The overall goal of this procedure is to obtain native oral biofilm from palatal expanders for fluorescence in situ hybridization. This is accomplished by first removing the palatal expander, carrying the four month old biofilm from the patient. Next, the acrylic resin flakes containing the biofilm are scraped off from the palatal expander.
Then fluorescence in C two hybridization of the fixed biofilm is carried out directly on coated slides. Finally, analysis of the stained biofilm is performed using confocal laser scanning microscopy P.In the end, the structure and composition of bacterial biofilm communities are shown through fluorescence in situ hybridization and confocal laser scanning microscopy. We first had the idea for this method when we encountered thick biofilms on orthodontic expanders visible to the naked eye.
They made us think of possible associations between them and periodontal diseases. This method can help answer key questions in the field of oral microbiology, such as the spatial arrangement and composition of native oral biotherms. The method is interdisciplinary and has to be done by different specialists.
So Elizabeth is an orthodontist and Barbara is a molecule lab biologist To begin. After four months of intraoral surface, using the NOLA dry field system, isolate the fixed palatal expander to avoid contamination. Remove the appliance from the patient's mouth using sterilized pliers gloves and trays.
Store the objects in 120 milliliter vials at minus 20 degrees Celsius. Take them to the laboratory on ice and process within 24 hours to fix the specimens. Scrape off biofilm flakes with a sterile scalpel or collect pieces with sterile forceps.
It is crucial to scrape off acrylic resin flakes of adequate thickness to ensure that the biofilm structure remains intact at ice cold. 4%paraldehyde solution until the sample is well covered. Incubate the mixture at four degrees Celsius for three to 12 hours.
Longer fixation times or higher fixation temperatures may render the cell envelopes of gram-negative cells less permeable to oligonucleotide probes. After the incubation, remove the paraldehyde solution and wash with ice cold one times PBS. Repeat this step two to three times to remove residual paraldehyde, Reese has bend the sample in one volume of ice cold one times PBS and add one volume of ice cold 96%ethanol store the sample at minus 20 degrees Celsius.
Samples can be stored for several months to years to dehydrate the sample. First, apply five to 30 microliters of paraldehyde fixed sample material to a microscope. Slide dry the sample for 15 minutes at 46 degrees Celsius or for longer at room temperature while the sample is drying for the lysozyme and the for maide to partially break down the cell walls.
For better entry of the probes, add 250 microliters of a one milligram per milliliter solution of lysozyme for 10 minutes of room temperature, dip the slides into 50 80 and 96%ethanol for three minutes each. The dehydrating effect of the ethanol concentration series will disintegrate the membranes for better oligonucleotide probe uptake. Finally, dry the slides at 46 degrees Celsius to perform in situ U hybridization.
Start by preparing one milliliter of fresh hybridization buffer and prewarm it to 47 degrees Celsius. Thaw oligonucleotide Probe solutions on ice and keep them protected from light. Place a piece of tissue paper into a square Petri dish and pipette 800 microliters of the hybridization buffer onto the tissue paper.
Close the chamber again immediately as the buffer contains form aide, add two microliters of each probe to 200 microliters of the rest of the hybridization buffer. Mix well and apply the mixture to the dehydrated sample on a microscope slide immediately place the slide horizontally into the dish and cover it. Incubate the dish in an oven for one to five hours at 46 degrees Celsius.
The dish functions as a moisture chamber preventing evaporation of hybridization solution from the slide. In particular, the evaporation of form amide can cause non-specific probe binding to non-target cells. Prepare 50 milliliters of washing buffer and preheat it to 48 degrees Celsius in a water bath.
Remove the dish with the slide from the hybridization oven. Immediately wash away the hybridization buffer with a small volume of prewarm washing buffer and transfer the slide into the remaining washing buffer. Place the tube containing the washing buffer and the slide back into the water bath and incubate for 10 to 15 minutes of 48 degrees Celsius.
To stain the samples with more than one probe. Repeat the earlier steps after the washing buffer has been removed, take care to start with the highest form a mild concentration. Remove the slide from the tube and dip into ice.
Cold doubled distilled water for two to three seconds. To eliminate residual washing buffer air dry the slide as quickly as possible as fast drying reduces. Probed dissociation.
Store the dried slides in the dark at minus 20 degrees Celsius for several weeks without any significant loss of probe conferred fluorescence signal. To image the samples, apply two drops of Antifa reagent close to the left and right ends of a slide, and then add a cover slip. Observe the samples under a confocal laser scanning microscope equipped with suitable filters or lasers, the slides can be stored at four degrees Celsius for up to seven days, or can be washed with double distilled water and stored at minus 20 degrees Celsius for longer periods.
It's graving biofilm of fixed orthodontic appliances. You'll suitable flakes that can be hybridized directly on coated glass slides for microscopy, such different groups of oral microbiome, bacteria in three dimensional configuration can be identified through specific probes. In this figure, the biofilm was stained with EUB mix and LGC mix firm acutes appearing green as they were stained with yellow and blue.
Here the biofilm was stained with EUB mix back 3 0 3 and pogi. Bacteria of the group bacteria dts appear in pink and poor fire. Ramons gingivalis is shown in yellowish white as all three probes bind into its DNA and the overlap of colors results in a white signal Morphological.
Differences between groups of bacteria can also be identified as well as certain structures like large clusters of coco bacteria. Examples of such clusters are shown here where EUB mix was used for staining. In addition, a typical mushroom like structure of the biofilm can be processed via 3D modeling of the CLSM data.
This is a 3D animation of the EUB mixed stained biofilm. This method can provide insight into orthodontically induced biofilm formation. It can also be applied to any other artificial surface in the human body that carries complex biofilm structures with possible pathogenicity.
After watching this video, you should have a good understanding of how to prepare native oral biofilms for fluorescence in C two hybridization and confocal laser scanning microscopy. It's important to remember that this is an interdisciplinary field and you can only be successful in a team. So always treat your colleagues nicely.
