MICROBIOLOGY 101 LABORATORY MANUAL

EXERCISE #2: EUKARYOTES/PROKARYOTES AND MEDIA PREPARATION

NAME, ID #:_______________________________________________

NAME of TA: ______________________________

REVISED: 08/16/99

INTRODUCTION TO EXERCISE:

As you learned in lecture there are #two cell types, the PROKARYOTES, which include the bacteria, and the EUKARYOTES. The eukaryotes tend to be larger and more complex than the prokaryotes. There is good evidence to support the theory that the eukaryotes arose from SYMBIOTIC RELATIONSHIPS between prokaryotes that MERGED or FUSED into larger cooperative complexes that eventually became the eukaryote cell. The eukaryotes are often multicellular organisms, but a significant percentage are either single cellular or small multicellular microscopic forms. The microbial eukaryotes fit within the confines of a MICROBIOLOGY course. The most prominent of the microbial eukaryotes include the PROTOZOA, the ALGAE (another set of algae images) and the MOLDS AND FUNGI. These microbes exist in prodigious numbers in the soil and natural waters and our lives depend upon their biochemical activities. The microbial algae are PHOTOSYNTHETIC organisms that live on simple minerals and use LIGHT as an energy source and produce oxygen as a waste product. Most of the non-photosynthetic microbes are SAPROPHYTES that live on DEAD ORGANIC MATTER or PREDATORS that live on other microbes. However, a number of protozoan and fungal forms are serious human, plant and animal pathogens. Such diseases as MALARIA, SLEEPING SICKNESS, SCHISTOSOMIASIS and LEISHMANIA kill and debilitate millions of humans every year. Fungi and yeast pathogens, are responsible for causing such diseases as THRUSH and VALLEY FEVER. However, fungi probably do their greatest damage through the destruction of food crops. A number of multicellular microbes exist including nematodes, PINWORMS, TAPEWORMS, HOOKWORMS, TRICHINOSIS and FLUKES. These multicellular forms often can produce stages that are large enough to see without the aid of a microscope. Nematodes are often seen in the pond and aquarium samples you will view.

In today's exercise you will continue learning how to use the microscope by observing some common, harmless eukaryotes and prokaryotes that are found in the water and soil around us. You will observe and draw what you see, paying particular attention to the organelles within the eukaryotes while becoming more familiar with the size differences between the eukaryotes and prokaryotes. You should relate this information to that you have heard in the lecture. Please note that there are TWO correct spellings of eukaryotes (eucaryotes) and prokaryotes (procaryotes); I may use both.

You will also visit the media room and see how microbial media is made and sterilized.

Figure 1. Eukaryotic cells and cell structures. Here are 3 electron micrographs of EUKARYOTIC cells. In the upper left is a section of a cell showing a portion of the nucleus and several mitochondria laying just outside of the nucleus. On the upper right yeast cell note the beginnings of a daughter bud and on the bottom left note the numerous bud scars left on the mother yeast cells after daughter cells have split off.

PURPOSE OF EXERCISE:

To continue improving your skill with the microscope by viewing more eukaryote and prokaryote microbes.
To enhance your observational skills while learning something about the eukaryote cellular organization as discussed in lecture and in NetText Chapter 2.
To learn the differences, in size and structures between protozoa, algae, fungi and bacteria.
- To identify some eukaryotic organelles.
To learn how media is made, what an autoclave is and how it works.

RELATIONSHIP TO LECTURE MATERIAL

NetText101/102: CHAP. II Cells; eukaryotes vs. prokaryotes.

GENERAL INSTRUCTIONS:

Read and examine pg. 143-169 in A Photographic Atlas for the Microbiology Laboratory. Failure to bring your Atlas to class or a copy of the lab exercise will cost you points after the first week.
Label the drawings clearly so the TA can easily read them. The TA will spot-check to verify the accuracy of their drawings, in which case they will initial them.
Observe the material shown on the TV monitor and relate it to what you are seeing at your desk.

PROCEDURE:

Divide your slides into 2 to 4 sections (depending on the size of the cover slips) and prepare wet mounts of the respective samples in the center of each section on the slides as illustrated below. Do not allow the liquid from the various samples to mix; use the corner of a tissue to soak up excess liquid. If an aquarium is available view samples from different sites such as the algae on a rock or on the side or the muck at the bottom (not the sand) etc.

Figure 2. Wet mount procedure. See Exercise #1 for explanation.

4. Place the slide in the microscope and examine a sample first with the 10X objective. Draw what you see; label it "10X ________". Then switch to the 40X objective and repeat the process, drawing proportionally in the circles below.

5. Estimate the size of the eukaryotes you draw by assuming that the diameter of the field of view for the 10 & 40X objectives are 1,600 & 440 micrometers respectively. Beside each drawing indicate: SIZE _______.

Protozoa
Figure 2. Several species of paramecium (40X). You will be viewing the far left large one in the paramecium culture, but there may be other species in the samples from the pond and the aquarium. Note the fine hairs that beat and move the paramecium; they are called CILIA.

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Figure 3. Fungi Components. The individual filaments of a mold are call HYPHAE and the mass of hyphae that is visible to the eye is called the MYCELIUM. At the end of the reproductive hyphae are one type of reproductive spores.

6. To view the fungi:

First examine the fungi under the dissecting scope on the side bench. Draw a section at the edge of the colony of what you see there, labeling the parts and record the magnification.
Choose one of the mold colonies on a plate and do the following.
- Add a SMALL DROP of Cotton Blue to the center of a clean slide.
- Take a piece of cellophane tape slightly shorter in length than you glass slide.
- Holding the tape so the sticky side is away from you, touch the sticky side lightly to the top of the mold colony.
- Place the tape/mold, sticky side down, over the blue dye with the mold directly in the stain & stick the tape to the slide. Press down gently and soak up any extra dye that runs out.
- View with the microscope, beginning with the 10X objective. Look for "hair-like" strands of broken individual hyphae and for unstained spores which are usually round, or oval shape.
- See if you can identify some of the mold structures shown in the ATLAS and below. Draw and label them. Show them to the instructor.
  - Excellent site for viewing fungi: Choose Aspergillus, and then take a look at some of the others.

7. Pick up your Rodac plates from the TA (along with some tape), label the bottom with your name and the date, and follow the TA's instructions on how to inoculate them to see what sort of microbes you have lurking in your house or on your body.

SAMPLE QUESTIONS: You should be able to answer these questions at the conclusion of this laboratory.

What is a saprophyte?
Name two protozoan diseases.
Describe three major differences between prokaryotes and eukaryotes.
Give the size, in micrometers, of a typical eukaryote and prokaryote.
Define mycelium and hyphae.
What do cilia do?
Draw a eukaryotic algae and a prokaryotic algae in proportion to their size.
Name two diseases caused by multicellular microbes.
Describe how an autoclave works and what temperature (Centigrade) is required to sterilize medium.
Describe how you would make a solid bacterial medium from common ingredients found in most kitchens.

Copyright © Dr. R. E. Hurlbert, 1999.
This material may be used for educational purposes only and may not be duplicated for commercial purposes.
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