Marine Invertebrate Zoology
Protocol Laboratory
Introduction
This lab is designed to familiarize students with common procedures and protocols in a scientific laboratory
setting. In addition, we will make modifications to certain tools in our dissection kit and create additional
microdissection needles. Much of this lab has been adopted from Dr. Richard Fox from Lander University.
Observational Records
Students in the sciences should develop the habit of keeping accurate, up to date, and informative records of
observations. It is a mistake to rely on memory for recollections of even the most impressive events fade
rapidly and become unreliable.
Throughout this class you will be required to make sketches of dissections and organisms. Recognizing
important and noteworthy features is an important skill to develop. During class if you have specific questions
on what is required for your drawing be sure to ask.
The primary objective of drawings is to render an accurate image of the organism or structure under
observation thereby providing a reliable record for future reference. Drawing may be difficult for you at first
but, regardless of talent, you should be able to execute useful drawings by the end of the course.
General Rules for Scientific Drawings
1. Make a general inspection of the object to be drawn and plan the layout of your
drawing. The drawing should fit comfortably in the space available on the paper. You may
need to enlarge or reduce it to fit it onto the page. It should not crowd the space nor should
it be lost in it. Be sure to save room for labels. Drawings should always be made in pencil.
2. Make the final drawing using firm, strong, solid lines made with a sharp, hard pencil. Avoid
hesitant, timid, sketchy, or fuzzy lines. Make sure all lines connect with something and that
the connections are clear and distinct. Avoid hiding your confusion about relationships in a
tangle of lines.
3. Label the drawing carefully and completely. Be sure they are written horizontally. Connect
the labels with the appropriate structure in the drawing with a ruled straight line. Do not
use arrowheads at the end of the lines. Do not let any of the lines cross each other on the
way to the object.
4. Label the entire drawing at the center of the top or bottom of the drawing. For microscopic
animals include an indication of the magnification at which the drawing was made.
5. Keep in mind that your goal is to produce an accurate record of your observations, not
simply to render an impression of an object's appearance. Consequently, you should draw
what you see and should not adopt conventions or symbols that represent structures
without depicting them accurately.
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Dissection
Invertebrate zoology is one of the few opportunities in the modern undergraduate biology curriculum to
develop the skills needed to conduct a firsthand study of animal morphology.
Dissecting Techniques
In general, the object of any dissection, whether of a large or small animal, is to expose tissues, organs, and
organ systems in their natural condition and with their natural relationships intact. Realize that the tissues,
especially those of living animals, are very delicate so you must develop the habit of handling them
carefully. Avoid making random, vague, and unnecessary movements with your tools and do not introduce a
tool into the specimen unless you have a specific task for it.
Contrary to a common misconception, good dissection involves minimal cutting. Instead, you should strive to
expose structures intact, without cutting them or nearby tissues, whenever possible. Most dissection consists
of separating the tissue around organs to free it from nearby structures using teasing needles, forceps, and a
blunt probe. When you are finished, you should be able to see clearly the organ of interest and perhaps lift it
and look at all its surfaces. The organ can be moved aside (reflected) to reveal deeper organs but can be
returned to its original position when desired. A good dissection reveals, accentuates, and clarifies, but does
not alter, the natural spatial relationships between structures.
The blunt probe in your dissecting kit is one of its most useful instruments. Its major use is to manipulate
structures, move organs aside, or lift them. It is ideal for separating adjacent organs as it is least likely to
puncture or tear delicate tissues. It is especially useful for tracing channels and passageways. Its tip should
not be over 1 mm in diameter.
The scalpel is the favorite instrument of beginning students, perhaps because it symbolizes the glamour of
surgery. In reality it is the least important tool in your kit and the one with which you can do the most
damage to the natural shape and relationships of the animal. It is a very specialized tool with specific
functions. Neither of your two cutting instruments, scissors nor scalpel, should be used often, but when
cutting is necessary, scissors are almost always the tool of choice. With scissors you have greater control over
direction and depth of cut than with the scalpel. In addition, scissors provide their own firm cutting surface
whereas a scalpel pushes soft, unsupported tissue out of its way instead of cutting it. Never use the scalpel as
a probe or pointer.
When it is necessary to cut an organ in order to expose underlying structures, it should not be removed
entirely. Instead, use needles and forceps to free the superficial organ from the surrounding tissues. Once it
is free, lift it in its middle and cut cleanly across it with the scissors. The two opposite ends will retain their
natural connections with the body but can be reflected to expose deeper tissues. When it becomes desirable
to see the superficial organ again in its original condition, the two ends can be moved back into place.
In any dissection the directions “right” and “left” ALWAYS refer to the animal's right and left. This may or may
not correspond to your own right and left depending on your orientation with respect to the specimen.
In almost all cases, specimens should be dissected under liquid rather than dry. The liquid should completely
cover the specimen. This supports the tissues, prevents desiccation, and eliminates reflections from glistening
surfaces. Obviously, the fluid should be appropriate to the circumstances. Living or fresh marine animals
should be in seawater or whatever relaxant, usually isotonic magnesium chloride, is used to anesthetize
them. Living freshwater and terrestrial animals should be in tap water, relaxant, or isotonic saline. Preserved
specimens should be in tap water.
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Dissecting Tools
The dissecting set for invertebrate zoology should contain the standard instruments found in high quality sets
used in freshman biology or comparative vertebrate anatomy plus a few tools suitable for small
specimens. The tools should be stainless steel when possible. The standard dissecting set should include:
1 pair 6" surgical scissors with one blunt, one pointed blade
1 pair heavy forceps
1 # 3 scalpel handle with disposable # 10 blades. (Numbers 11, 12, 15 also fit the # 3 handle.)
1 straight teasing needle with wooden or plastic handle
1 bent teasing needle with wooden or plastic handle
1 15cm plastic rule
2 1 plastic or wooden box to contain the tools
In addition you will need the following:
1 pair fine-pointed watchmaker's or microdissection forceps
2 2 minuten nadeln on applicator sticks, one with straight point, one bent
Cloth towels (provided by the college)
1 pkg #1 insect pins, preferably stainless steel (provide by the college)
The minuten nadeln on sticks will be made in class.
The microdissecting forceps will be provided by the college as needed, and are expensive and delicate and
should be handled carefully. The points must be protected from damage and you should make every effort to
avoid dropping them. With the tips open, push them into a #1 cork. The forceps should always be stored with
the cork in place.
After each use, your tools should be washed with warm water, rinsed, and dried thoroughly. Properly cared
for, a dissecting set with high quality tools will easily last through your student years and beyond.
Absorbent terrycloth towels are useful in the laboratory. A towel spread flat beside the dissecting microscope
serves as a staging area for wet dishes before they are placed on the microscope. It will absorb any liquid, such
as seawater or magnesium chloride and prevent its coming in contact with the microscope.
Before beginning work, remove all of your tools from their case and array them on the towel ready for
use. Another towel can be used to dry your hands and dissecting tools.
Wetmounts
Some samples can be placed directly under the microscope; however, many samples are more easily viewed as
wet mounts. In a wet mount, the specimen is placed in a drop of water or other liquid held between the slide
and the cover slip by surface tension. This method is commonly used, for example, to view microscopic
organisms that live in water or other liquid media, especially when studying their movement and behavior.
Preparation of a Wetmount
Place one or two drops of liquid in the center of a clean glass microscope slide. Add stain or other materials as
appropriate and mix thoroughly with a teasing needle. Do not add more than a total of 2-3 drops of liquid.
Carefully lower a clean coverslip onto the liquid in such a way that no air bubbles are trapped beneath the
coverslip. This is best accomplished by placing one edge of the coverslip on the slide touching the
liquid. Support the opposite edge of the coverslip with a teasing needle or forceps and slowly lower it to the
surface of the slide.
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Use # 1½ or # 2 square 22mm coverslips for ordinary microscopic examination. Use # 0 coverslips if you plan
to use oil immersion. These are thinner and much easier to break so you must be more careful with them.
It will probably be necessary to adjust the amount of liquid on the slide. There should be no liquid outside the
coverslip but there should be enough under the coverslip to support it so it does not stick tenaciously to the
slide and crush or distort organisms. If you have too much liquid, use a tissue to absorb the excess. If you do
not have enough, use a pipet to place a drop or two on the slide next to the edge of the coverslip. The liquid
will run beneath the coverslip if it is needed. If it is not needed, you will have to remove it with tissue.
Excess liquid on the slide will run off the slide onto the stage where it will form a thin film between the slide
and stage. This film acts like glue and its presence makes it impossible to move the slide smoothly across the
stage and almost impossible to move it at all. It may also damage the microscope, especially if it is a salt
solution.
Alteration of a Wetmount without Removing Coverslip
It is frequently desirable to change the liquid below the coverslip of a wetmount without removing the
coverslip. This is easily accomplished and can (should) be done while you observe the organism.
To accomplish such a change, place a drop of the new fluid on the slide next to and touching one edge of the
coverslip. Touch the edge of a piece of absorbent tissue paper to the opposite edge of the coverslip so it is in
contact with the old liquid beneath the coverslip. The old fluid will be absorbed by the paper and removed
from the wetmount. The new fluid will move under the coverslip to replace the old as it is removed.
Micro Teasing Needles
Very fine needles, known by the German minuten nadeln, or the English microneedles, mounted at the ends of
wooden applicator sticks (Fig 1) are essential for manipulation and dissection of small animals and are easily
prepared. They are not indestructible and must be replaced periodically.
Fabrication of a Microneedle
1. Split, with the grain if possible, 2-3mm of the tip of an applicator stick with a scalpel. Do this safely by
placing the stick on the surface of the table and pushing the blade of your scalpel into the side of the
end of the stick (not the end of the end). Do not hold the stick in your hand while splitting it and do
not cut toward yourself.
2. Insert about 2 mm of the blunt end of a 0.2mm microneedle into the cleft, applying a drop of waterinsoluble glue to the end of the stick around the cleft and base of the needle, and wrapping it tightly
with sewing thread. Twirl the end of the stick between your thumb and forefinger to spread the glue
over the thread, end of the stick, base of the needle and into the cleft.
3. Make at least two miocroneedles, one with a straight tip and the other bent at a 45° angle (Fig 1).
Color-code your two miocroneedles. This makes it easy to select the desired needle without having to
look closely at the tip.
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Figure 1. A micro teasing needle made from a minuten nadel and an applicator stick.
Be careful when working with these tiny needles. Microneedles are tiny and are easily lost at the workbench.
Lost needles have a habit of ending up later sticking into fingers or elbows.
The delicate needles are easily bent but are almost as easily restored to their original condition. If the tip gets
turned over into a tiny hook, it can be straightened by pulling it between the nail of your forefinger pinched
tightly against the ball of the thumb, or between the points of your forceps.
Forceps
The tips of new forceps (especially inexpensive ones) often require modification before they are suitable for
use. This is accomplished by grinding excess metal from the tips with a sharpening stone such as an Ouachita
or Arkansas stone (available from Fine Science Tools or a hardware store).
Modification of Forceps
1. Hold the blades of the forceps together lightly so the opposing tips touch each other but do not press
firmly against each other.
2. Look at them with the dissecting microscope. Do they meet each other in perfect alignment or do they
pass each other or attempt to do so? Are the two tips the same length? Are the tips as sharp as you
would like? Perhaps they are too sharp?
3. You can correct the alignment and produce the shape you want with the sharpening stone. Hold the
tips of the forceps lightly together and slide the point back and forth over the surface of the stone
against the metal you want to remove. These stones will remove metal rapidly so check the tips
frequently to be sure you are not removing too much.
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4. If the tips meet each other at a spot behind the tip, it will help to bend the tips a little. Never try to
remove metal from the inside of the tips.
5. If one tip is longer than the other the forceps will be unusable until corrected. Hold the tips together
and rub the point of the combined tips together on the stone. Check the lengths frequently and stop
grinding when the two are equal.
6. For storage push the tips into the end of a small cork and keep them there when not being used.
Dissecting Pans
Standard dissecting pans available from biological supply companies are too large to be used conveniently with
most invertebrates. Dissecting pan wax can be purchased from Wards Natural Science Establishment. The
wax is melted in a saucepan dedicated to that purpose (it will be ruined for other uses) and the molten wax
poured to an appropriate depth into the chosen container.
The wax should be poured so there is ample freeboard above the wax to more than accommodate the
thickness of the specimen and the fluid in which it will be immersed. Because it does not rust, aluminum pans
are preferable to steel. The college will provide pans and wax for this lab; you will be required to make the
pans.
Pinning
Most dissections of soft-bodied invertebrates require that you pin the body wall aside to reveal the body cavity
and its viscera. In most instances # 1 insect pins are appropriate but for very small or delicate animals # 000
may be preferable whereas for larger specimens # 4 may be best. The pins should be stainless steel, especially
if they are used in seawater or magnesium chloride. High quality stainless steel insect pins are available from
Fine Science Tools.
Insert the pins at 45 ° angles through the body wall and push them firmly into the wax of the dissecting pan.
Think ahead before you insert the first pin and arrange the position of the specimen in the pan so it, the
specimen, will be conveniently located after all pins are in place. The animal should be positioned so it will be
visible with the dissecting microscope and so none of it extends beyond the edges of the pan.
Compound Microscope
Light Adjustment
When using the compound microscope, the light intensity must be carefully adjusted. There is a tendency
among beginning students to use too much light. Transparent objects, such as Amoeba, will be invisible if the
light intensity is too high.
In general, light intensity should be adjusted using the iris diaphragm, not the electrical rheostat control. Set
the rheostat control at about 75% of maximum and leave it there. Use the iris diaphragm to adjust the light
intensity. Sometimes it will be necessary to use the rheostat, as for example, when using the scanning lens.
Initial Focus
Finding the correct level at which to focus is difficult when most of the material under the coverslip is
water. In the absence of a conspicuous object, there is nothing on which to focus. Use the edge of the
coverslip as an easily-found "object" at the correct level and focus on it first, then look for the small objects
you are really interested in, secure in the knowledge that you are focused at about the right level.
Systematic Scan
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Finding small and/or scarce organisms on a slide is most efficient if done systematically. First, be sure the light
is adjusted properly and the microscope is focused at the correct height (i.e. between the coverslip and the
slide). Organisms are difficult or impossible to see if the light is too strong.
Scanning Guidelines:
1. Using the scanning lens, find one corner of the coverslip, say the upper left.
2. Move the slide so you follow the upper edge of the coverslip to the upper right corner.
3. Move down a distance equivalent to a little less than the diameter of one field and then move
back to the left side of the coverslip. Your new field should just overlap the already scanned strip
next to the edge of the coverslip. Be sure there is no unexamined space between the two strips.
4. Continue moving back and forth across the slide, neglecting no part of it, until you find what you
are looking for or you reach the lower right corner.
Optical Sections
When observing organisms with the compound microscope, keep the fine focus in nearly continual motion,
focusing up and down through the object. Each position of the fine focus gives you a crisp focused image of
one level of the object, much as if you had sliced, or sectioned, the object at that level. The resulting view is
known as an optical section. Other levels are out of focus and cannot be seen clearly. To get an accurate
impression of the entire organism you must take optical sections at all levels by continual refocusing up and
down.
Oil Immersion
To use the oil immersion lens, focus on the object of interest with the high dry lens and be sure the object is
centered in the middle of the field. Rotate the nosepiece half way to the oil immersion (100X) objective. Place
a drop of immersion oil on the slide directly atop the center of the hole in the stage. Slowly move the
nosepiece around until the oil immersion lens clicks into position.
Be sure you rotate the nosepiece directly to the oil immersion lens and not to the high dry. If you do the latter,
you will get oil on the high dry lens. Should this happen, clean it immediately with lens paper (only) and no
harm will be done but do not allow oil to remain on this lens.
Look through the eyepiece and focus on the object very carefully using fine adjustment only. When finished
with the oil immersion lens clean it carefully with lens paper. If you are using a prepared slide, clean it
also. Coverslips contaminated with oil are usually not reused and should be discarded. Oil immersion is
usually used without a coverslip or with a # 0 coverslip.
Cleaning Lenses
You should keep the lenses of your microscopes clean. Make it a point to clean the ocular lenses (eyepieces)
of your compound and dissecting microscopes at the start of each laboratory session. The objectives,
however, rarely need cleaning unless they have been accidentally smeared with the liquid from a
wetmount. Use nothing but fresh, unused lens paper to clean lenses and discard it after use.
If you notice specks or smears in your field of view, it means at least one of the many lenses in the system
needs cleaning. You can identify the offending lens using a simple protocol:
1.
2.
3.
4.
5.
Look through the eyepiece so you can see the spots.
Move the slide.
If the spots move, they are on the slide, which should then be cleaned.
If the spots don't move with the slide, try rotating the eyepieces one at a time.
If the spots rotate, they are on one of the lenses of that particular eyepiece.
In this case they are almost always on the outer surface of the upper lens because it is the one
exposed to oily eyelashes, dust, and the occasional fingerprint.
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6. If the dirt did not rotate, it must be somewhere else. Raise or lower the condenser.
7. If the spots go out of focus they could be either on the top of the substage lamp or the condenser
lens. Clean both.
8. If the contamination manifests itself as a blurred image that cannot be corrected by focusing, the
objective is probably fouled. Moisten a piece of lens paper with water and clean the outer surface of
its lens.
Incident and Transmitted Light
Dissecting microscopes are usually equipped with two light sources. The incident light shines down on the
object from above the stage whereas transmitted light illuminates the object from below and passes through
it. The two lights have different effects and different uses and are not interchangeable.
In general, incident light should be used for opaque objects and transmitted light for transparent objects.
Avoid using transmitted light on opaque objects as the contrast between the bright light and the dark object
makes it difficult to see the features of the object.
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Name __________________________________
Review Questions: Protocol Laboratory
1. What critical piece of information should always be included when drawing organisms under
magnification?
2. When using a microscope, what is the difference between transmitted and incident light?
3. What is the best method for removing excess water from a wet mount preparation?
4. As a general rule, when conducting a dissection which instrument is used most frequently?
5. When using the microscope and obtaining your initial focus, what object should you focus on to ensure
that you are on the correct plane?
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