cheo. Web viewExamine and identify various prokaryotic and eukaryotic microorganisms using the light microscope

REMOTE LAB ACTIVITY

SUBJECT SEMESTER: ________________

TITLE OF LAB: Cell Types Prokaryote, Protista and Fungi

Lab format: This lab is a remote lab activity.

Relationship to theory (if appropriate): In this lab you will be examining the underlying processes that make up the cell cycle.

Instructions for Instructors: This protocol is written under an open source CC BY license. You may use the procedure as is or modify as necessary for your class. Be sure to let your students know if they should complete optional exercises in this lab procedure as lab technicians will not know if you want your students to complete optional exercise.

Instructions for Students: Read the complete laboratory procedure before coming to lab. Under the experimental sections, complete all pre-lab materials before logging on to the remote lab, complete data collection sections during your on-line period, and answer questions in analysis sections after your on-line period. Your instructor will let you know if you are required to complete any optional exercises in this lab.

Remote Resources: Primary - Microscope; Secondary – Microorganism Slides.

CONTENTS FOR THIS NANSLO LAB ACTIVITY:

Learning Objectives................................................................................................. 2Background Information ........................................................................................ 2 - 8Equipment .............................................................................................................. 9Pre-lab Exercise 1: Single Cellular Organisms Bacteria and Protists ...................... 9Pre-lab Exercise 2: Comparing Protists .................................................................. 9 – 10Pre-lab Exercise 3: Organisms Fungi ...................................................................... 10 - 11Experimental Procedure ......................................................................................... 11Exercise 1: Single Cellular Organisms Bacteria and Protists .................................. 11 - 12Exercise 2: Comparing Protists .............................................................................. 12 - 13Exercise 3: Organisms Fungi .................................................................................. 13Summary Questions: .............................................................................................. 13 - 14Preparing for this NANSLO Lab Activity .................................................................. 15 - 29

1 | P a g e L a s t U p d a t e d A p r i l 3 0 , 2 0 1 4

LEARNING OBJECTIVES:

After completing this lab, you should be able to do the following things:

1. Distinguish between prokaryotes and eukaryotes cellular structures.2. Describe the basic cellular structures and characteristics of Protista.3. Examine and identify various prokaryotic and eukaryotic microorganisms using the light

microscope.4. Identify and describe the three basic shapes of bacteria.5. Explain the significance of the bacterial cell wall structures (such as the peptidoglycan) and the

Gram staining differences.6. Identify two free-living protozoan an: Amoeba and Paramecium.7. Differentiate between yeasts and molds.

BACKGROUND INFORMATION:

All living things are composed of one or more cells, which make cells the basic units of life. Most cells are microscopic but some, such as a human ovum, may be seen by the naked eye. While cells can vary in size, they generally range from 10 to 100 micrometers. Figure 1 shows the relative sizes of the different types of cells and the components that they are made up of.

Figure 1: Chart showing relative size of different times of cells.

The cell theory describes some basic concepts that we understand about cells. Historically, it stems from work done in the mid-1800’s in Germany. Three scientists, all working independently, came up with the following statements:

1. All animals are composed of cells. 2. All plants are composed of cell. 3. All cells come from pre-existing cells.

According to the cell theory, the cell is the base unit of life. A single cell possesses all of the requirements for life. Therefore, to begin the study of biology, one must have a good understanding of a cell. Cells are broken into two main groups: prokaryotic and eukaryotic. Pro as a prefix means “before” and karyon refers to nucleus (kernel). Therefore, a prokaryotic cell is one without a nucleus or “pre-nucleus.”


On the other hand, eu means “true,” so eukaryotic cells are the cells with a true membrane bound nucleus.

Prokaryotic (bacterial types) cells are more primitive. They have been identified in geologic strata dating back 3.5 billion years. These are also considered to be the simplest life forms because their cells lack membrane bound organelles. See Figure 2. Note that there are no internal membranes within the cell.

Figure 2: Prokaryotic Cell

Eukaryotic cells appear in the fossil record about 2 billion years after prokaryotic cells. Eukaryotic cells are found in plants, animals, protists, and fungi. They show a higher level of complexity in that they have internal, membrane bound organelles. This complexity has led to greater specialization, allowing this group of cells to have success in both single and multi-cellular organisms.

All eukaryotic cells have internal organelles. Each organelle has a specific function that relates to the overall function of the cell. With a greater understanding of the specific function of each organelle, one can start to see structure-function relationships which are inherent in biology. A good example of this would be a cell that has a role in the production of proteins needed for export. It would have a greater amount of rough endoplasmic reticulum (ER). The rough ER functions to produce proteins, and then the protein move into the membrane channels for transport.

Study the list below to become familiar with all of the different types of organelles and other structures in a cell. Figure 3a shows a generalized animal cell, and Figure 3b shows a generalized plant cell.


Figure 3a: Generalized animal cell.

Figure 3b: Generalized plant cell.

Centrioles – Centrioles are self-replicating organelles made up of microtubules and are found only in animal cells. They have a role in cell division.

Cilia and Flagella – Cilia and flagella are microtubule based structures that function in the locomotion of single-celled organisms. In multi-cellular organisms, cilia can function to move fluid or materials.

Endoplasmic Reticulum – Extending from the outside of the nucleus into the cytoplasm, the ER is a system of internal membranes within the cytoplasm of the cell. Some parts of the ER are covered with protein-making ribosomes. The presence of the ribosomes makes this the Rough ER. Smooth ER does not have ribosomes that attach to the surface but are involved in the chemical activities of fat metabolism and the detoxification of toxic substances (alcohol & drugs).

Golgi Apparatus – The Golgi apparatus is the distribution and shipping department for the cell's chemical products. Think of it as the “Mailboxes Etc.” store of a cell. It modifies substances made at the endoplasmic reticulum and prepares them for export to the outside of the cell.


Intermediate Filaments, Microfilaments, and Microtubules – We have mentioned these structures before in reference to cilia, flagella, and centrioles. These are long protein structures that provide a structural framework for the inside of the cell membranes – the cytoskeleton. They provide the cell with shape, support and the ability to move about the cytoplasm. They also serve to transport materials from place to place. Additionally, these fibers help with the process of cell division by contracting together in the middle of the cell to assist in cell division.

Lysosomes – Lysosomes break down macromolecules. They digest worn-out cell components to make way for newly formed ones. These organelles also eliminate particles the cell has taken in. For example: white blood cells (WBCs) engulf pathogens such as bacteria that are disease causing. The presence of lysosomes in plant cells is being debated.

Mitochondria – Mitochondria have a unique double-membrane structure, and they have their own DNA. The membrane systems function in a complex series of reactions to extract energy from organic molecules (i.e. food.) They are the site of cell metabolism. The inner membrane is bent into numerous folds called cristae. The cristae separate the mitochondrion into two compartments, an inner matrix and an outer compartment.

Nucleus – The nucleus like the mitochondria has a double membrane structure. It contains the genetic material of the cell (DNA) and separates the nucleoplasm from the cytoplasm. This membrane has pores, nucleopores, which allow for the passing of molecules. One nuclear component within this membrane is chromatin which is a mass of loosely organized DNA and proteins. When tightly coiled into shorter, denser structures, chromatin forms chromosomes. Another component of the nucleus is the nucleolus which is the site of ribosome manufacturing. Lastly, the nucleoplasm refers to the liquid matrix on the inside of the nucleus. The nucleus has two major functions: (1) it stores the cell's hereditary material, or DNA, and (2) it coordinates the cell's activities. All cells have DNA in a prokaryotic cell but it is not bound by a membrane. In a eukaryotic cell, the DNA is found in the nucleus.

Plasma Membrane – All living cells (both prokaryotic and eukaryotic) have plasma membranes that enclose their contents. The plasma membrane is a phospholipid bilayer. This means that there are two layers of lipids that create a sort of sandwich. These membranes regulate the passage of molecules into and out of the cells.

Ribosomes – All living cells contain ribosomes or tiny organelles composed of approximately 60 percent ribonucleic acid (RNA) and 40 percent protein. They are constructed from two oddly-shaped subunits which serve to manufacture proteins. Cells that produce large amounts of protein have great numbers of free and attached ribosomes. Remember, they can be attached to the endoplasmic reticulum creating the rough endoplasmic reticulum.

Cytoplasm – Also known as cytosol, this is the fluid portion of the cell. It is found in all cell types. In a eukaryotic cell, it is between the nuclear membrane and the plasma membrane. In a prokaryotic cell, it is found inside the plasma membrane.

Cell Wall – Plant, fungal cells, and some bacterial cells have a rigid wall surrounding the plasma membrane. The cell walls are composed of complex carbohydrates and serve a variety of functions from protecting the cell to regulating the life cycle of the plant organism.

Chloroplasts – The chloroplast is a saclike organelle that contains chlorophyll, a green pigment that carries out photosynthesis. Chloroplasts are found in plants. Some prokaryotic organisms have chlorophyll, but it is not contained in an organelle. Photosynthesis produces sugars which are used as an energy source for cells. Like the mitochondrion, chloroplasts have complex double membrane structures, DNA, and are energy organelles.

Vacuole –Vacuole is a membrane bound storage cavity in a cell. In a plant cell, it is a large, single central vacuole that stores compounds, helps in plant growth, and plays an important structural


role for the plant. In other organisms, it can function for storage ingestion, digestion, excretion, and expulsion of excess water.

Classifying Organisms

Living things are classified into groups based on their relatedness. Originally this relatedness was determined by physical or biochemical similarities. As technology changes, we are able to get a deeper understanding of the relationships between organisms. For example, in Darwin’s time he was simply looking at the visual and structural similarities and the differences between organisms. Today, we can use DNA analysis to explore the interrelationships between the organisms. This better understanding of the molecular similarities of organisms has led to some changes in how we organize life. Currently, there are three domains and six kingdoms. This three-domain system, which classifies life on the planet into three different domains - Archaea, Bacteria and Eukaryote, was put forth by American microbiologist and physicist Carl Woese in 1990 by looking at differences in organisms rRNA1. Therefore, the current taxonomic system has eight main levels which are:

Of the three domains, two are made up of prokaryotic cells- the archea and the bacteria. At present, there are no agreed kingdom level classifications in the archea and bactierial domains, something which may change as technology and our understanding continues to improve. The archea are a group that like all prokaryotic cells lack a nuclear membrane. They do have distinct biochemistry and RNA markers from bacteria. Often these archea are called extremophiles, because they can inhabit areas with extreme environmental conditions such as in the hot sulfur springs and under the polar ice caps. The characteristics and habitats of these organisms have led scientists to consider the archea some of the oldest species of organisms on Earth2.

The domain bacteria contain the kingdom Bacteria, and these are the bacteria you are probably more familiar with as many of them are pathogenic to humans. This group is made up of prokaryotic cells possessing primarily diacyl glycerol diester lipids in their membranes, bacterial rRNA, and no nuclear membrane2. Bacteria have a very simple cell design and can be classified according to shape, cell wall structure, and Gram staining.


Most Bacteria have a thick outer covering called the cell wall (refer back to Figure 2). Just within the cell wall is the cell membrane. Along the surface of the bacteria cell you might encounter structures called pilus, whose job is to help transfer DNA during conjugation. Additionally bacteria have flagella for motility.

The shape of a bacterial cell affects its survival and activity in the environment. As an example, cocci or spherical shaped bacteria have less surface area than a bacilli (rod shaped) or spirillum (spiral shaped.) As a result, the cocci can survive more severe desiccation or dehydration. Bacilli, however, have a greater surface area to volume ratio and can take up nutrients from dilute solutions more efficiently. Shape of a bacterium will also affect motility. For instance, spirillum are spiral cells and move with a corkscrew motion3.

Another way to classify bacteria is based on differences in the composition of cell walls. The difference becomes clear by means of a technique called the Gram stain which identifies bacteria as either Gram positive or Gram negative. In the staining process, Gram positive bacteria will hold the dye and appear purple. This is due to the thicker peptidoglycan layer just outside of the cell membrane3. On the other hand, Gram negative bacteria have a much thinner peptidoglycan layer and will release the first dye used and appear red from the second dye3. Knowing whether a disease-causing bacterium is Gram positive or Gram negative is important in identifying the bacterium and then prescribing antibiotics. The stain is named for H. C. J. Gram for a Danish physician who invented it in 18843.

Figure 4: Peptidoglycan layer and gram staining.

The domain eukarya contains all of the organisms that are composed of eukaryotic cells. These are the cells characterized by having a true nucleus (one in which the nuclear material is surrounded by a membrane) and membrane-bound organelles. In this domain, we have the following kingdoms: Protista, Fungi, Plantae and Animalia.

The Protista kingdom is a mixed collection of organisms that are placed together because they are not plants, animals, or fungi. It is rather a mixed bag. These organisms can be heterotrophic or autotrophic, motile, or sessile and single or multicellular.


The kingdom Fungi are commonly known as fungus and molds. These organisms are primarily multicellular with cell walls composed of chitin. Yeast is the exception. It is a single celled fungus. Fungi are heterotrophic with extra cellular digestion and primarily sessile but may have a motile life stage.

The kingdom Plantae is composed of the plants you are familiar with – everything from moss and ferns to pine trees and corn. These organisms are multicellular, autotrophic, and sessile.

Lastly, the kingdom Animalia is composed of all of the animals. These are all heterotrophic, multicellular, and motile. For a minute, just think about the diversity found in this group of organisms – from sponges and insects to elephants and whales. Figure 4 gives a simple visual to help you recognize the groups.

Figure 4: Organization scheme for living things.

References:

1. Three Domains of Life, date of publication unknown, PDF - http://www.usc.edu/org/cosee-west/Nov30_2011/Three%20domains%20of%20life.pdf

2. Three-domain system, Retrieved from http://en.wikipedia.org/wiki/Three-domain_system3. http://www.albany.edu/sph/coned/lesson3.pdf


http://en.wikipedia.org/wiki/Three-domain_system

http://www.usc.edu/org/cosee-west/Nov30_2011/Three%20domains%20of%20life.pdf

http://www.usc.edu/org/cosee-west/Nov30_2011/Three%20domains%20of%20life.pdf

EQUIPMENT:

Paper Pencil/pen Slides

o Mixed protistso Human epithelial cellso Bacterial smearo Yeasto Mushroom/fungus

Computer (access to remote laboratory

PRE-LAB EXERCISE 1: Single Cellular Organisms

The cell is the base unit of life. From this statement, we understand that all living things are composed of one or more cells. This lab exercise will explore single celled prokaryotic and eukaryotic cells. We will not make the distinction between the Archea and the Bacteria domains in this lab. Bacteria are often recognized based on shape. The following images in Figure 6 give you an idea of the 3 shapes- rod shaped (Bacillus), spherical (Coccus) and spiral (Spirillum).

Figure 6: Bacterial shapes

The protists are in the domain eukarya, kingdom Protista. While there are some types of protistas that are multicellular, the ones observed today are all single celled.

Pre-Lab 1 Questions:

1. Based on your pre-lab reading do you think the cells you will be observing will be prokaryotic or eukaryotic? Explain your reasoning.

2. Rewrite your answer to question one in the form of an If … Then … hypothesis.3. What structural differences do you expect to see in these two groups of organisms?

PRE-LAB EXERCISE 2: Comparing Protists

Protists are eukaryotic organisms that cannot be classified as animal, plants or fungi. They are not prokaryotes because they have a membrane bound nucleus and internal organelles. Yet they do not have the specialized features that characterize the other three eukaryotic kingdoms.


Protists are single celled or very simple multi-cellular creatures with specialized internal structures, and they only function as individuals. They only act alone, even if they live in very large colonies. They do not help other similar cells of share any of life’s needs with each other. This unique kingdom is divided into three groups: (1) the animal like protists; (2) the plant like protists; and (3) the fungi like protists.

Protists are important to humans in a variety of ways. There are a number of protists that can infect humans. Amoebic dysentery, Giardiasis, Leishmaniasis, Chagas' disease, Toxoplasmosis, Plasmodium, and others can be a concern to humans. Many other protists are used in filtration systems (diatoms) and as thickening agents (alginate (produced by brown sea weeds, also called kelp and brown algae), agar and carageenan (red sea weeds). In this lab you will look at two animal like protist (protozoans) the Amoeba and Paramecium.


1. While we know that there are three major groups of protistas, do additional research into how the organisms in these groups move and obtain nutrients.

2. Based on your pre-lab reading, what structural differences do you think you will see between the protozoan cells? Explain your reasoning.

3. Rewrite your answer to question one in the form of an If … Then … hypothesis?

PRE-LAB EXERCISE 3: Organisms Fungi

All multi-cellular organisms are composed of eukaryotic cells. In Exercise 2, protists were examined, which make up one of the four kingdoms of eukaryotic cells. Protists are generally single-celled, and the remaining three kingdoms of eukaryotes (plantae, animalia and fungi) are all multi-cellular organisms. In this lab exercise, we will investigate the kingdom Fungi.

The kingdom Fungi is composed of eukaryotic cells. They are nonvascular and heterotrophic with external digestion. What this means is that the fungi organism will secrete enzymes into the substrate they are growing on and then absorb the broken down nutrients. Other types of fungi are parasitic and obtain nutrients from their hosts. As we saw in the prokaryotic organisms, the composition of the cell wall is an important identifier of cells. In the fungi, the cell wall is composed of the carbohydrate chitin rather than the cellulose we see in plant cell walls, and they store glycogen rather than starch making them more closely related to animals than plants.

Fungi reproduce both sexually and asexually. An example of a fruiting body of a fungus is the mushroom. The mushroom forms when a fungi is ready to release spores. The hyphae (long thin strands) make up a mycelia mat (a mass of hyphae) which is the vegetative portion of the organism. As the mycelium grows, it will produce the fruiting body (mushroom) which will then release spores.

Fungi and molds are not the same. While both are part of the Fungi kingdom, the term fungus is more general, and the term mold refers to a particular group of fungi called Hyphomycetes, which are characterized with having filamentous hyphae and producing airborne spores. Yeast, while still a member of the Fungi kingdom, is a single celled fungi.



1. Based on your pre-lab reading, what structural differences do you think you will see between the Fungi and the bacterial cells? Explain your reasoning.

2. Rewrite your answer to question one in the form of an If … Then … hypothesis.3. In Exercise 2, you explored the Protista kingdom. Some protistas can be made up of more than

one cell and function as colonial organisms. What is the difference between a colonial organism and a multi-cellular organism?

EXPERIMENTAL PROCEDURE:

Once you have logged on to the remote lab system, you will perform the following laboratory procedures. See Preparing for this NANSLO Lab Activity below.

EXERCISE 1: Single Cellular Organisms Bacteria and Protists

DATA COLLECTION:

1. Select the Bacteria forms, smear slide (Slide Cassette 2: #19) from the microscope interface. Using the 10X objective, locate the bacterial sample and bring it into focus.

2. Carefully work your way through all the objectives, focusing with each one until you reach the 60X objective and capture an image of the three bacterial shapes. Insert the images below.

3. Measure the size of one type of bacterial cell. To determine the size of the cells, we are going to use the ratio method. In order to do this, you will need one piece of information which is the width of your field of view. On our microscopes, the field of view is 305µm at 40X magnification and 205µm at 60X magnification.

4. Using the image in Figure 7 as an example, we can see that the total width of the field of view is 13.6 cm or 136 mm (Image A). The cell (Gray) is 3.7 cm or 37mm (Image B).

Figure 7: Measurements

5. Dividing 37mm/136mm = 0.272 which we multiply by the total length of the field of view so (40X; 0.272 * 305µm = 82.96µm or 60X; 0.272 * 205µm = 55.76µm) rounded for significant figures gives us a cell size of (40x; 83µm or 60X; 56µm).

6. Using your image of a bacterial cell and a ruler, determine the size of the bacterial cell. 7. Record your bacterial cell size in a data table. You will need a data table with columns for

bacteria cell size, Protista cells size, plant cell size, and animal cell size.


8. Select the Mixed Protozoa Whole Mount slide (Slide Cassette 1: #6) from the microscope interface. Using the 10X objective, locate the bacterial sample and bring it into focus.

9. Carefully work your way through all the objectives, focusing with each one, until you reach the 60X objective and capture an image of several different types of protists.

10. Repeat steps 3 – 7 for one protista cell.11. Record your Protista cell size in the data table.

ANALYSIS:

12. Based on your observations, which cells were prokaryotic? eukaryotic? Use your observations to support your claim.

13. Research the two prokaryotic kingdoms. What characteristics caused these organisms to be separated into two kingdoms? Do you agree with the separation? Explain your answer.

14. Research the endosymbiotic theory at the following websites. Keep in mind that sometimes websites stop working. If this is the case, do a web search with the key word “Endosymbiosis”:

http://www.biology.iupui.edu/biocourses/N100/2k2endosymb.htmlhttp://evolution.berkeley.edu/evolibrary/article/_0/endosymbiosis_01http://evolution.berkeley.edu/evosite/history/endosym.shtml

Write a two to three paragraph summary discussing this theory and if there is evidence to support it. Can you propose an alternate theory?

15. Based on the claim made above do you accept or reject your initial hypothesis? Use evidence gathered to explain why you would accept or reject your initial hypothesis

16. (Optional) Rewrite your hypothesis based on the evidence form analysis question 2.

EXERCISE 2: Comparing Protists

DATA COLLECTION:

1. Select the Paramecium Slide: (Slide Cassette __: #__) from the microscope interface. Using the 10X objective, locate the yeast sample and bring it into focus. A Paramecium is a single celled organism in the protista kingdom.

2. Carefully work your way through all the objectives focusing with each one until you reach the 60x objective. Select an area on the slide so that the cells completely fill the field of view.

3. Position the slide so that the edge of a cell is at the left edge of your field of view. Capture an image of the Paramecium. Insert your Paramecium image below.

4. Select the Amoeba, Section slide (Slide Cassette __: #__) from the microscope interface. Using the 10X objective, locate the Amoeba sample and bring it into focus.

5. Carefully work your way through all the objectives focusing with each one until you reach the 60x objective. Select an area on the slide so that the cells completely fill the field of view.

6. Position the slide so that the left edge of a cell is at the left edge of your field of view. Capture an image of the Amoeba cells. Insert your Amoeba images below.

ANALYSIS:

1. After looking at these protozoan specimens, what are the visible differences in motility?


http://evolution.berkeley.edu/evosite/history/endosym.shtml

http://evolution.berkeley.edu/evolibrary/article/_0/endosymbiosis_01

http://www.biology.iupui.edu/biocourses/N100/2k2endosymb.html

2. Based on your understanding of the how these two organisms move, predict how the cell membranes would differ. Do additional research and see if your prediction is correct.

3. Based on the prediction made above, do you accept or reject your initial hypothesis (in the prelab portion)? Use evidence gathered to explain why you would accept or reject your initial hypothesis.

4. (Optional) Rewrite your hypothesis based on the evidence from analysis question 2.

EXERCISE 3: Organisms Fungi

DATA COLLECTION:

1. Select the Ascomycetes: Yeast three types Yeast slide (Slide Cassette 2: #42) from the microscope interface. Using the 10X objective, locate the yeast sample and bring it into focus. Yeast is a single celled organism in the Fungi kingdom.

2. Carefully work your way through all objectives, focusing with each one until you reach the 60x objective. Select an area on the slide so that the yeast cells completely fill the field of view.

3. Position the slide so that the left edge of a cell is at the left edge of your field of view. Capture an image of the yeast. Insert your yeast images below.

4. Select the Wood rot fungus, Section slide (Slide Cassette 2: #46) from the microscope interface. Using the 10X objective, locate the fungus sample and bring it into focus.

5. Carefully work your way through all objectives, focusing with each one until you reach the 60x objective. Select an area on the slide so that the yeast cells completely fill the field of view.

6. Position the slide so that the left edge of a cell is at the left edge of your field of view. Capture an image of the yeast. Insert your fungus images below.

ANALYSIS:

1. Fungi reproduce by spores. How are spores structurally different from seeds? Is a sport asexual or sexual?

2. List and describe the main characteristics of fungi. 3. Are fungi autotrophic or heterotrophic? Explain your reasoning. 4. Many fungi are parasitic or disease causing. Research a fungus of your choice and write a

paragraph summing up your findings. 5. What is the difference between hyphae and mycelia?

SUMMARY QUESTIONS:

1. Based on your observations, what structural differences did you see between the Protista, Fungi and Bacterial groups? Use your observations to support your claim.

2. After completing the lab, what surprised you the most about the organisms you observed? 3. Fill out the following chart. Some have been completed to get you started.

Kingdom Not Applicable

Not Applicable Fungi

Motility motile Food Production Both autotrophic and heterotrophic Type of Cell Prokaryotic Domain Bacteria Eukarya


4. Of the cell types you studied, which ones do you think would be most successful in harsh conditions? Defend your answer.

5. What characteristics do plants and fungi share? Plants and fungi were initially classified as one group. What characteristics would have led early taxonomists to do this? Do you agree with the separation of these organisms into two separate kingdoms? Explain your reasoning.

6. Research the importance of members of the Protista and Fungi Kingdom to man. Write a two to three paragraph mini-essay on one of these organisms and their importance to the ecosystem.

7. Compare and contrast the following features in prokaryotic and eukaryotic microorganisms: size, DNA location, ribosomes, cell wall, and ability to move.

8. Research the most common bacteria found on the human skin and how their population is controlled. (Hint: Think of the chemicals produced by human skin cells.).


PREPARING FOR THIS NANSLO LAB ACTIVITY:

Read and understand the information below before you proceed with the lab!

Scheduling an Appointment Using the NANSLO Scheduling System

Your instructor has reserved a block of time through the NANSLO Scheduling System for you to complete this activity. For more information on how to set up a time to access this NANSLO lab activity, seewww.wiche.edu/nanslo/scheduling-software.

Students Accessing a NANSLO Lab Activity for the First Time

You must install software on your computer before accessing a NANSLO lab activity for the first time. Use this link to access instructions on how to install this software based on the NANSLO lab listed below that you will use to access your lab activity – www.wiche.edu/nanslo/lab-tutorials

1. NANSLO Colorado Node -- all Colorado colleges.2. NANSLO Montana Node -- Great Falls College Montana State University, Flathead Valley

Community College, Lake Area Technical Institute, and Laramie County Community College.3. NANSLO British Columbia Node -- Kodiak College.

Using the Microscope for a NANSLO Remote Web-based Science Lab Activity

We've provided you with three ways to learn how to use the microscope for this NANSLO lab activity:

1. Read these instructions. 2. Watch this short video - https://www.youtube.com/watch?

feature=player_embedded&v=m7w9ssIgVdw. 3. Print off these instructions to read ( PDF version of the instructions.)

NOTE: The conference number in this video tutorial is an example. See “Communicating with Your Lab Partners” below to determine the toll free number and pin to use for your NANSLO lab activity.

MICROSCOPE RWSL LAB INTERFACE INSTRUCTIONS

The Remote Web-based Science Lab (RWSL) microscope is a high quality digital microscope located at the NANSLO Node. Using a web interface as shown below, you can control every function of the microscope just as if you were sitting in front of it.

The equipment control software shown below is written using the LabVIEW software from National Instruments. The user interface is presented as a LabVIEW control panel which will be referred to as the lab interface for the remainder of the document.


http://www.wiche.edu/info/nanslo/creative_science/Using_the_Microscope_for_a_NANSLO_RWSL_Lab_Activity_for_NANSLO_labs.pdf

http://www.wiche.edu/nanslo/lab-tutorials

http://www.wiche.edu/nanslo/scheduling-software

Figure 1: Remote Web-based Science Lab (RWSL) Microscope Lab Interface

COMMUNICATING WITH YOUR LAB PARTNERS

As soon as you have accessed this lab interface, call into the toll free conference number shown on the control panel to communicate with your lab partners and with the Lab Technicians. Use the PIN code noted to join your lab partners. Only one person can be in control of the equipment at any one time so talking together on a conference line helps to coordinate control of the equipment and creates a more collaborative environment for you and your lab partners.

GAINING CONTROL OF THE MICROSCOPE

Right click anywhere in the grey area of the lab interface and choose “Request Control of VI” from the dialogue box that appears when multiple students are using the microscope at the same time,. After you request control, you may have to wait a short time before you actually receive control and are able to use the features on this lab interface.


Figure 2: Selecting "Request Control of VI"

RELEASING CONTROL OF THE MICROSCOPE

To release control of the microscope so that another student can use it, right click anywhere in the grey area of the lab interface and choose "Release Control of VI" from the dialogue box that appears.

Figure 3: Selecting "Release Control of VI"


MICROSCOPE CONTROLS

The Stage Controls allow you to adjust the visual of the specimen that has been placed on the stage of the microscope, select lenses with various magnifications, and select whether or not the condenser lens is in the light beam. Below are more specific instructions on using these controls. When using the arrows on this lab interface, click and hold the arrow until the desired effect is achieved or click and wait to view the result before clicking again. Quick clicks on the arrows may cause the system to lock up.

Figure 4: Microscope Controls - Stage, Objective & Condenser

Stage Controls: Using the left and right and up and down arrows found to the right of the microscope image in the Stage Control area, moves the microscope stage which holds the specimen. These arrows allow you to precisely control the position of the specimen on the stage.

1. Use the "Right" and "Left" arrows to move the Stage so that you can view the specimen from left to right.

2. Use the "Backward" and "Forward" arrows to move the Stage so that you can view the top, middle or bottom of the specimen.

3. Use the "Up" and "Down" arrows to move the stage closer or farther away from the objective lens to bring a specimen into focus. BE CAREFUL! Don't move the stage too close to the lens.

When selecting the button between the "Up" and "Down" arrows, you can toggle between “Coarse” and “Fine” focus. When the button is dark green and “Coarse/Fine” is displayed to the right of the button, the microscope is in “Coarse” focus. When the button is bright green and “Fine” is displayed, the microscope is in “Fine” focus. Typically, you will start with coarse focus which moves the stage in large


increments and then use fine focus to complete your final focusing as it moves the stage in smaller increments. There is no difference between the course and fine focus when using the 60X objective

NOTE: When you click on these arrows, the specimen appears to move in the opposite direction. Since the objective stays fixed, the image moves in the opposite direction of the stage. This is how these controls work on most microscopes so the "feel" of the microscope is preserved over the web.

Figure 5: Right/Left & Backward/Forward Stage Controls Figure

6: Up/Down Stage Controls & Coarse/Fine Focus Control

Objective: A microscope mounts an objective lens very close to the object to be viewed. Depending on need, different lenses with different power will be used on the microscope. This microscope feature multiple objectives, each with different power, mounted on a rotating turret. The larger the magnification numbers the greater the magnification. For example, if a specimen is viewed through a 40X objective lens, the magnifier in that lens displays the specimen 40 times larger than an equivalent view as seen by the unaided eye. Remember that the ocular or other lenses also add to the magnification.

This microscope has five lenses – 4X, 10X, 20X, 40X, and 60X. Use the arrows below the objective lens box that indicates the magnification of the current objective lens to move to a higher or lower magnification lens. If you have activated the “Picture-in-Picture” Preset 2 (see below) you will be able to see the objective lens move when you select a new magnification.

Condenser: The condenser controls whether or not the condenser lens is in the light beam. You want to have the condenser OUT for the 4x objective but IN for all the others.

SELECTING A CASSETTE AND LOADING SLIDES ONTO THE STAGE

There are two tabs on the lab interface. When you first access the lab interface, the "Microscope" tab is displayed by default. Click on the Slide Loader tab at the top of the screen to access the controls for the Slide Loader robot. There can be up to four cassettes available on the Slide Loader. These cassettes are used to store slides, and each can hold up to 50 slides. The cassettes available to you are dependent on the lab activity to be completed. Once a cassette has been selected, you will use the drop-down list to select your slides.


Figure 7: Select the Slide Loader Tab to select a cassette and slides.

EXAMPLE OF HOW TO LOAD SLIDES

In this example, we have selected Cassette #1. Using the drop-down menu, we have selected "1: Colored Threads Whole Mount." Then, we selected the "Load" button. A message indicates that the slide is loading. Using the picture-in-picture camera, you can watch this happening. The robotics selects the slide and places it on the microscope stage.

Figure 8: Selecting the slide "1: Colored Threads Whole Mount" from Cassette #1


Notice that when a slide is actually on the microscope (or when it is being loaded or unloaded), the cassette controls are grayed out so you cannot load a second slide until the first is removed. Once the slide is on the microscope stage, it will be listed in the "Current Slide on Stage" box. The only thing that the Slide Loader robot can do is return it to the cassette when the "Return Slide to Cassette" button is selected.

Figure 9: "LOADING SLIDE ... PLEASE WAIT" is displayed in the "Current Slide on Stage" window

Select the "Microscope" tab to perform the NANSLO lab activity. Once you are finished with the slide, select the "Slide Loader" tab and select "Return Slide to Cassette" button. Once the slide is returned to the cassette, the Slide Loader controls are again available to select another slide from the cassette.

ENHANCING THE MICROSCOPE IMAGE

The digital camera mounted on the microscope has a camera control unit that is equipped with a series of image processing functions that enable you to quickly and easily correct imaging problems that arise from low or high contrast, poor focus, insufficient or uneven illumination, sample shading or discoloration and noise. The most common reason for uneven elimination is a light source that does not completely fill the field of view on lower magnifications. The White Balance should be used only if the image appears to be brown or gray, and you think you might need to adjust it (although it won't hurt anything to click this button).

A choice of color modes can be selected in the Microscope Image area and are used to display the image in different color palettes in order to highlight certain features. The default setting is "Normal."


Figure 10: Microscope Image Special Effects and Other Image Controls for Camera

Here is a description of each option:

1. In the “Normal” mode, the sample is displayed in its true colors.2. In the “Negative” mode, the sample is displayed in a color-inverted form, where red, green, and

blue values are converted into their complementary colors. The technique is useful in situations when color inversion can be of benefit in exposing subtle details or in quantitative analysis of samples.

3. In the “Blue Black” mode, the black portions of a grayscale negative sample are displayed in blue. This mode is often useful to reveal details in samples having a high degree of contrast. The “Blue Black” filter can aid you in examining a wide spectrum of difficult samples.

4. In the “Black & White” mode, a grayscale image of the sample is displayed.5. In the “Sepia” mode, a brown scale (black and white) image of the sample is displayed. Although

typically this filter is of little utility, it can be employed to alter image color characteristics to improve the visualization of sample detail.

6. At times, the sample may have an unacceptable color quality. Use “White Balance” calibration to remove the color cast. This process is often referred to as white balancing.

7. Auto Exposure is on automatically. You do not need to do anything with Auto Exposure unless you are adjusting the luminance. If you are doing so, you should turn off Auto Exposure by clicking on the button. The green light is now off. Now adjust the luminance. See explanation below.

Reference: http://www.microscopyu.com/articles/digitalimaging/dn100/correctingimages.html


Auto Exposure is normally turned on, but you can turn it off if you want to play around with the brightness of the light source and not have the microscope camera automatically adjust it. It is usually best, though, to leave it turned on.

When you turn off the Auto Exposure, the button turns dark green. Some new controls appear that let you turn the LED off or on, and also adjust the intensity of the light source. The intensity of the light source can be increased or decreased manually with the dial that now appears next to the Objective control when Auto Exposure is turned off.

Figure 11: Additional controls available when Auto Exposure is turned off

CAPTURING AND SAVING A MICROSCOPE IMAGE

When the “Capture Image” button is pressed, a high-resolution image of what is currently in the field of view of the objective is captured. While the image is being captured, the button will be illuminated bright green. The capture is complete when the light turns off. Be patient as this may take several seconds to complete.

After the Capture Image light turns off, select the “View Captured Image” tab on the bottom of this control panel to view the image.


Figure 12: Click the capture image button (#1), wait till the green light goes off, and then select the View Captured Image tab (#2)

After opening this image through the View Captured Image tab, you will need to take a snapshot of it and save it to your computer. There are several ways to do this, depending on your operating system.

WINDOWS:

1. Pressing the two keys ALT and Print Screen simultaneously will copy the active window into your computer clipboard. Then you can past it into a document.

2. Windows 7 and above has a Snipping Tool program under Programs/Accessories which can capture selected areas of the screen.

3. Right click on it and select "Copy" from the menu presented. After right clicking and selecting Copy, just open a document and right click and select Paste. You can either paste it directly into your lab report document or into another one for safe keeping until you use it later. You can use drawing tools in your word processing editor to annotate this image so you can show your instructor that you know what you were suppose to be looking for!


Figure 13: Right click and select Copy to paste the image into a document.

MAC:

1. Press these three keys simultaneously – . This will change your cursor icon into a little cross.

2. Now press the spacebar, and the icon becomes a camera. Click in the image window you want to take a snapshot of, and it will save the image to a file on your desktop.

There are lots of free screenshot utilities you can also use to capture this image.

If you are familiar with saving a document to your computer, you also can select “Save Image As” from the pop-up menu, give the image a name and then select a location on your computer where you want this image to be saved for future use.


MICROSCOPE IMAGE VIEW WINDOW

The Image View Window displays the real-time video feed from the digital camera “looking through” the microscope.

Figure 14: Image View Window

PICTURE-IN-PICTURE CONTROLS - CAMERA PRESET POSITIONS AND PAN-TILT-ZOOM CONTROLS

When you click on the "Picture-in-Picture" button, it turns bright green. A second real-time video feed from another digital camera appears in the Image View Window. The controls shown in Figure 15 are all operational when the Picture-in-Picture feature is selected.


Figure 15: Picture-in-Picture Image Controls

CAMERA PRESETS

There are six camera preset positions.

Figure 16: Picture-in-picture Camera Preset 1 and 6 - Displays the microscope, microscope camera, and a camera control unit projecting the sample on the Stage.

Figure 17: Picture-in-picture Camera Preset 2: Displays a closeup of the objective lens.


Figure 18: Picture-in-picture Camera Preset 3 - Displays a closeup of the camera control unit projecting the sample on the Stage.

Figure 19: Picture-in-picture Camera Preset 4 - Displays the microscope eye piece and the camera mounted to the microscope.

Figure 20: Picture-in-picture Camera Preset 5 - Displays the Condenser Lens underneath the Stage that focuses the light on the sample. The Condenser Lens controls the width of the beam. In some instances you will want a tighter beam while in other cases you will want a broader beam to control the image quality. This setting has been optimized for you.


PAN, TILT, ZOOM CONTROLS FOR PICTURE-IN-PICTURE

For each camera preset view, additional camera options are available.

1. Use the up and down arrows to tilt the camera up or down. 2. Use the right and left arrows to pan right or left.3. Use the left "Zoom OUT" arrow and right "Zoom IN" arrow to zoom out and in.

Figure 21: Picture-in-picture Camera - Example of "Zoom In" capability

For more information about NANSLO, visit www.wiche.edu/nanslo.

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