Lab 9
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LABORATORY NINE

 THE KINGDOMS OF MONERA AND PROTISTA

 OVERVIEW:

            In order to make it easier to study all the various organisms that have been identified and many more organisms that have not been identified, biologists have grouped the living world into five kingdoms.  See Laboratory Five for the characteristics of the Five Kingdoms.  These five kingdoms are separated by fundamental characteristics such as cellular qualities and modes of nutrition.  In this laboratory we will be studying two kingdoms that are very diverse yet have two major characteristics in common — the organisms in these kingdoms are usually microscopic and many are solitary cells.  To be totally accurate, we must admit that there are some filamentous or colonial groups in these kingdoms, and even some are multicellular.  The Kingdom Monera contains prokaryotic life forms, such as the true bacteria and the blue-green bacteria or cyanobacteria, the latter formerly known as blue-green algae.  Prokaryotic cells lack a nuclear envelope; that is, the genetic material is not doubly membrane-bound but loose in the cytoplasm.  The Kingdom Protista is a diverse kingdom that contains the single-celled eukaryotic organisms, such as protozoa and some algae. Eukaryotic cells have a nuclear envelope that separates the genetic material from the rest of the cytoplasm.  Many biologists place all of the seaweeds, for example, sea lettuce, a green alga; and kelp, a brown alga, in this diverse Kingdom.

 

OBJECTIVES OF LABORATORY:

• To review the characteristics of the five kingdoms of life.

• To learn the major characteristics of the Kingdoms Monera and Protista.

• To review the steps of the Gram stain technique and to understand how it is used.

• To use observed characteristics and a dichotomous key to identify organisms.

• To be familiar with examples of the Kingdoms Monera and Protista that are available in the

   laboratory.

 

KINGDOM MONERA:

            Among the most commonly studied Monera are some of the bacteria that are medically significant.  We will be looking at a technique today called the Gram stain that is used to separate disease-causing organisms into two large groups on the basis of the staining characteristics of the cells.  Once the bacterial cells' particular Gram staining characteristic is known, medical personnel often can proceed with that information alone to prescribe an appropriate antibiotic.  It turns out that the two large groups of bacteria have different cell wall qualities, and those qualities are what make the cells stain differently as well as cause the cells to respond to some antibiotics differently.  Also, with the Gram stain, the clinician can describe the shape and possible grouping of the bacterial cells.  Morphology and cell wall staining qualities are a great help in both identifying any pathogenic organism and then choosing the correct treatment for the condition.

Choose one of the four kinds of cultures available on petri plates in the laboratory.  Watch as your lab instructor demonstrates proper technique for using the inoculating loop to make a bacterial smear.  Draw a dime-sized circle on the underside of a clean slide.  Place a small drop of water in the circle on the topside of the slide.  With the loop take a small, very small, amount of the culture from the plate and stir it into the drop of water.  Allow the drop to

dry without heating.  When the drop is dry, heat fix the smear by passing the slide through the flame of the burner with the smear side up once or twice.  Proceed with the following steps of the Gram stain.  See the diagrams on the facing page.

 

  1. Place the slide in a clothespin and cover the smear with crystal violet for 30 seconds.

 

  1. Wash the smear with a stream of distilled water from the wash bottle, a squirt bottle labeled "water", very gently.

 

  1. Without drying the slide, cover the smear with Gram's iodine for 10 seconds.

 

  1. Wash with distilled water.

 

  1. Remove the iodine by washing with 95% Ethanol (EtOH).  Continue to decolorize the smear as long as crystal violet dye (purple) washes from the smear, or about 10 seconds or so.  How much you decolorize depends on how thick the smear is, and smears are best when they are thin.  Remember we said use a very small amount of bacteria in making the smear! 

 

  1. Gently wash off the alcohol again with distilled water.    

   

  1. Stain the smear with safranin for 30 seconds.

 

  1. Wash with distilled water again.

 

  1. Blot the slide dry with the bibulous paper (in a booklet) provided.  Do not rub the smear, just blot by placing the slide inside the pages and pressing down.

 

  1. Observe the smear with the use of the oil immersion objective of your microscope.  Do not use a cover slip, but do place the drop of oil directly on the smear.  Your instructor will assist with this operation.  Be sure that you understand what to do before you use the oil immersion objective at this point.

 

            You should see purple or pink (reddish) cells.  You should be able to describe the morphology of the cells and perhaps how the cells are grouped.  Those that are purple are said to be Gram-positive, and those cells that are pink are referred to as Gram-negative.  Think back to the procedure that you performed.  Cells that are purple, Gram-positive, retained the crystal violet stain throughout the whole procedure.  Cells that are pink, Gram-negative, were decolorized by the alcohol wash even though they had been treated by the iodine, a mordant in stain technology terms.  Those cells that retain the purple dye when treated with iodine have a thick cell wall of a material called peptidoglycan, polymerized sugar molecules linked with short amino acid chains.  Those that are easily decolorized have only a thin layer of peptidoglycan but also an additional lipid bilayer in their cells walls; referred to as an “outer membrane” that does not hold the crystal violet dye.  These latter cells are stained with the safranin in the last step so that we can better see their morphology and arrangement.

            In our work today you had four possibilities of cultures.  Make a decision about the Gram reaction of the cells that you stained.  Look at the smears of your neighbors to see other forms.

 

 

 

 

 

 

            The antibiotic penicillin is much more effective against Gram-positive organisms than Gram-negative ones.  Gram-positive organisms have cell walls rich in peptidoglycan. Penicillin prevents the formation of peptidoglycan cell walls, and as a result the cells die or are unable to divide.  If you have a Gram-positive smear, then you as the clinician might prescribe penicillin or a related antibiotic. But you are not completely finished here.  There are two groups of Gram-positive organisms that we have in the lab today, and in order to tell your patient how to avoid contracting this disease again, you need to know which of these two groups this Gram-positive organism belongs.  These two groups contain organisms that either have the enzyme catalase or lack that enzyme; that is, they are catalase-positive or catalase-negative.  You can determine the presence or absence of the catalase enzyme by taking a small amount of the culture and testing it with hydrogen peroxide.  To do this place a loopful of the bacteria from the original culture on a clean slide and place a drop of peroxide on the culture.  If you see bubbles, your culture is catalase-positive.  Catalase-positive organisms use oxygen in their metabolism and need to remove some of the poisonous products produced during their metabolic processes.  Catalase-negative organisms do not use oxygen in their metabolism and therefore produce no such poisonous byproducts.

            Look back at your Gram stain.  If your Gram-positive cells are in clusters, this bacterium is likely to be catalase-positive.  If your Gram-positive cells are in chains, this culture is likely to be catalase-negative.  Your Gram stain can give you confirming information about some of the qualities of bacterial cells.  If your organism is Gram-positive and catalase-positive, you are dealing with organisms that are found on the skin, and you likely will advise greater care in bathing and the use of an anti-bacterial soap.  If your organism is Gram-positive and catalase-negative, you are dealing with an organism that inhabits the major openings of the body.  This may require further treatment and the use of an appropriate washing solution.

            The antibiotic streptomycin is an antibiotic of choice for Gram-negative organisms. Penicillin is not as useful for Gram-negative organisms, because their cell walls contain much less peptidoglycan.  Streptomycin interferes with DNA transcription and replication. You as the clinician likely will prescribe this antibiotic, but you need a little more information about these Gram-negative organisms that all look alike.  Gram-negative organisms are likely to have come from the patient’s digestive tract, from an environmental source, or by direct transmission from another infected individual.  There is a particularly hard to treat Gram-negative organism that you wish to rule out in this patient's case.  You can separate this more "difficult" organism from other more easily treatable Gram-negative forms by checking for the presence of the oxidase enzyme, an enzyme involved in an important energy-yielding metabolic pathway.  Place a small amount of the original culture on a clean slide.  Place an oxidase disk that has been dipped into distilled water, on the smear that you have made with part of the smear left uncovered by the disk.  Place the slide on the slide warmer set at 37º C for five minutes.  When the time has passed, the gray disk will be a purple color where it touches the bacterial smear if the culture is oxidase-positive.  There will be no color change if the culture is oxidase-negative.  Discard this slide, disk and all, in the biohazard box.

             If your culture is Gram-negative and oxidase-negative, then streptomycin alone is probably appropriate.  If your culture is Gram-negative and oxidase-positive, then you likely will consider a different antibiotic (polymyxin B, perhaps) as appropriate treatment and realize that you are dealing with an infectious agent that may be more difficult to treat.

 

 

 

MONERA DEMONSTRATIONS:

            On a bench in the laboratory you will find several demonstration set-ups to show the diversity within the Kingdom Monera.  Particularly notice the prepared slide that displays the major shapes of the bacteria—coccus (spherical), bacillus (rod-shaped), and spirillum (spiral-shaped).  Among the demonstrations is an example of the Cyanobacteria or blue-green bacteria.  These organisms are important producers (carry on photosynthesis) in aquatic ecosystems.  Many cyanobacteria are capable of "fixing nitrogen," converting molecular nitrogen into a form

that can be used by other living things.  Record information about the demonstrations in your notebook.

 

KINGDOM PROTISTA:

            The Kingdom Protista is usually studied by grouping the organisms into three categories:  the animal-like, the fungus-like, and the plant-like protists.  Today we will look most closely at the organisms that are animal-like, commonly called protozoa.  We will be using a tool, a dichotomous key, which is often developed by biologists to aid other biologists and beginning researchers in the study of specific groups of organisms.

            To begin your study of the protozoa, make a wet-mount slide of the "protozoan survey mixture" culture that is available on the front bench.  To make this slide, first make a small ring of "protoslo" on the slide, and then put a drop of the culture in the ring.  Add the cover glass carefully.  Starting with the scanning lens of your microscope, examine the slide to find a protozoan, and then move to the low-power objective and then finally to the high-power objective.  Use the dichotomous key to identify the organism.  Make a drawing of the protozoan.  Repeat this procedure several times to find and identify other protozoa in the survey mixture.

 

USING A DICHOTOMOUS KEY:

            To use the key, begin at the first couplet and decide which of the two statements matches up better with a characteristic of the specimen that you are observing.  After you make that decision, move to the couplet that is referred to by number at the end of the chosen statement.  After several couplets are passed, you will choose a statement that has a name instead of a number after it.  That is the possible name of a specimen.  The simple key that we are using today provides us with drawings of these protozoa.  Dichotomous keys do not always have drawings so it is a good idea to always start at the top of a key and work through it to make sure that you are properly identifying the specimen.

            As a thought exercise make a dichotomous key for a set of objects:  a nickel, a quarter, a paper clip, a thumbtack, a penny, a small nail, a needle, and a plastic poker chip.  This could be a good quiz!

 

PROTISTA DEMONSTRATIONS:

            On a bench you will find demonstrations of the diversity of the Kingdom Protista.  Record in your notebook all information about these varied and interesting forms of life.  Pay close attention to organisms that are plant-like and fungus-like which we did not study in lab today.

            Sketches of demonstrations and the accompanying information should be recorded by you for later review.

 

Assignment

 

 Next week we will be studying plants-particularly adaptations of plants to land existence.  Read Laboratory Ten, Plant Structure and Evolution, before you come to class.

 

 

Notes: