BIO112 Laboratory Guide #3   DIVERSITY OF THE BACTERIA AND PROTISTS
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INTRODUCTION. Monera is the former kingdom of the prokaryotes -- those single-celled organisms without a nucleus or other membrane-bound organelles. Recent research on bacterial systematics indicates that the bacteria actually belong in two distinct domains (a category higher than the kingdom): Archaea and Bacteria. The prokaryotes are the most ancient lineage of living organisms, with evidence of fossil bacteria being found in rocks 3.5 billion years old. The earth was ruled by bacteria and bacteria alone for at least 1.5 billion years! The bacteria are very diverse metabolically, but two main functional divisions can be made: some are heterotrophic, and others are autotrophic. The photosynthetic prokaryotes are the cyanobacteria, formerly called blue-green algae because they resemble the aquatic eukaryotic algae with which they occur. Due to the small size of bacteria and the lack of distinguishing characteristics visible under a light microscope, bacteria are often classified based on both the shape of their cells (e.g., sperical vs. rod-shaped) as well as their response to the Gram stain. All bacteria have cell walls, which stain differently depending on their chemical makeup. All eukaryotes are classified in the domain Eukarya. The protists are single-celled eukaryotes, formerly all lumped into the kingdom Protista. Currently there are a number of classification schemes for the protists, reflecting the dynamic state of protist biology: some schemes even recognize multiple “candidate kingdoms.” This is a very diverse group because it includes both autotrophic and heterotrophic organisms. The autotrophs (“self-feeders”) are photosynthetic, manufacturing their own food by capturing the energy of sunlight in the chemical bonds of carbohydrates. Photosynthetic organisms can be readily recognized by the presence of pigments in their cells. Protists also include "mixotrophs," organisms such as Euglena that are both heterotrophic and autotrophic. Mode of locomotion is also diverse among the protists. They move via flagella (whip-like tails), cilia (short, hair-like structures that beat in unison), or pseudopods (“false foot” -- as in the oozing amoeba); some have no means of locomotion.   In this lab you will encounter representatives of both of these large and diverse groups and will learn about their basic biology and ecology. Antibiotics are bioctive agents which inhibit the growth and ultimately kill bacteria. The molds (fungi) are often in direct competition with bacteria for resources or are subject to bacterial infection. As a consequence, many molds produce potent antibiotics. Several of these have been adapted for medical use in treating bacterial diseases, for example the antibiotic penicillin was originally isolated from the bread mold Penicillium. In this lab you will test the several antibiotics for their effectiveness in inhibiting growth of two bacteria, Escherichia coli and Bacillus cereus. In the process you will learn and practice several common techniques used in microbiology - the study of microbes.   After completing this laboratory you should be able to:   1)   List and define the distinguishing characteristics of members of the domains Archaea, Bacteria, and Eukarya; and the candidate kingdoms for the protists.   2)   Identify and correctly classify the specimens seen in this laboratory.   3)   Discuss the basic biology and ecology of the bacteria and protists.   4)  Understand and demonstrate sterile technique as applied to the establishment and handling of bacterial cultures.   5)  Make reliable, quantitative measures of bacterial antibiotic sensitivity form cultured plates.                       PART I. DIVERSITY OF THE BACTERIA AND PROTISTS   Materials      Specimens at numbered stations arranged on the lab benches.  These consist of fresh materials and live specimens as well as preserved specimens, prepared microscope slides, and other written and illustrated materials.        Microscopes, slides, cover slips, forceps, lens paper.        Dissecting stereomicroscopes.   Procedure   1.   View the specimens of bacteria and protists, noting their key identifying features.  A great idea for future studying would be to make a drawing of each specimen, labeling all of the characteristic structural features.     2.   For each specimen, be sure to record its common name as well as its formal classification (e.g., domain, kingdom or candidate kingdom, phylum and species) 3.   Be able to recognize all specimens as well as their distinguishing characteristics. 4.   For the live specimens, make a wet mount of  each, or examine a prepared slide, or look at an entire specimen, depending on the organism.   Study Suggestions   1.   Make detailed sketches and notes on specimens.  This will help you in two ways: 1) when you attempt to draw a specimen, you are forced to look at it more closely, and 2) the drawings will help you study later. 2.   Plan to come view the specimens once or twice more before the lab test.  Test yourself by attempting to identify the specimens without first looking at their labels.   A. BACTERIA Bacterial diversity includes the domains Archaea and Bacteria, both of which are characterized by having prokaryotic cells.  Only specimens of domain Bacteria are presented in this lab exercise; however, pictures of typical archaean habitats and bacteria are provided. The extant members of Domain Archae (archaebacteria) fall into three groups - the methanogens, the extreme halogens, and the extreme thermophiles (or thermoacidophiles).  Review each of these groups in the Bacterial Diversity powerpoint at the Bacterial table.  Make sure that you can distinguish the conditions under which each group lives, where on earth those conditions are found, and what makes those conditions "extreme".  Note that these are not phylogenetically valid groups; the true internal phylogeny of the Archea has not been definitively established. The specimens of domain Bacteria (eubacteria) in today's laboratory were selected to illustrate the diversity in bacterial shape, response to Gram stain, and mode of nutrition.  The so-called “gram-positive” bacteria retain a Gram stain in the thick layers of peptidoglycan in their cell walls and therefore will appear purple/violet.  In contrast, the “gram-negative” bacteria have less peptidoglycan (and it is beneath an outer membrane of their cell wall) and therefore do not take up the stain; they appear red.  The Gram stain is an important way to differentiate among different types of bacteria that are otherwise difficult to distinguish under a microscope.  View the slides of Bacillus cereus var. mycoides and E. coli and note the color of each: which species is Gram + and which is Gram -?  Broadly speaking, bacterial cells may be classified into three groups based on their morphology: rod-shaped (bacilli; singular = bacillus), spiral (i.e., spirilli/helices) and  spherical (cocci, sing. = coccus).  Review these forms in the Bacterial Diversity PowerPoint at the Bacteria table. All of the above bacteria are heterotrophic, but the next specimen is photosynthetic and therefore belongs to the group of bacteria known as cyanobacteria.  View the specimen of Oscillatoria sp., noting the presence of photosynthetic pigments that make the cells appear green.  How would you tell Oscillatoria from a green alga (Chlorophyta)?     B. PROTISTS All protists are in Domain Eukarya, meaning that their cells are eukaryotic.  The vast majority of protists are single-celled, though there are some exceptions, especially among the "algae."   Protists are an extremely diverse group of organisms, and their classification seems to be in a constant state of flux.  Just as an example of protistan diversity, their modes of nutrition range from ingesting food (the "protozoans") to absorbing food (fungus-like protists) to photosynthesis ("algae").  As was pointed out earlier, there are also "mixotrophs" than can both ingest food and carry out photosynthesis.  Unfortunately, protistan systematics is not a straightforward affair: the various algae, for example, are not always closely related (i.e., monophyletic).   In today's lab we will survey representatives from many of the "candidate kingdoms" or "superclades" of protists, using the systematics that adopted by your textbook. ---------------- CLADE EXCAVATA Diplomonadida and Parabasala The diplomonads (and their relatives the parabasalids) represent a group of eukaryotes that probably diverged very early from the rest of Domain Eukarya.  They have multiple flagella, two nuclei, and cytoskeltons that are simple compared to those of most eukaryotes.  View the prepared slide showing cysts of Giardia lamblia, the parasitic protist that causes giardiasis, which is characterized by severe diarrhea and cramping.  Giardia is transmitted via these cysts in infected waterways, even in wilderness areas; therefore, campers and backpackers should always boil their water or treat it chemically with iodine to ensure that they don't pick up this parasite.  Although probably not visible in this specimen, a key feature of this species and other members of its candidate kingdom is that they lack mitochondria. Euglenozoa All members of Euglenozoa are single-celled protozoans with flagella.  Their mode of nutrition varies: some species are heterotrophic, some are photosynthetic, and some are mixotrophic.  Furthermore, some euglenozoans are free-living and others are parasitic. In addition to the Euglenoids (next paragraph), candidate kingdom Euglenozoa also includes Typanosoma (a kinetoplastid), the parasitic organism that causes sleeping sickness. Prepare a slide with live specimens of Euglena -- you may wish to add a drop of  "Protoslo" on the slide to slow down the Euglena so you can view them closely. Euglena has an unusual flexible pellicle of protein embedded within its cell membrane -- this is quite unlike the solid cell walls of some protists.  Can you detect changes in the shape of Euglena as it moves around?  The two flagella -- one long, one short -- are located at the anterior end of the cell.  Look for the light-sensitive stigma, also located near the anterior end.  What color is Euglena, and what does this tell you about its mode of nutrition?   ---------------- SAR GROUP CLADE STRAMENOPILA  The stramenopiles are a diverse clade of algae and mold-like protists united by common structural features of their flagella (some of which bear fine hairlike projections) and their chloroplasts, if present (these are derived from endosymbiotic eukaryotes).  Diatoms (Bacillariophyta) Diatoms are single-celled photosynthetic organisms with silica shells.  Diatoms are found in both freshwater and saltwater.  They lack flagella.  Like their relatives the brown algae, diatoms store food in the form of the carbohydrate laminarin.  Prepare a slide of water from Foster Lake and search for live diatoms -- their geometric siliceous shells are distinctive.  See also the slides of preserved diatoms. Brown Algae (Phaeophyta) The brown algae are all multicellular "seaweeds," but range in size from microscopic to enormous (e.g., Macrocystis, the giant Pacific kelp, may be 100 meters long!).  Brown algae are largely marine organisms, and are particularly common in the cold waters of the temperate zone.  Their characteristic brown or olive-green color comes from the presence of the pigment fucoxanthin (a kind of xanthophyll); brown algae also have chlorophylls a and c.  Phaeophytes store food as laminarin (a carbohydrate), in contrast to the storage molecules of the green and red algae. Examine the preserved specimens of brown algae.  Laminaria has a flattened leaf-like structure as well as a stalk and a "holdfast" which helps to secure it to its substrate (usually rocks).  This differentiation into specialized structures is characteristic of many brown algae.  Both Sargassum (of Sargasso Sea fame) and Fucus have air bladders that help to keep them afloat; see the plastomount of Fucus to get a good sense of the three-dimensional nature of these bladders. Golden Algae (Chrysophyta) The golden algae are much smaller colonial forms, as illustrated in the photo. Water molds (oomycotes) The oomycotes are single-celled fungus-like protists    Their thread-like hyphae are similar to fungal hyphae, but have cellulosic cell walls (not chitinous, as in kingdom Fungi); furthermore, the diploid condition dominates in oomycote life cycles, whereas the haploid condition dominates in the fungi.  View the prepared slide of the water mold Achyla, noting the hyphae -- what advantage does the thread-like shape of the hyphae confer?  (Hint: most oomycotes are either parasites or saprobes.) SAR GROUP CLADE ALVEOLATA    This is a diverse clade including both autotrophs and heterotrophs.  All are single-celled and are characterized by "alveoli" (sub-surface, membrane-bound cavities), but their mode of nutrition and locomotion varies from group to group.  There are three distinctive groups of alveolates:   Dinoflagellates (Dinoflagellae)   Dinoflagellates are mostly photosynthetic marine phytoplankton with two flagella; they also have distinctive internal armor of cellulose plates.   See the prepared slide of Ceratium.  Note the armored appearance and look for the flagella -- can you see the transverse groove that houses one of the flagella?  (The other flagellum would be sticking out from the body of the dinoflagellate, perpendicular to the first flagellum.) Ciliates (Ciliata) The ciliates are unicellular heterotrophs with cilia, fine hairlike projections used for locomotion.  Prepare a slide from the culture of live Paramecium -- as with Euglena, you may need to use "Protoslo" to slow them down enough to observe them carefully.   Paramecium, like most ciliates, is a freshwater organism.  Try to locate the following structures on Paramecium: cilia, oral groove, macronucleus and micronucleus, and contractile vacuole. Observe the contractile vacuole closely under the oil immersion lens on your microscope -- what does it appear to be doing?  What is its function? Apicomplexans (Apicomplexa) The organism that causes malaria (Plasmodium) is an apicomplexan, one of the three sub-groups within Alveolata.  This group is characterized by the presence of an "apical complex" of organelles that is used to penetrate the tissues of the host organism.  (No specimen today in lab.) SAR GROUP CLADE RHYSARIA   The Rhizaria are a three taxa of planktonic protists with fine, thread-like pseudopods (axiopodia) which radiate out from the center.  Radiolarians have spherical shells made of silica and/or chiton.  Forams (Foraminifera) have irregularly-shaped shells made of calcium carbonate.  As the photo illustrates the “White Cliffs of Dover” on the English Channel are made largely of these foram “tests”.  The Cercozoans have no shells.   Review the photomicrographs of these three groups on display. ---------------- CLADE ARCHAEPLASTIDA The Archaeplastida clade includes the red algae (Rhodophyta) and the green algae (Chlorophyta).  The latter group are the ancestral group to all of the members of the Kingdom Plantae. Red Algae (Rhododophyta) Rhodophyta includes the red algae, so-called because of their red color, imparted by the pigment phycoerythrin (they also have chlorophyll a).  Red algae may be differentiated from brown and green algae not only by their color, but also because they store food as floridean starch (another kind of carbohydrate) and because they have no flagellated cells at any point in their life cycle.  Like phaeophytes, red algae are primarily multicellular and mostly marine organisms, but unlike the brown algae, they are most abundant in the warm waters of tropical regions. View the preserved specimens of Polysiphonia, a red alga.  See also the package of roasted seaweed -- the red alga Porphyra ("nori"), is used as a wrapper for sushi.  Agar and carrageenan are two other products derived from red algae that are very valuable commercially.  Agar is used in the lab to grow bacterial cultures, and carrageenan is found in a wide range of products, from paint to ice cream. Green Algae (Chlorophyta and Charophyta) The green algae, Chlorophyta, are the protistan group most closely related to the Kingdom Plantae (true plants).  Like true plants, the color of green algae is imparted by chlorophyll a and b; also like plants, their food is stored as starch (how do these two features differ from those of red algae and brown algae?)  Green algae may be single-celled, colonial, or multicellular; they are mostly freshwater, but some are marine or even terrestrial.  View the live and preserved specimens of Volvox, a freshwater, colonial green alga.  Describe the shape and motility of Volvox.  Can you see "daughter colonies" within a colony?  See also the preserved specimens of Ulva (sea lettuce), an edible seaweed that is only two cell layers thick! The Charophytes are the most direct ancestors of the plants.  Chorophytes have stem-like structures,, with radial branches at each node.  Each of the intermodal “stem” regions is aingle giant cell.  The Charophytes also show true alternation of generations, as seen in the plants. ---------------- CLADE UNIKONTA AMOEBOZOA The Amoebozoans have heteotrophic, single-celled amoeba forms with thick protoplasmic psuedopods as at least part of their life cycle.  They feed by engulfing small particles (phagocytosis). Slime Molds (Myxogastrica)  The slime molds are superficially similar to true fungi. However, slime molds have amoeboid stages that are much more similar to amoebas than they are to fungi.  Both plasmodial and cellular slime molds have complex life cycles that involve both feeding and reproductive stages.  A key distinction is that the feeding stage (plasmodium) of the plasmodial slime molds is a massive multinucleate cell, whereas the feeding stage of the cellular slime molds is made up of many independent amoeboid cells.  When the cellular slime mold cells start running low on food, they form an aggregate colony that looks like a plasmodium, but it is composed of many cells separated by plasma membranes. Examine the prepared slide of the cellular slime mold Dictydium -- this is a whole mount of the sporangium, or spore-producing structure, of this organism. Tubulinids and Entamoebas (Gymnamoebae and Entamoebae) Both tubulinids and entamoebas have lobe-shaped protoplasmic pseudopods which they use for locomotion and feeding.  The tubulinids are primarily free-living, while the entamoebas typically live within the gut of vertebrate hosts. Prepare a slide with a single live Amoeba proteus, a common pond tubulinid.  Use either a depression slide or support the coverslip or a regular flat slide with a few grains of sand so as not to crush the amoeba.   Observe the locomotion of the amoeba.  How do the pseudopods work?  Try to locate food vacuoles within the cell.  How does an amoeba ingest its food?   Make a labeled diagram of an amoeba. The "protoplasmic amoebas" were historically lumped with the "filamentous amoebas" into a single phylum called Sarcodina".  It is now recognized that this was a decidedly polyphyletic group.  Which protists clades now contain the filamentous amoebas (which have fine, ray-like pseudopods - see groups above)? CLADE UNIKONTA OPISTHOKONTA The opisthokonts are the other main group of Clade Unikonta.  Although there are few surviving species of nucleariids or choanoflagellate protists, you should recognize that these groups are thought to be ancestral to Kindoms Fungae and Animalia, respectively.  Review the bases for this classification in your text.       PART II: ANTIBIOTIC SENSITIVITY IN BACTERIA   In this part of the lab you will test the ability of six antibiotics to inhibit growth in two bacterial cultures.  Escherichia coli (E. coli) is a gram-positive bacillus and Bacillus cereus (B. cereus) is a gram-negative bacillus.   Materials   nutrient agar plates broth cultures of E. coli and B. cereus antibiotic disks – Penicillin, Gentomycin, Ampicillin, Chloramphenicol, Streptomycin,                             Tetracycline control disks disk dispenser Bunsen burner and striker sterile swabs forceps Sharpie pen incubator set to 37ºC   Procedure          BE SURE TO WEAR AN APRON, GLOVES, AND SAFETY GOGGLES        WHEN WORKING WITH LIVE BACTERIAL CULTURES.  USED        GLOVES AND SWABS GO IN THE ORANGE BAG BIOHAZARD        CONTAINER.  CONTAMINATED METAL SURFACES, SUCH AS        FORCEPS TIPS MUST BE FLAMED WHEN YOU ARE FINISHED.         WASH YOUR HANDS AFTERWARDS USING ANTIBACTERIAL        SOAP.   1.  On your desk, you will find two plates of nutrient agar. Label the bottom of one of      these plates with your initials and E. coli. Label the second plate with your initials      and B. cereus.   2.  Practice streaking the pretend plates at the end of your worksheet with your pencil      following the sequence in #3 below. Be sure to cover the entire plate with each streaking.   3.  Inoculate your plate labeled E. coli by the following procedure:        a. prepare a sterile swab, making sure not to touch the tip to any surfaces      b. unscrew the E.coli broth culture tube and flame the lip for 1-2 seconds      c. dip the sterile swab into the culture tube – DO NOT TOUCH THE RIM      d. remove the top of the E. coli plate      e. carefully streak the entire surface of the agar with the swab      f. rotate the plate 90º and streak it again      g. rotate the plate 45º and streak it a third time      h. cover the plate      i. recap the culture tube.   4.  After you have streaked the E. coli plate, invert the plate and leave it on your desk top.   5.  Repeat steps 3&4 with the plate and liquid culture labeled B. cereus. Be sure to use      a new sterile swab for this inoculation.   6.  Check the antibiotic disk dispenser to make sure that all six antibiotic vials are      securely mounted.   7.  Uncover the E. coli plate, position the disk dispenser over it, and dispense the      six disks onto the plate.   8.  Use flamed forceps to deposit a control disk in the center of the plate.   9.   Press each disc gently with the wooden end of a sterile swab so that it makes       good contact with the agar surface.   10. Recover the plate and invert it.   11. Repeat steps 7-10 with the B. cereus plate.   12. Give both plates to the instructor or TA. The plates will be incubated for       24 hours at 37ºC.          BE SURE TO WEAR AN APRON, GLOVES, AND SAFETY GOGGLES        WHEN WORKING WITH LIVE BACTERIAL CULTURES.  USED        GLOVES AND SWABS GO IN THE ORANGE BAG BIOHAZARD        CONTAINER.  CONTAMINATED METAL SURFACES, SUCH AS        FORCEPS TIPS MUST BE FLAMED WHEN YOU ARE FINISHED.         WASH YOUR HANDS AFTERWARDS USING ANTIBACTERIAL        SOAP.   13. About 24 hours later, remove both plates from the incubator.   14. Carefully measure the clear circle of inhibited growth around each of the antibiotic       disks, as well as the control disk in the center.  Use an electronic caliper set to       the mm scale.  Be sure to zero the caliper with the jaws closed before each       measurement.   15. Enter your measured values in the table in your lab worksheet.  The antibiotic codes       on the disks are:        P-10  Penicillin               GM-10 Gentomycin     AM10  Ampicillin      C-30 Chloramphenicol   S-10 Streptomycin       TE-30 Tetracycline   16. Return the plates to the incubator.          BE SURE TO WEAR AN APRON, GLOVES, AND SAFETY GOGGLES        WHEN WORKING WITH LIVE BACTERIAL CULTURES.  USED        GLOVES AND SWABS GO IN THE ORANGE BAG BIOHAZARD        CONTAINER.  CONTAMINATED METAL SURFACES, SUCH AS        FORCEPS TIPS MUST BE FLAMED WHEN YOU ARE FINISHED.         WASH YOUR HANDS AFTERWARDS USING ANTIBACTERIAL        SOAP.   17. After another 24 hours (48 hours total elapsed time) repeat steps13-15.   18. WHEN YOU HAVE FINISHED WITH BOTH PLATES, PLACE THEM       IN THE ORANGE BAG BIOHAZARD CONTAINER AT THE FRONT       OF THE ROOM.
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