The overall goal of the following experiment is to isolate new potential intracellular pathogens present in environmental or clinical samples. This is achieved by inoculation of the sample of interest into a culture of AME in order to select bacteria that are able to enter and multiply in amobi. Next, the amobi that are lysed, or in which a clear intracellular growth is observed are further analyzed in order to identify the microorganism at the species level.
Then if no bacterial growth is obtained using the ameba co-culture method, ameba enrichment is performed to detect endogenous amobi that might contain specific intracellular bacteria.Obtained. Results are the isolation of new bacterial species and novel viruses with this innovative method, which may be further characterized using approaches including microscopy and genome sequencing. The main advantages of these techniques over other exist methods such as cenic culture or mammalian cell culture, is that it can be applied to isolate strict intracellular bacteria or that you can even apply this technique to easily contaminated samples that contain complex microbial communities.
Let's me introduce Sebastian BA technician from my laboratory who will demonstrate the procedure To test a water sample filter a 500 to 1000 milliliter sample through a 0.22 micrometer pore size membrane. Then place the membrane in pages, amoeba, saline, or pass and shake it for solid samples such as soil, sand, or activated sludge. Resuspend them in distilled water or PBS and filter them through a 0.22 micrometer pore size membrane.
Shake the membrane in pass, cultivate a ebe in cell culture flask containing 30 milliliters of PYG medium at 25 degrees Celsius overnight to harvest the amobi vigorously. Shake the flask and centrifuge the cell suspension for 10 minutes at 1500 G.Use past medium to wash the pellet twice. Then use a covis slide to count the cells before adjusting the volume to obtain a suspension of five times 10 to the fifth cells per milliliter.
Transfer one milliliter per well of the suspension into 24. Well microplate and incubate them for at least two hours at 25 degrees Celsius to allow the sedimentation and attachment of Abe to the bottom of the well. Beginning with a hundred microliters of undiluted samples.
Inoculate each plate with tenfold serial dilution of the filtered water resuspended and filtered soil solution, or the culture to me to increase contact and phagocytosis of microorganisms by ABI centrifuge, the microplate at 1800 G for 10 minutes. Incubate the plates for 45 minutes at 25 degrees Celsius before using pass to wash them three times. Then add one milliliter per well of pass with or without antibiotics.
Next, incubate the Microplate at 32 degrees Celsius in a humidified atmosphere. To avoid insist of the amobi, then use a 20 x objective to observe each well daily to detect the presence of bacteria invading and lysing amobi. In the case of lysis, perform a subculture on fresh amobi by inoculating 100 milliliters of co cultures to a monolayer of about 10 to the fifth abi per centimeter squared.
To specifically isolate a given bacterial species inoculate specific agar media designed for particular bacteria. In the absence of lysis, four to seven days after the first inoculation transfer 100 milliliters of co cultures to a fresh amiable culture of 900 milliliters. In a 24 well plate to carry out modified romanowski staining.
After co culturing samples on cover slips allow them to dry before immersing them in fixative solution five times. Next, immerse the cover slips five times in staining solution one then immerse the cover slip five times in staining solution two. Finally use distilled water to rinse the cover slips.
Let them dry, melt them and observe by microscopy to perform immunofluorescent staining. Fix the cover slips by incubating and methanol for five minutes or 4%para formaldehyde for 10 minutes. Use PBS to wash the samples three times before incubating and blocking solution.
For two hours, prepare a box containing blotting paper. Add distilled water to obtain a humid chamber. Cover the blotting paper with paraform.
Place drops of antibodies on the paraform after incubating the cover, slips for one hour in blocking solution containing antibodies raised against the microorganism of interest. Use PBS 0.1%saponin to wash the samples three times. Then incubate with a fluorescent secondary antibody for one hour.
Wash the slides three times with PBS 0.1%saponin one time with PBS and one time with distilled water before mounting the cover slip and observing by fluorescence microscopy. To prepare samples for amoeba enrichment, add pass to solid and semi-solid samples and resuspend by vortexing to allow enrichment of free living amoeba in the pellet centrifuge, the suspension at low speed. Next, inoculate a non-nutritive agar or NNA plate with two milliliters of a 10 sigma diluted overnight culture of escherichia coli and let it dry for 45 minutes.
Then add a drop of the amiable sample on one side of an NNA plate and let it flow over the Petri dish to form a line in the center of the dish. Observe the Petri dish daily if an amiable migration front is detected. Cut out a small piece of agar at the migration front and inoculate a fresh NNA petri dish covered with a lawn of e coli.
Repeat the re inoculation several times or as necessary to obtain a pure amoeba culture as shown in this table. Using amoeba co culture and enrichment, a whole range of environmental and or pathogenic bacteria were discovered. The most common bacteria isolated by ameba coal culture are members of the mycobacterium genus that were recovered from water treatment plants and water networks.
Legionella and a proteobacteria species were isolated from water treatment plants and hospital water networks. Several chlamydia related species, including Estella la sinensis, were also isolated from river water and water treatment facilities. This image shows another example of bacteria found in a specific amoeba called para chlamydia.
A cant amobi. This chlamydia related bacterium was isolated from nasal mucosa of female volunteers by amiable enrichment and is a potential agent of pneumonia further demonstrating the importance of amobi in the maintenance and dispersion of bacterial pathogens that might be especially pathogenic for immunocompromised patients. Following this procedure, other methods such as electron microscopy, immunofluorescence sequencing of core genes of all genome sequencing may be performed in order to fully characterize the newly discovered strain.
In addition, it is possible to test the ity of this strain by inoculation to animals or to mammalian cell lines. Don't forget that working with new potential pathogens can be extremely hazardous, and that precautions such as working in a P two laboratory with sufficient equipment should be taken to perform this experiment.
