우리는 그들의 기술 지원에 대 한 응용된 생물 과학 부, 도요 대학, 군마 농업 기술 센터의 씨 Koudai 타 니 구치의 씨 아키라 무라카미에 감사. 우리는 또한 그들의 제안에 대 한 교수 Shosaku Kashiwada 박사 우 Maheswari Rajagopalan, 도요 대학에 감사입니다. 이 연구는 생명 및 환경 과학, 도요 대학에 대 한 연구 센터에 의해 부분에서 지원 되었다.

참고: Ipecac (C. ipecacuanha)에 사용 되었다이 연구 생 phytohormones의 분석을 용이 하 게 하기 때문에.
1. 성장 조건 Ipecac의 우발적인 촬영 유도
<ol><li>PH 5.710, 조정 phytohormone 무료 B5 매체를 준비 하 고 0.2 %gellan 껌을 추가. 압력가 마로 소독 하 여 소독.</li>
	<li>멸 균 페 트리 접시 (90 m m × 20 m m)에 압력가 하는 매체의 25 mL를 붓으십시오.</li>
	<li>Ipecac plantlets 수술 메스를 사용 하 여 살 균 아크릴 플레이트에 블레이드 번호 22의 8 밀리미터 internodal 세그먼트를 잘라내어 매체 (그림 1)에 배치.</li>
	<li>24 ° C 15 µmol m−2의 − 1 5 주 동안 14 h 빛/10-h 어두운 주기에서 빛의 아래에 B5 매체 phytohormone 무료에 문화.</li>
	<li>지역 식별 난 (꼭대기) 4 (기저) 각 세그먼트 (그림 1B). 일주일에 한 번 우발적인 촬영 > 0.3 m m 길이 현미경으로 각 지역에서의 수를 계산 합니다.</li>
</ol>2. 추출 및 Phytohormones의 정화
<ol><li>각 4 개의 2 mL 샘플 튜브 5 m m 지 르 코니 아 비드를 넣어.</li>
	<li>지역으로 세그먼트를 잘라 iv (각 2 m m에 길이) 수술 메스를 사용 하 여 살 균 아크릴 판에 나.</li>
	<li>별도 샘플 튜브 (10-30 mg 신선한 무게)에 각 지역의 8 개 세그먼트를 수집 합니다.</li>
	<li>무게, 그리고 그 후 액체 질소에 샘플을 동결.</li>
	<li>비드 기반 균질 화기를 사용 하 여 얼음된 샘플을 짝사랑.</li>
	<li>Auxin의 각 내부 표준의 500 pg와 紀 (d5-IAA, d5tZ, d5tZR, d6-iP, d6-지적재산권)를 포함 하는 1 mL 이기에 짓 눌린된 샘플 일시 중지와 동 믹서를 사용 하 여.</li>
	<li>1 시간에 4 ° C에서 누른 다음 실 온에서 5 분 동안 3500 × g 에서 원심.</li>
	<li>1% (v/v) 초 산을 포함 하는 80% (v/v) 이기에 펠 릿을 세척. 원심 분리기 다시 3500 × g 에서 실 온에서 5 분. 일회용 유리 튜브에서 (2.7, 2.8 단계)에서 supernatants 결합.</li>
	<li>추가 600 µ L 각 결합된 상쾌한을 포함 1% (v/v) 초 산 물이 고 이기는 진공 집중 장치를 사용 하 여 증발.</li>
	<li>이기, 메탄올, 및 1% (v/v) 초 산을 포함 하는 물의 1 mL를 각각 적용 하 여 친수성 닥터지-평형 (HLB) 열 카트리지를 equilibrate.</li>
	<li>Equilibrated HLB 카트리지 당 하나의 샘플 솔루션을 적용 합니다.</li>
	<li>1 mL 물 1% (v/v) 초 산을 포함 하는 카트리지를 씻어.</li>
	<li>포함 하는 유리 튜브에 1% (v/v) 초 산 2 mL 80% (v/v) 이기와 모든 호르몬을 elute.</li>
	<li>포함 하는 1% (v/v) 초 산 진공 집중 장치를 사용 하 여 물에 추출 eluate에 이기를 증발. 참고:이 완전히 건조 하지 않습니다.</li>
	<li>1 mL 이기, 1 mL 메탄올, 0.5 mL를 적용 하 여 혼합 모드, 강한 양이온 교환 (MCX) 열 카트리지 equilibrate 0.1 M HCl, 그리고 포함 하는 1% (v/v) 초 산 1 mL 물.</li>
	<li>Equilibrated MCX 카트리지 당 하나의 샘플 솔루션을 적용 합니다.</li>
	<li>1 mL 물 1% (v/v) 초 산을 포함 하는 카트리지를 씻어.</li>
	<li>포함 하는 유리 튜브에 1% (v/v) 초 산 2 mL 30% (v/v) 이기와 IAA elute.</li>
	<li>씻어 카트리지 포함 하는 1% (v/v) 아세트산 2 mL 80% (v/v) 이기.</li>
	<li>씻으십시오 2 mL 물, 1 mL 물 5% 수성 암모니아를 포함 하는 카트리지.</li>
	<li>유리 튜브에 5% 수성 암모니아를 포함 하는 2 mL 60% (v/v) 이기와는 CKs을 elute.</li>
	<li>진공 농축 기를 사용 하 여 각 호르몬 분수의 용 매를 증발 하 고 LC-MS/MS 분석까지 −30 ° C에 저장 합니다.</li>
</ol>3. LC-MS/MS 분석 IAA 및 CKs
<ol><li>각 호르몬 추출 메탄올의 600 µ L 튜브에 녹이 고 나사 목 총 복구 유리병에 솔루션을 전송. 진공 집중 장치를 사용 하 여 용 매를 증발.</li>
	<li>20 µ L 30% (v/v) 이기, IAA 분수와 CK 분수 나사 목 총 복구 튜브에 1% (v/v) 초 산을 포함 하는 20 µ L 물에 용 해.</li>
	<li>HPLC 시스템 트리플 4 극 자 MS 시스템에서 긍정적인 이온 모드에서 샘플을 분석 합니다. 표 1에 나열 된 참고: 우리 HPLC 조건을 설정 합니다.<ol>
<li>IAA 차입에 대 한 5%-50% 용 매 B 7 분 이상의 이진 그라디언트를 사용 하 여 다음 98% 용 매 B에 의해 증가 1 분 동안 누른 다음 둥지를 주입 하 여 5% 용 매 B에 2 분 동안 equilibrate.</li>
		<li>CK 차입에 대 한 2%-40% 용 매 B 5 분 이상 40%-70% 용 매 B 7 분에서의 이진 그라디언트를 사용 하 여 다음 95% 용 매 B 늘려 1 분 동안 누른 다음으로 2% 용 매 B에 2 분 동안 equilibrate 주입을 중첩.</li>
		<li>분무 이온화 (ESI) 설정-MS 이온 소스 표 2에 나열 된 매개 변수.</li>
	</ol>
</li>
	<li>여러 반응 (MRM) 전환 표 3에 나열 된 각 분석의 정량화에 대 한 모니터링을 사용 합니다.</li>
	<li>계량 중수소 라는 표준 레이블 없는 비율의 표준 곡선에 대 한 내 생 IAA 및 CK 수준.</li>
</ol>

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저자 들은 관심 없음 충돌 선언 합니다.

Phytohormones의 분포를 식별 하기 위해 organogenesis에 관련 된 그것은 적용 될 때 phytohormones는 것 유도 위한 explants 때문에 있는 organogenesis phytohormone 무료 매체에 관찰 될 수 있다 식물 재료를 사용 하는 것이 중요 촬영 또는 뿌리, 그들은 전체 explant을, 세포 분화 및 organogenesis에 있는 식물의 고유한가 소성을 평가 하 고 어려운 영향을 미칩니다. 우발적인 촬영 phytohormone 무료 문화 미디어 Dianthus caryophy...

고트 Haberlandt (1854-1945 년) "totipotency", 어떤 식물에 의해 세포 수, 차별화, 분열과 성숙한 식물1에서 특정 세포 유형으로 차별화 그들의 이전 후에 전체 식물을 재생성의 개념을 제안 했다. 조직 문화에서 조합 및 성장 매체에 적용 된 것 phytohormones의 농도 의해 결정 됩니다 여부 식물 재생 여부 유도 될 수 있다. Skoog와 밀러 우발적인 뿌리 매체 포함 된 낮은 비율2에 유도 될 수 있는 반면 우발적인 촬영에 auxins, CKs의 높은 비율을 포함 하는 문화 매체에 굳은 살 담배에서에서 유도 될 수 있는 발견. 그 발견 이후 조직 문화는 널리 이용 된다 경제적으로 중요 한 작물의 전파 및 유전자 변형 식물3의 재생. 우발적인 촬영 촬영 정점 분열 조직, 잎, 뿌리, internodes 등 다른 조직에서 유도 될 수 있다. Phytohormone 치료는 대부분의 식물 종에서 우발적인 촬영의 유도 필요 합니다. 그러나, 최적 농도 조합 다 종에 의해 및 explants로 사용 하는 조직 중. 따라서, 많은 노력 결정 최적 농도 실험에 대 한 phytohormones의 조합으로 간다.
Carapichea ipecacuanha (Brot) L. 앤더슨 (ipecac)는 emetine 등 뿌리4에 주로 cephaeline, 알 카 로이드를 포함 하는 약용 식물 이다. 루트 추출 물 expectorant, 토 제, 그리고 amoebicide5로 사용 됩니다. 비록 ipecac 브라질의 열 대 우림에 자연적으로 성장, 문화, 씨앗을 설정 하기를 꺼 려 이며 발 아 율 감소는 추운 기후6일본, 씨앗 보관. 대신, 조직 문화에 의해 전파, 어떤 우발적인 촬영 internodes에 가장 효율적인 방법7,8. 흥미롭게도, 우발적인 촬영 phytohormone 치료8없이이 종에서 유도 될 수 있다.
우발적인 촬영 표 피 기저 지역9에 callusing, 없이 internodal 세그먼트의 꼭대기 지역에 형성 된다. 이 차이 아마도 phytohormonal 규정 internodal 세그먼트에서 조직 극성을 나타냅니다. Ipecac 문화 시스템 우발적인 촬영 형성 동안 내 생 phytohormone 레벨에 변화를 분석 하는 독특한 기회를 허용 합니다. 한 auxin (indole-3-아세트산 (IAA)) 및 4 개의 CKs 생 레벨의 분석에 대 한 우리의 방법을 소개 (isopentenyl 아데닌 (iP), isopentenyl 아데닌 아 (iPR), 트랜스-제 (tZ), 및 트랜스-제 아 (tZR)) 액정-MS/MS 사용 internodal 세그먼트.

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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">[2H5]indole-3-acetic acid</td>
			<td style="width:176px;">Olchemlm Ltd</td>
			<td style="width:119px;">031 1531</td>
			<td style="width:167px;">Internal standard for LC-MS/MS</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">[2H5]trans-zeatin</td>
			<td style="width:176px;">Olchemlm Ltd</td>
			<td style="width:119px;">030 0301</td>
			<td>Internal standard for LC-MS/MS</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">[2H5]trans-zeatin riboside</td>
			<td style="width:176px;">Olchemlm Ltd</td>
			<td style="width:119px;">030 0311</td>
			<td>Internal standard for LC-MS/MS</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">[2H6]N6-isopentenyl adenine</td>
			<td style="width:176px;">Olchemlm Ltd</td>
			<td style="width:119px;">030 0161</td>
			<td>Internal standard for LC-MS/MS</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:216px;">[2H6]N6-isopentenyl adenosine</td>
			<td style="width:176px;">Olchemlm Ltd</td>
			<td style="width:119px;">030 0171</td>
			<td>Internal standard for LC-MS/MS</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">indole-3-acetic acid</td>
			<td style="width:176px;">Wako</td>
			<td>098 00181</td>
			<td>standard for LC-MS/MS</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">trans-zeatin</td>
			<td style="width:176px;">SIGMA-ALDRICH</td>
			<td style="width:119px;">Z0876 5MG</td>
			<td>standard for LC-MS/MS</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">trans-zeatin riboside</td>
			<td style="width:176px;">Wako</td>
			<td>262 01081</td>
			<td>standard for LC-MS/MS</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">N6-isopentenyl adenine</td>
			<td style="width:176px;">SIGMA-ALDRICH</td>
			<td style="width:119px;">D7674 1G</td>
			<td>standard for LC-MS/MS</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">N6-isopentenyl adenosine</td>
			<td style="width:176px;">ACROS ORGANICS</td>
			<td style="width:119px;">22648 1000</td>
			<td>standard for LC-MS/MS</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:216px;">acetonitrile hypergrade for LC-MS LiChrosolv</td>
			<td style="width:176px;">MERCK</td>
			<td style="width:119px;">1.00029.1000</td>
			<td>solvent for LC-MS/MS</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:216px;">Water for chromatography LiChrosolv</td>
			<td style="width:176px;">MERCK</td>
			<td style="width:119px;">1.15333.1000</td>
			<td>solvent for LC-MS/MS</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">HPLC</td>
			<td style="width:176px;">SHIMADZU</td>
			<td style="width:119px;">Prominence</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">MS</td>
			<td style="width:176px;">Sciex</td>
			<td style="width:119px;">3200QTRAP</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Oasis HLB 30 mg/1 cc</td>
			<td style="width:176px;">Waters</td>
			<td style="width:119px;">WAT094225</td>
			<td>cartridge column</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Oasis MCX 30 mg/1 cc</td>
			<td style="width:176px;">Waters</td>
			<td style="width:119px;">186000252</td>
			<td>cartridge column</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">screw neck total recovery vial</td>
			<td style="width:176px;">Waters</td>
			<td style="width:119px;">186002805</td>
			<td></td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;width:216px;">blue, 12 x 32mm screw neck cap and PTFE/silicone septum</td>
			<td style="width:176px;">Waters</td>
			<td style="width:119px;">186000274</td>
			<td></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:216px;">Acquity UPLC BEH C18, 2.1x100 mm</td>
			<td style="width:176px;">Waters</td>
			<td style="width:119px;">186002350</td>
			<td>UPLC column</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:216px;">Proshell 120 EC-C18, 2.1x50 mm</td>
			<td style="width:176px;">Agilent</td>
			<td style="width:119px;">699775-902</td>
			<td>UPLC column</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Digital microscope</td>
			<td style="width:176px;">Leica</td>
			<td style="width:119px;">DHS1000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">TissueLyser II</td>
			<td style="width:176px;">QIAGEN</td>
			<td style="width:119px;">85300</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Surgical blade</td>
			<td style="width:176px;">Feather</td>
			<td style="width:119px;">No. 22</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Scalpel handle</td>
			<td style="width:176px;">Feather</td>
			<td style="width:119px;">No. 4</td>
			<td></td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;width:216px;">Savant SpeedVac/Refregerated vapor trap</td>
			<td style="width:176px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">SPD111/RVT4104</td>
			<td>vacuum concentrartor</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:216px;">Disposable glass tobe (13x100 mm)</td>
			<td style="width:176px;">IWAKI</td>
			<td style="width:119px;">9832-1310</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Sterile petri dish</td>
			<td style="width:176px;">INA OPTICA</td>
			<td style="width:119px;">I-90-20</td>
			<td></td>
		</tr>
	</tbody>
</table>

quantification of endogenous auxin and cytokinin during internode culture of ipecac

우발적인 촬영 형성 유전자 변형 식물의 재생 및 경제적으로 중요 한 작물의 전파에 대 한 중요 한 기술입니다. Phytohormone 치료는 대부분의 종에서 우발적인 촬영의 유도 필요 합니다. Auxin과 시 토 키 닌 (CK) 사이의 균형에 의해 결정 됩니다 여부 우발적인 촬영 유도 될 수 있는 수준. 많은 노력 결정 최적 농도 및 explants로 사용 하는 각 조직에 각 식물 종에 phytohormones의 조합으로 간다. 그러나 Ipecac에서, 우발적인 촬영 수 유도 될 phytohormone 치료 없이 문화 매체에서 internodal 세그먼트에. 평가할 셀 차별화 ipecac의 고유한 소성 수 있습니다. Ipecac 우발적인 촬영 유도, 우리 phytohormone 무료 B5 중간 5 주 동안 0.2 %gellan 껌으로 고형화에 14 h 빛/10-h 어두운 주기에 internodal 세그먼트 15 µmol m−2의 − 1 의 빛에서 24 ° C에서 경작. 우발적인 촬영 형성 동안 phytohormone 역학 조사, 우리 생 indole-3-아세트산 및 CKs 세그먼트에 의해 측정 액체 크로마토그래피 탠덤 질량 분석 LC-MS/MS. 이 메서드는 간단한 방식으로 생 indole-3-아세트산 및 CKs 레벨의 분석을 수 있습니다. 그것은 다른 식물 종에 organogenesis 동안 내 생 auxin과 CK의 역학 조사에 적용할 수 있습니다.

1세인트 주에서 아니 우발적인 촬영 형성 했다. 2노스다코타 주에 작은 쏘고 나타났다. 3rd , 4번째 주에 수 꼭대기 지역 (I 및 II)에서 주로 증가 쏘고 (그림 2A). 5일 주에 수 촬영의 되었고 약 7 시내 지역 나 지역 II (그림 2B) 5. 대조적으로, 단지 몇 가지 촬영 영역 II...

Watch this Scientific Journal Video about Quantification of Endogenous Auxin and Cytokinin During Internode Culture of Ipecac at JoVE.com

Quantification of Endogenous Auxin and Cytokinin During Internode Culture of Ipecac

Ipecac의 위치인 문화 중 생 Auxin 및 시 토 키 닌의 정량화

우발적인 촬영 phytohormone 치료 없이 ipecac의 internodal 세그먼트에 유도 될 수 있다. 우발적인 촬영 형성 동안 phytohormone 역학을 평가 하기 위해 내 생 auxin 및 LC-MS/MS에 의해 internodal 세그먼트에서 시 토 키 닌을 측정 했습니다.

Adventitious shoot formation is an important technique for the propagation of economically important crops and for the regeneration of transgenic plants. ...

quantification-endogenous-auxin-cytokinin-during-internode-culture

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8.9K 조회수.  Toyo University. 우발적인 촬영 phytohormone 치료 없이 ipecac의 internodal 세그먼트에 유도 될 수 있다. 우발적인 촬영 형성 동안 phytohormone 역학을 평가 하기 위해 내 생 auxin 및 LC-MS/MS에 의해 internodal 세그먼트에서 시 토 키 닌을 측정 했습니다.

비디오: Quantification of Endogenous Auxin and Cytokinin During Internode Culture of Ipecac

EPA Method 1615. Measurement of Enterovirus and Norovirus Occurrence in Water by Culture and RT-qPCR. I. Collection of Virus Samples

EPA Method 1615 was developed with a goal of providing a standard method for measuring enteroviruses and noroviruses in environmental and drinking waters. The standardized sampling component of the method concentrates viruses that may be present in water by passage of a minimum specified volume of water through an electropositive cartridge filter. The minimum specified volumes for surface and finished/ground water are 300 L and 1,500 L, respectively. A major method limitation is the tendency for the filters to clog before meeting the sample volume requirement. Studies using two different, but equivalent, cartridge filter options showed that filter clogging was a problem with 10% of the samples with one of the filter types compared to 6% with the other filter type. Clogging tends to increase with turbidity, but cannot be predicted based on turbidity measurements only. From a cost standpoint one of the filter options is preferable over the other, but the water quality and experience with the water system to be sampled should be taken into consideration in making filter selections.

EPA Method 1615 uses an electropositive filter to concentrate enteroviruses and noroviruses in environmental and drinking waters. This manuscript describes the procedure for collecting samples for Method 1615 analyses.

엔테로 바이러스 및 문화 RT-qPCR에 의해 물에서 노로 바이러스의 발생의 EPA 방법 1615 측정. 바이러스 샘플의 I. 컬렉션

EPA Method 1615 was developed with a goal of providing a standard method for measuring enteroviruses and noroviruses in environmental and drinking waters. ...

연구

환경과학

Quantification of Heavy Metals and Other Inorganic Contaminants on the Productivity of Microalgae

Increasing demand for renewable fuels has researchers investigating the feasibility of alternative feedstocks, such as microalgae. Inherent advantages include high potential yield, use of non-arable land and integration with waste streams. The nutrient requirements of a large-scale microalgae production system will require the coupling of cultivation systems with industrial waste resources, such as carbon dioxide from flue gas and nutrients from wastewater. Inorganic contaminants present in these wastes can potentially lead to bioaccumulation in microalgal biomass negatively impact productivity and limiting end use. This study focuses on the experimental evaluation of the impact and the fate of 14 inorganic contaminants (As, Cd, Co, Cr, Cu, Hg, Mn, Ni, Pb, Sb, Se, Sn, V and Zn) on Nannochloropsis salina growth. Microalgae were cultivated in photobioreactors illuminated at 984 &#181;mol m-2 sec-1 and maintained at pH 7 in a growth media polluted with inorganic contaminants at levels expected based on the composition found in commercial coal flue gas systems. Contaminants present in the biomass and the medium at the end of a 7 day growth period were analytically quantified through cold vapor atomic absorption spectrometry for Hg and through inductively coupled plasma mass spectrometry for As, Cd, Co, Cr, Cu, Mn, Ni, Pb, Sb, Se, Sn, V and Zn. Results show N. salina is a sensitive strain to the multi-metal environment with a statistical decrease in biomass yieldwith the introduction of these contaminants. The techniques presented here are adequate for quantifying algal growth and determining the fate of inorganic contaminants.

Integration of microalgal cultivation with industrial flue gas will ultimately introduce heavy metals and other inorganic compounds into the growth media. This study presents a procedure used to determine the end fate and impact of heavy metals and inorganic contaminants on the growth of Nannochloropsis salina grown in photobioreactors.

중금속과 미세 조류의 생산성에 다른 무기 오염 물질의 정량화

Increasing demand for renewable fuels has researchers investigating the feasibility of alternative feedstocks, such as microalgae. Inherent advantages ...

Simultaneous DNA-RNA Extraction from Coastal Sediments and Quantification of 16S rRNA Genes and Transcripts by Real-time PCR

Real Time Polymerase Chain Reaction also known as quantitative PCR (q-PCR) is a widely used tool in microbial ecology to quantify gene abundances of taxonomic and functional groups in environmental samples. Used in combination with a reverse transcriptase reaction (RT-q-PCR), it can also be employed to quantify gene transcripts. q-PCR makes use of highly sensitive fluorescent detection chemistries that allow quantification of PCR amplicons during the exponential phase of the reaction. Therefore, the biases associated with &#39;end-point&#39; PCR detected in the plateau phase of the PCR reaction are avoided. A protocol to quantify bacterial 16S rRNA genes and transcripts from coastal sediments via real-time PCR is provided. First, a method for the co-extraction of DNA and RNA from coastal sediments, including the additional steps required for the preparation of DNA-free RNA, is outlined. Second, a step-by-step guide for the quantification of 16S rRNA genes and transcripts from the extracted nucleic acids via q-PCR and RT-q-PCR is outlined. This includes details for the construction of DNA and RNA standard curves. Key considerations for the use of RT-q-PCR assays in microbial ecology are included.

A protocol to quantify bacterial 16S rRNA genes and transcripts from coastal sediments via real-time PCR is provided. The methodology includes the co-extraction of DNA and RNA; preparation of DNA-free RNA; and 16S rRNA gene and transcript quantification via RT-q-PCR, including standard curve construction.

실시간 PCR에 의한 연안 퇴적물 및 정량 16S의 rRNA의 유전자 및 성적 증명서에서 동시 DNA-RNA 추출

Real Time Polymerase Chain Reaction also known as quantitative PCR (q-PCR) is a widely used tool in microbial ecology to quantify gene abundances of ...

Capturing Flow-weighted Water and Suspended Particulates from Agricultural Canals During Drainage Events

The purpose of this study is to describe the methods used to capture flow-weighted water and suspended particulates from farm canals during drainage discharge events. Farm canals can be enriched by nutrients such as phosphorus (P) that are susceptible to transport. Phosphorus in the form of suspended particulates can significantly contribute to the overall P loads in drainage water. A settling tank experiment was conducted to capture suspended particulates during discrete drainage events. Farm canal discharge water was collected in a series of two 200 L settling tanks over the entire duration of the drainage event, so as to represent a composite subsample of the water being discharged. Imhoff settling cones are ultimately used to settle out the suspended particulates. This is achieved by siphoning water from the settling tanks via the cones. The particulates are then collected for physico-chemical analyses.

Nutrients present in particulate form can contribute significantly to the overall loads in agricultural drainage waters. This study describes a novel method to capture flow-weighted water and suspended particulates from farm canal drainage over the entire duration of the drainage event.

배수 이벤트 기간 동안 흐름 중 물과 농업 운하에서 일시 중단 된 입자를 캡처

The purpose of this study is to describe the methods used to capture flow-weighted water and suspended particulates from farm canals during drainage ...

The Effect of the Application of Thyme Essential Oil on Microbial Load During Meat Drying

Meat is a high protein meal that is used in the preparation of jerky, a popular food snack, where preservation and safety are important. To assure food safety and to extend the shelf life of meat and meat products, the use of either synthetic or natural preservatives have been applied to control and eliminate foodborne bacteria. A growing interest in the application of natural food additives for meat has increased. Microorganisms, such as Escherichia coli, contaminate meat and meat products, causing foodborne illnesses. Therefore, it is necessary to improve the meat conservation process. However, the use of essential oils when the meat is being dried has not been deeply studied. In this regard, there is an opportunity to increase the value of dried meat and reduce the risk of foodborne illnesses by applying essential oils during the drying process. In this protocol, we present a novel method of applying thyme essential oil (TEO) during meat drying, specifically in vapor form directly in a drying chamber. For evaluation, we use Minimal Inhibitory Concentration (MIC) to detect the number of harmful bacteria in the treated samples compared to raw samples. The preliminary results show that this method is a viable and alternative option to synthetic preservatives and that it significantly reduces microbial load in dried meat.

Microorganisms such as Escherichia coli that contaminate meat products cause foodborne illnesses. The use of essential oils in the meat drying process has not been deeply studied. Here, we present a novel method of applying thyme essential oil to meat during drying to reduce the microbial load in dried meat.

고기 건조 하는 동안 미생물 부하에 백 리 향 에센셜 오일의 응용 프로그램의 효과

Meat is a high protein meal that is used in the preparation of jerky, a popular food snack, where preservation and safety are important. To assure food ...

Stress Distribution During Cold Compression of Rocks and Mineral Aggregates Using Synchrotron-based X-Ray Diffraction

We report detailed procedures for performing compression experiments on rocks and mineral aggregates within a multi-anvil deformation apparatus (D-DIA) coupled with synchrotron X-radiation. A cube-shaped sample assembly is prepared and compressed, at room temperature, by a set of four X-ray transparent sintered diamond anvils and two tungsten carbide anvils, in the lateral and the vertical planes, respectively. All six anvils are housed within a 250-ton hydraulic press and driven inward simultaneously by two wedged guide blocks. A horizontal energy dispersive X-ray beam is projected through and diffracted by the sample assembly. The beam is commonly in the mode of either white or monochromatic X-ray. In the case of white X-ray, the diffracted X-rays are detected by a solid-state detector array that collects the resulting energy dispersive diffraction pattern. In the case of monochromatic X-ray, the diffracted pattern is recorded using a two-dimensional (2-D) detector, such as an imaging plate or a charge-coupled device (CCD) detector. The 2-D diffraction patterns are analyzed to derive lattice spacings. The elastic strains of the sample are derived from the atomic lattice spacing within grains. The stress is then calculated using the predetermined elastic modulus and the elastic strain. Furthermore, the stress distribution in two-dimensions allow for understanding how stress is distributed in different orientations. In addition, a scintillator in the X-ray path yields a visible light image of the sample environment, which allows for the precise measurement of sample length changes during the experiment, yielding a direct measurement of volume strain on the sample. This type of experiment can quantify the stress distribution within geomaterials, which can ultimately shed light on the mechanism responsible for compaction. Such knowledge has the potential to significantly improve our understanding of key processes in rock mechanics, geotechnical engineering, mineral physics, and material science applications where compactive processes are important.

We report detailed procedures for compression experiments on rocks and mineral aggregates within a multi-anvil deformation apparatus coupled with synchrotron X-radiation. Such experiments allow quantification of the stress distribution within samples, that ultimately sheds light on compaction processes in geomaterials.

We report detailed procedures for performing compression experiments on rocks and mineral aggregates within a multi-anvil deformation apparatus (D-DIA) ...

Chemical Isolation, Quantification, and Separation of Skin Lipids from Reptiles

Reptiles signal to conspecifics using lipids in their skin, primarily to enable mate tracking and assessment. The isolation of these lipids has utility in research focused on evolutionary patterns and mechanisms of chemical communication, in addition to understanding the waterproofing role of lipids in the evolution of terrestrial life. In an applied approach, such skin-based cues have potential use for wildlife managers dealing with invasive species. The main steps for quantifying reptile skin lipids in the protocol presented here include extraction, total lipid determination, and fractionation via column chromatography, the latter process resulting in purified eluates of compounds which can then either be analyzed to assign compound identifications (e.g., gas chromatography-mass spectrometry [GC-MS]) and/or used directly in more refined bioassays. Skin lipids can be extracted from living skin, shed skin, or dead whole animals, using nonpolar organic solvents (e.g., hexane, benzene, toluene). Extraction solubilizes the lipids and, then, the solvent can be evaporated to yield a measurable lipid-only extract. Fractionation involves the separation of the total lipid extract into specific eluates via traditional column chromatography. The total lipid extract is first bound to a substrate-based column (e.g., alumina) and, then, individual eluates (&#34;fractions&#34;) of solvent at specific volumes are passed sequentially through the column to elute sets of compounds from the lipid mixture based on common polarity. The fractions progress in polarity at a standardized sequence by increasing the relative amount of polar solvent (e.g., diethyl ether) in nonpolar solvent. In this manuscript, we describe several methods for extracting skin lipids of reptiles and, then, provide a standard protocol for isolating different sets of compounds based on polarity, using traditional column chromatography. Whole lipid extracts or specific fractions can, then, be used in bioassays to determine any biological activity elicited by the compounds therein.

In reptiles, skin lipids from conspecifics are crucial for sexual signaling, with potential use in invasive species management. Here, we describe protocols for extracting skin lipids from shed skin or whole animals, determining and analyzing the total lipid mass, and separating the lipids using fractionation via column chromatography.

화학, 정량화, 고립과 파충류의 피부 지질 분리

Reptiles signal to conspecifics using lipids in their skin, primarily to enable mate tracking and assessment. The isolation of these lipids has utility in ...

Split Point Analysis and Uncertainty Quantification of Thermal-Optical Organic/Elemental Carbon Measurements

Researchers from myriad fields frequently seek to quantify and classify concentrations of carbonaceous aerosols as organic carbon (OC) or elemental carbon (EC). This is commonly accomplished using thermal-optical OC/EC analyzers (TOAs), which enable measurement via controlled thermal pyrolysis and oxidation under specific temperature protocols and within constrained atmospheres. Several commercial TOAs exist, including a semi-continuous instrument that enables on-line analyses in the field. This instrument employs an in-test calibration procedure that requires relatively frequent calibration. This article details a calibration protocol for this semi-continuous TOA and presents an open-source software tool for data analysis and rigorous Monte Carlo quantification of uncertainties. Notably, the software tool includes novel means to correct for instrument drift and identify and quantify the uncertainty in the OC/EC split point. This is a significant improvement on the uncertainty estimation in the manufacturer&#8217;s software, which ignores split point uncertainty and otherwise uses fixed equations for relative and absolute errors (generally leading to under-estimated uncertainties and often yielding non-physical results as demonstrated in several example data sets). The demonstrated calibration protocol and new software tool enabling accurate quantification of combined uncertainties from calibration, repeatability, and OC/EC split point are shared with the intent of assisting other researchers in achieving better measurements of OC, EC, and total carbon mass in aerosol samples.

This article presents a protocol and software tool for the quantification of uncertainties in the calibration and data analysis of a semi-continuous thermal-optical organic/elemental carbon analyzer.

열 광학 유기/원소 탄소 측정의 분할 점 분석 및 불확실성 정량화

Researchers from myriad fields frequently seek to quantify and classify concentrations of carbonaceous aerosols as organic carbon (OC) or elemental carbon ...

Processing Embryo, Eggshell, and Fungal Culture for Scanning Electron Microscopy

Although scanning electron microscopy (SEM) is being widely used for the ultra-structural analysis of various biological and non-biological samples, methods involved in processing different biological samples involve unique practices. All conventional practices described in the literature for processing samples still find useful applications, but subtle changes in the sample preparation can alter image quality, as well as, introduce artifacts. Hence, using a unique sample preparation technique specific to the type of tissue analyzed is required to obtain a good quality image with ultrastructural resolution. The focus of this study is to provide the optimal sample preparation protocols for imaging embryos, rigid eggshells, and fungal cultures using SEM. The following optimizations were recommended to yield good results for the three different delicate biological samples studied. Use of milder fixatives like 4% paraformaldehyde or 3% glutaraldehyde followed by dehydration with ethanol series is mandatory. Fungal mycelium on agar blocks obtained by slide cultures yields a better ultrastructural integrity compared to cultures taken directly from agar plates. Chemical drying of embryos with HMDS provides drying without introducing surface tension artifacts compared to critical point drying. HMDS prevents cracking caused by shrinkage as samples are less brittle during drying. However, for fungal culture, critical point drying provides acceptable image quality compared to chemical drying. Eggshells can be imaged with no special preparation steps except for thorough washing and air drying prior to mounting. Preparation methodologies were standardized based on acceptable image quality obtained with each trial.&#160;

Here, we present detailed processing protocols for imaging delicate tissue samples using scanning electron microscopy (SEM). Three different processing methods, namely, hexamethyl disilazana (HMDS) chemical drying, simple air drying, and critical point drying are described for preparing rigid eggshells, embryos at early developmental stages, and fungal cultures respectively.

전자 현미경 검사법을 검사하기위한 배아, 달걀 껍질 및 곰팡이 배양 처리

Although scanning electron microscopy (SEM) is being widely used for the ultra-structural analysis of various biological and non-biological samples, ...

Microalgae Cultivation and Biomass Quantification in a Bench-Scale Photobioreactor with Corrosive Flue Gases

Photobioreactors are illuminated cultivation systems for experiments on phototrophic microorganisms. These systems provide a sterile environment for microalgal cultivation with temperature, pH, and gas composition and flow rate control. At bench-scale, photobioreactors are advantageous to researchers studying microalgal properties, productivity, and growth optimization. At industrial scales, photobioreactors can maintain product purity and improve production efficiency. The video describes the preparation and use of a bench-scale photobioreactor for microalgal cultivation, including the safe use of corrosive gas inputs, and details relevant biomass measurements and biomass productivity calculations. Specifically, the video illustrates microalgal culture storage and preparation for inoculation, photobioreactor assembly and sterilization, biomass concentration measurements, and a logistic model for microalgal biomass productivity with rate calculations including maximum and overall biomass productivities. Additionally, since there is growing interest in experiments to cultivate microalgae using simulated or real waste gas emissions, the video will cover the photobioreactor equipment adaptations necessary to work with corrosive gases and discuss safe sampling in such scenarios.

Bench-scale, axenic cultivation facilitates microalgal characterization and productivity optimization before subsequent process scale-up. Photobioreactors provide the necessary control for reliable and reproducible microalgal experiments and can be adapted to safely cultivate microalgae with the corrosive gases (CO2, SO2, NO2) from municipal or industrial combustion emissions.

부식성 연도 가스가 있는 벤치 스케일 광생물반응기에서 미세조류 재배 및 바이오매스 정량화

Photobioreactors are illuminated cultivation systems for experiments on phototrophic microorganisms. These systems provide a sterile environment for ...