Photographing arbuscules is among the most demanding challenges in mycological photography. You must first locate them, then succeed in preparing a slide that produces at least a convincing image — ideally of publication quality. This page describes and illustrates each step in detail, using Rhizophagus irregularis and the host plant Plantago lanceolata.
Menu
- Required tools and instrumentation
- Preparation and staining
- Root selection and examination
- Final mounting
- Photography
Tools and instrumentation used
- Stereo microscope and compound microscope with at least a 40X objective
- Micro-tools: Mini-scissors, micro-needles and fine forceps
- Camera
- Slide, cover slips, and mounting media
- Colonized and stained roots – Ink and vinegar method
Preparation and staining
Extracting colonized roots
Extraction begins with a well-colonized but relatively young Petri dish, chosen to show roots that were recently colonized by the fungus. In this example, the entire root network that grew across into the polymer side is removed. These relatively young roots offer good potential for finding arbuscules. Under the stereo microscope, vesicles can already be seen in the roots — a sign of good colonization. Note that the author has never been able to spot arbuscules in a living root, despite many attempts.
Petri dish containing well-colonized roots. Under the stereo microscope, spores are visible in the polymer as well as vesicles in the roots.
In this example, all of the roots present in the polymer are sampled. Roots from vermiculite would also work well.
It is best to check under the stereo microscope that the roots are well colonized. The presence of numerous vesicles indicates good potential for finding arbuscules.
The bundle of roots, with the polymer still attached, is ready for staining. It will successively be treated with KOH, rinsed, then subjected to staining with ink and vinegar. Most of the polymer will detach from the roots during the treatment.
Warning: The effects of KOH or vinegar on the polymer have not been formally evaluated. It is always preferable to carry out these operations under a chemical fume hood.
Staining
The hyphae must then be stained so they can be distinguished from the root tissue. The method used is the ink and vinegar method, described in detail on the corresponding page.
Root selection and examination
After staining, select the roots that show the best photographic potential. Arbuscules must be distinguished from vesicles, which are not of interest at this stage. Rhizophagus irregularis produces large intracellular vesicles that stain dark blue, almost black. Arbuscules, which are much less contrasted, show a faint medium blue — this is the subtle signal to look for under the stereo microscope.
In Rhizophagus irregularis, segments rich in vesicles usually contain few or no arbuscules, and their abundance hinders observation. The following photo illustrates three roots of different photographic potential. Only the root indicated by the green arrow is a good candidate for photographing arbuscules.
The following examination takes place under the compound microscope, with a 10X objective.
No arbuscules found? Look for other segments in the batch of stained roots. Depending on the fungal species, the age of the root, and the degree of colonization, arbuscules can be abundant or, on the contrary, very rare.
Back under the stereo microscope, the root is leaf-peeled: that is, torn into sheets a single cell layer thick. The arbuscules exposed in these thin sheets are then suitable for very high-quality photography.
To carry out this leaf-peeling: Gently remove the cover slip and keep only the promising segment on the slide. The initial water mount makes this operation easier.
Place a new drop of water on the root segment. All of the following manipulations are carried out in a liquid medium, inside this drop of water, under the stereo microscope.
Using fine forceps and micro-needles (acupuncture needles preferably), under the stereo microscope, proceed with leaf-peeling the root as illustrated.
Final mounting
After leaf-peeling, very gently blot the excess water before the final mount. Use the needles to reposition the sheets to optimize their arrangement on the slide and to move aside uninteresting debris.
At this stage, three mounting options are possible:
- Water mount: place a drop of water, position the cover slip while moving the segments as little as possible, and photograph immediately. The cells are still turgid and the arbuscules are close to their natural shape. Several photos at different depths of field will be necessary. The sample will not keep for the long term.
- PVLG mount (PolyVinyl-lacto-glycol): let the sheets dry, then mount with PVLG. This is a “classic” method. Contrast seems to improve after a few days, but excellent photos are possible right from mounting. These slides can be kept for a very long time.
- UV glue mount: let dry, then fix the cover slip with a transparent glue that hardens under ultraviolet light. The cover slip is permanently fixed within a few seconds, which allows the use of immersion oil for 100X images immediately. The storage duration of slides prepared this way has not been validated.
Photography:
Place the slide under the microscope and methodically explore in search of the best-placed arbuscules. If none is sufficiently photogenic, restart the process from the segment selection step. In this example, the sample produced good results.
Here are some arbuscules photographed from this slide with a 40X objective, after digital zoom and cropping on the area of interest.
Same sample and same preparation, but on another root segment.
