GENERAL BIOLOGY LAB II (BSC1011L) Lab #2: Bacteria and Protista ________________________________________________________________________ OBJECTIVES: Examine different types of bacteria.

Compare and contrast the different Protist phyla. Examine the similarities and differences between members of Kingdoms Protista and Fungi. Describe the significance of the Volvocine line. ________________________________________________________________________ INTRODUCTION: Kingdom Bacteria Organisms classified into Domains Eubacteria and Archaea, i.e. the prokaryotes, are the oldest and most abundant organisms on Earth. Present for over 1 billion years before the evolution of eukaryotes, cyanobacteria produced enough oxygen in the atmosphere to promote the development of diverse eukaryotic species including protists, fungi, animals and plants. Bacteria are ubiquitous in nature, inhabiting environments that range from the frozen ice in Antarctica to the digestive tracts of ruminant animals. To date, greater than 7,000 bacterial species have been identified, some of which are pathogenic to humans (e.g. Yersinia pestis, the causative agent of plague or Helicobacter pylori, which promotes ulcer formation) while others are important for food production, manufacturing pharmaceuticals and the decomposition of dying/decaying material (saprophytes). In addition, some bacterial species form symbiotic (mutalistic) associations with other organisms in which both partners benefit from the relationship. For instance, lichens are a mutalistic pairing of a fungus and a green alga or cyanobacterium. In this partnership, the fungus provides the green alga/cyanobacterium with protection while the green alga/cyanobacterium provides the fungus with food. Overall, bacteria are a diverse group of organisms that play vital roles in the ecosystem. Bacteria, and prokaryotes in general, are smaller and have a much simpler internal organization, lacking the membrane-bound organelles (Fig. 1) characteristic of eukaryotes. The composition of the genetic material in bacteria is also very different; in contrast to the multiple linear chromosomes found in the nucleus of eukaryotes, bacteria possess a single, circular chromosome in the nucleoid region of the cell. The main differences between prokaryotic and eukaryotic organisms are summarized in Table 1.

Figure 1. General structure of a prokaryotic cell

Table 1. Main differences between prokaryotic and eukaryotic organisms Characteristic Unicellularity Cell size Chromosomes Cell division/genetic recombination Compartmentalization Mitochondria Nucleus Ribosomes Flagella Photosynthesis Cell wall Prokaryotic Yes 1m or less Single, circular Binary fission asexual Conjugation Absent Absent Absent Present Present Yes Peptidoglycan Eukaryotic No 10m or more Multiple, linear Mitosis Meiosis - sexual Present Present Present Present Present Yes Absent in animals, present in plants, fungi and some protists

Bacteria are generally single-celled (unicellular) organisms; however, some species (primarily the cyanobacteria) are multicellular, forming associations of various sizes, i.e. filaments or colonies. Their classification is usually based on morphology and biochemistry. The morphological characteristics include (1) shape [coccus (spherical, Fig. 2a), bacillus (rod-shaped, Fig. 2b), and spirillium (helical, Fig. 2c)] and (2) the differential thickness of their cell wall (gram positive vs. gram negative see below) while the biochemical characteristics refer to (1) whether or not they use oxygen for cellular respiration (aerobic vs. anaerobic) and (2) whether they can use light to generate their own carbon source (autotrophy) or if they require organic molecules to obtain carbon (heterotophy).

Figure 2. Types of Bacteria: a) cocci, b) bacillus and c) sprillium The role of the bacterial cell wall is to maintain the shape of the cell. Depending upon the thickness of the peptidoglycan (protective) layer in the cell wall, bacteria are classified as either gram-positive or gram-negative. Gram-positive bacteria (e.g. Streptococcus and Micrococcus) have much simpler cell walls with a very thick peptidoglycan layer that is capable of retaining the Crystal violet dye (purple) used during the gram-staining procedure. Gram-negative bacteria (e.g. Escherichia coli and Serratia), on the other hand, possess a much thinner layer of peptidoglycan in their cell wall and thus, a reduced affinity for Crystal violet. Instead, these bacteria stain pink from the Safranin dye also added during the gram staining process (Fig. 3). Although gramnegative bacteria have less peptidoglycan, their cell walls are more complex due to the presence of lipopolysaccharides which secrete potent toxins.

Figure 3. Gram-negative vs. Gram-positive bacteria ________________________________________________________________________ TASK 1: Using a compound microscope NOTE: This task is intended to serve as a quick review of how to use a microscope. Your TA will assess whether your group needs to perform this exercise or if you can skip to Task 2.

Microscopes are used to examine specimens too small to be observed with the naked eye. There are two major types of microscopes that you will use in this lab, compound light and dissecting microscopes. In general, a compound light microscope is used to visualize very small items (e.g. blood cells) while a dissecting microscope is used for observing much larger ones (e.g. mouthparts of a grasshopper).

A. Familiarize yourself with the use of the light microscope 1. Obtain TWO compound microscopes per group. Make sure to record which microscopes your group will be responsible for on the sign-in sheet at the front of the room. You will use these microscopes for the remainder of the semester. 2. Identify each part labeled on the compound microscope in Figure 3 and note its function in Table 2.

Oculars

Body Tube

Nosepiece Arm Objective Slide Holder Stage Clip Stage Condenser iris diaphragm Substage Lamp Base Field Iris Diaphragm Coarse Focus Adjustment Fine Focus Adjustment

Figure 4. Main parts of a compound light microscope

Table 2: Identifying parts of a compound microscope Part Nosepiece Objective Stage Clip Stage Condenser Iris Diaphragm Substage Lamp Base Oculars Body Tube Arm Slide Holder Coarse Focus Adjustment Fine Focus Adjustment Field Iris Diaphragm Function

3. Plug in your microscope and turn the light source on. Rotate the objective lens to the 4X power. It should click into place. Note: You should always start at the lowest power available on a microscope. 4. Locate the coarse adjustment. Turn it while watching the stage. See how fast or slow it allows you to move the stage compared with the fine adjustment. 5. Adjust the ocular lenses so that they fit the width between your eyes. 6. Obtain the letter e slide from your slide box and place it on the stage (make sure it is held by the clip). Move the stage back and forth (left and right, forward or backward) so that the e is directly beneath the objective lens.

7. Use the coarse adjustment to move the slide to about 1 cm from the objective lens. Looking through the oculars, move the coarse adjustment until you can see the e through the lens. Only use the coarse adjustment when you are viewing the specimen with the 4X or 10X objective lens. 8. Use the fine adjustment to get the e into sharp focus. 9. Move the e left and right. And then forward and backwards. 10. Change the objective lens to 10X.

Questions: a. As you view the letter e, how is it oriented? Upside down or right side up? What does that tell you about how the microscope processes the image?

b. How does the image move when the slide is moved to the left or right?

c. What happens to the brightness of the view when you switch from the 4X to the 10X objective?

B. Magnification 1. Calculate the total magnification (objective magnification x ocular magnification) for each objective and record in the table below. The oculars magnify the specimen 10 times. Record this information in Table 3.

Table 3: Calculating magnification of a compound microscope Objective Magnification Ocular Magnification Total Magnification

Questions: a. How many times is the image of the e magnified when it is viewed through the highest power objective lens?

b. If you didnt know what you had on your slide (an e) and you began examining it at the highest power, how could you determine it was an e?

C. Field of View The field of view is the area you can see when you look through the lens of a microscope (Fig. 5). Understanding the size of this field under different magnifications is important because it allows you to be able to estimate the size of objects in your view.

Figure 5. Field of view (FOV) under various magnifications

Questions: a. Discuss the advantage and limitation of viewing specimens under highest magnification.

b. What about the low-power objective?

c. Which magnification provides the largest FOV? Which provides the smallest?

D. Preparing Wet Mounts of Biological Specimens 1. Place a drop of pond water on a clean slide. Position the edge of a coverslip against the water drop (at a 45o angle) and then slowly lower the coverslip onto the slide. This is called a wet mount. 2. Once prepared, view the slide with your microscope. Try to locate and identify any microorganisms present on your slide and draw these in the space provided. Use the Microorganisms Appendix below to identify some of the organisms you see. Take note of the microscope tips as you begin to look for organisms.

Magnification: __________

Microorganisms Appendix
All pictures are courtesy of South Florida Periphyton Research Laboratory at FIU

Gamphonema coronatum

Frustulia rhomboides Chlamydomonas Nitzschiae

Spirillium

Oscillatoria Nitzschiad Ulothrix Desmidium baileyii

Fragilaria synegrotesca

Gamphonema cf. vibriodes

Bulbochaete Navicula 10

________________________________________________________________________ TASK 2: Bacterial Identification A. Identifying bacterial types View prepared slides of the three bacterial shapes (bacillus, coccus, and spirillum) as well as the gram stained bacteria located in the slide box at your table. Draw what you see in the spaces provided below.

Magnification: ________

Magnification: ________

Magnification: ________

bacillus

coccus

spirillum

Magnification: ________

Magnification: ________

Gram negative bacteria

Gram positive bacteria

B: Identification of bacteria cultured from hands Different bacterial species require different environments for growth. In fact, there are over 100 trillion bacteria that live on/in humans (Costello et al., 2009), some of which aid in nutrition while others resist pathogens and maintain a normal, healthy flora. Listed in Figure 6 are a few of the most commonly found bacteria that inhabitat the human body. You can also see pictures of some of these bacteria on p. 42 of your dissection atlas. 11

Figure 6. Bacteria commonly found on/in humans In addition to environment, bacterial species also differ in their nutrient requirements. This factor makes it is possible to isolate bacterial species by growing them on agar that has or is missing a particular nutrient necessary for growth of specific bacterial species. During last weeks lab, you cultured bacteria present on your hands before and after washing them with soap or disinfecting them with Purell. Both agents are advertised as effective means of removing dirt, grease and certain bacterial strains, the main difference being that soap requires water for use while Purell, an alcohol based hand sanitizer, does not. The purpose of the experiment you setup last week is twofold: (1) to compare the effectiveness of both disinfecting agents in killing bacteria and (2) to identify the different strains of bacteria that are present on your hands when they have not been washed/sanitized. Procedure: 1. Obtain your groups plates from the refrigerator. 2. Observe the growth and appearance of the colonies on all plates (See Fig 7). 3. Identify the bacterial strains present on your groups plates using Table 4. Note the number and type of bacterial strains present on your groups plates on the petri dish schematics below. a. Using a sharpie marker, draw a line down the center of a new slide. b. On each half of the slide, draw a circle. c. Label each half of the slide with a number (i.e. 1 or 2).

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d. Add one drop of water to the circles on each side of the slide. e. Using a toothpick, transfer a small amount of one colony of a particular strain of bacteria present on the plate and add it to a drop of water on a slide. Mix well. f. Allow to air-dry for about 5-10 minutes and then pass three-four times over an ethanol lamp to heat fix the bacteria to the slide.

g. Flood the slide with methylene blue and leave for about 1 min. h. Pick up the slide with forceps and rinse off the excess dye with distilled water. i. Place the slide on a paper towel and fold over as seen in the picture below. j. Examine the slide under the microscope. k. Repeat steps a-c for each of the bacterial strains present on your plates.

A.

B.

Figure 7. A. Blood agar and B. MacConkey agar plates after a 24 hour incubation period

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Table 4. Comparison of different bacteria strains

Bacterial Strain

Shape

Gram Stain (+/-) +

Occurrence on the skin nearly 100%

Appearance on Blood Agar small white colonies

Appearance on MacConkey Agar no growth

Staphylococcus spherical epidermidis

Staphylococcus spherical aureus

~ 25%

"gold," or yellowish-white colonies

no growth

Streptococcus pyogenes

spherical

rare, > 5%

colonies exhibit large zones of -hemolysis

no growth

Corynebacteria

rodshaped

nearly 100%

colonies exhibit a small zone of -hemolysis

no growth

Escherichia coli

rodshaped

rare, > 5%

medium to large grayish-white colonies

pink colonies

Blood agar plates

Before

After Soap

Before

After Purell

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MacConkey agar plates

Before

After Soap

Before

After Purell

Questions: a. What ingredient (s) present in MacConkey agar inhibits the growth of gram positive bacteria?

b. Since Streptococcus pyogenes and Corynebacteria both exhibit -hemolysis on sheep blood agar, what other type of media could you use to distinguish between the two bacterial strains?

c. Did each group members before plates contain all the same bacterial strains? If not, which strain(s) was common to all group members?

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d. Which treatment, hand washing or Purell, was more effective at eliminating the bacteria on the hands?

i. Could eliminating the bacteria on the hands be harmful? Explain.

________________________________________________________________________ Kingdom Protista Members of the Kingdom Protista are the earliest known eukaryotes, with fossils estimated to be 1.5 billion years old. Although it is not possible to know exactly how eukaryotic cells arose, the endosymbiotic theory (Fig. 8) proposes that a primitive eukaryotic cell engulfed an aerobic bacterium that had the necessary enzymes to derive energy from oxygen. In the increasingly oxygenated Earth, aerobic respiration conferred a selective advantage on the eukaryotic host. Similarly, other cells may have also engulfed photosynthetic bacteria, enabling them to become autotrophic. These aerobic and photosynthetic bacteria gave rise to modern-day mitochondria and chloroplasts. Evidence in favor of the endosymbiotic theory is compelling since both mitochondria and chloroplasts possess traits characteristic of bacteria, i.e., they have their own circular DNA, are surrounded by double membranes and divide by binary fission. It is believed that a series of endosymbiotic events gave rise to the different organelles that characterize eukaryotic cells today.

Figure 8. Endosymbiotic theory

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Of all the eukaryotic kingdoms, Kingdom Protista is the most diverse, consisting of organisms that lack distinguishing characteristics of fungi, animals or plants. Members of this group are unicellular and multicellular organisms that vary in size, means of reproduction, locomotion and nutritional strategies. Protists are also broadly separated into three main groups, (1) algae (plant-like), (2) slime molds (fungus-like) and (3) protozoans (animal-like), as illustrated in Figure 9. In the upcoming exercises you will examine representative species from these three groups.

Figure 9: Kingdom Protista cladogram ________________________________________________________________________ TASK 3: Examining members of the Kingdom Protista A. ALGAE Algae are an aquatic group of autotrophic organisms that commonly occupy marine and freshwater environments. Algae are classified into 5 phyla, Chlorophyta, Phaeophyta, Rhodophyta, Chrysophyta and Euglenophyta, and can be differentiated based on the types of pigments that each possesses. In addition to pigmentation, the different algal species also have disparate modes of cellular organization (ranging from 17

unicellular, filamentous to colonial), reproductive mechanisms (some reproduce sexually, while others can reproduce both sexually and asexually) as well as the composition of their cell walls (See Table 5).

Table 5. Different Types of Algae Phylum Common name Pigments present Cell wall composition Distinctive structures present Cellular organization Movement Example(s) Chlorophyta Green algae Chlorophyll a, b Cellulose Stigma (some species) Unicellular filamentous colonial Sessile & motile Chlamydomonas Cladophora Phaeophyta Brown algae Fucoxanthin Rhodophyta Red algae Phycobilins Chrysophyta Diatoms Chlorophyll a,c Xanthophyll Silicon dioxide (glass) Euglenophyta Euglenoids Chlorophyll a,b Protein Stigma Filamentous Unicellular Unicellular

Sessile Kelp Fucus

Sessile attached Polysiphonia Porphyra

Motile diatomaceous earth diatoms

Motile flagella Euglena

The differences between algal groups are enormous, but in this task you will focus on traits present in select members of Phylum Chlorophyta, also known as the Volvocine line. The Volvocine line includes five genera (Chlamydomonas, Gonium, Pandorina, Eudorina, and Volvox) of related organisms that show progressive changes in cell aggregation and specialization (see pgs. 43-44 in your dissection atlas for images). Chlamydomonas, for example, is a single celled, motile alga with a stigma (eyespot) that functions in the absorption of light. Reproduction in Chlamydomonas is usually asexual except during times of environmental stress, when the organism produces identically sized and shaped gametes (isogamy) for sexual reproduction. At the other end of the spectrum is Volvox, which in contrast to Chlamydomonas, is colonial, has specialized cells for reproduction and is oogamous, (i.e. gametes produced are not identical; one gamete is small and motile while the other is large and non-motile). While all members of the Volvocine line can reproduce both sexually and asexually, oogamy is unique to Volvox. Similarly, while each genus possesses eyespots to sense light and flagella for movement, cell polarity only becomes evident later in the Volvocine line, beginning with Pandorina.

NOTES: Do NOT contaminate the living samples by mixing the caps between the samples.

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 Do NOT completely close the caps on any of the living samples!!

Procedure 1: 1. Prepare wet mounts of the protists listed in Table 6. 2. Observe the slides under a compound microscope. 3. Complete Table 6 below.

Table 6: The Volvocine line Genus Characteristic Number of cells Chlamydomonas Gonium Pandorina Eudorina Volvox

Colony size

Cell specialization/ differentiation

Isogamy vs. oogamy

Drawing (note magnification)

Questions: 1. Explain the significance of the increased cell specialization of the Volvocine line?

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2. What are some of the possible advantages of oogamy?

3. How does the stigma help algae survive?

-----------------------------------------------------------------------------------------------------------B. Protozoa Protozoans (proto = first and zoa = animal) are unicellular, heterotrophic organisms that occupy marine, freshwater and terrestrial environments. Members of this group are generally characterized by their mode of locomotion; (1) ameboid use psuedopods (Phylum Rhizopoda e.g. Amoeba), (2) ciliate use cilia (Phylum Ciliophora e.g. Paramecium) and (3) flagellate use flagella (Phylum Sarcomastigophora e.g. Trypanosoma). In addition, some protozoans also possess a food vacuole which is used to digest and absorb ingested materials and contractile vacuoles that function in expelling water. Reproduction in these organisms varies, but most genera reproduce asexually and sexually. Procedure 2: 1. Prepare wet mounts of the Amoeba and Paramecium. 2. View each specimen under the microscope. 3. Complete Table 7 below. 4. If prepared slides are available, compare these to your wet mounts.

Table 7: Protozoa

Phylum

Genus

Description

Drawing (note magnification)

Rhizopoda

Amoeba

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Ciliophora

Paramecium

Questions: 1. Why is a contractile vacuole harder to see than a food vacuole?

2. Compare and contrast the movement of Amoeba and Paramecium.

-----------------------------------------------------------------------------------------------------------C. Myxomycota Myxomycota, more commonly known as slime molds, are brightly-colored (yellow or orange), heterotrophic organisms that exhibit amoeboid movement. Like fungi (mushrooms), slime molds are multinucleate, feed on dead/decaying material (i.e. they are decomposers) and reproduce via spores produced in sporangia (see Figure 8.5 in your dissecting atlas). However, in contrast to fungi, the cell walls of slime molds are not made of chitin but instead are composed of cellulose.

NOTES: Obtain TWO dissecting microscopes per group. Make sure to record which microscopes your group will be responsible for on the sign-in sheet at the front of the room. You will use these microscope numbers for the remainder of the semester. Before you begin Procedure 3, your TA will need to demonstrate to your group the proper procedure for using a dissecting microscope.

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Procedure 3: 1. Examine both Physarum and Coprinus (i.e., the button mushroom) with a dissecting microscope. 2. View the prepared slide of Coprinus. Compare what you see to Figure 8.5 (pg. 53) in your dissecting atlas.

Phylum

Genus

Description

Drawing (note magnification)

Myxomycota

Physarum

Basidiomycota

Coprinus

Question: What is the significance of spores forming on the ends of upright filaments in the Coprinus, rather than closer to the protective substrate?

________________________________________________________________________ Task 4: Check your fast plants and record any changes in your Fast Plant appendix _______________________________________________________________________ Task 5: Check and water your basil plants ________________________________________________________________________

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LOOK AHEAD: Before coming to lab next week, make sure to read the Photosynthesis task sheet. ________________________________________________________________________ REFERENCES: Costello EK, Lauber CL, Hamady M, Fierer N, Gordon JI, Knight R. 2009. Bacterial community variation in human body habitats across space and time. Science 326: 16941697.

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