Elisabeth H Laban Biological Science Technician NOAA/NOS/CCEHBR and SC DNR MRRI

1. Adaptations and Modifications of methods used by GSFMC processors for sectioningGray triggerfish spines Elisabeth H Laban Biological Science Technician NOAA/NOS/CCEHBR and SC DNR MRRI

2. It is important to track your sample through the process of mounting and cutting sections. Flagging each spine with a numbered label is one way to accomplish this.

3. spine morphology LC Dorsal tip of spine CG CG Lateral or medial left Lateral or medial right medial right medial left Posteromedian groove PCP Ventral base of spine Part of head that was removed with spine TCG Anterior view of triggerfish spine Posterior view of triggerfish spine Posterior/lateral view of base of dorsal spine. LC, lateral condyle; CG, condyle groove; PCP, posterior condyle process; and TCG, tip of condyle groove.

4. Some samples may bereturned to the lab with part of the skull cap still attached to the dorsal spines. Lateral or medial right Lateral or medial left medial left medial right Part of skull Excess material can be removed using nippers

5. Dried tissue, muscle and fibers are scraped clean from the area of interest, from the base of the spine to at least a centimeter above the tip of the condyle groove, using an X-acto knife, forceps, razor blade, and/or a nylon bristle brush. The posterior condyle process is “shaved” to allow the spine to lie “flat” mounting the spine laterally for cutting. CG CG TCG PCP Posteromedian groove

6. Using a black marker, a cut line is marked on the cleaned spine at the dorsal tip of the condyle groove. This is done to assist in aligning the spine when mounting and cutting, as the best sections for age determination are cut dorsal to the tip of the condyle groove. Posterior views of cleaned spine l v d l a p CG d v p a Tip of CG PCP CG Lateral views of cleaned spine a=anterior; p=posterior; d=dorsal; v=ventral; l=lateral

7. After cleaning and marking the cutting line on the spine, it is flagged with a label containing enough of the sample identification number to uniquely distinguish it from the other samples to be processed.

8. For convenience, the flagged spines are placed on cafeteria tray, or other storage container, until “enough” spines have been cleaned and flagged and you are ready to mount samples for cutting. This way the spine does not need to be returned to the sample storage envelope before mounting and cutting.

9. Currently, GSFMC processors are gluing the spine to an index card and securing the index card (ic), and a piece of cardboard (cb), used as a cushion, to the metal cutting plate (mcp) with binder clips. Note that the GSFMC blade assembly consists of four blades and three spacers, resulting in three sections of a cut spine mcp cb ic b To reduce the waste created by single use index cards and limited use cardboard “cushions”; which become water logged during cutting; the GSFMC mounting materials were replaced with a heavy gauge frosted plastic notebook cover (or heavy gauge 3-ring notebook dividers) and craft foam sheets. The frosted plastic and the craft foam are waterproof and thus can be reused many times. Above images by Carrie Fioramonti; University of West Florida

10. a b a b b a a b b b Both the craft foam sheet (“a” on the images) and the heavy gauge frosted plastic notebook cover (“b” on the images) have been cut to the size of the metal cutting plate. Alignment lines are marked on the craft foam piece (see arrows). When the plastic cutting plate is placed on the craft foam, the alignment lines are clearly visible. The cutting line on the spine is also visible through the plastic.

11. The plastic cutting plate is placed on the craft foam. The spine is oriented with the alignment lines on the craft foam, and an additional mark is placed on the spine near the “cushion rest” (reducing the amount of glue used). High temperature hot melt glue is then applied to either the spine or the plastic and the spine secured in place along the alignment lines.

12. These images show a flagged spine mounted, medial right, to the frosted plastic cutting plate. The cutting mark on the spine is visible through the glue and plastic. The yellow craft foam “cushion” shows how it has been used to aid in the horizontal alignment of the spine and to reduced amount of glue used.

13. This series of images shows a flagged, marked spine, mounted with the anterior surface against the plastic cutting plate. The alignment lines on the craft foam and the craft foam “cushion” help in assuring a perpendicular cut.

14. Once the spines have been mounted on the plastic cutting plates, they can be stored for later processing. As you can see in the picture below, some samples are stored in a box and others have been stored flat and overlapping on a cafeteria lunch tray. 2 2 1 2

15. The swing arm is then moved so the outside of the parallel alignment mark is just touching the edge of the first, or “inside”, blade. With this alignment and care during mounting, little to no adjustment to the swing arm is necessary when mounting the cutting plates for processing. The craft foam cushion can be temporarily held in place, against the metal cutting plate with a few drops of water. The swing arm is lowered and allowed to rest on the blade assembly to adjust the cutting chuck so the perpendicular alignment mark is resting directing against the blades.

16. A plastic cutting plate with a mounted spine is placed against the craft foam cushion, making sure the alignment is correct, and the entire assembly is clamped in place to the metal cutting plate with 3 binder clamps. mcp cf pcp

17. The sample is lowered near the blade to adjust the swing arm if necessary. The swing arm is lifted and the saw is turned on and the dial set between 7 and 8 (210 and 240 rpm), the sample is lowered onto the moving blades and the sample is cut. mcp cf pcp cm 1st cb ba

18. Bending the cutting plate to see if the cut is complete. The cut is not complete there is material still visible against plastic cutting plate. The cut is complete, cut spaces are visible against plastic cutting plate. If you are not sure you have completely cut through the spine; you can lift the swing arm, turn the speed dial to zero, remove the front clamps, bend the plastic cutting plate down and away to check if the cut is visible on the backside of the cutting plate. You can also bend the plate back on itself to see if the spine and sections “separate”. Care should be taken to avoid breaking the spine before the cutting is complete. If there is no indication that the cut is complete, re-clamp the assembly and continue cutting.

19. When cutting is complete, remove the plastic cutting plate from the saw. Using forceps, remove the sections from the cutting plate, bending the cutting plate backwards can help expose the sections for easy removal. Place the spine sections on a microscope slide labeled with the sample’s complete unique identification number.

20. This is an image of dorsal spine sections from Gray triggerfish, mounted with CytoSeal, on glass slides, labeled with the unique identification number. The slides are stored on the trays of an Eberbach slide cabinet until the CytoSeal has dried. The slides are then transferred to a slide box for about two weeks to allow the CytoSeal to completely cure. Finally, the slides are transferred to slide drawers for storage until the samples are analyzed.

21. Using a single edge razor blade, and care, the glue is “scraped” off the plastic cutting plate, allowing the plate to be used again and again. Even if you nick or “cut” a small hole in the plastic plate during glue removal, the plate is still reusable. Simply reorient the plate for the next spine mounting session. I have found it easier to remove the glue, without nicking the plastic, by doing this step during the cutting process (i.e. While one sample is being cut; the sections from the previous sample are adhered to the labeled slide, remnants are removed and returned to the sample envelope and the glue is removed from the plastic cutting piece. ) The small amount of water remaining from the cutting process acts as a bit of lubricant during the glue removal. This step can be completed when all the samples have been cut, just use a bit more care.