This work was supported by the Auburn University fund FOP 101002-139294-2050.

All experimental procedures were approved by IACUC of Auburn University, Auburn, Alabama.
1. Preparation of Culture Media, Dishes and Bacterial Culture
<ol>
	<li>Inoculate 10 ml of nutrient broth in a flask with a single colony of Bacillus subtilis BSB3 culture, grown overnight on a nutrient agar plate.</li>
	<li>Make 500 ml of sporulation medium19 (per liter: Bacto Nutrient Broth, 8 g; 10% (w/v) KCl, 10 ml; 1.2% (w/v) MgSO4 x 7H2O, 10 ml; agar, 15 g) and autoclave at 121 oC for 20 min. Allow to cool to 50 oC and add the following sterile solutions:1 M Ca(NO3)2, 1 ml; 0.01 M MnCl2, 1 ml; 1 mM FeSO4, 1 ml.</li>
	<li>Cool prepared medium to 40 oC for 20 min at room temperature and pour 25 ml into each of 20 sterile Petri dishes in the sterile cabinet. Let the medium solidify overnight.</li>
	<li>Spread 0.5 ml of overnight culture of Bacillus subtilis BSB3 onto the surface of the medium in the Petri dishes. Incubate culture at 37 oC for 5 days until 90% sporulation. Reveal phase-bright spores by high resolution microscopy.</li>
	<li>Harvest bacteria by adding 1 ml of sterile phosphate buffered saline (PBS) to the plate and scraping bacteria from a surface using a sterile cell spreader. Store the suspension in the refrigerator until needed.</li>
	<li>Confirm viability of bacteria by plating 0.1 ml of the appropriate dilution (10-5 to 10-6) of a bacterial suspension on sporulation medium plates followed by incubation for 18 - 24 hr at 37 oC.</li>
	<li>Count bacterial colonies on the plates using a colony counter and calculate the titer of bacteria in the tested suspension. Prepare bacterial suspension with the final concentration 1 x 108 CFU/ml in PBS.</li>
</ol>
2. Animals
<ol>
	<li>Use 32 male Sprague&#8211;Dawley rats (250 - 300 g). Maintain animals under specific pathogen-free conditions with free access to standard pellet chow and water. Establish a mean temperature (21 &#177; 1 oC), humidity (50 &#177; 5%) and a 12 hr light-dark cycle.</li>
</ol>
3. Experimental Design
<ol>
	<li>Start treatment of animals by oral gavage11, after 2 days of acclimatization. Use syringe with the oral gavage needle. Treat 16 rats with B. subtilis BSB3 suspension (1 ml per rat) twice a day for two days (B. subtilis group). Treat 16 rats with PBS (1 ml per rat) twice a day for two days (PBS group) (Figure 1).</li>
	<li>After treatment, subdivide each group (8 rats per group): Group 1- control (PBS/no stress), Group 2- B. subtilis (B. subtilis /no stress), Group 3- stress (PBS/heat stress) and Group 4- B. subtilis + stress (B. subtilis/heat stress).</li>
	<li>Measure the rectal temperature of each rat using an electronic digital thermometer. Keep rats of groups 1 and 2 at room temperature for 25 min. Place animals of groups 3 and 4 in a climate chamber at 45 oC and relative humidity 55% for 25 min.</li>
	<li>Afterwards, measure the rectal temperature of each rat in all groups using an electronic digital thermometer. Place all animals at room temperature for 4 hr.</li>
	<li>Anesthetize rats with isoflurane (2 - 4%) in a sealable anesthesia jar and observe until the rats are deeply anesthetized (absence of response to toe pinch). Euthanize rats by rapid decapitation with a guillotine.</li>
	<li>Collect trunk blood from each rat20 (15 - 16 ml) with the apyrogenic pipettes into apyrogenic tubes to obtain serum and immediately take samples of blood (7 &#181;l from each rat) with a pipette for microscopic examination.</li>
	<li>Put tubes with the blood in a refrigerator for at least 30 min to allow clotting. Centrifuge tubes at 20 oC, 7,000 x g for 10 min and quickly remove serum with a pipette. Aliquot serum obtained from each rat into 50 &#181;l volumes and store at -20 oC until assay.</li>
</ol>
4. Surgical Procedure
<ol>
	<li>Cut/trim all fur from the abdomen and thorax of the underside of the carcass with electric shears. Place the carcass in a sterile cabinet and treat the shaved surface of the carcass with 70% alcohol.</li>
	<li>Use a sterile scalpel to create a midline incision of the abdomen. Remove liver, mesenteric lymph nodes, spleen and small intestine from each rat using sterile tweezers.</li>
</ol>
5. Analysis of Bacterial Translocation&#160;
<ol>
	<li>Place each sample of liver, mesenteric lymph nodes and spleen into pre-weighed sterile tubes and weigh. Calculate the weight of the sample as a difference between the weight of a tube with a sample and the weight of an empty tube.</li>
	<li>Add sterile PBS to the tube to obtain a 1:10 dilution (w/v) of the sample and homogenize each sample with a sterile glass tissue homogenizer to obtain a homogeneous suspension21.</li>
	<li>Make serial dilutions (10-1 to 10-4) of each sample in sterile PBS. Plate 0.1 ml of all dilutions of each tissue onto surface of MacConkey&#39;s and 5% blood agar and Brucella blood agar with hemin (0.005 g/L) and vitamin K1 (0.01 g/L) plates.</li>
	<li>Incubate MacConkey&#39;s and 5% blood agar plates aerobically and Brucella blood agar plates under anaerobic conditions at 37 oC22.</li>
	<li>Count colonies of aerobic bacteria after 24 hr, and colonies of anaerobic bacteria after 48 hr of incubation using a colony counter. Express the results as the number of colony-forming units (CFU) in a gram of tissue.</li>
</ol>
6. Histological Analysis&#160;
<ol>
	<li>Remove small intestine samples from each rat by the surgical procedure (steps 4.1 - 4.2). Fix samples in Bouin&#8217;s Fixative, embed in paraffin, prepare 6 &#181;m sections and stain sections with hematoxylin and eosin using standard procedures23.</li>
	<li>Use a high-resolution microscope system for measuring height of intestinal villi and total mucosal thickness in each sample (magnification 100X)24. Analyze 4 samples from each rat and make at least twenty measurements in each sample. Express results in micrometers.</li>
</ol>
7. Cytokine Assay
<ol>
	<li>Use commercial rat ELISA kits for IL-1&#946;; IL-6; TNF-&#945;; INF-&#947;, and IL-10 to measure levels of cytokines in serum according to manufacturer&#8217;s protocol.</li>
	<li>Generate standard curves for each set of samples assayed using standards for the relevant kit. Define the level of cytokines in each sample by comparison of experimental data with the appropriate standard curve.</li>
</ol>
8. Lipopolysaccharide Assay
<ol>
	<li>Analyze level of LPS in serum using a rat LPS ELISA kit according to manufacturer&#8217;s protocol. Estimate concentration of LPS in samples by comparison of the obtained values with the values of the standard curve.</li>
</ol>
9. High Resolution Light Microscopy of Blood
<ol>
	<li>Use the microscope system described previously25,26. Use a vibration isolation platform as a base for the microscope system and video camera and computer25 to record the live images. Calibrate test images using a Richardson slide as previously described27.</li>
	<li>Place 7 &#181;l of freshly drawn blood from each rat on a glass slide, coverslip, photograph and record ten image frames of 72 &#215; 53.3 &#181;m2 in each sample (magnification 1,700X).</li>
	<li>Measure the concentration of vesicles using software which provides high-resolution direct-view optical images in real time. Examine a minimum of 20 image frames for each experimental condition28.</li>
</ol>
10. Statistics
<ol>
	<li>Express all values as mean and standard deviation. Perform statistical analysis and graph plotting. Analyze the results with the two sample t-test, set the significance level at 0.05 to define statistical significance.</li>
</ol>

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The authors declare that they have no competing financial interests.

Exposure to high temperature results in serious health conditions29. Prevention and early detection of complications related to heat stress are of vital importance30. The presented protocol can be used to evaluate the efficacy of various approaches for the prevention of heat stress adverse effects.
Critical steps within this protocol include: the heat treatment procedure (45 oC, 25 min); the usage of apyrogenic materials during collection and p...

Different stressors affect human and animal health. Temperature is one of the most stressful factors, causing chronic, acute and even lethal illnesses1. Changes in intestinal morphology and a loss of gut barrier integrity after heat stress were documented in many cases2,3. This protective barrier is responsible for defense against translocation of gut bacteria and their toxins, in particular lipopolysaccharides (LPS), from the lumen to the internal circulation4,5. Stability of the gut microbiota significantly influences intestinal barrier function, the immune response of the host, and tolerance to stress conditions6. Thus, modulation of the intestinal microbiota can provide a novel approach for prevention of stress-related adverse effects. 
Beneficial bacteria have been used as a promising strategy to modify the gut microbiota for successful management of various clinical conditions, such as diarrhea, inflammatory bowel disease, atopic dermatitis, metabolic disorders7-10. Probiotic effects of beneficial bacteria include production of essential metabolites to support intestinal health, stimulation of the immune system, promotion of lactose tolerance, restoration of epithelial dysfunction. Probiotics were effective in prevention of the stress-related complications in vitro and in animal models11,12. 
Researchers have given more attention to Bacillus bacteria as probiotics because these bacteria support intestinal homeostasis13 and have beneficial effects on the host14. Our previous data demonstrated high efficacy of Bacillus probiotics against pathogens in vitro15,16 and in clinical trials17,18. Here, we aim to evaluate the preventive effect of B. subtilis BSB3 strain against complications after heat stress.

&#160;
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td rowspan="2">Phosphate buffered saline (PBS)</td>
			<td>Sigma-Aldrich,&#160;St. Louis, MO</td>
			<td rowspan="2">P4417</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Ethyl Alcohol&#160;</td>
			<td>Pharmco products Inc. Brookfield, CT, USA&#160;</td>
			<td>64-17-5</td>
			<td></td>
		</tr>
		<tr>
			<td>Agar</td>
			<td>VWR</td>
			<td>97064-334</td>
			<td></td>
		</tr>
		<tr>
			<td>MacConkey agar plates</td>
			<td>VWR</td>
			<td>470180-742</td>
			<td></td>
		</tr>
		<tr>
			<td>5% blood agar plates</td>
			<td>VWR</td>
			<td>89405-024</td>
			<td></td>
		</tr>
		<tr>
			<td>Brucella blood with Hemin, and Vitamin K, 
			&#160;plates</td>
			<td>VWR</td>
			<td>89405-032</td>
			<td></td>
		</tr>
		<tr>
			<td>Bouin&#8217;s Fixative</td>
			<td>Electron Microscopy Sciences, Hatfield, PA, USA</td>
			<td>15990</td>
			<td></td>
		</tr>
		<tr>
			<td>IL-10 Rat ELISA kit</td>
			<td>Invitrogen, Camarillo, CA, USA</td>
			<td>KRC0101</td>
			<td></td>
		</tr>
		<tr>
			<td>IL-1beta Rat ELISA Kit</td>
			<td>Invitrogen, Camarillo, CA, USA</td>
			<td>KRC0011</td>
			<td></td>
		</tr>
		<tr>
			<td>IL-6 Rat ELISA Kit</td>
			<td>Invitrogen, Camarillo, CA, USA</td>
			<td>KRC0061</td>
			<td></td>
		</tr>
		<tr>
			<td>INF-gamma Rat ELISA Kit</td>
			<td>Invitrogen, Camarillo, CA, USA</td>
			<td>KRC4021</td>
			<td></td>
		</tr>
		<tr>
			<td>TNF-alpha Rat ELISA Kit</td>
			<td>Invitrogen, Camarillo, CA, USA</td>
			<td>KRC3011</td>
			<td></td>
		</tr>
		<tr>
			<td>Rat LPS ELISA Kit</td>
			<td>NeoBioLab, MA, USA</td>
			<td>RL0275</td>
			<td></td>
		</tr>
		<tr>
			<td>Environmental Chamber 6020-1</td>
			<td>Caron, Marietta, OH, USA</td>
			<td>6020-1</td>
			<td></td>
		</tr>
		<tr>
			<td rowspan="2">Centrifuge&#160;</td>
			<td rowspan="2">Beckman Coulter, Indianapolis, IN, USA</td>
			<td>Optima L-90K</td>
			<td rowspan="2"></td>
		</tr>
		<tr>
			<td>Ultra Centrifuge</td>
		</tr>
		<tr>
			<td>Light microscope optical system</td>
			<td>CitoViva Technology Inc., Auburn, AL</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Colony counter</td>
			<td>Fisher Scientific , Pittsburgh, PA, USA</td>
			<td>RE-3325</td>
			<td></td>
		</tr>
		<tr>
			<td>Freezer</td>
			<td>Haier, Brooklyn, NY, USA</td>
			<td>HCMO50LA</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultra microplate reader</td>
			<td>Bio-Tek Instrument, Winooski, VT, USA</td>
			<td>ELx 808</td>
			<td></td>
		</tr>
		<tr>
			<td>Auto Strip Washer</td>
			<td>Bio-Tek Instrument, Winooski, VT, USA</td>
			<td>ELx50</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipettes</td>
			<td>Gilson, Pipetman, France</td>
			<td>P100, P200, P1000</td>
			<td></td>
		</tr>
		<tr>
			<td>C24 Incubator Shaker</td>
			<td>New Brunswick Scientific, Enfield, CT, USA</td>
			<td>Classic C24</td>
			<td></td>
		</tr>
		<tr>
			<td>Rocking Shaker&#160;</td>
			<td>Reliable Scientific, Inc., Nesbit, MS, USA</td>
			<td>55</td>
			<td></td>
		</tr>
		<tr>
			<td>Petri dishes</td>
			<td>Fisher Scientific, Pittsburgh, PA, USA</td>
			<td>875713</td>
			<td>100mmX15mm</td>
		</tr>
		<tr>
			<td>SterilGard III Advance</td>
			<td>The Baker Company, Sanford, ME, USA</td>
			<td>SG403</td>
			<td></td>
		</tr>
		<tr>
			<td>Culture Growing Flasks</td>
			<td>Corning Incorporated, Corning, NY, USA</td>
			<td>4995</td>
			<td>PYREX 250mL Erlenmeyer flasks</td>
		</tr>
		<tr>
			<td>Optical Spectrometer Genesys 20</td>
			<td>Thermo Scientific, Waltham, MA, USA.</td>
			<td>4001</td>
			<td></td>
		</tr>
		<tr>
			<td>Sony DXC-33 Video camera</td>
			<td>Sony</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Richardson test slide</td>
			<td>Electron Microscopy Science, Hatfield, PA, USA</td>
			<td>80303</td>
			<td></td>
		</tr>
		<tr>
			<td>Millipore water purification system</td>
			<td>Millipore</td>
			<td>Direct-Q</td>
			<td></td>
		</tr>
		<tr>
			<td>Image-Pro Plus software</td>
			<td>Media Cybernetics, MD, USA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Triple Beam Balance</td>
			<td>OHAUS Corporation, Parsippany, NJ, USA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td rowspan="3">Tissue Homogenizer, Dounce</td>
			<td rowspan="3">VWR</td>
			<td rowspan="3">71000-518</td>
			<td rowspan="3"></td>
		</tr>
		<tr>
		</tr>
		<tr>
		</tr>
	</tbody>
</table>

prevention of heat stress adverse effects in rats by bacillus subtilis strain

This study was designed to evaluate the protective effect of the Bacillus subtilis strain against complications related to heat stress. Thirty-two Sprague-Dawley rats were used in this study. Animals were orally treated twice a day for two days with B. subtilis BSB3 strain or PBS. The next day after the last treatment, each group was divided and two experimental groups (one treated with PBS and one treated with B. subtilis) were placed at 45 oC for 25 min. Two control groups stayed for 25 min at room temperature. All rats were euthanized and different parameters were analyzed in all groups. Adverse effects of heat stress are registered by the decrease of villi height and total mucosal thickness in the intestinal epithelium; translocation of bacteria from the lumen; increased vesiculation of erythrocytes and elevation of the lipopolysaccharides (LPS) level in the blood. The protective efficacy of treatment is evaluated by prevention of these side effects. The protocol was set up for the oral treatment of rats with bacteria for prevention of heat stress complications, but this protocol can be modified and used for other routes of administration and for analysis of different compounds.

The mean body temperature of animals before and immediately after heat stress was 36.7 &#177; 0.07 oC and 40.3 &#177; 0.17 oC, respectively (P &#60; 0.05). Exposure of rats to heat (group 3) resulted in significant inhibition of villi height and total mucosal thickness (Figures 2, 3). Oral administration of BSB3 strain before stress protected intestine from the harmful effect of heat.
Trans...

Watch this Scientific Journal Video about Prevention of Heat Stress Adverse Effects in Rats by Bacillus subtilis Strain at JoVE.com

Prevention of Heat Stress Adverse Effects in Rats by Bacillus subtilis Strain

We described a protocol for prevention of heat stress effects in rats by oral pre-treatment with beneficial bacteria. This protocol can be modified and used for various routes of administration and for analysis of different compounds.

Prevention of Heat Stress Adverse Effects in Rats by Bacillus subtilis Strain

This study was designed to evaluate the protective effect of the Bacillus subtilis strain against complications related to heat stress. Thirty-two ...

prevention-heat-stress-adverse-effects-rats-bacillus-subtilis

Research

JoVE Journal

Immunology and Infection

7.4K Views.  Auburn University. We described a protocol for prevention of heat stress effects in rats by oral pre-treatment with beneficial bacteria. This protocol can be modified and used for various routes of administration and for analysis of different compounds.

Video: Prevention of Heat Stress Adverse Effects in Rats by Bacillus subtilis Strain

Orthotopic Hind-Limb Transplantation in Rats

Composite tissue allotransplantation (CTA) now represents a valid therapeutic option after the loss of a hand, forearm or digits and has become a novel therapeutic entity in reconstructive surgery. However, long term high-dose multi-drug immunosuppressive therapy is required to ensure graft survival, bearing the risk of serious side effects which halters broader application. Further progression in this field may depend on better understanding of basic immunology and ischemia reperfusion injury in composite tissue grafts.
To date, orthotopic hind limb transplantation in rats has been the preferred rodent model for reconstructive transplantation (RT), however, it is an extremely demanding procedure that requires extraordinary microsurgical skills for reattachment of vasculature, bones, muscles and nerves.
We have introduced the vascular cuff anastomosis technique to this model, providing a rapid and reliable approach to rat hind limb transplantation. This technique simplifies and shortens the surgical procedure and enables surgeons with basic microsurgical experience to successfully perform the operation with high survival and low complication rates. The technique seems to be well suited for immunological as well as ischemia reperfusion injury (IRI) studies.

Here we describe the orthotopic rat hind-limb transplantation procedure, which seems to be the gold standard in vivo model for composite tissue allotransplantation research.

Composite tissue allotransplantation (CTA) now represents a valid therapeutic option after the loss of a hand, forearm or digits and has become a novel ...

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.

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

Introducing Shear Stress in the Study of Bacterial Adhesion

During bacterial infections a sequence of interactions occur between the pathogen and its host. Bacterial adhesion to the host cell surface is often the initial and determining step of the pathogenesis. Although experimentally adhesion is mostly studied in static conditions adhesion actually takes place in the presence of flowing liquid. First encounters between bacteria and their host often occur at the mucosal level, mouth, lung, gut, eye, etc. where mucus flows along the surface of epithelial cells. Later in infection, pathogens occasionally access the blood circulation causing life-threatening illnesses such as septicemia, sepsis and meningitis. A defining feature of these infections is the ability of these pathogens to interact with endothelial cells in presence of circulating blood. The presence of flowing liquid, mucus or blood for instance, determines adhesion because it generates a mechanical force on the pathogen. To characterize the effect of flowing liquid one usually refers to the notion of shear stress, which is the tangential force exerted per unit area by a fluid moving near a stationary wall, expressed in dynes/cm2. Intensities of shear stress vary widely according to the different vessels type, size, organ, location etc. (0-100 dynes/cm2). Circulation in capillaries can reach very low shear stress values and even temporarily stop during periods ranging between a few seconds to several minutes 1. On the other end of the spectrum shear stress in arterioles can reach 100 dynes/cm2 2. The impact of shear stress on different biological processes has been clearly demonstrated as for instance during the interaction of leukocytes with the endothelium 3. To take into account this mechanical parameter in the process of bacterial adhesion we took advantage of an experimental procedure based on the use of a disposable flow chamber 4. Host cells are grown in the flow chamber and fluorescent bacteria are introduced in the flow controlled by a syringe pump. We initially focused our investigations on the bacterial pathogen Neisseria meningitidis, a Gram-negative bacterium responsible for septicemia and meningitis. The procedure described here allowed us to study the impact of shear stress on the ability of the bacteria to: adhere to cells 1, to proliferate on the cell surface 5and to detach to colonize new sites 6 (Figure 1). Complementary technical information can be found in reference 7. Shear stress values presented here were chosen based on our previous experience1 and to represent values found in the literature. The protocol should be applicable to a wide range of pathogens with specific adjustments depending on the objectives of the study.

During the infection process, a key step is the adhesion of pathogens with host cells. In most instances this adhesion step occurs in the presence of mechanical stress generated by flowing liquid. We describe a technique that introduces shear stress as an important parameter in the study of bacterial adhesion.

During bacterial infections a sequence of interactions occur between the pathogen and its host. Bacterial adhesion to the host cell surface is often the ...

Single-cell Analysis of Bacillus subtilis Biofilms Using Fluorescence Microscopy and Flow Cytometry

Biofilm formation is a general attribute to almost all bacteria 1-6. When bacteria form biofilms, cells are encased in extracellular matrix that is mostly constituted by proteins and exopolysaccharides, among other factors 7-10. The microbial community encased within the biofilm often shows the differentiation of distinct subpopulation of specialized cells 11-17. These subpopulations coexist and often show spatial and temporal organization within the biofilm 18-21.
Biofilm formation in the model organism Bacillus subtilis requires the differentiation of distinct subpopulations of specialized cells. Among them, the subpopulation of matrix producers, responsible to produce and secrete the extracellular matrix of the biofilm is essential for biofilm formation 11,19. Hence, differentiation of matrix producers is a hallmark of biofilm formation in B. subtilis.
We have used fluorescent reporters to visualize and quantify the subpopulation of matrix producers in biofilms of B. subtilis 15,19,22-24. Concretely, we have observed that the subpopulation of matrix producers differentiates in response to the presence of self-produced extracellular signal surfactin 25. Interestingly, surfactin is produced by a subpopulation of specialized cells different from the subpopulation of matrix producers 15.
We have detailed in this report the technical approach necessary to visualize and quantify the subpopulation of matrix producers and surfactin producers within the biofilms of B. subtilis. To do this, fluorescent reporters of genes required for matrix production and surfactin production are inserted into the chromosome of B. subtilis. Reporters are expressed only in a subpopulation of specialized cells. Then, the subpopulations can be monitored using fluorescence microscopy and flow cytometry (See Fig 1).
The fact that different subpopulations of specialized cells coexist within multicellular communities of bacteria gives us a different perspective about the regulation of gene expression in prokaryotes. This protocol addresses this phenomenon experimentally and it can be easily adapted to any other working model, to elucidate the molecular mechanisms underlying phenotypic heterogeneity within a microbial community.

Single-cell Analysis of Bacillus subtilis Biofilms Using Fluorescence Microscopy and Flow Cytometry

Microbial biofilms are generally constituted by distinct subpopulations of specialized cells. Single-cell analysis of these subpopulations requires the use of fluorescent reporters. Here we describe a protocol to visualize and monitor several subpopulationswithin B. subtilis biofilms using fluorescence microscopy and flow cytometry.

Biofilm formation is a general attribute to almost all bacteria 1-6. When bacteria form biofilms, cells are encased in extracellular matrix that is mostly ...

Flow Cytometric Analysis of Particle-bound Bet v 1 Allergen in PM10

Flow cytometry is a method widely used to quantify suspended solids such as cells or bacteria in a size range from 0.5 to several tens of micrometers in diameter. In addition to a characterization of forward and sideward scatter properties, it enables the use of fluorescent labeled markers like antibodies to detect respective structures. Using indirect antibody staining, flow cytometry is employed here to quantify birch pollen allergen (precisely Bet v 1)-loaded particles of 0.5 to 10 &#181;m in diameter in inhalable particulate matter (PM10, particle size &#8804;10 &#181;m in diameter). PM10 particles may act as carriers of adsorbed allergens possibly transporting them to the lower respiratory tract, where they could trigger allergic reactions.
So far the allergen content of PM10 has been studied by means of enzyme linked immunosorbent assays (ELISAs) and scanning electron microscopy. ELISA measures the dissolved and not the particle-bound allergen. Compared to scanning electron microscopy, which can visualize allergen-loaded particles, flow cytometry may additionally quantify them. As allergen content of ambient air can deviate from birch pollen count, allergic symptoms might perhaps correlate better with allergen exposure than with pollen count. In conjunction with clinical data, the presented method offers the opportunity to test in future experiments whether allergic reactions to birch pollen antigens are associated with the Bet v 1 allergen content of PM10 particles &#62;0.5 &#181;m.

Here, we present a protocol to quantify allergen-loaded particles by flow cytometry. Ambient particulate matter particles may act as carriers of adsorbed allergens. We show here that flow cytometry, a method widely used to characterize suspended solids &#62;0.5 &#181;m in diameter, can be used to measure these allergen-loaded particles.

Flow cytometry is a method widely used to quantify suspended solids such as cells or bacteria in a size range from 0.5 to several tens of micrometers in ...

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

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

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

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

Effects of Exposure of Formaldehyde to a Rat Model of Atopic Dermatitis Induced by Neonatal Capsaicin Treatment

Atopic dermatitis is chronically relapsing pruritic eczema and prevails around the world especially in developed countries. Complex interactions between genetic and environmental factors are known to play an important role in the pathophysiology of atopic dermatitis. However, we still lack a detailed picture of the pathogenesis of this disease. Thus, it is of importance to develop appropriate animal models for elucidating the progression of atopic dermatitis. Moreover, investigating the effect of environmental factors such as air pollutants on atopic dermatitis expands understanding of the disease. Here, we describe a method for inducing atopic dermatitis in rats with neonatal capsaicin treatment and a protocol for exposure of a constant concentration of formaldehyde to rats to reveal effects on the development of atopic dermatitis in infantile and adolescent periods. These protocols have been successfully applied to several experiments and can be used for other substances.

We describe here methods for neonatal capsaicin treatment to induce atopic dermatitis in rats and exposure of vaporized formaldehyde to investigate the effects of formaldehyde inhalation on atopic dermatitis.

Atopic dermatitis is chronically relapsing pruritic eczema and prevails around the world especially in developed countries. Complex interactions between ...

Measuring Influenza Neutralizing Antibody Responses to A(H3N2) Viruses in Human Sera by Microneutralization Assays Using MDCK-SIAT1 Cells

Neutralizing antibodies against hemagglutinin (HA) of influenza viruses are considered the main immune mechanism that correlates with protection for influenza infections. Microneutralization (MN) assays are often used to measure neutralizing antibody responses in human sera after influenza vaccination or infection. Madine Darby Canine Kidney (MDCK) cells are the commonly used cell substrate for MN assays. However, currently circulating 3C.2a and 3C.3a A(H3N2) influenza viruses have acquired altered receptor binding specificity. The MDCK-SIAT1 cell line with increased &#945;-2,6 sialic galactose moieties on the surface has proven to provide improved infectivity and more faithful replications than conventional MDCK cells for these contemporary A(H3N2) viruses. Here, we describe a MN assay using MDCK-SIAT1 cells that has been optimized to quantify neutralizing antibody titers to these contemporary A(H3N2) viruses. In this protocol, heat inactivated sera containing neutralizing antibodies are first serially diluted, then incubated with 100 TCID50/well of influenza A(H3N2) viruses to allow antibodies in the sera to bind to the viruses. MDCK-SIAT1 cells are then added to the virus-antibody mixture, and incubated for 18 - 20 h at 37 &#176;C, 5% CO2 to allow A(H3N2) viruses to infect MDCK-SIAT1 cells. After overnight incubation, plates are fixed and the amount of virus in each well is quantified by an enzyme-linked immunosorbent assay (ELISA) using anti-influenza A nucleoprotein (NP) monoclonal antibodies. Neutralizing antibody titer is defined as the reciprocal of the highest serum dilution that provides &#8805;50% inhibition of virus infectivity.

Measuring Influenza Neutralizing Antibody Responses to AH3N2 Viruses in Human Sera by Microneutralization Assays Using MDCK-SIAT1 Cells

Influenza neutralizing antibodies correlate with protection of influenza infections. Microneutralization assays measure neutralizing antibodies in human sera and are often used for influenza human serology. We describe a microneutralization assay using MDCK-SIAT1 cells to measure neutralizing antibody titers to contemporary 3C.2a and 3C.3a A(H3N2) viruses following influenza vaccination or infection.

Neutralizing antibodies against hemagglutinin (HA) of influenza viruses are considered the main immune mechanism that correlates with protection for ...

Isolation of Salmonella typhimurium-containing Phagosomes from Macrophages

Salmonella typhimurium is a facultative intracellular bacterium that causes gastroenteritis in humans. After invasion of the lamina propria, S. typhimurium bacteria are quickly detected and phagocytized by macrophages, and contained in vesicles known as phagosomes in order to be degraded. Isolation of S. typhimurium-containing phagosomes have been widely used to study how S. typhimurium infection alters the process of phagosome maturation to prevent bacterial degradation. Classically, the isolation of bacteria-containing phagosomes has been performed by sucrose gradient centrifugation. However, this process is time-consuming, and requires specialized equipment and a certain degree of dexterity. Described here is a simple and quick method for the isolation of S. typhimurium-containing phagosomes from macrophages by coating the bacteria with biotin-streptavidin-conjugated magnetic beads. Phagosomes obtained by this method can be suspended in any buffer of choice, allowing the utilization of isolated phagosomes for a broad range of assays, such as protein, metabolite, and lipid analysis. In summary, this method for the isolation of S. typhimurium-containing phagosomes is specific, efficient, rapid, requires minimum equipment, and is more versatile than the classical method of isolation by sucrose gradient-ultracentrifugation.

Isolation of Salmonella typhimurium-containing Phagosomes from Macrophages

We describe here a simple and quick method for the isolation of Salmonella typhimurium-containing phagosomes from macrophages by coating the bacteria with biotin and streptavidin.

Salmonella typhimurium is a facultative intracellular bacterium that causes gastroenteritis in humans. After invasion of the lamina propria, S. ...

Assessment of the Cytotoxic and Immunomodulatory Effects of Substances in Human Precision-cut Lung Slices

Respiratory diseases in their broad diversity need appropriate model systems to understand the underlying mechanisms and enable development of new therapeutics. Additionally, registration of new substances requires appropriate risk assessment with adequate testing systems to avoid the risk of individuals being harmed, for example, in the working environment. Such risk assessments are usually conducted in animal studies. In view of the 3Rs principle and public skepticism against animal experiments, human alternative methods, such as precision-cut lung slices (PCLS), have been evolving. The present paper describes the ex vivo technique of human PCLS to study the immunomodulatory potential of low-molecular-weight substances, such as ammonium hexachloroplatinate (HClPt). Measured endpoints include viability and local respiratory inflammation, marked by altered secretion of cytokines and chemokines. Pro-inflammatory cytokines, tumor necrosis factor alpha (TNF-&#945;), and interleukin 1 alpha (IL-1&#945;) were significantly increased in human PCLS after exposure to a sub-toxic concentration of HClPt. Even though the technique of PCLS has been substantially optimized over the past decades, its applicability for the testing of immunomodulation is still in development. Therefore, the results presented here are preliminary, even though they show the potential of human PCLS as a valuable tool in respiratory research.

In view of the 3Rs principle, respiratory models as alternatives to animal studies are evolving. Especially for risk assessment of respiratory substances, there is a lack of appropriate assays. Here, we describe the use of human precision-cut lung slices for the assessment of airborne substances.