width:119px;">20-021-CV</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:391px;">1XPBS</td>
			<td style="width:195px;">Dot Scientific</td>
			<td style="width:119px;">30042-500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:391px;">RIPA Lysis</td>
			<td style="width:195px;">Life Technologies</td>
			<td style="width:119px;">89901</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:391px;">Staining</td>
			<td style="width:195px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:391px;">Methanol</td>
			<td style="width:195px;">Fisher Scientific</td>
			<td style="width:119px;">A433P-4</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:391px;">HSV 1&#38;2, specific for gD</td>
			<td style="width:195px;">ViroStat</td>
			<td style="width:119px;">196</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:391px;">DAPI</td>
			<td style="width:195px;">SIGMA</td>
			<td style="width:119px;">D8417-5MG</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:391px;">Gram Crystal Violet</td>
			<td style="width:195px;">Troy Biologicals</td>
			<td style="width:119px;">212527</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:391px;">Supplies</td>
			<td style="width:195px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="29">
			<td height="29" style="height:29px;width:391px;">Petri dish 100X15</td>
			<td style="width:195px;">Dot Scientific</td>
			<td style="width:119px;">229693&#160;</td>
			<td></td>
		</tr>
		<tr height="29">
			<td height="29" style="height:29px;width:391px;">Petri dish 150X15</td>
			<td style="width:195px;">Kord Valmark</td>
			<td style="width:119px;">2902</td>
			<td></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;width:391px;">96-Well plates</td>
			<td style="width:195px;">Evergreen Scientific</td>
			<td style="width:119px;">222-8030-01F</td>
			<td></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;width:391px;">24-well plates</td>
			<td style="width:195px;">Evergreen Scientific</td>
			<td style="width:119px;">222-8044-01F</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:391px;">Culture tubes 100x13</td>
			<td style="width:195px;">Thomas Scientific</td>
			<td style="width:119px;">9187L61</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:391px;">Cover slip circles, 12mm</td>
			<td style="width:195px;">Deckglaser</td>
			<td style="width:119px;">CB00120RA1</td>
			<td></td>
		</tr>
	</tbody>
</table>

determination of biofilm initiation on virus-infected cells by bacteria and fungi

곰팡이, 박테리아와 바이러스를 포함 분류학 왕국에서 polymicrobial 상호 작용의 연구는 이전에 마이크로 바이 옴의 바이러스 성 회원이 바이러스에 감염된 숙주 세포와 후속 미생물의 상호 작용에 미치는 영향에 대해 조사되지 않았다. 세균 및 진균과 바이러스의 공동 주거 구강 및 생식기의 점막 표면에 주로 존재한다. 점막 세포, 지속적인 만성 또는 지속적인 잠재 바이러스 감염 특히이 표현 수용체의 상당수에서 바이러스 변경을 통해 마이크로 바이 옴의 구성원에 미치는 영향과 유형을 가질 수있다. 숙주 세포의 막 구조의 변형이 바이오 필름의 형성, 즉 부착하는 첫 번째 단계를 개시하기 위해 상주하고 기회 병원균 후속 부재 변형 기능을 초래할 것이다. 이 연구는 biofil의 시작에 HSV의 효과의 정량 및 육안 검사하는 방법을 설명합니다S. m의 형성 (부착) 구균 및 C. 알비 칸스.

이 보고서에 설명 된 시스템에서 얻을 수있는 데이터의 안정성의 수준은 그림 2 AF (38)에 표시됩니다. 이 시스템의 사용을 통해 바이러스로 감염된 세포와의 상호 접착에 미치는 효과 포도상 진균 작용의 조절을 묘사 할 수있다. polymicrobial 상호 작용은 동일한 세포에서 발생되는지 여부를 결정하기 위해도 3 및도 4에 도시 된 ?...

Watch this Scientific Journal Video about Determination of Biofilm Initiation on Virus-infected Cells by Bacteria and Fungi at JoVE.com

Determination of Biofilm Initiation on Virus-infected Cells by Bacteria and Fungi

A method is described herein for the determination of inter-Kingdom association and competition (bacterial and fungal) for adherence to virus-infected HeLa cell monolayers. This protocol can be extended to multiple combinations of prokaryotes, eukaryotes, and viruses.

박테리아와 균류에 의해 바이러스에 감염된 세포에 바이오 필름 개시의 결정

The study of polymicrobial interactions across the taxonomic kingdoms that include fungi, bacteria and virus have not been previously examined with ...

determination-biofilm-initiation-on-virus-infected-cells-bacteria

Research

JoVE Journal

Immunology and Infection

9.6K 조회수.  Midwestern University. A method is described herein for the determination of inter-Kingdom association and competition (bacterial and fungal) for adherence to virus-infected HeLa cell monolayers. This protocol can be extended to multiple combinations of prokaryotes, eukaryotes, and viruses.

비디오: Determination of Biofilm Initiation on Virus-infected Cells by Bacteria and Fungi

Pseudomonas aeruginosa and Saccharomyces cerevisiae Biofilm in Flow Cells

Many microbial cells have the ability to form sessile microbial communities defined as biofilms that have altered physiological and pathological properties compared to free living microorganisms. Biofilms in nature are often difficult to investigate and reside under poorly defined conditions1. Using a transparent substratum it is possible to device a system where simple biofilms can be examined in a non-destructive way in real-time: here we demonstrate the assembly and operation of a flow cell model system, for in vitro 3D studies of microbial biofilms generating high reproducibility under well-defined conditions2,3.
The system consists of a flow cell that serves as growth chamber for the biofilm. The flow cell is supplied with nutrients and oxygen from a medium flask via a peristaltic pump and spent medium is collected in a waste container. This construction of the flow system allows a continuous supply of nutrients and administration of e.g. antibiotics with minimal disturbance of the cells grown in the flow chamber. Moreover, the flow conditions within the flow cell allow studies of biofilm exposed to shear stress. A bubble trapping device confines air bubbles from the tubing which otherwise could disrupt the biofilm structure in the flow cell.
The flow cell system is compatible with Confocal Laser Scanning Microscopy (CLSM) and can thereby provide highly detailed 3D information about developing microbial biofilms. Cells in the biofilm can be labeled with fluorescent probes or proteins compatible with CLSM analysis. This enables online visualization and allows investigation of niches in the developing biofilm. Microbial interrelationship, investigation of antimicrobial agents or the expression of specific genes, are of the many experimental setups that can be investigated in the flow cell system.

Pseudomonas aeruginosa and Saccharomyces cerevisiae Biofilm in Flow Cells

Protocol describing the application of a flow cell system for growing and analyzing microbial biofilms for Confocal Laser Scanning Microscopy (CLSM).

흐름 세포 녹농균 및 Saccharomyces cerevisiae의의 Biofilm

Many microbial cells have the ability to form sessile microbial communities defined as biofilms that have altered physiological and pathological ...

연구

면역학 및 감염병학

Microtiter Dish Biofilm Formation Assay

Biofilms are communities of microbes attached to surfaces, which can be found in medical, industrial and natural settings. In fact, life in a biofilm probably represents the predominate mode of growth for microbes in most environments. Mature biofilms have a few distinct characteristics. Biofilm microbes are typically surrounded by an extracellular matrix that provides structure and protection to the community. Microbes growing in a biofilm also have a characteristic architecture generally comprised of macrocolonies (containing thousands of cells) surrounded by fluid-filled channels. Biofilm-grown microbes are also notorious for their resistance to a range of antimicrobial agents including clinically relevant antibiotics.
The microtiter dish assay is an important tool for the study of the early stages in biofilm formation, and has been applied primarily for the study of bacterial biofilms, although this assay has also been used to study fungal biofilm formation. Because this assay uses static, batch-growth conditions, it does not allow for the formation of the mature biofilms typically associated with flow cell systems. However, the assay has been effective at identifying many factors required for initiation of biofilm formation (i.e, flagella, pili, adhesins, enzymes involved in cyclic-di-GMP binding and metabolism) and well as genes involved in extracellular polysaccharide production. Furthermore, published work indicates that biofilms grown in microtiter dishes do develop some properties of mature biofilms, such a antibiotic tolerance and resistance to immune system effectors.
This simple microtiter dish assay allows for the formation of a biofilm on the wall and/or bottom of a microtiter dish. The high throughput nature of the assay makes it useful for genetic screens, as well as testing biofilm formation by multiple strains under various growth conditions. Variants of this assay have been used to assess early biofilm formation for a wide variety of microbes, including but not limited to, pseudomonads, Vibrio cholerae, Escherichia coli, staphylocci, enterococci, mycobacteria and fungi.
In the protocol described here, we will focus on the use of this assay to study biofilm formation by the model organism Pseudomonas aeruginosa. In this assay, the extent of biofilm formation is measured using the dye crystal violet (CV). However, a number of other colorimetric and metabolic stains have been reported for the quantification of biofilm formation using the microtiter plate assay. The ease, low cost and flexibility of the microtiter plate assay has made it a critical tool for the study of biofilms.

The assay describes a rapid means to measure early biofilm formation in bacteria and fungi. This method uses a microtiter plate as the substratum for microbial biofilm formation, and the biofilm is visualized using crystal violet strain. The assay provides either a qualitative or quantitative assay for early biofilm formation.

Microtiter 디시 Biofilm 형성 분석

Biofilms are communities of microbes attached to surfaces, which can be found in medical, industrial and natural settings.  In fact, life in a biofilm ...

Following Cell-fate in E. coli After Infection by Phage Lambda

The system comprising bacteriophage (phage) lambda and the bacterium E. coli has long served as a paradigm for cell-fate determination1,2. Following the simultaneous infection of the cell by a number of phages, one of two pathways is chosen: lytic (virulent) or lysogenic (dormant)3,4. We recently developed a method for fluorescently labeling individual phages, and were able to examine the post-infection decision in real-time under the microscope, at the level of individual phages and cells5. Here, we describe the full procedure for performing the infection experiments described in our earlier work5. This includes the creation of fluorescent phages, infection of the cells, imaging under the microscope and data analysis. The fluorescent phage is a "hybrid", co-expressing wild- type and YFP-fusion versions of the capsid gpD protein. A crude phage lysate is first obtained by inducing a lysogen of the gpD-EYFP (Enhanced Yellow Fluorescent Protein) phage, harboring a plasmid expressing wild type gpD. A series of purification steps are then performed, followed by DAPI-labeling and imaging under the microscope. This is done in order to verify the uniformity, DNA packaging efficiency, fluorescence signal and structural stability of the phage stock. The initial adsorption of phages to bacteria is performed on ice, then followed by a short incubation at 35&deg;C to trigger viral DNA injection6. The phage/bacteria mixture is then moved to the surface of a thin nutrient agar slab, covered with a coverslip and imaged under an epifluorescence microscope. The post-infection process is followed for 4 hr, at 10 min interval. Multiple stage positions are tracked such that ~100 cell infections can be traced in a single experiment. At each position and time point, images are acquired in the phase-contrast and red and green fluorescent channels. The phase-contrast image is used later for automated cell recognition while the fluorescent channels are used to characterize the infection outcome: production of new fluorescent phages (green) followed by cell lysis, or expression of lysogeny factors (red) followed by resumed cell growth and division. The acquired time-lapse movies are processed using a combination of manual and automated methods. Data analysis results in the identification of infection parameters for each infection event (e.g. number and positions of infecting phages) as well as infection outcome (lysis/lysogeny). Additional parameters can be extracted if desired.

Following Cell-fate in E. coli After Infection by Phage Lambda

This article describes the procedure for preparing a fluorescently-labeled version of bacteriophage lambda, infection of E. coli bacteria, following the infection outcome under the microscope, and analysis of infection results.

에 셀 운명을 따라<em&gt; E. 대장균</em&gt; 파지 람다에 의해 감염 후

The system comprising bacteriophage (phage) lambda and the bacterium E. coli has long served as a paradigm for cell-fate determination1,2. Following the ...

Purification and Visualization of Lipopolysaccharide from Gram-negative Bacteria by Hot Aqueous-phenol Extraction

Lipopolysaccharide (LPS) is a major component of Gram-negative bacterial outer membranes. It is a tripartite molecule consisting of lipid A, which is embedded in the outer membrane, a core oligosaccharide and repeating O-antigen units that extend outward from the surface of the cell1, 2. LPS is an immunodominant molecule that is important for the virulence and pathogenesis of many bacterial species, including Pseudomonas aeruginosa, Salmonella species, and Escherichia coli3-5, and differences in LPS O-antigen composition form the basis for serotyping of strains. LPS is involved in attachment to host cells at the initiation of infection and provides protection from complement-mediated killing; strains that lack LPS can be attenuated for virulence6-8. For these reasons, it is important to visualize LPS, particularly from clinical isolates. Visualizing LPS banding patterns and recognition by specific antibodies can be useful tools to identify strain lineages and to characterize various mutants. 
In this report, we describe a hot aqueous-phenol method for the isolation and purification of LPS from Gram-negative bacterial cells. This protocol allows for the extraction of LPS away from nucleic acids and proteins that can interfere with visualization of LPS that occurs with shorter, less intensive extraction methods9. LPS prepared this way can be separated by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) and directly stained using carbohydrate/glycoprotein stains or standard silver staining methods. Many anti-sera to LPS contain antibodies that cross-react with outer membrane proteins or other antigenic targets that can hinder reactivity observed following Western immunoblot of SDS-PAGE-separated crude cell lysates. Protease treatment of crude cell lysates alone is not always an effective way of removing this background using this or other visualization methods. Further, extensive protease treatment in an attempt to remove this background can lead to poor quality LPS that is not well resolved by any of the aforementioned methods. For these reasons, we believe that the following protocol, adapted from Westpahl and Jann10, is ideal for LPS extraction.

We describe a modified hot aqueous-phenol extraction method for purifying lipopolysaccharide (LPS) from Gram-negative bacteria. Once extracted, the LPS can be subsequently analyzed by SDS-PAGE and visualized by direct staining or Western immunoblot.

핫 수성 - 페놀 추출에 의한 그람 음성 박테리아에서 정화와 Lipopolysaccharide의 시각화

Lipopolysaccharide (LPS)  is a major component of Gram-negative bacterial outer membranes. It is a tripartite molecule consisting of  lipid A, which is ...

Use of Image Cytometry for Quantification of Pathogenic Fungi in Association with Host Cells

Studies of the cellular pathogenesis mechanisms of pathogenic yeasts such as Candida albicans, Histoplasma capsulatum, and Cryptococcus neoformans commonly employ infection of mammalian hosts or host cells (i.e. macrophages) followed by yeast quantification using colony forming unit analysis or flow cytometry. While colony forming unit enumeration has been the most commonly used method in the field, this technique has disadvantages and limitations, including slow growth of some fungal species on solid media and low and/or variable plating efficiencies, which is of particular concern when comparing growth of wild-type and mutant strains. Flow cytometry can provide rapid quantitative information regarding yeast viability, however, adoption of flow cytometric detection for pathogenic yeasts has been limited for a number of practical reasons including its high cost and biosafety considerations. Here, we demonstrate an image-based cytometric methodology using the Cellometer Vision (Nexcelom Bioscience, LLC) for the quantification of viable pathogenic yeasts in co-culture with macrophages. Our studies focus on detection of two human fungal pathogens: Histoplasma capsulatum and Candida albicans. H. capsulatum colonizes alveolar macrophages by replicating within the macrophage phagosome, and here, we quantitatively assess the growth of H. capsulatum yeasts in RAW 264.7 macrophages using acridine orange/propidium iodide staining in combination with image cytometry. Our method faithfully recapitulates growth trends as measured by traditional colony forming unit enumeration, but with significantly increased sensitivity. Additionally, we directly assess infection of live macrophages with a GFP-expressing strain of C. albicans. Our methodology offers a rapid, accurate, and economical means for detection and quantification of important human fungal pathogens in association with host cells.

Here, we demonstrate how image cytometry can be used for quantification of pathogenic fungi in association with host cells in culture. This technique can be used as an alternative to CFU enumeration.

숙주 세포와 관련하여 병원성 곰팡이의 정량화를 위해 이미지 계측법의 사용

Studies of the cellular pathogenesis mechanisms of pathogenic yeasts such as Candida albicans, Histoplasma capsulatum, and Cryptococcus neoformans ...

Application of Fluorescent Nanoparticles to Study Remodeling of the Endo-lysosomal System by Intracellular Bacteria

Fluorescent nanoparticles (NPs) with desirable chemical, optical and mechanical properties are promising tools to label intracellular organelles. Here, we introduce a method using gold-BSA-rhodamine NPs to label the endo-lysosomal system of eukaryotic cells and monitor manipulations of host cellular pathways by the intracellular pathogen Salmonella enterica. The NPs were readily internalized by HeLa cells and localized in late endosomes/lysosomes. Salmonella infection induced rearrangement of the vesicles and accumulation of NPs in Salmonella-induced membrane structures. We deployed the Imaris software package for quantitative analyses of confocal microscopy images. The number of objects and their size distribution in non-infected cells were distinct from the ones in Salmonella-infected cells, indicating extremely remodeling of the endo-lysosomal system by WT Salmonella.

This article describes methods for the synthesis and fluorescent labeling of nanoparticles (NPs). The NPs were applied in pulse-chase experiments to label the endo-lysosomal system of eukaryotic cells. Manipulation of the endo-lysosomal system by activities of the intracellular pathogen Salmonella enterica were followed by live cell imaging and quantified.

형광 나노 입자의 응용 프로그램은 세포 내 세균에 의해 엔도 - 리소좀 시스템의 리모델링을 연구하기 위해

Fluorescent nanoparticles (NPs) with desirable chemical, optical and mechanical properties are promising tools to label intracellular organelles. Here, we ...

T Cells Capture Bacteria by Transinfection from Dendritic Cells

Recently, we have shown, contrary to what is described, that CD4+ T cells, the paradigm of adaptive immune cells, capture bacteria from infected dendritic cells (DCs) by a process called transinfection. Here, we describe the analysis of the transinfection process, which occurs during the course of antigen presentation. This process was unveiled by using CD4+ T cells from transgenic OTII mice, which bear a T cell receptor (TCR) specific for a peptide of ovoalbumin (OVAp), which therefore can form stable immune complexes with infected dendritic cells loaded with this specific OVAp. The dynamics of green fluorescent protein (GFP)-expressing bacteria during DC-T cell transmission can be monitored by live-cell imaging and the quantification of bacterial transinfection can be performed by flow cytometry. In addition, transinfection can be quantified by a more sensitive method based in the use of gentamicin, a non-permeable aminoglycoside antibiotic killing extracellular bacteria but not intracellular ones. This classical method has been used previously in microbiology to study the efficiency of bacterial infections. We hereby explain the protocol of the complete process, from the isolation of the primary cells to the quantification of transinfection.

Here a protocol is presented to measure bacterial capture by CD4+ T cells which occurs during antigen presentation via transinfection from pre-infected dendritic cells (DC). We show how to perform the necessary steps: isolation of primary cells, infection of DC, DC/T cell conjugate formation, and measurement of bacterial T cell transfection.

T 세포는 수지상 세포에서 Transinfection에 의해 박테리아를 캡처

Recently, we have shown, contrary to what is described, that CD4+ T cells, the paradigm of adaptive immune cells, capture bacteria from infected dendritic ...

Phenotypic and Functional Analysis of Activated Regulatory T Cells Isolated from Chronic Lymphocytic Choriomeningitis Virus-infected Mice

Regulatory T (Treg) cells, which express Foxp3 as a transcription factor, are subsets of CD4+ T cells. Treg cells play crucial roles in immune tolerance and homeostasis maintenance by regulating the immune response. The primary role of Treg cells is to suppress the proliferation of effector T (Teff) cells and the production of cytokines such as IFN-&#947;, TNF-&#945;, and IL-2. It has been demonstrated that Treg cells&#39; ability to inhibit the function of Teff cells is enhanced during persistent pathogen infection and cancer development. To clarify the function of Treg cells under resting or inflamed conditions, a variety of in vitro suppression assays using mouse or human Treg cells have been devised. The main aim of this study is to develop a method to compare the differences in phenotype and suppressive function between resting and activated Treg cells. To isolate activated Treg cells, mice were infected with lymphocytic choriomeningitis virus (LCMV) clone 13 (CL13), a chronic strain of LCMV. Treg cells isolated from the spleen of LCMV CL13-infected mice exhibited both the activated phenotype and enhanced suppressive activity compared with resting Treg cells isolated from na&#239;ve mice. Here, we describe the basic protocol for ex vivo phenotype analysis to distinguish activated Treg cells from resting Treg cells. Furthermore, we describe a protocol for the measurement of the suppressive activity of fully activated Treg cells.

Here, we describe a protocol to analyze the phenotype of regulatory T (Treg) cells isolated from na&#239;ve and chronic lymphocytic choriomeningitis virus-infected mice. In addition, we provide a process to evaluate the suppressive activity of the Treg cells.

표현형 및 만성 림프 구성 맥락 수막염 바이러스에 감염된 마우스에서 고립 활성화 규제 T 세포의 기능 분석

Regulatory T (Treg) cells, which express Foxp3 as a transcription factor, are subsets of CD4+ T cells. Treg cells play crucial roles in immune tolerance ...

Visualizing the Effects of Sputum on Biofilm Development Using a Chambered Coverglass Model

Biofilms consist of groups of bacteria encased in a self-secreted matrix. They play an important role in industrial contamination as well as in the development and persistence of many health related infections. One of the most well described and studied biofilms in human disease occurs in chronic pulmonary infection of cystic fibrosis patients. When studying biofilms in the context of the host, many factors can impact biofilm formation and development. In order to identify how host factors may affect biofilm formation and development, we used a static chambered coverglass method to grow biofilms in the presence of host-derived factors in the form of sputum supernatants. Bacteria are seeded into chambers and exposed to sputum filtrates. Following 48 hr of growth, biofilms are stained with a commercial biofilm viability kit prior to confocal microscopy and analysis. Following image acquisition, biofilm properties can be assessed using different software platforms. This method allows us to visualize key properties of biofilm growth in presence of different substances including antibiotics.

This protocol describes the visualization of biofilm development following exposure to host-factors using a slide chamber model. This model allows for direct visualization of biofilm development as well as analysis of biofilm parameters using computer software programs.

챔버의 coverglass 모델을 사용하여 바이오 필름 개발에 객담의 효과를 시각화

Biofilms consist of groups of bacteria encased in a self-secreted matrix. They play an important role in industrial contamination as well as in the ...

Optical Screening of Novel Bacteria-specific Probes on Ex Vivo Human Lung Tissue by Confocal Laser Endomicroscopy

Improving the speed and accuracy of bacterial detection is important for patient stratification and to ensure the appropriate use of antimicrobials. To achieve this goal, the development of diagnostic techniques to recognize bacterial presence in real-time at the point-of-care is required. Optical imaging for direct identification of bacteria within the host is an attractive approach. Several attempts at chemical probe design and validation have been investigated, however none have yet been successfully translated into the clinic. Here we describe a method for ex vivo validation of bacteria-specific probes for identification of bacteria within the distal lung, imaged by fibered confocal fluorescence microscopy (FCFM). Our model used ex vivo human lung tissue and a clinically approved confocal laser endomicroscopy (CLE) platform to screen novel bacteria-specific imaging compounds, closely mimicking imaging conditions expected to be encountered with patients. Therefore, screening compounds by this technique provides confidence of potential clinical tractability.

Optical Screening of Novel Bacteria-specific Probes on Ex Vivo Human Lung Tissue by Confocal Laser Endomicroscopy

This technique describes an efficient screening process for evaluating bacteria-specific optical imaging agents within ex vivo human lung tissue, by fibered confocal fluorescence microscopy for the rapid identification of small molecule chemical probe-candidates with translatable potential.

새로운 박테리아 특정의 광학 검사 Confocal 레이저 Endomicroscopy에 의해 전 비보 인간의 폐 조직에 프로브

Improving the speed and accuracy of bacterial detection is important for patient stratification and to ensure the appropriate use of antimicrobials. To ...