Name: 视频: 评估细菌来源的抗菌化合物效果的延迟生长抑制试验 - 概念
Uploaded: 2024-02-29T13:13:40.000+00:00
Duration: 1 min 16 s
Description: 168 次观看. 来源:Moran， J. C. et al.， 延迟生长抑制测定，用于量化细菌衍生的抗菌剂对竞争的影响。J. Vis. Exp.（2016 年） 该视频展示了延迟生长抑制测定，用于研究一种细菌产生的抗菌化合物如何影响另一种竞争细菌菌株。

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The following method is adapted&#160;to test competitor strains with inhibitor-producing strains.
NOTE: This protocol involves aerosol generation with bacterial cultures. This can only be performed in a contained space with the implementation of locally approved safety considerations. Spraying of the culture onto the agar plates within a level 2 safety cabinet will minimize contamination of the agar plates and the surrounding environment. This will also protect the researcher from aerosol inhalation of the sprayed bacteria, this is a major concern if the researcher is using containment level 2 organisms. Appropriate personal protective equipment must be worn. The area surrounding the petri dish is best covered with disposable absorbent tissue. Post-spray decontamination using UV light is advised.
1. Preparing the Agar Plates
<ol>
	<li>Prepare brain heart infusion (BHI) agar in accordance with the manufacturer&#39;s instructions.</li>
	<li>Autoclave the BHI agar at 121 &#176;C, 15 Pound-force per square inch (psi) for 20 min, or according to the autoclave manufacturer&#39;s recommendations, to sterilize.</li>
	<li>Pour 15 ml aliquots of sterilized BHI agar into sterile petri dishes and allow it to set. 
	NOTE: Ensure every agar plate to be used within the assay originates from the same stock of agar and that there is the same amount of agar in each plate. This limits the variability in available nutrients and inhibitor diffusion between plates allowing comparability.</li>
</ol>
2. Preparing Sterile Broth
<ol>
	<li>Prepare BHI broth in accordance with the manufacturer&#39;s instructions.</li>
	<li>Add 10 ml aliquots of BHI to 28 ml glass universal bottles.</li>
	<li>Autoclave the BHI broth at 121 &#176;C, 15 psi for 20 min, or according to the autoclave manufacturer&#39;s recommendations, to sterilize.&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; 
	NOTE: Ensure all broth used in the assay originates from the same batch of broth autoclaved at the same time to limit variation in nutrients which could cause variation in results.</li>
</ol>
3. Preparing Sterile Vaporizer Bottle(s)
<ol>
	<li>Dismantle the vaporizer bottle.</li>
	<li>Spray 5% surface active cleaning agent through the vaporizer to eliminate internal contaminants, and soak the vaporizer bottle, lid, and vaporizer in the 5% surface active cleaning agent overnight.&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; 
	NOTE: Gloves and appropriate personal protective equipment should be used when handling surface active cleaning agents.</li>
	<li>Dry the vaporizer, lid, and bottles under a sterile air flow hood.</li>
	<li>Fill the bottle with 70% ethanol, reattach the vaporizer, and spray some of the ethanol through the vaporizer.</li>
	<li>Put the lid on the vaporizer and store the ethanol inside until use.</li>
	<li>Immediately prior to use, aseptically rinse the bottle with sterile BHI broth and spray the broth through the vaporizer.&#160;&#160; 
	NOTE: This prevents the ethanol from affecting the inoculum added to the bottle in section 6.3.</li>
</ol>
4. Preparing the Bacterial Overnight Culture(s)
<ol>
	<li>Streak the competitor and inhibitor strains onto a sterile BHI agar plate and incubate at 37 &#176;C aerobically for approximately 18 hr (overnight). 
	NOTE: This plate produces a stock of bacteria that can be used for multiple replicates within the same week when stored at 4 &#176;C.</li>
	<li>Inoculate 10 ml of sterile BHI broth with a single colony of the required competitor bacterial strain within a 28 ml universal bottle.</li>
	<li>Incubate the universal bottle at 37 &#176;C aerobically overnight by shaking at 250 rpm in an orbital shaker.</li>
</ol>
5. Spot Culture of the Inhibitor-producing Isolate
<ol>
	<li>Prepare the overnight culture(s) as described in section 4.</li>
	<li>Ensure agar plates are completely dry, with no condensation on the lid, prior to inoculation (e.g.&#160;by incubating the plate for 60 min at 37 &#176;C); this prevents the inoculum from spreading across the plate from the inoculation site.</li>
	<li>Pipette 25 &#181;l of the overnight culture onto the center of the agar plates. Avoid completely depressing the pipette plunger to prevent air bubbles that spray culture across the agar.</li>
	<li>Allow the culture to dry at room temperature to limit the spread of the culture. Incubate the agar plates at 37 &#176;C aerobically overnight.</li>
</ol>
6. Preparing the Spray Inoculum of the Test Competitor Strain(s)
<ol>
	<li>Prepare the overnight culture as described in section 4. Dilute the overnight culture 10-fold in BHI broth to yield approximately 4 x 106 &#160;colony forming unit (CFU) ml-1 and a suspension of OD600&#160;0.3 &#177;0.05. 
	NOTE: A 10-fold dilution will not give 4 x 106&#160;CFU ml-1&#160;for all bacterial species, calculate the necessary dilution factor by spread plating serial dilutions between 1 x 10-1-1 x 10-6.</li>
	<li>Pour the diluted culture into a sterile plastic perfume vaporizer bottle. 
	NOTE: The total volume must be more than is necessary for the number of agar plates being sprayed. Approximately 250 &#181;l per plate is suitable for a 9 cm diameter Petri dish.</li>
</ol>
7. Deferred Growth Inhibition Assay
<ol>
	<li>Spray the culture through each of the vaporizer bottles to ensure that the culture is loaded in the spray mechanism prior to spraying onto the agar.</li>
	<li>From a distance of approximately 15 cm above the agar, spray approximately 250 &#181;l of diluted culture (3 sprays of a typical 50 ml vaporizer) over the entire agar surface. Incubate the agar plates at 37 &#176;C aerobically overnight. Repeat for other plates with the same number of sprays.</li>
</ol>
8. Interpreting Deferred Growth Inhibition Assay Results
<ol>
	<li>Measure the growth inhibition zone of the sprayed competitor strain (Figure 1A-C, &#34;X&#34; to &#34;X&#34;) in millimeters, subtracting the diameter of the central spot of the inhibitor-producer (Figure 1A-C, &#34;Y&#34; to &#34;Y&#34;).&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; 
	NOTE:&#160;Figure 1E&#160;shows how this data could be presented</li>
	<li>Record the clarity score for zones of inhibition.&#160; 
	NOTE: This indicates the degree of inhibition observed. Examples are shown in&#160;Figure 1A-D, strains that can be used for standardization are suggested in the representative results section. The following scale can be used for clarity scoring: a clarity score of 0 to describe a zone of complete inhibition of the competitor strain&#39;s growth. A score of 1 describes a zone of almost complete inhibition of growth, with only individual colonies present in the zone of clearing. A score of 2 describes a visible zone of reduced growth, growth within this zone is confluent or near confluent but reduced in density by &#62;50 %. A score of 3 describes only minimal growth reduction, growth is confluent and reduced by less than 50 %, and a zone is still visible and measurable. A score of 4 describes no visible inhibition of growth. A score of 5 describes that the growth of the competitor strain was increased in the proximity of the test inhibitor-producing strain. See&#160;Figure 1&#160;for examples. These scores are subjective to the experimenter.</li>
</ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Brain heart infusion broth</td>
			<td>Lab M</td>
			<td>lab049</td>
			<td>Rich general purpose media</td>
		</tr>
		<tr>
			<td>Agar-agar</td>
			<td>Merck Milipore</td>
			<td>101614</td>
			<td></td>
		</tr>
		<tr>
			<td>Petri dishes</td>
			<td>Sarstedt</td>
			<td>82.1473.001</td>
			<td></td>
		</tr>
		<tr>
			<td>Plastic perfume vaporizer</td>
			<td>not known</td>
			<td>-</td>
			<td>10 cm high with 3.5 cm diameter clear plastic spray bottle with lid, purchased from the travel section of a supermarket</td>
		</tr>
		<tr>
			<td>Universal bottles</td>
			<td></td>
			<td>no longer in production, alternative SLS BOT5006</td>
			<td>28 ml glass cylindrical bottle (approx 280 mm diameter, 850 mm height), flat bottomed with plastic screw cap</td>
		</tr>
		<tr>
			<td>Decon 90</td>
			<td>Decon</td>
			<td></td>
			<td>surface active cleaning agent</td>
		</tr>
	</tbody>
</table>

a deferred growth inhibition assay to evaluate the effect of bacteria-derived antimicrobial compounds

Source: Moran, J. C. et al., <a href="https://app.jove.com/v/54437">Deferred Growth Inhibition Assay to Quantify the Effect of Bacteria-derived Antimicrobials on Competition.</a> J. Vis. Exp.&#160;(2016)
This video showcases the deferred growth inhibition assay, used to investigate how antimicrobial compounds produced by one type of bacteria affect another competing bacterial strain.

<img alt="Figure 1" src="/files/ftp_upload/21993/21993_Figure1.jpg" /> 
Figure 1:&#160;The range of qualitative and quantitative results that can be obtained from the deferred growth inhibition assay.&#160;Results for the growth inhibition can range from (A) complete inhibition (score of 0), (B) partial inhibition (score of 1), (C) partial inhibition (score of 2) to (D) no inhibition...

A Deferred Growth Inhibition Assay to Evaluate the Effect of Bacteria-Derived Antimicrobial Compounds

The following method is adapted&#160;to test competitor strains with inhibitor-producing strains.
NOTE: This protocol involves aerosol generation with ...

Begin with a nutrient-containing agar plate and place a drop of an overnight culture of inhibitor-producing primary bacteria at the center.
Allow the culture to dry, ensuring minimum spreading of bacteria, and incubate.
Individual bacteria form distinct colonies and secrete antimicrobial compounds &#8212; inhibitor molecules that diffuse through the agar to a certain distance.
Spray a diluted culture of a competitor bacterial strain evenly onto the agar surface that contains colonies of primary bacteria.
During incubation, the antimicrobial compounds interact with the competitor bacteria and disrupt their essential cellular processes.
This results in the death of competitor bacteria in that specific area, causing a significant decline in bacterial growth.
In the area without antimicrobial compounds, competitor bacteria utilize the nutrients and form distinct colonies.
This creates a differential growth pattern with a distinct zone of inhibition. Determine the diameter of this zone and zone clarity to evaluate the effect of bacteria-derived antimicrobial compounds.

a-deferred-growth-inhibition-assay-to-evaluate-effect-bacteria

Universidad San Sebastian

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Immunodiagnostics

Specialized Assays

168 次观看. 来源:Moran， J. C. et al.， 延迟生长抑制测定，用于量化细菌衍生的抗菌剂对竞争的影响。J. Vis. Exp.（2016 年） 该视频展示了延迟生长抑制测定，用于研究一种细菌产生的抗菌化合物如何影响另一种竞争细菌菌株。 

视频: 评估细菌来源的抗菌化合物效果的延迟生长抑制试验 - 概念

Inhibition of Aspergillus flavus Growth and Aflatoxin Production in Transgenic Maize Expressing the &#945;-amylase Inhibitor from Lablab purpureus L.

Aflatoxin contamination in food and feed crops is a major challenge worldwide. Aflatoxins, produced by the fungus Aspergillus flavus (A. flavus) are potent carcinogens that substantially reduce crop value in maize and other oil rich crops like peanut besides posing serious threat to human and animal health. Different approaches, including traditional breeding, transgenic expression of resistance associated proteins, and RNA interference (RNAi)-based host-induced gene silencing of critical A. flavus gene targets, are being evaluated to increase aflatoxin resistance in susceptible crops. Past studies have shown an important role of &#945;-amylase in A. flavus pathogenesis and aflatoxin production, suggesting this gene/enzyme is a potential target to reduce both A. flavus growth and aflatoxin production. In this regard, the current study was undertaken to evaluate heterologous expression (under control of the constitutive CaMV 35S promoter) of a Lablab purpureus L. &#945;-amylase inhibitor-like protein (AILP) in maize against A. flavus. AILP is a 36-kDa protein, which is a competitive inhibitor of A. flavus &#945;-amylase enzyme and belongs to the lectin&#8211;arcelin&#8211;&#945;-amylase inhibitor protein family in common bean. In vitro studies prior to the current work had demonstrated the role of AILP in inhibition of A. flavus &#945;-amylase activity and fungal growth. Fungal growth and aflatoxin production in mature kernels were monitored in real time using a GFP-expressing A. flavus strain. This kernel screening assay (KSA) is very simple to set up and provides reliable and reproducible data on infection and the extent of spread that could be quantified for evaluation of germplasm and transgenic lines. The fluorescence from the GFP strain is closely correlated to fungal growth and, by extension, it is well-correlated to aflatoxin values.&#160; The goal of the current work was to implement this previous knowledge in a commercially important crop like maize to increase aflatoxin resistance. Our results show a 35%&#8211;72% reduction in A. flavus growth in AILP-expressing transgenic maize kernels which, in turn, translated into a 62%&#8211;88% reduction in aflatoxin levels.

Inhibition of Aspergillus flavus Growth and Aflatoxin Production in Transgenic Maize Expressing the &#945;-amylase Inhibitor from Lablab purpureus L.

Here we present a protocol to analyze Aspergillus flavus growth and aflatoxin production in maize kernels expressing an antifungal protein.&#160; Using a GFP-expressing A. flavus strain we monitored the infection and spread of the fungus in mature kernels in real time. The assay is rapid, reliable, and reproducible.

转基因玉米中黄曲霉生长和黄曲霉毒素的产生表达表达.

Aflatoxin contamination in food and feed crops is a major challenge worldwide. Aflatoxins, produced by the fungus Aspergillus flavus (A. flavus) are ...

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环境科学

Use of the Soft-agar Overlay Technique to Screen for Bacterially Produced Inhibitory Compounds

The soft-agar overlay technique was originally developed over 70 years ago and has been widely used in several areas of microbiological research, including work with bacteriophages and bacteriocins, proteinaceous antibacterial agents. This approach is relatively inexpensive, with minimal resource requirements. This technique consists of spotting supernatant from a donor strain (potentially harboring a toxic compound(s)) onto a solidified soft agar overlay that is seeded with a bacterial test strain (potentially sensitive to the toxic compound(s)). We utilized this technique to screen a library of Pseudomonas syringae strains for intraspecific killing. By combining this approach with a precipitation step and targeted gene deletions, multiple toxic compounds produced by the same strain can be differentiated. The two antagonistic agents commonly recovered using this technique are bacteriophages and bacteriocins. These two agents can be differentiated using two simple additional tests. Performing a serial dilution on a supernatant containing bacteriophage will result in individual plaques becoming less in number with greater dilution, whereas serial dilution of a supernatant containing bacteriocin will result a clearing zone that becomes uniformly more turbid with greater dilution. Additionally, a bacteriophage will produce a clearing zone when spotted onto a fresh soft agar overlay seeded with the same strain, whereas a bacteriocin will not produce a clearing zone when transferred to a fresh soft agar lawn, owing to the dilution of the bacteriocin.

We describe a simple method for screening bacterial cultures for the production of compounds inhibitory towards other bacteria.

软琼脂覆盖技术使用筛选细菌产生的抑制化合物

The soft-agar overlay technique was originally developed over 70 years ago and has been widely used in several areas of microbiological research, ...

免疫与感染

Coincubation Assay for Quantifying Competitive Interactions between Vibrio fischeri Isolates

This manuscript describes a culture-based, coincubation assay for detecting and characterizing competitive interactions between two bacterial populations. This method employs stable plasmids that allow each population to be differentially tagged with distinct antibiotic resistance capabilities and fluorescent proteins for selection and visual discrimination of each population, respectively. Here, we describe the preparation and coincubation of competing Vibrio fischeri strains, fluorescence microscopy imaging, and quantitative data analysis. This approach is simple, yields quick results, and can be used to determine whether one population kills or inhibits the growth of another population, and whether competition is mediated through a diffusible molecule or requires direct cell-cell contact. Because each bacterial population expresses a different fluorescent protein, the assay permits the spatial discrimination of competing populations within a mixed colony. Although the described methods are performed with the symbiotic bacterium V. fischeri using conditions optimized for this species, the protocol can be adapted for most culturable bacterial isolates.

Coincubation Assay for Quantifying Competitive Interactions between Vibrio fischeri Isolates

Bacteria encode diverse mechanisms for engaging in interbacterial competition. Here, we present a culture-based protocol for characterizing competitive interactions between bacterial isolates and how they impact the spatial structure of a mixed population.

用于量化Vibrio Fischeri分离物之间竞争交互的联合孵化测定

This manuscript describes a culture-based, coincubation assay for detecting and characterizing competitive interactions between two bacterial populations. ...

A Deferred Growth Inhibition Assay to Evaluate the Effect of Bacteria-Derived Antimicrobial Compounds

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A Deferred Growth Inhibition Assay to Evaluate the Effect of Bacteria-Derived Antimicrobial Compounds