Visualization of Candida albicans in the Murine Gastrointestinal Tract Using Fluorescent In Situ Hybridization

This protocol has yielded some of the best insights we have so far into how Candida albicans inhabits the mammalian gastrointestinal tract. For example, until recently, we didn't really know where Candida is localized within the gut or which of its many morphological forms it assumes within this niche. The main advantage of this technique is that it allows one to visualize Candida within its natural environment without disrupting the three-dimensional interactions the fungus has with host structures or with bacteria in the microbiota.

Because the gut is a complex environment, it's very difficult to model in the laboratory and therefore it's been very satisfying to find a way to probe Candida biology directly in a host model. Certain components of the gut microbiota including Candida albicans are suspected to play roles in diseases such as inflammatory bowel disease. This technique can potentially be used to diagnose the presence of specific microorganisms in biopsy specimens obtained from patients.

Mastery of the gavage technique and the ability to dissect gastrointestinal segments without tearing the organs is important for obtaining optimal results. To start, gavage each animal with the calculated volume of a given inoculum. Attach an autoclaved animal feeding needle to a one milliliter syringe and fill the syringe with one gavage volume making sure to remove all bubbles.

Place the syringe on a sterile surface. To secure the animal, hold it at the base of the tail with the dominant hand and slide the nondominant hand up the animal's back to the loose skin just behind the ears. Gather the scruff together tightly with the thumb and forefinger.

Pick up the animal by the scruff, flip it over and loop the small finger of the same hand around the tail to immobilize the animal against the palm of the hand. Gently extend the animal's head so that it is in a straight line with the body. Then pick up the syringe with the dominant hand and hold it perpendicular to the animal with the curved needle pointing down.

Use the ball of the needle to gently hook an inside corner of the mouth and advance the needle along the side of the mouth to the back of the throat, then move the needle to the center. Once the ball of the needle is at the back of the animal's throat, tilt the needle up so that it is parallel with the animal's body line and allow it to slide smoothly down the throat. If the needle does not slide smoothly down the throat, the needle may not be properly positioned and should be withdrawn to reposition and try again.

Smoothly advance the needle allowing the animal to swallow the needle until only a few millimeters remain visible. Test that the ball of the needle is in the stomach by gently advancing the plunger and if there is no resistance, continue delivering the inoculum. Slowly withdraw the needle once the inoculum has been administered and place the animal in its cage.

Continue monitoring the animal for five to 10 minutes for signs of labored breathing or distress and verify that it resumes normal activity soon after gavage. If the same inoculum will be used for the next animal, clean the needle with an alcohol wipe. If a different inoculum will be used, replace the needle.

Use blunt-ended forceps to pinch a section of the abdominal skin and incise the skin and the underlying fascia near the base of the pelvis using scissors. Extend the incision in a U-shape along each side of the peritoneal cavity and up to the ribcage, then lift the skin out of the way. Use blunt-ended forceps to gently extract the cecum from the peritoneal cavity using scissors to sever the connections between the cecum and the small and large intestines.

Place the entire cecum into a histology cassette so that it is untwisted and lays flat. Place the second foam pad on top and close the cassette. Then place the cassette into a screw cap jar containing methacarn.

Excise the section of the large intestines that contains one to two fecal pellets and has a length less than that of the cassette. Place the tissue into the cassette keeping it flat and untwisted. Overlay it with the second foam pad, close the cassette and place it into the methacarn.

For each section of the small intestine to be sampled, excise a one to two centimeter segment that contain some digestive material. Place the tissue into the cassette, overlay with the second foam pad, close the cassette and put it into the methacarn. When excising the stomach, sever the connections to the esophagus and the small intestines.

Place the tissue into the cassette and place the cassette in the methacarn. Leave the cassettes in the methacarn solution at room temperature for at least three hours but less than two weeks. After fixation, remove the foam pads and return each intact tissue section into its cassette.

Then wash the tissues twice for 35 minutes in 100%methanol, twice for 25 minutes in 100%ethanol, and twice for 20 minutes in xylene. Pat the cassettes dry on a paper towel and place them in a pre-melted paraffin wax for two hours at 70 degrees Celsius in a hybridization oven. Then remove the cassettes from the wax, allow the excess wax to drain and store them at room temperature until they are embedded in wax blocks.

De-wax the paraffin embedded histological sections by inserting the slides into a pre-warmed jar filled with xylene. Leave the jar at 60 degrees Celsius for 10 minutes, then discard the xylene wash. Pour fresh room temperature xylene into the jar and repeat the incubation.

Then fill the jar with 100%ethanol and incubate it at room temperature for five minutes. After the incubation, remove the slides from the jar and air dry them. To stain the C.albicans with a fish probe, start by drawing a circle around the tissue section using a Pat Pen.

Pipette 50 microliters of probe containing hybridization solution onto the fixed tissue and gently spread it with the pipette tip. Overlay the liquid with a hybridization coverslip making sure to prevent bubbles. Then seal the slides in a water tight hybridization chamber and incubate the sections for three hours at 50 degrees Celsius in a hybridization oven in the dark.

After the incubation, add the washing solution to a Coplin jar. Carefully remove the coverslip from the slide with forceps and place the slide in the washing solution. Cover the jar with aluminum foil and incubate it at 50 degrees Celsius for 20 minutes.

Next, wash the slides by pouring off the washing solution and refilling the jar with PBS. Immediately pour off and refill with fresh PBS repeating the process for a total of two washes. Wick away the PBS with a delicate task wipe and circle the intestinal tissue section with a Pat Pen.

Place the slides in an opaque plastic container and add 50 microliters of mucin nuclei staining solution to the tissue section. Cover the container with aluminum foil and incubate it at four degrees Celsius for 45 minutes. After the incubation, put the slides in a Coplin jar and wash them twice with PBS.

Tap away the excess PBS on a paper towel and then wick away the final drops with a tissue. Place one drop of mounting medium on each section and overlay it with a glass coverslip making sure to prevent bubbles. Let the mounting medium spread under the entire coverslip.

Anchor the coverslip in place with nail polish at the corners and image the slides using a fluorescent microscope. This protocol can be used to fluorescently label C.albicans in vitro and in host tissues. In vitro propagated hyphae, round yeast cells, gut cells, and opaque cells were fixed, permeabilized, and distinguished with fluorescence and phase contrast microscopy.

Round yeasts and highly elongated hyphae were imaged in the mouse large intestines. Host tissues were stained with the C.albicans specific probe or the panfungal probe. C.albicans was labeled red, host cell nuclei blue, and the mucus layer green.

C.albicans was detected in different segments of the murine GI tract including the regions immediately adjacent to the host mucosa and more central regions of the gut lumen. For scientists who have not previously performed the gavage technique, it is important to obtain specialized training before attempting this method. Following fish staining, immunofluorescent techniques can be used to stain additional features of the host mucosa.

This can allow for characterization of tissue damage or immune responses of the host. This technique has allowed us to characterize the relationship between fungal cell morphology of Candida albicans mutants and survival within the host furthering our understanding of Candida albicans biology that can be taken advantage of when developing therapies.