Exercise 2: Bacteria and Protozoans objectives




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Exercise 2: Bacteria and Protozoans

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OBJECTIVES:

  1. Understand the general characteristics of bacteria and cyanobacteria.

  2. Learn how to perform a Gram stain to identify bacteria.

  3. Understand the general characteristics of protozoans.

  4. Compare the differences between different protozoa phyla.

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INTRODUCTION TO BACTERIA

Cellular organism evolved along two lines: 1) species lacking membrane-bound organelles (prokaryotes), and 2) species with membrane-bound organelles (eukaryotes). Another major difference between these two lines is that prokaryotes have no nucleus, and genetic material resides in the cytoplasm of the cell, whereas eukaryotes have a nucleus that houses



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Figure 1: Three basic shapes of bacteria: a) bacillus, b) coccus,

and c) spirillum.


enetic material. Within prokaryotes, there are two distinct groups: Archaea and Bacteria. Species within the domain Archea often inhabit, but are not restricted to, extreme and stressful environments (e.g. areas with very high temperatures or pHs) where other organisms cannot reside. Species within the domain Bacteria exist in a variety of environments, and are the most abundant and widely distributed organisms on Earth. Individual bacterial cells are microscopic, and their cell walls give them three characteristic shapes: bacillus (rod-shaped), coccus (spherical), and spirillum (spiral) (Fig. 1). Most bacteria are heterotrophic - they derive their energy from organic molecules made by other organism. Heterotrophic bacteria are often decomposers, feeding on dead organic matter and releasing nutrients locked in dead tissue.
Question:
Why is it important that decomposers, such as bacteria, release nutrients?

Bacteria reproduce asexually via fission, in which a cell’s DNA replicates and the cell pinches in half without the nuclear and chromosomal events associated with mitosis (General Biology I) - this enables bacteria to synthesize DNA almost continuously. Some bacteria have genetic recombination via conjugation, in which all or part of the genetic material of one bacterium is transferred to another bacterium and a new set of genes is assembled.


Questions:


  1. Why do you think bacteria do not undertake mitosis in order to reproduce?




  1. Based on the information above, is there any chance for genetic variability among bacteria? Why or why not?

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Task 1 - GRAM STAIN: differentiating types of bacteria

Bacteria are microscopic, and can be difficult to identify, therefore there are several techniques to divide them into distinct groups; one of which is Gram staining. This technique divides bacteria into Gram-positive and Gram-negative strains based on differences in the structural and chemical composition of their cell wall (Fig. 2).


Figure 2: Structural differences between Gram-positive and Gram-negative bacteria.


Bacterial cell walls contain peptidoglycan, which consists of polymers of modified sugars cross-linked by short polypeptides. Gram-positive bacteria, like Staphylococcus, have simple, thick cell walls with high concentrations of peptidoglycan. Gram-negative bacteria, like E. coli, have thinner cell walls with much less peptidoglycan, and are structurally more complex than Gram-positive bacteria (the outer membrane on a gram-negative cell wall contains lipopolysaccarchides - carbohydrates bonded to lipids). Gram staining takes advantage of this difference by infusing bacterial cells with purple dye and determining the type of bacteria (+ or -) by how well the dye is retained in its cell walls. Gram-positive bacteria tend to retain more dye and appear purple because their thick cell walls trap the dye, and Gram-negative bacteria do not retain the dye well and appear pink in color. You will perform a Gram stain (Fig. 3) on some commonly encountered bacteria in order to see the differences between Gram-positive and Gram-negative strains.

Figure 3: Gram stain procedure
Procedure:


  1. Use a compound light microscope to observe slides of available bacteria prepared with a Gram stain. Remember to start with the lowest power objective and progress to the next highest objective after focusing. Oil-immersion may be needed to observe some of the bacteria. Follow these instructions exactly or you will damage the microscope:

    1. Focus at the lowest power and work your way up to the 40X objective, getting the slide as focused as possible

    2. Partially rotate the turret so that 40x and 100x objectives straddle the specimen

    3. Place a small drop of oil on the slide in the center of the lighted area

    4. Rotate turret so that the 100x oil immersion objective touches the oil and clicks into place

    5. Focus only with fine focus. Hopefully, the specimen will come into focus easily. Do not change focus dramatically. If you still have trouble, move the slide slightly left and right, looking for movement in the visual field, and focus on the object which moved.

    6. Never go back to the 10x or 40x objectives after you have applied oil to the specimen since oil can ruin the lower power objectives. The 4x objective can be used because it is high enough to be above the oil.

    7. When you have finished, wipe the 100x oil immersion objective carefully with lens paper to remove all oil. Wipe oil from the slide thoroughly with a Kimwipe. Clean the stage if any oil spilled on it.




  1. Obtain a clean slide and cover slip.




  1. Using a transfer loop, apply a small portion of a bacterial culture (only a tiny portion of the culture is needed - excess material will make the bacteria unviewable) to the clean slide with a drop of water. Prior to transferring the bacteria, make sure you sterilize the transfer loop by passing it over the flame of a lit Bunsen burner several times. It is important not to cross-contaminate the samples, or your sample may contain a mixture of Gram-positive and Gram-negative bacteria.




  1. After applying the bacteria to the slide, sterilize the transfer loop and return it to its case.




  1. Heat the slide gently (bacteria side up) by holding it with the slide holders and passing it over the top of the flame of a Bunsen burner several times to adhere the bacteria to the slide. Make sure you do not hold the slide directly over the flame or it will break.




  1. When the slide has cooled, hold the slide over the sink and gently drench the bacterial smear with drops of crystal violet for 20 seconds.




  1. Rinse the slide for 2 seconds with a gentle, but steady stream of deionized water.




  1. Gently drench the bacterial smear with drops of iodine for 1 minute.




  1. Drop 95% alcohol on the smear with a pipette until no purple shows in the alcohol coming off the slide. Quickly rinse the slide with water to remove the alcohol.




  1. Gently drench the bacterial smear with drops of safranin for 20 seconds.




  1. Gently rinse the slide with water and allow to air dry.




  1. Observe the smear with your microscope and determine if the bacteria is Gram-positive or negative, its relative size (in comparison to the other bacterial species), and its shape.




  1. Repeat the Gram stain procedure using the other bacterial cultures available.




  1. Record your observations in Table 1 below.

Table 1: Results from Gram stains



Bacterial species

Gram Stain (+/-)

Relative size

Shape












































































  1. Obtain a clean slide and cover slip.




  1. Using the wide end of a toothpick, scrape your teeth near the gum line.




  1. Thoroughly mix the contents of the toothpick with a drop of water on the microscope slide.




  1. Repeat steps 3-12 and record your results in Table 1.



Questions:


  1. Which type of bacteria is most prevalent in the sample from your teeth?


  1. What is an advantage of bacteria being Gram-positive or Gram-negative?

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Task 2 - COMPARE CYANOBACTERIA

Cyanobacteria (Fig. 4) are photosynthetic bacteria that harness light energy to drive the synthesis of organic compounds. Most cyanobacteria are free-living, but some live symbiotically with plants and other organisms. The photosynthetic pigments found in cyanobacteria include chlorophyll a, phycocyanin, and phycoerythrin, and the mixture of pigments they house influences the color of cyanobacteria (blue-green to brown to dark green). Cyanobacteria are the base of the food web in many ecosystems, and in addition to producing biomass and energy that is consumed by heterotrophs, cyanobacteria also produce oxygen as a byproduct of photosynthesis. Despite their similarities to algae (Labs 3 & 4), cyanobacteria are prokaryotes, whereas algae are eukaryotes.


Figure 4: Cyanobacteria a) Oscillatoria, b) Nostoc, c) Gleocapsa, and d) Merismopedia



Procedure B:


  1. Using your compound light microscopes, observe the prepared slides of Oscillatoria, Nostoc, and other available cyanobacteria, and compare these to available live cultures.




  1. Fill in the appropriate information about cyanobacteria in your Taxa organization chart (if the subject does not apply to the organisms place a large “X” in the box or write NA).


Question:
How does the structure and relative size of cyanobacteria compare to the bacteria observed during the Gram stains?

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INTRODUCTION TO PROTOZOA

Protozoans are part of a group of organisms called protists. This group is comprised of eukaryotic organisms that lack the features of animals, plants, and fungi, but may exhibit characteristics similar to one or more of these groups. The current classification of protists divides the group into three supergroups, each of which share several characteristics with either plants, animals, or fungi: protozoa are free ranging heterotrophs, slime molds are decomposers like fungi, and non-green algae are generally autotrophic. The next lab will focus on slime molds and algae, but here we will learn about protozoans. Protozoans are single-celled eukaryotes with animal-like, heterotrophic ecology. Protozoans typically have food vacuoles to enclose food particles for digestion and contractile vacuoles to expel excess water.

Body plan and locomotion are the major differences across protozoan groups. Amoebas (Rhizopoda) live in marine, freshwater, and terrestrial ecosystems, and are unified by the presence of pseudopodia - moveable extensions of cytoplasm used for locomotion and gathering food. When Amoebas move, they extend a pseudopodium, anchor it, cytoplasm streams into the pseudopodium, and the process is repeated. In addition to movement, pseudopodia can also be used to acquire food. Amoebas use phagocytosis to engulf food particles and form food vacuole surrounded by membranes. Enzymes are secreted into food vacuoles for intracellular digestion. Contractile vacuoles maintain a cell’s water balance by accumulating and expelling excess water. Other common amoebas include Difflugia, which creates a protective case of sand grains that surround its body.

Figure 5: Ameoba.



F

Figure 6: Foraminifera


oraminifera
(Fig. 6) surround themselves with a secreted test/shell made of calcium carbonate. These tests are often perforated with pores that allow pseudopods to extend into the environment and are used for food acquisition and movement. Some species float in the water column, but most live on or within sediment in marine ecosystems ranging from deep ocean trenches to salt marshes and everywhere in between. Foraminifera are important food sources for organisms such as snails, sand dollars, and fishes, and the tests of Foraminifera often accumulate in sediment and can be used to examine past conditions by paleontologist who use the tests to investigate the geologic age of sediments and the conditions under which it was formed.


Figure 7: Paramecium body plan



Paramecium (Fig. 7) have a submembrane system of microtubules that can coordinate the movement of thousands of small cilia (hair-like projections) used for locomotion. The specific arrangements of cilia adapt ciliates for their particular life cycle. Paramecium, like most ciliates, can undergo both asexual and sexual reproduction, and unique to ciliates is the presence of two types of nuclei: a large macronucleus and several tiny micronuclei. The macronucleus has 50 or more copies of the genome, and the genes are not distributed in typical chromosomes, but are instead packaged into a much larger number of small units, each with hundreds of copies of just a few genes. The macronucleus controls the everyday functions of the cell. During conjugation (Fig. 8a; sexual reproduction), individuals from two different strains align longitudinally and exchange genetic material. Mitosis then occurs in each cell, during which the micronuclei either remain in the parent cell or are transferred to the other cell. Asexual reproduction is achieved through mitosis by transverse fission during which the macronucleus splits, rather than undergoing mitotic division (Fig. 8b).

Figure 8: During conjugation (sexual reproduction) (a), Paramecium individuals exchange genetic material. Paramecium divide asexually by transverse fission (b).



Vorticella (Fig. 9) are sessile (non-motile) freshwater ciliates with two notable features: 1) a contractile stalk that attaches the organism to the substrate, and 2) a cell body with a corona of cilia. To feed, Vorticella extends its contractile stalk to push the cell body as far as possible from the substrate and from other individuals, and then beats its cilia to capture food particles. Similar to Paramecium, Vorticella reproduces sexually via conjugation or asexually via fission. Vorticella can also reproduce asexually by budding, when the cell undergoes longitudinal fission. One daughter keeps the stalk and the free daughter swims until finding suitable substrate to fix and develop its own stalk. Vorticella may be found in groups considered colonies, but they are not true colonies because each cell has its own individual stalk, which allows each cell to detach from the cluster by reverting to a free swimming cell. This often occurs when environmental conditions are unfavorable for survival.

Figure 9: Vorticella

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Task 3 - COMPARE PROTOZOANS

You will investigate a variety of protozoans, each with unique characteristics. Make sure you note the major structural and behavioral similarities and differences between each organism.


Procedure:


  1. Using your compound light microscope, observe each prepared (Amoeba, Foraminifera, Paramecium, and Vorticella) and living (Amoeba, Paramecium, and Vorticella) protozoa available.




  1. Note how the movement of Amoeba and Paramecium are different, and how the structure of each protozoa enables it to perform its desired functions.




  1. Fill in the appropriate information about Amoeba and ciliates in your Taxa organization chart.

Questions:


  1. Do Amoeba and Paramecium move at similar rates, or does one move much faster than the other? What advantage(s) are there to moving fast or slow? What is an advantage to being sedentary?



  1. What are the advantages and disadvantages of asexual and sexual reproduction in Paramecium?



  1. How fast can Vorticella contract and extend its stalk? What advantage/disadvantage does this provide?



  1. How does the beating of cilia along the cell body of Vorticella enable it to capture food? Based on Vorticella’s size, what type of food would you expect it to eat?

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EXERCISE SUMMARY
Bacteria and protozoans are two groups of organisms with relatively simple morphologies and physiologies compared to animals, plants, and fungi, but possess the necessary structures to thrive in their respective environments. In this lab, you have observed different strains of bacteria, and investigated how differences in their cell walls lead to variability in their form and function. These prokaryotes lack many of the complex cellular structure that eukaryotes posses, but are the most successful group of organisms on the planet, and will continue to thrive and be important members of ecosystems worldwide indefinitely. Protozoans, one group of protists, are more complex than bacteria in that they have membrane-bound organelles, and a central structure (nucleus) that houses and organizes genetic material. Each of the model protozoans you have observed differs in structure, reproductive method(s), and/or function within its aquatic environment. It is important that you understand the similarities and differences between these groups, and as this course progresses, you will learn about more advanced autotrophic and heterotrophic organisms, and elucidate how each is morphologically, physiologically, and embryologically more complex than the previous groups. This understanding will provide insight into how life has evolved on this planet and why certain organisms are more successful than others.




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