يرغب JH أن نعترف وأشكر معلمه، صديق والمدير السابق فاليري بيري لتعليم لنا لأول مرة هذا النموذج. وغاري Woodnutt، الذي علمنا أسس وألهم التزامنا مجال مضاد للجراثيم PK / PD. ترغب جميع المؤلفين أن أشكر ديفيد باين على دعمه وتقديره للعلم لدينا. ونود أن نعترف لدينا سابقا في الجسم الحي الزملاء علم الأحياء المجهرية الذين ساهموا في الجسم من البيانات التي تم إنشاؤها باستخدام هذه الطرق، وخاصة بيت DeMarsh، روب ستروب، روني صفحة ونيريسا سيمون. وأخيرا، نتوجه بالشكر لدينا في المختبر الزملاء علم الأحياء المجهرية للحصول على المساعدة، وخاصة لتوفير كافة البلدان المتوسطة الدخل نطلب (لين مكلوسكي، جوش الغربية وشارون مين)؛ ولفريق العمل لدينا علم الأحياء الدقيقة السريرية الذي يقدم مشورة الخبراء والدعم (ليندا ميلر، نيكول Scangarella-عمان وديبورا بتلر).

<ol>
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الكتاب والعاملين في شركة جلاكسو سميث كلاين للادوية وتملك أسهم من الأسهم دفع جميع داخل الشركة.

تقييم فعالية المرشحين المحتملين المخدرات في النماذج الحيوانية للعدوى هو عنصر حاسم في عملية اكتشاف الأدوية المضادة للبكتيريا. في الجسم الحي توفر دراسات الفعالية البيانات الهامة من مختلف أنحاء مدى الجهود الاكتشاف، من توضيح العلاقات هيكل النشاط (SAR) وتحسين سلسلة الكيميائية الرائدة من خلال تحديد الدوائية، الدوائية (PK / PD) خصائص المركبات ودعم اختيار الجرعة لصنع القرار التطوير السريري وموافقة الجهات التنظيمية. ووصف العديد من النماذج عدوى الحيوان في الأدب لهذه الأغراض. واحد من النماذج الأكثر شيوعا واستخداما هو العدلات عدوى الماوس الفخذ، الذي كان رائدا من قبل نسر 1 و 2 و توسعت بنسبة Vogelman وكريغ 3 و 4 في وقت لاحق. وكان هذا النموذج لا تقدر بثمن لدعم تحديد نقاط قابلية البكتيرية وتقديم أهداف PK / PD لتوجيه الجرعات الإنسان. هناك العديد من الامتحانPLES حيث ثبتت القدرة التنبؤية لهذا النموذج 4-7. ومع ذلك، واحدة من عيوب نموذج الإصابة في الفخذ هو عدم وجود صلة مباشرة للالتهابات الرئة. الآن هناك تركيز أكبر على مطابقة موقع الإصابة في النماذج الحيوانية إلى موقع الإصابة في البشر. وبالتالي، لدراسة المركبات لعلاج الالتهاب الرئوي، وهو مثالي للاستفادة من نموذج عدوى الرئة في الحيوانات. وقد وصفت العديد من الطرق لإحداث التهابات الرئة في الحيوانات المختبرية وتستخدم للدراسات فعالية مضادة للجراثيم بما في ذلك استنشاق الأنف، والطموح من طريق الفم والبلعوم، والتعرض الهباء الجوي، والعمليات الجراحية التلقيح داخل القصبة الهوائية 8. في كثير من الحالات، والحيوانات (وخاصة الفئران) يجب أولا المقدمة العدلات من أجل تحقيق التهاب في الرئتين قوية. وعلى الرغم من هذه الدولة المناعة بشدة، وعدد من مسببات الأمراض البكتيرية والسلالات التي تنتج العدوى قابلة للحياة في القوارض هومحدودة. على سبيل المثال، يمكن أن يكون من الصعب تحديد بنجاح المستدمية النزلية أو راكدة بومانية في نماذج الالتهاب الرئوي 9 و 10، وحتى أكثر ضراوة مسببات الأمراض، مثل المكورات العقدية الرئوية، الزائفة الزنجارية والكلبسيلة الرئوية، يمكن أن تشكل تحديات 11-13. في عام 1991، وصف سميث نموذجا التهاب في الرئتين في فئران فطيم مناعيا التي تأسست العدوى عن طريق غرس البكتيريا مباشرة إلى الرئتين عبر بسيطة غير الجراحية طريقة التنبيب داخل الرغامى 14. التوت وآخرون. في وقت لاحق تعديل طريقة لاصابة الفئران 15. باستخدام اللقاح على أساس أجار، هذه التقنية التلقيح تحرض التهابات الرئة قوية في كل من الفئران مناعيا والفئران مع S. الرئوية، المستدمية النزلية، K. الرئوية، الزائفة الزنجارية وA. baumannii. فمن قابلة للمجموعة واسعة من العزلات، بما في ذلك إيواء مختلف مقاومةمحددات تعصب وتلك التي لا تنتج العدوى قابلة للحياة عبر طرق التلقيح أخرى. كما يسمح فعالية يتم تحديدها في الحيوانات مناعيا، وهو الشرط الذي هو أكثر أهمية من نموذج الفأر العدلات التقليدية للسكان المريض التي ليست المناعة بشدة. الاتساق والموثوقية من هذا النموذج عبر مسببات الأمراض وعزلة متعددة يجعلها مناسبة تماما للدراسات فعالية مضادة للجراثيم.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>TSA II Trypticase Soy Agar with 5% Sheep Blood</td>
			<td>Becton Dickinson and Company</td>
			<td>4321261</td>
			<td></td>
		</tr>
		<tr>
			<td>Chocolate II Agar</td>
			<td>Becton Dickinson and Company</td>
			<td>4321267</td>
			<td></td>
		</tr>
		<tr>
			<td>BBL Brain Heart Infusion Broth Modified</td>
			<td>Becton Dickinson and Company</td>
			<td>299070</td>
			<td></td>
		</tr>
		<tr>
			<td>BBL Trypticase Soy Broth with 20% Glycerol</td>
			<td>Becton Dickinson and Company</td>
			<td>297808</td>
			<td>storage media for frozen stock</td>
		</tr>
		<tr>
			<td>Inoculating loop</td>
			<td>Nunc</td>
			<td>251586</td>
			<td></td>
		</tr>
		<tr>
			<td>0.9% Sodium Chloride, USP</td>
			<td>Baxter Healthcare Corporation</td>
			<td>NDC 0338-0048-04</td>
			<td></td>
		</tr>
		<tr>
			<td>Difco Nutrient Agar</td>
			<td>Becton Dickinson and Company</td>
			<td>213000</td>
			<td></td>
		</tr>
		<tr>
			<td>30 ml free standing centrifuge tube with cap</td>
			<td>EverGreen Scientific</td>
			<td>222-3530-G80</td>
			<td></td>
		</tr>
		<tr>
			<td>50 ml Polypropylene Conical Tube 30 x115mm</td>
			<td>Falcon, a Corning Brand</td>
			<td>352070</td>
			<td></td>
		</tr>
		<tr>
			<td>14 ml Polypropylene Round-Bottom Tube 17 x 100mm</td>
			<td>Falcon, a Corning Brand</td>
			<td>352059</td>
			<td></td>
		</tr>
		<tr>
			<td>Guide Tool</td>
			<td>Intek Services Ltd</td>
			<td>GT01</td>
			<td>custom-made metal cannula for rats</td>
		</tr>
		<tr>
			<td>90&#39; Portex tubing</td>
			<td>SAI</td>
			<td>POR-080-100</td>
			<td>plastic cannula for rats</td>
		</tr>
		<tr>
			<td>1 ml TB syringe slip tip</td>
			<td>Becton Dickinson and Company</td>
			<td>309659</td>
			<td></td>
		</tr>
		<tr>
			<td>PrecisionGlide Needle 25G x 5/8</td>
			<td>Becton Dickinson and Company</td>
			<td>305122</td>
			<td></td>
		</tr>
		<tr>
			<td>Animal feeding needle - straight 20x3&#34; 2-1/4</td>
			<td>Popper and Sons, Inc</td>
			<td>7903</td>
			<td>used to create metal cannula for mice</td>
		</tr>
		<tr>
			<td>Intramedic polyethylene tubing</td>
			<td>Clay Adams brand - Becton Dickinson and Company</td>
			<td>427401</td>
			<td>plastic cannula for mouse&#160;</td>
		</tr>
		<tr>
			<td>75 TN 5.0ul syringe 26s/2&#34;/2</td>
			<td>Hamilton</td>
			<td>87930</td>
			<td>HPLC glass injection syringe</td>
		</tr>
		<tr>
			<td>Pyrex tube, culture 25x150 screwcap with rubber liner</td>
			<td>Corning</td>
			<td>9825-25</td>
			<td>to autoclave/store metal cannulae</td>
		</tr>
		<tr>
			<td>70% isopropyl alcohol</td>
			<td>Vi-Jon (Swan)</td>
			<td>NDC 0869-0810-43</td>
			<td></td>
		</tr>
		<tr>
			<td>Isoflurane, USP</td>
			<td>Piramal Healthcare</td>
			<td>NDC 66794-013-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Stomacher&#160;</td>
			<td>Seward</td>
			<td>Stomacher80</td>
			<td></td>
		</tr>
		<tr>
			<td>Stomacher 80 classic bags</td>
			<td>Seward</td>
			<td>BA6040</td>
			<td></td>
		</tr>
		<tr>
			<td>Assay plate, 96 well, round bottom</td>
			<td>Costar, Corning Inc</td>
			<td>3795</td>
			<td>optional, for serial diluting</td>
		</tr>
		<tr>
			<td>Blood Omni Plates</td>
			<td>Hardy</td>
			<td>#A127</td>
			<td>optional, rectangular blood plates</td>
		</tr>
	</tbody>
</table>

a robust pneumonia model in immunocompetent rodents to evaluate antibacterial efficacy against s. pneumoniae, h. influenzae, k. pneumoniae, p. aeruginosa or a. baumannii

فلا بد من إثبات فعالية من العلاجات المضادة للبكتيريا مرشح في النماذج الحيوانية للعدوى كجزء من عملية الاكتشاف والتطوير، ويفضل أن يكون ذلك في النماذج التي تحاكي إشارة السريري المقصود. ووصف طريقة لإحداث التهابات الرئة قوية في الفئران مناعيا والفئران والذي يسمح لتقييم العلاجات في نموذج من التهاب رئوي خطير تسببه S. الرئوية، المستدمية النزلية، الزائفة الزنجارية، K. الرئوية أو A. baumannii. يتم تخدير الحيوانات، ويترسب على اللقاح استنادا أجار في عمق الرئة عن طريق إدخال أنبوب داخل الرغامى غير الجراحية. العدوى الناجمة يتسق، قابلة للتكرار، ومستقر لمدة 48 ساعة على الأقل، ويصل إلى 96 ساعة بالنسبة لمعظم العزلات. وقد أظهرت الدراسات التي أجريت على مضادات الجراثيم تسويقها علاقة جيدة بين في نجاعة فيفو وفي قابلية المختبر، والتوافق بين الدوائية الأهداف / الدوائية تحديدفي هذا النموذج، والأهداف المقبولة سريريا وقد لوحظ. على الرغم من وجود استثمار الوقت الأولي عندما تعلم هذه التقنية، فإنه لا يمكن أن يؤديها بسرعة وكفاءة مرة واحدة ويتم تحقيق الكفاءة. وتشمل الفوائد من طراز إلغاء شرط العدلات، وزيادة متانة والتكاثر، والقدرة على دراسة المزيد من مسببات الأمراض ويعزل، وتحسين المرونة في تصميم الدراسة وإنشاء العدوى تحديا في مضيف مناعيا.

Watch this Scientific Journal Video about A Robust Pneumonia Model in Immunocompetent Rodents to Evaluate Antibacterial Efficacy against S. pneumoniae, H. influenzae, K. pneumoniae, P. aeruginosa or  

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.

نموذج الالتهاب الرئوي القوي في مناعيا القوارض لتقييم مضاد للبكتيريا فعالية ضد<i&gt; S. الرئوية</i&gt;،<i&gt; H. النزلية</i&gt;،<i&gt; K. الرئوية</i&gt;،<em&gt; P. الزنجارية</em&gt; أو<em&gt; A. baumannii</em

Efficacy of candidate antibacterial treatments must be demonstrated in animal models of infection as part of the discovery and development process, ...

a-robust-pneumonia-model-immunocompetent-rodents-to-evaluate

Universidad San Sebastian

Research

JoVE Journal

Immunology and Infection

A Robust Pneumonia Model in Immunocompetent Rodents to Evaluate Antibacterial Efficacy against S. pneumoniae, H. influenzae, K. pneumoniae, P. aeruginosa or A. baumannii

14.5K Views.  GlaxoSmithKline. 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.

Video: A Robust Pneumonia Model in Immunocompetent Rodents to Evaluate Antibacterial Efficacy against S. pneumoniae, H. influenzae, K. pneumoniae, P. aeruginosa or  A. baumannii

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.

باستخدام التصوير Bioluminescent بالتحقيق في التآزر بين العقدية الرئوية والأنفلونزا من الفيروسات في الفئران الرضع

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

Live Cell Imaging of Bacillus subtilis and Streptococcus pneumoniae using Automated Time-lapse Microscopy

During the last few years scientists became increasingly aware that average data obtained from microbial population based experiments are not representative of the behavior, status or phenotype of single cells. Due to this new insight the number of single cell studies rises continuously (for recent reviews see 1,2,3). However, many of the single cell techniques applied do not allow monitoring the development and behavior of one specific single cell in time (e.g. flow cytometry or standard microscopy).
Here, we provide a detailed description of a microscopy method used in several recent studies 4, 5, 6, 7, which allows following and recording (fluorescence of) individual bacterial cells of Bacillus subtilis and Streptococcus pneumoniae through growth and division for many generations. The resulting movies can be used to construct phylogenetic lineage trees by tracing back the history of a single cell within a population that originated from one common ancestor. This time-lapse fluorescence microscopy method cannot only be used to investigate growth, division and differentiation of individual cells, but also to analyze the effect of cell history and ancestry on specific cellular behavior. Furthermore, time-lapse microscopy is ideally suited to examine gene expression dynamics and protein localization during the bacterial cell cycle. The method explains how to prepare the bacterial cells and construct the microscope slide to enable the outgrowth of single cells into a microcolony. In short, single cells are spotted on a semi-solid surface consisting of growth medium supplemented with agarose on which they grow and divide under a fluorescence microscope within a temperature controlled environmental chamber. Images are captured at specific intervals and are later analyzed using the open source software ImageJ.

Live Cell Imaging of Bacillus subtilis and Streptococcus pneumoniae using Automated Time-lapse Microscopy

This protocol provides a step-by-step procedure to monitor single cell behavior of different bacteria in time using automated fluorescence time-lapse microscopy. Furthermore, we provide guidelines how to analyze the microscopy images.

تعيش خلية من التصوير<emالعصوية الرقيقة> و العقدية الرئوية الآلي باستخدام الميكروسكوب مرور الزمن

During the last few years scientists became increasingly aware that average data obtained from microbial population based experiments are not ...

Use of Artificial Sputum Medium to Test Antibiotic Efficacy Against Pseudomonas aeruginosa in Conditions More Relevant to the Cystic Fibrosis Lung

There is growing concern about the relevance of in vitro antimicrobial susceptibility tests when applied to isolates of P. aeruginosa from cystic fibrosis (CF) patients. Existing methods rely on single or a few isolates grown aerobically and planktonically. Predetermined cut-offs are used to define whether the bacteria are sensitive or resistant to any given antibiotic1. However, during chronic lung infections in CF, P. aeruginosa populations exist in biofilms and there is evidence that the environment is largely microaerophilic2. The stark difference in conditions between bacteria in the lung and those during diagnostic testing has called into question the reliability and even relevance of these tests3.
 Artificial sputum medium (ASM) is a culture medium containing the components of CF patient sputum, including amino acids, mucin and free DNA. P. aeruginosa growth in ASM mimics growth during CF infections, with the formation of self-aggregating biofilm structures and population divergence4,5,6. The aim of this study was to develop a microtitre-plate assay to study antimicrobial susceptibility of P. aeruginosa based on growth in ASM, which is applicable to both microaerophilic and aerobic conditions.
 An ASM assay was developed in a microtitre plate format. P. aeruginosa biofilms were allowed to develop for 3 days prior to incubation with antimicrobial agents at different concentrations for 24 hours. After biofilm disruption, cell viability was measured by staining with resazurin. This assay was used to ascertain the sessile cell minimum inhibitory concentration (SMIC) of tobramycin for 15 different P. aeruginosa isolates under aerobic and microaerophilic conditions and SMIC values were compared to those obtained with standard broth growth. Whilst there was some evidence for increased MIC values for isolates grown in ASM when compared to their planktonic counterparts, the biggest differences were found with bacteria tested in microaerophilic conditions, which showed a much increased resistance up to a &gt;128 fold, towards tobramycin in the ASM system when compared to assays carried out in aerobic conditions.
 The lack of association between current susceptibility testing methods and clinical outcome has questioned the validity of current methods3. Several in vitro models have been used previously to study P. aeruginosa biofilms7, 8. However, these methods rely on surface attached biofilms, whereas the ASM biofilms resemble those observed in the CF lung9 . In addition, reduced oxygen concentration in the mucus has been shown to alter the behavior of P. aeruginosa2 and affect antibiotic susceptibility10. Therefore using ASM under microaerophilic conditions may provide a more realistic environment in which to study antimicrobial susceptibility.

Use of Artificial Sputum Medium to Test Antibiotic Efficacy Against Pseudomonas aeruginosa in Conditions More Relevant to the Cystic Fibrosis Lung

Current diagnostic antimicrobial susceptibility testing relies on the planktonic growth of isolates in nutrient rich, aerobic conditions. Here, we employ an alternative artificial sputum medium to study antimicrobial susceptibility of Pseudomonas aeruginosa biofilms under both aerobic and microaerophilic conditions more representative of the cystic fibrosis lung.

استخدام متوسط ​​البلغم الاصطناعي لاختبار فعالية المضادات الحيوية لمكافحة<em&gt; الزائفة الزنجارية</em&gt; في شروط أكثر ملاءمة لالرئة التليف الكيسي

There  is growing concern about the relevance of in vitro antimicrobial  susceptibility tests when applied to isolates of P. aeruginosa from  cystic ...

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.

توصيف الاستجابات الالتهابية خلال الاستعمار داخل الشرج مع الالتهاب الرئوي العقدي

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.

رصد التغيرات في غشاء التقاطب، غشاء النزاهة، وبين الخلايا ايون التركيزات في<em&gt; العقدية الرئوية</em&gt; استخدام الأصباغ نيون

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 Using the Quellung Reaction

There are over 90 different capsular serotypes of Streptococcus pneumoniae (the pneumococcus). As well as being a tool for understanding pneumococcal epidemiology, capsular serotyping can provide useful information for vaccine efficacy and impact studies.&#160;The Quellung reaction is the gold standard method for pneumococcal capsular serotyping. The method involves testing a pneumococcal cell suspension with pooled and specific antisera directed against the capsular polysaccharide. The antigen-antibody reactions are observed microscopically. The protocol has three main steps: 1) preparation of a bacterial cell suspension, 2) mixing of cells and antisera on a glass slide, and 3) reading the Quellung reaction using a microscope. The Quellung reaction is reasonably simple to perform and can be applied wherever a suitable microscope and antisera are available.

Capsular Serotyping of Streptococcus pneumoniae Using the Quellung Reaction

The Quellung reaction is the gold standard technique for serotyping Streptococcus pneumoniae. This technique utilizes a microscope and specific pneumococcal antisera and is commonly used in reference and research laboratories worldwide.

المحفظة المصلي من<em&gt; العقدية الرئوية</em&gt; باستخدام تفاعل الانتباج

There are over 90 different capsular serotypes of Streptococcus pneumoniae (the pneumococcus). As well as being a tool for understanding pneumococcal ...

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.

المحفظة المصلي من<em&gt; العقدية الرئوية</em&gt; من جانب تراص اللاتكس

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

Image-based Flow Cytometry Technique to Evaluate Changes in Granulocyte Function In Vitro

Granulocytes play a key role in the body&#8217;s innate immune response to bacterial and viral infections. While methods exist to measure granulocyte function, in general these are limited in terms of the information they can provide. For example, most existing assays merely provide a percentage of how many granulocytes are activated following a single, fixed length incubation. Complicating matters, most assays focus on only one aspect of function due to limitations in detection technology. This report demonstrates a technique for simultaneous measurement of granulocyte phagocytosis of bacteria and oxidative burst. By measuring both of these functions at the same time, three unique phenotypes of activated granulocytes were identified: 1) Low Activation (minimal phagocytosis, no oxidative burst), 2) Moderate Activation (moderate phagocytosis, some oxidative burst, but no co-localization of the two functional events), and 3) High Activation (high phagocytosis, high oxidative burst, co-localization of phagocytosis and oxidative burst). A fourth population that consisted of inactivated granulocytes was also identified. Using assay incubations of 10, 20, and 40-min the effect of assay incubation duration on the redistribution of activated granulocyte phenotypes was assessed. A fourth incubation was completed on ice as a control. By using serial time incubations, the assay may be able to able to detect how a treatment spatially affects granulocyte function. All samples were measured using an image-based flow cytometer equipped with a quantitative imaging (QI) option, autosampler, and multiple lasers (488, 642, and 785 nm).

Image-based Flow Cytometry Technique to Evaluate Changes in Granulocyte Function In Vitro

This method demonstrates a technique for assessing granulocyte function by simultaneously measuring phagocytosis of bacteria and oxidative burst. Image-based flow cytometry allowed for the identification of three distinct subsets of activated granulocytes that all differed in their relative functional capacity.

الصورة القائمة على التدفق الخلوي وتقنيات لتقييم التغيرات في الحبيبية وظيفة<em&gt; في المختبر</em

Granulocytes play a key role in the body&#8217;s innate immune response to bacterial and viral infections. While methods exist to measure granulocyte ...

Intra-tracheal Administration of Haemophilus influenzae in Mouse Models to Study Airway Inflammation

Here, we describe a detailed procedure to efficiently and directly deliver Haemophilus influenzae into the lower respiratory tracts of mice. We demonstrate the procedure for preparing H. influenzae inoculum, intra-tracheal instillation of H. influenzae into the lung, collection of broncho-alveolar lavage fluid (BALF), analysis of immune cells in the BALF, and RNA isolation for differential gene expression analysis. This procedure can be used to study the lung inflammatory response to any bacteria, virus or fungi. Direct tracheal instillation is mostly preferred over intranasal or aerosol inhalation procedures because it more efficiently delivers the bacterial inoculum into the lower respiratory tract with less ambiguity.

Intra-tracheal Administration of Haemophilus influenzae in Mouse Models to Study Airway Inflammation

We demonstrate the procedure for intra-tracheal inoculation of Haemophilus influenzae into the lower respiratory tracts of mice. This is a very useful tool to study signaling pathways that regulate airway inflammation in mouse models.

الإدارة داخل القصبة الهوائية لل<em&gt; المستدمية النزلية</em&gt; في نماذج الماوس لدراسة التهاب الشعب الهوائية

Here, we describe a detailed procedure to efficiently and directly deliver Haemophilus influenzae into the lower respiratory tracts of mice. We ...

A High-throughput Compatible Assay to Evaluate Drug Efficacy against Macrophage Passaged Mycobacterium tuberculosis

The early drug development process for anti-tuberculosis drugs is hindered by the inefficient translation of compounds with in vitro activity to effectiveness in the clinical setting. This is likely due to a lack of consideration for the physiologically relevant cellular penetration barriers that exist in the infected host. We recently established an alternative infection model that generates large macrophage aggregate structures containing densely packed M. tuberculosis (Mtb) at its core, which was suitable for drug susceptibility testing. This infection model is inexpensive, rapid, and most importantly BSL-2 compatible. Here, we describe the experimental procedures to generate Mtb/macrophage aggregate structures that would produce macrophage-passaged Mtb for drug susceptibility testing. In particular, we demonstrate how this infection system could be directly adapted to the 96-well plate format showing throughput capability for the screening of compound libraries against Mtb. Overall, this assay is a valuable addition to the currently available Mtb drug discovery toolbox due to its simplicity, cost effectiveness, and scalability.

A High-throughput Compatible Assay to Evaluate Drug Efficacy against Macrophage Passaged Mycobacterium tuberculosis

New models and assays that would improve the early drug development process for next-generation anti-tuberculosis drugs are highly desirable. Here, we describe a quick, inexpensive, and BSL-2 compatible assay to evaluate drug efficacy against Mycobacterium tuberculosis that can be easily adapted for high-throughput screening.