by Bob Harms ()

I am grateful to Professor Jim Mauseth for guiding me through the process of clearing and staining the Evolvulus leaves for this project. Without his patience and help it couldn't have happened.

Four of the best clearings. Click for enlargement.

E. alsinoides	E. nuttallianus	E. sericeus	E. discolor

1. Leaves in individual capsules were placed overnight in a 1% aqueous solution of basic fuchsin to which 10 gm of solid NaOH had been added and left overnight at 60° C. The NaOH digests most issue and renders the pubescence transparent. Basic fuchsin stains the veins (lignin) red.
2. The NaOH/Fuchsin solution was removed and the leaves were washed with multiple changes of tap water over several days.
3. Half the water was removed and replaced with ETOH. This process was repeated over several days.
4. All liquid was removed and replaced with absolute ETOH.
5. ETOH was gradually replaced with xylene over several days.
6. Permount was added to the xylene prior to mounting.
7. Leaves were removed from the capsules onto a drop of pure Permount on a glass slide and covered with a cover glass.

The leaves are small and thin and step 1, treating with NaOH, digests most issue making them very fragile. During steps 2—6 the capsules had to be gently agitated. Several leaves suffered minor fractures during these steps.

The three worst results. Click for enlargement.

E. alsinoides	E. discolor (previously as E. nuttallianus)

Permount has a high degree of viscosity, and the mounting step 7, removing the leaves from the capsules, was difficult — in large part because the capsules I had chosen narrowed to a neck and the leaves tended to hang at this point and had to be prodded out so that I could move them onto slides. One leaf was totally destroyed, 2 leaves folded over, several broke into pieces. E. alsinoides leaves suffered the most damage, and indication that they were the most fragile. E. nuttallianus fared better than the others. In all, only a third survived intact or with only minor damage — but in spite of this the partials that remained provided clear evidence as to the nature of the venation of that leaf.