Lab Notes

Lab 1.2 Tools I and 2.2 Tools II

Learning Objectives

• Describe basic anatomy and physiology of suspension feeding in blue mussels.
  • Anatomy:
    • Belongs to the bivalve class of Mollusca.
    • Possesses two hinged valves (shells).
    • Has a mantle that surrounds its body cavity and secretes its shell.
    • Has a foot that allows it to dig into the slit before it is anchored by secreted byssal threads.
    • The mussel opens and closes its shells by contracting its adductor muscles to suspension feed.
  • Feeding:
    • Blue mussels feed by pumping water containing suspended particles (i.e., algae) through its inhalant siphon and across the ctenidia or gills.
    • Movement into the mussel is facilitated by a fringe of cilia called papilla.
    • As particles traverse grooves of the filamentous gills, the food is coated by mucus secreted by gland cells present on the filamentous gills.
    • Food is partitioned and shunted to the labial palps where it is sorted as food and non-food.
    • Food rejected is called pseudofeces; food that is directed from the labial palps to the mouth for processing through the digestive system exits as feces.
• List and describe the impact of biotic and abiotic factors on suspension feeding in blue mussels.
• Properly collect observations of blue mussels using the following laboratory tools: micropipette, compound microscope, balance, centrifuge, spectrophotometer, pH meter, and Vernier caliper.
• Describe how to make solutions from either solid chemicals or liquid stock solutions (includes serial dilutions and percent solutions). Include proper equipment, labware, and accurate quantities.
  • Dilutions:
    • How would you make 8 ml of a 1:20 dilution? Assume water is the solvent.
    • Record the volume of the stock solution and the volume of water in the boxes provided on your answer sheet. Show your work.
    • $C1V1=C2V2$
      • $C1$= 1 concentration of stock
      • $V1$= ? volume of stock to add to final solution
      • $C2$= 1/20 concentration of final solution
      • $V2$= 8 ml volume of solution
    • $(1)(v1)= (1/20)(8ml)$
    • $V1= 0.40 mL$
    • Solution: add .40 ml of stock (v1) to 7.6 ml of water (V2-V1) to make 8 ml (v2) of the final solution at a dilution of 1:20 (c2).
• Determine an appropriate hypothesis for a suspension feeding experiment based on relevant background information.
• Record, analyze, and plot data from a suspension feeding experiment using Excel.
• Produce a standard curve and use the curve to determine concentration of an unknown Solution.
  • Making a standard curve: concentration vs absorbance values
  • Absorbance vs time
  • $y= mx+ b$
  • $y$= absorbance (measured from unknown sample)
  • Shellfish diet concentration vs time standard curve: concentration (cells/mL) vs absorbance values (600 nm)
• Vocab:
  • Seston: particles suspended in water, includes: inorganic compounds, detritus (dead organic matter), various species of phytoplankton and plankton algae

Pipette Vocabulary

• Pipetting pushbutton: micropipettes plunge has “two stops” often
  • First stop: allows you to take in and expel solutions
  • Second stop: used to expel the last drop of solution from the tip
  • Ejector pushbutton: used to eject the tip from the end of the pipette into the waste receptacle

Spectrophotometer Vocabulary

• Spectrometer: produces flight of a selected wavelength
• Photometer: measures light intensity
• Monochromator: selection of light at one particular wavelength is created using a prism or diffraction grating
• Cuvette: made of glass for visible light and quartz for ultra violet light
• Detector: photodiode that measures the intensity of light transmitted and sends a voltage signal to a display device
• Transmitted light: amount of light that shines through the sample (scattered) light
• Absorbed light: amount of light that does not shine through the sample or is blocked by the sample

Microscope Vocab

• Ocular lens:
• Turret (nosepiece)
• Stage: where microscope slides are mounted
• Condenser lens: below the stage, focuses light from the illumination system
• Iris diaphragm: regulates the amount of light passing through the condenser lens (can be opened or closed)
• Arm: supports the body tube and objective lenses on the nosepiece and stage
• Base: stabilizes the microscope

Spectrophotometer

• Turn on and let warm up for 20-30
• Fill a blank control
• Set wavelength to 600
• Zero the spec
• Blank the spec
• Measure your absorbance
• Use kimwipes to keep the outside of the cuvettes clean
• Accuracy: +- 0.001 absorbance, usually only read to 3 decimal places

Microscope

• Objective: stage in lowest position and 4x lens
• Mounting: mount the slide by holding the slide amr open and placing the slide on the stage w coverslip up and use the stacked stage knows (x-y travel knobs) below the stage on left side of scope
• Illumination: turn on the light using brightness dial on the right of the microscope
• Close the diaphragm at lower magnification, open at higher magnification
• Focus the condenser lens: rotate the top of the condenser lens just below the stage
• Focusing: center the specimen always start w lowest power objective lens and observe the objective lens from the side as you raise the stage to highest position
• Use coarse knob to lower the stage and the fine to maximize resolution
• Ocular lens adjustment:
  • Interpupillary distance spacing adjustment
  • Left eyepiece diopter ring
• Brightness: adjust the diaphragm using the level
• Magnification: calculated by multiplying the ocular lens (10x) bt the objective lens (4x, 10x, 40x, 100x)
• Only go to higher magnification when well focused at low power
• Images are inverted and reflected, ‘image inversion’ and reversal

Lab Questions

• What is the conversion factor for μl to liters? ml to liters? Place your answers in scientific notation. For example, 1 μl is $10^{-3}$ ml; and 1 ml is $10^{3}$ μl.
  • 1 μl is $10^{-6}$ L; and 1 ml is $10^{-3}$ L
• Which micropipette(s) would you use to measure the following volumes?
  • 0. 018ml → P100
  • 0. 1ml → P100

• Describe the direction of movement of the dye particles. Use these terms in your response: anterior, posterior, demibranch, ctenidia, labial palps, mouth, margin, cilia, and filaments.
  • The dye particles move along the right and left ctenidia. The dye began in the crease of the gills, anterior to the labial palps, and began to travel downward along the outer and inner demibranch towards the mouth of the muscle. The dye also moved along the mantle margin toward the branchial septum, traveling posteriorly. The dye moved from the posterior to the anterior of the muscle, similarly moving in the ventral and dorsal direction along the demi branches. The dye is propelled by cilia along the muscle’s filaments.
• What is responsible for movement of the carmine dye across the ctenidia?
  • Diffusion from areas of high to low conversion powered by cilia.

Recitation Notes

• Recitation notes on mussel feeding behavior:
  • Suspension feed on plankton brought into mantle cavity using inhalant siphon
  • Sort food coated in mucus at labial palps; discard non-food as pseudofeces via inhalant siphon; food processed in GI tract; feces exits through anus into the exhalant siphon
  • Feeding and filtration rates are concentration dependent (1-5 mg/l) and temperature dependent (5-20 C)
• Match the tool and its purpose:
  • Spectrophotometer: measures turbidity or concentration (absorbance at specific wavelength)
  • Vernier caliper: measures the size of an object or organism to 0.01 cm
  • Graduated cylinder: measures the volume of a liquid
  • Centrifuge: separates mixtures based on density
  • Excel: analyze and visualize data, create graphs, stat calculations and generating trends
  • Microscope: view images at specific magnifications
  • Hydrogen ion concentration: pH meter

Clicker Questions

• Which structure in the Mytilus edulis serves mainly to sort food from non-food and send the food to the GI tract for digestion?
  • Cilia
  • Filaments
  • Demi Branches
  • Labial palps
  • Mantle

Lab 03: SLP and Intro to Isopods

SLP Learning Objectives

• Activity 1A: Locating your assigned SLP Article
  • Identify the components and the format of a BIO204 literature reference
  • Locate a peer-reviewed journal article using the SBU library eJournals.
• Activity 1B: Applying the Reading Strategy
  • Read scientific journal articles using a reading strategy.
  • Identify the sections in a scientific journal article (introduction, methods, results, discussion, conclusion, and literature cited) and describe their content.
• Activity 2: Introduction to Isopods
  • Identify and compare the major body parts of external gross isopod anatomy between terrestrial species.
  • Apply proper terminology to location and orientation of an organism's (i.e. isopod, rat, etc.) anatomy.

Isopods

• Evolved from aquatic environment to terrestrial environment, faces challenge of desiccation. Most groups evolved a waxy coating to protect themselves from water loss. When threatened, can roll into a ball and expose only hard chitinous dorsal surface. Many isopods are omnivorous scavengers.

Body

• Divided into three main regions or tagmata: the head (cephalothorax), the thorax (pereon) and the abdomen (pleon).
  • Pereon: contains seven segments, each with a set of jointed limbs called pereopods. The dorsal surface (tergite) of each segment of the pereon may be smooth, or have texted bumps (tubercles).
  • Pleon: contains 5 segments. The final segment of the pleon is the pleotelson which has a pair of uropods (good identifying characteristic- vary in shape and length).
• You should be able to apply the following directional planes and the anatomical terms to the images below: frontal plane, sagittal plane, transverse plane, anterior, posterior, dorsal, ventral, cephalic, rostral, caudal, medial, lateral, distal, proximal.
• Activity 3: Identification of Isopod Species
  • Apply a dichotomous key to determine the species of an isopod.

Dichotomous Key

• Tool used in science to identify and organisms by its specific epithet or genus species name. There are keys for living organisms and nonliving organism (rocks). Careful observation of gross anatomy is essential to the effective use of a dichotomous key. The presence or absence of characteristics direct the identification of the organism
• Activity 4: Exploring Isopods
  • Conduct humidity experiment using choice chamber and record observations (as a section)
  • Conduct an isopod humidity and thigmokinesis behavior experiment and record observations.
  • Measure the size of an organism using ImageJ software.
  • Terrestrial isopods are usually found in moist environments because they breathe through pseudo-lung structures that require moisture.
  • Thigmokinesis is a response to touch or contact with a predator, and may vary by organism within a species.

Clicker Questions

• What was the purpose of the standard curve in lab 2?
  • Convert from absorbance to transmittance using the beer-lambert law
    • False: beer lambert relates absorbance and concentration (not transmittance)
  • Determine the number of algal cells that mussels consume over time
    • Correct: standard curve quantify the concentration of algal cells in the sample and helps us calculate the consumed algal cells
  • Convert the absorbance of algal cells to concentrations of algal cells
    • Correct: Standard curve plots absorbance vs concentration of algal cells, using the equation of the standard curve we can determine the concentration of algal cells
  • Calculate the ratio of algal cells in the stock to the number of algal cells at time “0”
    • False: curve determines concentration first and foremost, calculations must be done with the standard curve equation to get algal cells
  • B and c
• What statement about SLP “citations” is incorrect?
  • A. Online images used in my poster should be added to my reference list.
  • B. Additional articles (other than my assigned paper) should be added to my reference list.
  • C. I can add my assigned paper’s citation on my title slide.
  • D. No in-text citations are required, if I only reference my assigned paper in my presentation.
  • E. If I use additional articles, I should include in-text citations for each.

Lab 4: Comparative Anatomy

• Demonstrate proper use of dissection tools and anatomical terminology to explore the anatomy of an organism.
• Locate and identify the major structures of the earthworm, rodent, and terrestrial isopod that are essential to GI, excretory, circulatory and respiratory systems and describe and compare the function of each.
• Discuss important structure-function relationships in the terrestrial isopod that will be important for behavioral studies later in the semester.
• Activity 1: Dissection Preparation
  • Dissection Strategy
  • Proper use of dissection tools
  • Body planes and anatomical directions
• Activity 2: Earthworm Dissection
  • Identify earthworm organs and their function
• Activity 3: Rodent Dissection and Exploration Modules
  • Identify rat organs and functions
  • Compare three systems (GI, excretory, circulatory) across three organisms (earthworm, rat, terrestrial isopod).

• Anatomical Planes and Directions:

• Match each organ system with its function:
  • Gastrointestinal: ingestion, digestion, absorption and egestion of food,
  • Excretory: production of urine, production and excretion of nitrogenous waste, salt and water balance, production of feces
  • Circulatory: distribution of molecules, transport materials through the body
  • Respiratory: gas exchange between $O2$ and $CO2$
• Comparison of Respiratory Systems:

• Dissecting Tools:
  • Dissecting needle: used to carefully separate, dissect and manipulate tissue without ripping. Needle is to be used secondary to blunt edged tools because it is more likely to cut the tissue. Good for tearing connective tissue/separating organs. This is better to use in dissections of a rat vs. an earthworm
  • Blunt probe: used for separating tissues, organs and glands from one another. Less likely to tear the tissue than a sharp tool (ie: needle). Good for probing and moving organs out of the way without tearing
  • Forceps: lift the body wall away from vital organs prior to curing, pull skin out of the way. Forceps are not sharp so the skin will not tear but be careful of pulling too hard
  • Scissors: superior to scalpels because they allow more controlled and precise incisions. Smaller set to be used for earthworms. Can cut skin/organs to gain access to other organs
  • Cutter: after initial incision with scissors cutters can be used to open the muscle and expose the internal organs. Used for rodent ribcage
• Anatomical Planes: * 3d organisms have 3 possible planes: * Transverse: called axial and horizontal plane (humans) * Sagittal: when positioned at human midline, is called the median (longitudinal) plane. When positioned to one side of the midline, is called a parasagittal plane * Frontal: called coronal plane in humans definition (and when applied to human/roden/earthworm//isopod) * Dorsal The back or the direction towards the back. The side that has a backbone for human and rodent. The top for an earthworm and isopod. Opposite of ventral * Ventral The front or the direction towards the front. The belly/underside for human, rodent, earthworm, and isopod. Opposite of dorsal * Distal For a limb or protruding structure, a position that is further away from the point of attachment. Opposite of proximal * Proximal For a limb or protruding structure, a position that is towards the point of attachment. Opposite of distal * Medial Towards a centerline or middle of a structure, typically of the body. Opposite of lateral * Lateral Away from a centerline or middle of a structure, typically the body. Opposite of medial * Anterior Confusing anatomical term and depends on animal. Opposite of posterior * Human: synonymous with ventral * Rodent: synonymous with rostral * Earthworm and Isopod: towards the mouth * Posterior Confusing anatomical term and depends on animal. Opposite of anterior * Human: synonymous with dorsal * Rodent: synonymous with caudal * Earthworm and Isopod: towards the tail or anus * Superior Used with humans to mean up, above, or to refer to structures that are higher. Opposite of inferior * Inferior Used with humans to mean down, below, or to refer to structures that are lower. Opposite of superior * Rostral Used with rodents to mean towards the nose. Opposite of caudal * Human: typically not used * Caudal Used with rodents to mean towards the tail. Opposite of rostral/cranial * Human: synonymous with inferior * Rodent: synonymous with cranial and anterior * Earthworm and Isopod: synonymous with anterior * Rodent: synonymous with posterior * Cranial Towards the head. Typically not used with earthworms or isopods. Opposite of caudal * Human: synonymous with superior * Rodent: synonymous with rostral and anterior
  • Deep Refers to the center of a structure or organ.
  • SuperficialRefers to the surface of a structure or organ.