SOP Histology PST

8/3/2019 SOP Histology PST

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SOP: Histology of Pyloric Stomach

Introduction

Repeat spawning in summer-run steelhead trout (Oncorhynchus mykiss) is a source of heightened attention in the area of

fisheries management and conservation efforts. Greatly limited to aquaculture (See Caballero 2001; Glencross 2004,

2006; Pereira 2002), the literature has largely failed to explore histological significance as it directly pertains to iteroparity

in kelts. Unlike analyzing blood plasma, to which chemical concentrations (triglycerides, lipids) can quickly fluctuate

depending on time and rate of metabolic activity, histology provides more of a static understanding of how external

factors directly impact specific organs; which are made up of tissues and highly indicative of health. Specifically, this

method has two aims; a conceptualization of which tissues are ultimately indicative of a kelts ability to spawn more than

once and to what extent, if any, can histological methods measure and explain the tendency for kelts to recover post-

spawn

Significance/Purpose

This SOP serves as a histological approach to understanding the changes in tissue microstructure of the pyloric stomach in

spawning and post-spawning in steelhead trout (Oncorhynchus mykiss).

Anatomy

Definitions

Esophagus- Short and expandable with muscled walls so that food can be swallowed

Cardiac Stomach- Aids inphysical digestion; storage for food as it exits esophagus, can be seen as little teeth whichchops food up

Pyloric Stomach- Aids in chemical digestion; produces enzymes from gastric glands that release substances which break

down food to prepare it for digestion

Pyloric Caeca- Absorbs nutrients and secretes digestive enzymes

Anterior Intestine-Digests and absorbs nutrients

Posterior Intestine-Absorbs water and excretiates residues from digestion

Credit: Zach Penney


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Histology

Pyloric Stomach- Attached to the cardiac stomach, the primary function of the pyloric stomach is its ability to produce

digestive enzymes which aids in digestion. The stomach is comprised of multiple rugaes or folds lining the stomach. The

rugaes expand providing space for food items without generating increased pressure on the stomach. Lining the rugae in

most stomach tissues is the surface epithelium comprised of mucous cells. The presence of epithelial cells tend to resist

attacks by digestive enzymes and acids. Specifically, the mucus secreted by the cells is compositional in a way to comba

digestion. Furthermore, this work postulates that the existence of epithelial cells and more importantly, the lamina

epithelialis, are a primary indicator of the level of metabolic activity in kelts. From a histological standpoint, the apical

mucous droplets secreted by the epithelial cells tend to show an affinity for eosin, specifically staining a light pink color.


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Artifacts

Artifacts such as tears and breaks in tissue sections, creases or folds in the tissue (darker lines across the slide), and

uneven stains (blotches of darker color) can be found on many prepared slides. If artifacts appear in selected images, it is

important to note and not to interpret as authentic histological structures. shows typical structures which demonstrate the

histological integrity of the specimen.

Materials/Equipment

1. Compound light microscope2. Lecia Application Suite LAZ camera and computer software3. Histology Slides; pyloric stomach, PST-2009,2010 (Prepared by Colorado Histoprep in Fort Collins, CO)

Parametersa. Female, good external conditionb. Sample size: 20

1. Lower Granite (2009;LG 816, 822, 823, 970, 975, 1001, 1003, 1004, 1005, 1201)2. Dworshak (2010;DWES 2-2, 5-2, 6-2, 9-2, 10-2, 11-2, 13-2, 14-2, 15-2, 17-2)

Hypothesis

1. The tissue structure (specifically the cross sectional area of the folds in the rugae comprising the epithelial cells aswell as the presence of atrophied epithelial cells) can serve as a distinction between spawning and post-spawning

kelts.

2. The tissue structure may correlate with a kelts potential to recover post-spawn.


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Procedures

1. Calibrate Camera for Histological Assessments (See

Appendix B for instructions)

2. Select histology slide (preferably starting with LG 816) and

place it on the stand

3. Considering the specimen cannot be viewed under the

microscope as a whole, it is best to move the knob (whichcontrols the X,Y movement of the stage) up and down in a zig

zag formation slowly across the slide.

4. Locate the rugae on the slide (Note: each slide ranges from 3-

6 ruga) and capture a picture of each.

5. Since this method primarily evaluates relative area, measure

length/width of each ruga and, if applicable, choose the three

largest areas out of the set for analysis.

5a. When measuring the ruga, begin by using thedistance line funciton (Appendix A) to measure the base

of the ruga (dotted line).

5b. Once the distance is calculated (microns), find the

midpoint of the line (total distance/2)

5c. Draw a line perpendicular to the midpoint of that

line. That will be the length of the ruga.

5d. Repeat step 5b. in order to calculate the width of the

line horizontally.

5e. Record these measurements on the data sheet


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6. Label the quadrants counterclockwise (shown below). This step is crucially important all of the quadrants must be

labeled counterclockwise.

7. Locate two suitable folds (as demonstrated earlier) within each quadrant for analysis. Although not histologically

necessary, it would be beneficial to empirically locate the longest and shortest (length-wise) in each quadrant to measure.

8. Once an appropriate section of histological integrity is

determined at a cross-section, the cross sectional area can be

measured. Choosing a section which has two visible pits

regardless of equalness in distance (Figure on Right PA, PB) can

aid in closely measuring distances as close to a quadrilateral withfour right angles (evidenced by dotted lines). The optimum

magnification for measurement of length and width should be in

the range of 100-300. Although the camera is calibrated in unit

microns to scale at lower magnifications (such as 40), the

degree of accurateness and the chance of human error in using the

distance line function can be lessened with an increase in

magnification.

9. Record all data and calculate areas on data sheet.


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Appendix A.

Distance Line

1. After the picture is captured, double check under the Browse option to make sure that the Calibration option has a

pixel set to unit microns ( ___ pixels = ___microns). If it does not show then the camera is not calibrated and no measures

will be calculated.


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2. Under Process, the Annotate tab will display a option for Line. Click the box next to Show

3. Next to Showon the right there will be various line options. Scroll down until Distance line appears.

4. One Distance line is clicked, a line can be drawn by clicking and dragging on the picture to measure desired length.

The measurements will appear as soon as the line is drawn

5. The color and width (in numerical measures) of line can be changed under the Line option as well


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Appendix B.

Instructions for Calibrating Microscope for Measurements

1. Startup Lecia Application Suite LAZ Camera and turnon microscope switch

2. Set light source on microscope (between 5-7)3. Optimum Exposure Adjustments

Exposure: < 50ms Gain: 1.2x Gamma: 1.20 Saturation: 220 24

4. White Balance Place a white item (preferably a fully white and

folded sheet of paper or back of a white businesscard) on the stage of microscope

Make sure the camera feed shows a white screen Press the button shown below to apply white

balance to entire screen

5. Calibration for MeasurementOptionsPreferencesMagnificationName (eg.; 10x, name of specific area)Magnification (total:

eyepiece x lens)

SOP Histology PST

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