• Day 1
• Day 2
• Day 3
• Day 4
• Day 5
• Day 6

Resources

• *LAB SAFETY**
• Laboratory notebook
• Graphing tutorial
• Molecular biol tips
• Writing assignment
• Bios 211 web site
• Searching literature

Other links

• Bios 111 honor code
• Graphing quiz (rice only)

Bios 111 Day 6 Assignments

Homework due

Today you are to turn in your final draft of materials and methods for days 1-4 (DNA work). The homework from day 5 is also due today. There will be no homework assignments for day 6. Enjoy!

Introduction

The compound light microscope is historically the single most important instrument toward the advancement of life sciences and related fields. Proficiency with a light microscope, including the ability to find any specimen and optimize its image, is by itself a critically important skill for a biologist or biochemist. Such proficiency is also a starting point from which to learn to use more sophisticated imaging instruments such as fluroescence, confocal, deconvolution, and even electron microscopes.

Background

You are probably familiar with the basic student model light microscope, in which one sees the image of one or more specimens as stained or pigmented objects against a white background. These are what we call "bright field" microscopes. The microscope field is the circle of light that one sees even if a specimen is not present.

A problem with a dedicated bright field microscope is that we can only see objects that intercept and modify light coming through an area of a slide. Many living organisms such as bacteria cannot be seen in a bright field microscope because they have no pigmentation and/or are so small that they simply disappear in the bright field of view. Our microscopes are equipped with special optics that not only magnify a specimen but also produce contrast. No living thing on earth is so small that it can escape our detection in one of our microscopes at a magnification of as little as 100x or even 40x.

Experimental overview

A tutorial will introduce you to the features of a research microscope. You will learn how to find specimens, optimize contrast and resolution, and bring images into a working magnification. You will learn to optimize bright field optics and to use dark field and phase contrast optics. You will learn to measure specimens using an eyepiece reticule and will learn to use a hemacytometer to determine cell concentrations in suspension.

Each of you will work individually with a microscope, prepare your own specimens, and practice finding and viewing microscopic targets. Your goal is to develop proficiency with this essential laboratory instrument.

• Tutorial on using the light microscope and its specialized optics to find, observe, and measure specimens
• Learn to prepare a wet mount of a living specimen
• Learn to prepare a cell suspension (Naegleria gruberi) and "charge" and use a cell counting chamber (hemacytometer)
• Examine individual specimens
• Examine a wet mount of Paramecium with stained yeast
• Examine a wet mount of Chaos carolinensis
• Prepare and examine a wet mount of Naegleria gruberi; follow the transformation from amoeba to flagellate
• Prepare and examine wet mounts of pond water specimens

A) Microscopy tutorial (guided session)

We will start with a "guided tour" of the compound microscope, with one microscope per person. We will ask that you not take notes during the tutorial itself. Instead, pay attention to the instructions and on operating the microscope. After the tutorial, instructors will provide the assistance you need, and you will have ample opportunity to take notes on finding specimens and using the specialized optics.

Features of a research compound microscope

We will go through the light path and point out adjustments that should be made along the way.

• Ocular reticule calibration (label on eyepiece tube)
• Working and storage positions
• Illuminator and field diaphragm
• Condenser and focusable rack
• Condenser turret, bright field position, and aperture diaphragm control
• Mounting and positioning a slide using the mechanical stage
• Objective turret and lenses
• Binocular eyepiece tube, focusable oculars, and ocular reticule

How to use the optics to find and view specimens

We will show you an effective, systematic approach to finding specimens and optimizing resolution and contrast for viewing.

• Scanning with the 4x lens, bright field
• Focusing and centering a specimen
• Working up to 100x magnification (10x objective)
• Aperture diaphragm control for contrast
• Dark field optics
• Working up to 400x magnification (40x "high dry" objective)
• Phase contrast optics

Measuring with a microscope – using an eyepiece reticule

A microscope is an effective measuring device, if you know how to calibrate and use it.

• Line up a specimen
• Convert tic marks to meaningful units
• Round results to reflect limits on instrument resolution

B) How to prepare a wet mount of a living specimen

To make observations on living specimens you either need to work very quickly, or you must learn to prepare sealed wet mounts so that the specimens do not dry up in the heat of the microscope lamp.

• Select single thickness slides and cover slips
• Safety, disposal, conservation of materials
• How to prepare a Vaseline chamber

C) How to prepare a cell suspension from an agar plate and estimate cell concentration

Your instructor and teaching assistants will demonstrate how to prepare a cell suspension and how to "charge" a hemacytometer. You will conduct the procedures yourself, and we will then have you count cells and use your cell counts to estimate cell concentration and total number of cells in your suspension.

D) Examine individual specimens

• Examine a wet mount of Paramecium with stained yeast
• Examine a wet mount of Chaos carolinensis
• Prepare and examine a wet mount of Naegleria gruberi; follow the transformation from amoeba to flagellate
• Prepare and examine wet mounts of pond water specimens

About your specimens

Paramecium with stained yeast

As you may know, living species are named using a binomial system. You can think of a species as having a first and last name, with last name listed first. Paramecium, the "last name" of this group of species, is a type of ciliate. The name Paramecium is the genus, or generic name. The second ("first") name is the species name. Notice that by convention scientific names are placed in italics.

Record the name of the species, which in our laboratory can be P. caudatum, P. multimicronucleatum, or P. bursaria. Observe motility and feeding behavior. Try to identify organelles in the ciliates, use the ocular micrometer scale to estimate length and width of a typical Paramecium, and estimate the diameter of a typical yeast cell. We recommend 40x dark field to find the cells initially, 100x bright or dark field to observe feeding behavior and color changes in food vacuoles, and 400x phase contrast for observing cilia and yeast.

Chaos (Pelomyxa) carolinensis

Chaos are the largest member of the Sarconid group, protists that move by amoeboid motion. They are so large that they are easily crushed by a cover slip, and collecting individuals from a culture takes practice. We will provide wet mounts in a proportion of 5-10 Paramecium to one Chaos.

Most amoebae shy away from light. Your Chaos will likely remain stationary until it is placed in the light path in bright field. As the amoeba moves it will shoot out pseudopodia that are well attached to a glass surface (the surface could be the slide or the underside of the cover slip). Pseudopodia are very thin, so one can see a lot of detail. Try using different optics to observe motility by pseudopodium formation and cytoplasmic streaming, including phase contrast.

Try to estimate the volume of a typical Chaos. What assumption or assumptions must be made? Can you measure a cell in three dimensions to come close to an accurate estimate? Hint: What if you know the volume of liquid that was placed on the cover slip? How would you define "accurate" in this context? Try determining the minimum and maximum possible volumes. If you had time, what might you do to narrow the range of possibilities (improve the accuracy of your estimate)? Sometimes accuracy is not possible and/or not necessary. Learn to work within limits. Consider that astronomers and cosmologists sometimes consider it to be a great accomplishment if they obtain an estimate that is accurate within an order of magnitude.

Naegleria gruberi

Naegleria has a remarkable life cycle that you can observe within 1 1/2 to 2 hours, provided that you can go back to the same slide at intervals to observe the progress of transformation. An instructor or teaching assistant will demonstrate how to use a "rubber policeman" or similar cell scraper to harvest Naegleria cysts and trophozooites from an agar surface.

Prepare a Vaseline mount from the Naegleria suspension, as instructed previously. The trophozooites resemble small, fast moving amoebae. You will need to observe them at 400x final magnification, using phase contrast. To obtain the best contrast make the Vaseline film as thin as you can get it.

You might want to take a quick look at your specimen so that you will know what to look for when you go back to it later. Start at 40x dark field and center up a group of cysts. Move up to 100x dark field and look for trophozooites. If the cysts have not been in suspension for too long you may see trophozooites emerging from the cysts. The trophozooites do not have the contrast that the cysts have, so they may be hard to spot at first. Look for slow changes in shape that are characteristic of amoeboid motion.

When you have an amoeba in your field center it up and move to 400x, phase contrast. You should clearly see the difference in contrast between pseudopodia and the main cell body. You may see vacuoles and possibly a nucleus. Once you can find trophozooites and optimize their contrast, please set the slide aside for 30 min or so. You will need to go back to this specimen several times at least, to observe the transformation. Each time, start with low power, looking for different shapes and means of motility. Part of the Naegleria life cycle is a flagellated form. Try to recognize and perhaps sketch each of the stages.

• Cyst – the dormant form is an opaque sphere of 12-15 µm diameter; you may be able to see one or more sealed pores from which a trophozooite will emerge; empty cysts are not as opaque and often have particulate matter vibrating inside them
• The trophozooite stage is the only one during which the organism feeds and divides; the irregularly shaped trophozooites move by pseudopodia, more rapidly than do most true amoebae
• Flagellate – After some time in liquid suspension a proportion of trophozooites transform into an elongated cell of fixed shape, with a pair of flagella; the flagellates move rapidly
• Intermediate stages – watch for trophozooites that have become stationary and rounded in shape – some will begin to wiggle as they develop flagella; watch for trophozooites that exhibit amoeboid motion but have flagella – these cells have reverted from the flagellate stage back to the trophozooite stage and will soon lose their flagella

Mixed freshwater cultures (collected specimens)

Our success in providing interesting materials from natural sources varies with time of the year and with the weather. However, even when a culture is not obviously teeming with organisms, there is plenty to see if you know how to look.

Estimate the dimensions and the volumes of the largest and smallest organisms you can find. What is the order of magnitude of the difference in the long dimension (an order of magnitude is a factor of ten). or example, if the longest species is a millimeter long and the shortest is 1 micrometer (0.001 mm) long, the difference is three orders of magnitude. What is the order of magnitude of the difference in volume? See if you can estimate the order of magnitude of the difference in magnitude between the volume of the smallest organism you observe, and your own volume. You might report the quantity as the number of organisms that could fit inside you if you were hollow. Such measurements are extremely imprecise, so you may find it necessary to round both the raw measurements and the estimates of order of magnitude to a single significant digit.

For several different organisms, use your present knowledge of general biology to assign them to appropriate kingdoms. Record a description of the organism, the kingdom, and your criteria for the assignment to that kingdom.

Epilogue

Why study invertebrates in the first place? Why not focus our studies on human tissues and human diseases? It its purest form, science seeks to understand nature regardless of the potential utility of such knowledge in solving immediate or foreseeable problems. However, for you pragmatists out there you have another very good reason for studying biological models that have no obvious relevance to human concerns.