Name: strategies to enhance cultivation of anaerobic bacteria from gastrointestinal tract of chicken
Uploaded: 2024-05-10T14:30:00.000+00:00
Duration: 4 min 36 s
Description: Read this Scientific Article Protocol about Author Spotlight: Advancing Intestinal Bacteria Cultivation for Poultry at JoVE.com

<meta charset="utf8"></head><body>鸡胃肠道中未培养微生物多样性的培养策略

<ol>
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<table><tbody><tr><td>Acetic acid</td><td>VWR</td><td>20104.334</td><td></td></tr><tr><td>Agar</td><td>VWR</td><td>97064-332</td><td></td></tr><tr><td>Ammonium chloride</td><td>Carl Roth</td><td>P726.1</td><td></td></tr><tr><td>Anaerobic station</td><td>Don Whitley Scientific</td><td>A35 HEPA</td><td></td></tr><tr><td>Butyric acid</td><td>Merck</td><td>8.0045.1000</td><td></td></tr><tr><td>Calcium chloride dihydrate&amp;nbsp;</td><td>VWR</td><td>97061-904</td><td></td></tr><tr><td>Centrifuge</td><td>Eppendorf</td><td>5424R</td><td></td></tr><tr><td>Chicken lysozyme (Muramidase)</td><td>VWR</td><td>1.05281.0010</td><td></td></tr><tr><td>Cysteine</td><td>VWR</td><td>97061-204</td><td></td></tr><tr><td>Dextrose</td><td>VWR</td><td>90000-908</td><td></td></tr><tr><td>Di-potassium hydrogen phosphate</td><td>Carl Roth</td><td>P749.1</td><td></td></tr><tr><td>EDTA</td><td>Carl Roth</td><td>8043.2</td><td></td></tr><tr><td>Legehennen/ Junghennenfutter</td><td>Deutsche Tiernahrung Cremer GmbH &amp;amp; Co. KG, D&amp;uuml;sseldorf, Germany</td><td>-</td><td></td></tr><tr><td>MagAttract HMW DNA Kit</td><td>Qiagen</td><td>67563</td><td></td></tr><tr><td>Magnesium chloride</td><td>Carl Roth</td><td>2189.1</td><td></td></tr><tr><td>Mixed gas (80% N&lt;sub&gt;2&lt;/sub&gt; (quality level 5.0), 15% CO&lt;sub&gt;2&lt;/sub&gt; (quality level 3.0) and 5% H&lt;sub&gt;2&lt;/sub&gt; (quality level 5.0))</td><td>Westfalen Gase GmbH, Germany</td><td>-</td><td></td></tr><tr><td>Mutanolysin, recombinant (lyophilisate)</td><td>A&amp;amp;A Biotechnology</td><td>1017-10L</td><td></td></tr><tr><td>Myo-inositol</td><td>Carl Roth</td><td>4191.2</td><td></td></tr><tr><td>PBS 1X</td><td>ChemSolute</td><td>8418.01</td><td></td></tr><tr><td>Potassium dihydrogen phosphate</td><td>Carl Roth</td><td>3904.2</td><td></td></tr><tr><td>Propionic acid</td><td>Carl Roth</td><td>6026.1</td><td></td></tr><tr><td>QuantiFluor dsDNA System</td><td>Promega</td><td>E2671</td><td></td></tr><tr><td>RNAse A&amp;nbsp;</td><td>QIAGEN Ribonuclease A (RNase A)&amp;nbsp;</td><td>19101</td><td></td></tr><tr><td>Sodium chloride</td><td>VWR</td><td>27800.291</td><td></td></tr><tr><td>Sodium resazurin</td><td>VWR</td><td>85019-296</td><td></td></tr><tr><td>Sodium thioglycolate</td><td>Sigma-Aldrich</td><td>102933</td><td></td></tr><tr><td>Soy Peptone, GMO-Free, Animal-Free</td><td>VWR</td><td>97064-186</td><td></td></tr><tr><td>Thermocycler</td><td>Bio-Rad</td><td>T100</td><td></td></tr><tr><td>Tryptone</td><td>Carl Roth</td><td>8952.1</td><td></td></tr><tr><td>Tween80</td><td>Carl Roth</td><td>9139.2</td><td></td></tr><tr><td>Vitamin mix (supplement)</td><td>VWR</td><td>968290NL</td><td></td></tr><tr><td>Vortex</td><td>Star Lab</td><td>07127/92930</td><td></td></tr><tr><td>Yeast Extract</td><td>Carl Roth</td><td>9257.05</td><td></td></tr><tr><td>&amp;beta;-D-Fructose</td><td>VWR</td><td>53188-23-1</td><td></td></tr></tbody></table>

strategies to enhance cultivation of anaerobic bacteria from gastrointestinal tract of chicken

Read this Scientific Article Protocol about Author Spotlight: Advancing Intestinal Bacteria Cultivation for Poultry at JoVE.com

<meta charset="utf8"></head><body>作者聚焦：推进家禽肠道细菌培养

加强鸡胃肠道厌氧菌培养的策略

The gastrointestinal tract (GIT) of chicken is a complex ecosystem harboring trillions of microbes that play a pivotal role in the host&#39;s physiology, ...

strategies-to-enhance-cultivation-anaerobic-bacteria-from

Research

JoVE Journal

Biology

Strategies to Enhance Cultivation of Anaerobic Bacteria from Gastrointestinal Tract of Chicken

664 次观看.  University of Hohenheim. 我们的目标是从鸡的肠道中分离和培养细菌，以提高我们对微生物网络和与宿主相互作用的了解。这些厌氧菌的表征支持肠道过程的知识，并为未来的研究丰富了细菌数据库。主要挑战之一是防止采样和培养过程中的氧气暴露和氧反应物质，以防止细菌灭活。根据我们的经验，这改善了厌氧菌的多样性恢复。我们的协议解决了了解家禽肌醇代谢...

视频: 作者聚焦：推进家禽肠道细菌培养

Windowing Chicken Eggs for Developmental Studies

The study of development has been greatly aided by the use of the chick embryo as an experimental model.  The ease of accessibility of the embryo has allowed for experiments to map cell fates using several approaches, including chick quail chimeras and focal dye labeling.  In addition, it allows for molecular perturbations of several types, including placement of protein-coated beads and introduction of plasmid DNA using in ovo electroporation.  These experiments have yielded important data on the development of the central and peripheral nervous systems.  For many of these studies, it is necessary to open the eggshell and reclose it without perturbing the embryo's growth.  The embryo can be examined at successive developmental stages by re-opening the eggshell.  While there are several excellent methods for opening chicken eggs, in this article we demonstrate one method that has been optimized for long survival times.  In this method, the egg rests on its side and a small window is cut in the shell.  After the experimental procedure, the shell is used to cover the egg for the duration of its development.  Clear plastic tape overlying the eggshell protects the embryo and helps retain hydration during the remainder of the incubation period.  This method has been used beginning at two days of incubation and has allowed survival through mature embryonic ages.

The ease of accessibility of the embryo has allowed for experiments to map cell fates using several approaches, including chick quail chimeras and focal dye labeling. In this article we demonstrate one egg preparation method that has been optimized for long survival times.

发育研究的窗口鸡蛋

The study of development has been greatly aided by the use of the chick embryo as an experimental model.  The ease of accessibility of the embryo has ...

科研

生物学

Culturing and Maintaining Clostridium difficile in an Anaerobic Environment

Clostridium difficile is a Gram-positive, anaerobic, sporogenic bacterium that is primarily responsible for antibiotic associated diarrhea (AAD) and is a significant nosocomial pathogen. C. difficile is notoriously difficult to isolate and cultivate and is extremely sensitive to even low levels of oxygen in the environment. Here, methods for isolating C. difficile from fecal samples and subsequently culturing C. difficile for preparation of glycerol stocks for long-term storage are presented. Techniques for preparing and enumerating spore stocks in the laboratory for a variety of downstream applications including microscopy and animal studies are also described. These techniques necessitate an anaerobic chamber, which maintains a consistent anaerobic environment to ensure proper conditions for optimal C. difficile growth. We provide protocols for transferring materials in and out of the chamber without causing significant oxygen contamination along with suggestions for regular maintenance required to sustain the appropriate anaerobic environment for efficient and consistent C. difficile cultivation.

Culturing and Maintaining Clostridium difficile in an Anaerobic Environment

Clostridium difficile is a pathogenic bacterium that is a strict anaerobe and causes antibiotic associated diarrhea (AAD). Here, methods for isolating, culturing and maintaining C. difficile vegetative cells and spores are described. These techniques necessitate an anaerobic chamber, which requires regular maintenance to ensure proper conditions for optimal C. difficile cultivation.

培养和维护<em&gt;难辨梭状芽孢杆菌</em&gt;在厌氧环境

Clostridium  difficile is a Gram-positive, anaerobic, sporogenic bacterium  that is primarily responsible for antibiotic associated diarrhea (AAD) and is ...

免疫与感染

Medium Preparation for the Cultivation of Microorganisms under Strictly Anaerobic/Anoxic Conditions

In contrast to aerobic organisms, strictly anaerobic microorganisms require the absence of oxygen and usually a low redox potential to initiate growth. As oxygen is ubiquitous in air, retaining O2-free conditions during all steps of cultivation is challenging but a prerequisite for anaerobic culturing. The protocol presented here demonstrates the successful cultivation of an anaerobic mixed culture derived from a biogas plant using a simple and inexpensive method. A precise description of the entire anoxic culturing process is given including media preparation, filling of cultivation flasks, supplementation with redox indicator and reducing agents to provide low redox potentials as well as exchanging the headspace to keep media free from oxygen. Furthermore, a detailed overview of aseptically inoculating gas tight serum flasks (by using sterile syringes and needles) and suitable incubation conditions is provided. The present protocol further deals with gas and liquid sampling for subsequent analyses regarding gas composition and volatile fatty acid concentrations using gas chromatography (GC) and high performance liquid chromatography (HPLC), respectively, and the calculation of biogas and methane yield considering the ideal gas law.

As obligate anaerobic organisms are unable to grow upon oxygen exposure, the use of anaerobic culturing techniques is indispensable. Here, we demonstrate a simple and effective method to cultivate a mixed culture derived from a biogas plant from media preparation to gas and volatile fatty acid quantification.

严格厌氧/缺氧条件下微生物培养的介质制备

In contrast to aerobic organisms, strictly anaerobic microorganisms require the absence of oxygen and usually a low redox potential to initiate growth. As ...

生物化学

用于培养环境微生物的原位模拟厌氧方法

A Set of In Situ Informed Simulated Medium Formats for Culturing Environmentally Acquired Anaerobic Microorganisms

Culture-dependent research of anaerobic microorganisms rests upon methodological competence. These methods must create and maintain suitable growth conditions (e.g., pH and carbon sources) for anaerobic microorganisms while also allowing samples to be extracted without compromising the artificial environment. To this end, methods that are informed by and simulate an in situ environment can be of great aid in culturing microorganisms from that environment. Here, we outline an in situ informed and simulated anaerobic method for culturing terrestrial surface and subsurface microorganisms, emphasizing anaerobic sample collection with minimal perturbation. This protocol details the production of a customizable anaerobic liquid medium, and the environmental&#160;acquisition and in vitro growth of anaerobic microorganisms. The protocol also covers critical components of an anaerobic bioreactor used for environmental simulations of sediment and anaerobic liquid media for environmentally acquired cultures. We have included preliminary Next Generation Sequencing data from a maintained microbiome over the lifespan of a bioreactor where the active culture dynamically adjusted in response to an experimental carbon source.

作者聚焦:通过原位培养揭开未知环境中陆地厌氧微生物的神秘面纱

The focus of this paper is to detail best practices for making media for fastidious anaerobic microorganisms acquired from an environment. These methods help manage anaerobic cultures and can be applied to support the growth of elusive uncultured microorganisms, the "microbial dark matter."

一套用于培养环境获得性厌氧微生物的 原位 信息模拟培养基格式

Culture-dependent research of anaerobic microorganisms rests upon methodological competence. These methods must create and maintain suitable growth ...

环境科学

Screening and Isolation of C-Glycoside-Cleaving Intestinal Bacteria

C-glycosides are commonly found in medicinal plants and exhibit extensive structural diversity along with various bioactivities, including antibacterial, anti-inflammatory, antiviral, antioxidant, and antineoplastic activities. In C-glycosides, the anomeric carbon of the sugar moiety is directly connected to an aglycone through carbon-carbon bonding. Compared with O-glycosides, C-glycosides are structurally stable and resistant to acids and enzymes. Consequently, they are typically unbreakable, resulting in poor absorbability and low bioavailability. Interestingly, some intestinal bacteria can cleave C-C glycosidic bonds, providing a specific and environmentally friendly biological approach to degrade C-glycosides. In this study, a set of standard operating procedures (SOPs) was developed for screening intestinal bacteria capable of cleaving C-C glycosidic bonds based on the biotransformation model of natural compounds. The SOPs include the preparation and enrichment of intestinal bacteria, activity-oriented screening, and activity validation in a low-carbon source medium. This methodology provides a foundational reference for researchers aiming to isolate and study these specialized functional bacteria.

This study establishes a set of standard operating procedures (SOPs) for efficiently screening and isolating intestinal bacteria capable of cleaving C-glycosides.

筛选和分离 C-糖苷裂解肠道细菌

C-glycosides are commonly found in medicinal plants and exhibit extensive structural diversity along with various bioactivities, including antibacterial, ...

Production of Germ-Free Fast-Growing Broilers from a Commercial Line for Microbiota Studies

Studies of the gut microbiota contribution to the host physiology and immunocompetence are facilitated by the availability of germ-free animal models, which are considered the gold standard. Nesting birds are ideal models for the production of germ-free animals since there is no need to raise their relatives under sterile conditions. Germ-free chickens are mainly generated from specific-pathogen-free (SPF) experimental lines, which are poorly representative of commercial chicken lines.&#160;The method proposed here allowed the production of germ-free chickens from the fast growing broiler line Ross PM3, commonly used by the poultry industry.&#160;Eggs were quickly collected after laying at a broiler breeder farm.&#160;They underwent a strict decontamination process from the collection to the introduction in a sterile egg hatching isolator.&#160;The chicks have been hatched and kept in these sterile isolators during the period necessary to control their sterility. Originally developed for an experimental SPF white leghorn line, the present protocol has been adapted not only to the Ross PM3 broiler line but also to quails. It therefore represents a robust and readily adaptable procedure to other poultry species and nesting birds of economic, biological or ecological relevance.

Here we describe a method for the generation of germ-free chicks from eggs of a commercial broiler line, the Ross PM3. This method can be adapted for the generation of germ-free animals from other poultry species.

从用于微生物群研究的商业生产线上生产无菌快速生长的肉鸡

Studies of the gut microbiota contribution to the host physiology and immunocompetence are facilitated by the availability of germ-free animal models, ...

Models of Murine Vaginal Colonization by Anaerobically Grown Bacteria

The mammalian vagina can be colonized by many bacterial taxa. The human vaginal microbiome is often dominated by Lactobacillus species, but one-in-four women experience bacterial vaginosis, in which a low level of lactobacilli is accompanied by an overgrowth of diverse anaerobic bacteria. This condition has been associated with many health complications, including risks to reproductive and sexual health. While there is growing evidence showing the complex nature of microbial interactions in human vaginal health, the individual roles of these different anaerobic bacteria are not fully understood. This is complicated by the lack of adequate models to study anaerobically grown vaginal bacteria. Mouse models allow us to investigate the biology and virulence of these organisms in vivo. Other mouse models of vaginal bacterial inoculation have previously been described. Here, we describe methods for the inoculation of anaerobically grown bacteria and their viable recovery in conventionally raised C57Bl/6 mice. A new, less stressful procedural method for vaginal inoculation and washing is also described. Inoculation and viable recovery of Gardnerella are outlined in detail, and strategies for additional anaerobes such as Prevotella bivia and Fusobacterium nucleatum are discussed.

The protocol presents a mouse model of vaginal colonization with anaerobically cultured human vaginal bacteria. We focus on Gardnerella vaginalis,&#160;while including suggestions for Prevotella bivia and Fusobacterium nucleatum. This protocol can also be used as a guide for vaginal inoculations and viable recovery of other anaerobically grown bacteria.

厌氧菌小鼠阴道定植模型

The mammalian vagina can be colonized by many bacterial taxa. The human vaginal microbiome is often dominated by Lactobacillus species, but one-in-four ...

A Hybrid DNA Extraction Method for the Qualitative and Quantitative Assessment of Bacterial Communities from Poultry Production Samples

The efficacy of DNA extraction protocols can be highly dependent upon both the type of sample being investigated and the types of downstream analyses performed. Considering that the use of new bacterial community analysis techniques (e.g., microbiomics, metagenomics) is becoming more prevalent in the agricultural and environmental sciences and many environmental samples within these disciplines can be physiochemically and microbiologically unique (e.g., fecal and litter/bedding samples from the poultry production spectrum), appropriate and effective DNA extraction methods need to be carefully chosen. Therefore, a novel semi-automated hybrid DNA extraction method was developed specifically for use with environmental poultry production samples. This method is a combination of the two major types of DNA extraction: mechanical and enzymatic. A two-step intense mechanical homogenization step (using bead-beating specifically formulated for environmental samples) was added to the beginning of the &#8220;gold standard&#8221; enzymatic DNA extraction method for fecal samples to enhance the removal of bacteria and DNA from the sample matrix and improve the recovery of Gram-positive bacterial community members. Once the enzymatic extraction portion of the hybrid method was initiated, the remaining purification process was automated using a robotic workstation to increase sample throughput and decrease sample processing error. In comparison to the strict mechanical and enzymatic DNA extraction methods, this novel hybrid method provided the best overall combined performance when considering quantitative (using 16S rRNA qPCR) and qualitative (using microbiomics) estimates of the total bacterial communities when processing poultry feces and litter samples.

A novel semi-automated hybrid DNA extraction method for use with environmental poultry production samples was developed and demonstrated improvements over a common mechanical and enzymatic extraction method in terms of the quantitative and qualitative estimates of the total bacterial communities.

一种混合DNA提取方法细菌群落的定性和定量评估，从家禽生产样品

The efficacy of DNA extraction protocols can be highly dependent upon both the type of sample being investigated and the types of downstream analyses ...

加强鸡胃肠道厌氧菌培养的策略

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发育研究的窗口鸡蛋

培养和维护

严格厌氧/缺氧条件下微生物培养的介质制备

一套用于培养环境获得性厌氧微生物的原位信息模拟培养基格式

一套用于培养环境获得性厌氧微生物的原位信息模拟培养基格式

一套用于培养环境获得性厌氧微生物的原位信息模拟培养基格式

筛选和分离 C-糖苷裂解肠道细菌

从用于微生物群研究的商业生产线上生产无菌快速生长的肉鸡

厌氧菌小鼠阴道定植模型

一种混合DNA提取方法细菌群落的定性和定量评估，从家禽生产样品

加强鸡胃肠道厌氧菌培养的策略

副本

摘要

探索更多视频

此视频中的章节

发育研究的窗口鸡蛋

培养和维护

严格厌氧/缺氧条件下微生物培养的介质制备

一套用于培养环境获得性厌氧微生物的 原位 信息模拟培养基格式

一套用于培养环境获得性厌氧微生物的 原位 信息模拟培养基格式

一套用于培养环境获得性厌氧微生物的 原位 信息模拟培养基格式

筛选和分离 C-糖苷裂解肠道细菌

从用于微生物群研究的商业生产线上生产无菌快速生长的肉鸡

厌氧菌小鼠阴道定植模型

一种混合DNA提取方法细菌群落的定性和定量评估，从家禽生产样品

一套用于培养环境获得性厌氧微生物的原位信息模拟培养基格式

一套用于培养环境获得性厌氧微生物的原位信息模拟培养基格式

一套用于培养环境获得性厌氧微生物的原位信息模拟培养基格式