3: Examination of Living Microbes and Environmental Plates

Learning Outcomes

By the end of the lab period you will be able to:

• Prepare wet mounts of live organisms including pond organisms and termites
• Identify Euglena, Paramecium, Amoeba, and Trichonympha by their morphology and how they move.
• Use our microscopes to follow a living organism as it moves through the field of view in the microscope.
• Inoculate agar plates with environmental samples and samples from the human body.

Introduction

Part I - Free Living Protozoa

For the most part, we will be using our time in the microbiology lab to explore prokaryotic cells (bacteria). They are easy to work with, visible under a light microscope, and many species are pathogenic organisms relevant to human health. In fact, many non-pathogenic bacteria are also relevant to human health, but in beneficial ways!

However, there are other groups of organisms that we won’t be able to work with this semester. These include viruses, which are too small to see using a light microscope. There are also much larger organisms including pathogenic worms such as tapeworms, which are visible to the naked eye.

The protozoans are a group of single-celled organisms that include both parasitic forms that cause disease in humans, and “free-living” forms that live in pond water. There are also protozoa that live in symbiosis with their hosts in a mutualistic manner (both the protozoan and the host benefit from the relationship). Free-living protozoa are readily available and really fun to look at using our microscopes because they can be viewed while they are alive which allows us to practice our microscopy skills as we use the scanning objective to both locate them and chase them around the field of view as they move!

Today we will be looking at 4 different types of protozoans. All of these organisms are eukaryotes and belong to the domain “Eukaryota”. Their cells are organized in a way that is very similar to your cells (as you are also in the domain Eukaryota). They have membrane-bound nuclei and organelles, and they are much larger than bacteria. A typical bacterial cell is only about 1-10 microns, while a large amoeba can be up to 750 microns!

Figure \(\PageIndex{1}\): The three domains of life. Single-celled eukaryotic organisms such as protozoa and fungi are in the domain Eukaryota. Chiswick Chap, CC BY-SA 4.0, via Wikimedia Commons

Amoeba

Have you ever heard of a brain-eating amoeba? Naegleria fowleri is the terrifying parasite that is being referred to! However, today in the lab, we can very safely look at the harmless Amoeba proteus. This organism moves around using extensions of the plasma membrane called “pseudopods” or “false feet” which stretch out to surround and engulf their food in the environment. The way amoebas move to trap and ingest their food is very similar to the way white blood cells trap and destroy invaders that have made their way into the human body.

Paramecium

The Paramecium has been so widely explored by scientists that it is often called the "white rats" of ciliates to highlight its role as a lab animal. Ciliates are organisms that are covered by cilia that cover their outer surface. The organism can move when the cilia move back and forth. You will be surprised to see how fast these organisms can get around when you try to chase them around the microscope slide.

Euglena

Euglena is the smallest of the protozoa that we will look at today and are unique in that they contain chloroplasts and can photosynthesize! This means they can make their own food in addition to eating and digesting food in the environment. Euglena is also different from Amoeba and Paramecium because they move by rotating their flagella located at either end of the organism.

Trichonympha

Trichonympha is not a free-living organism but instead is a symbiont with termites. Termites eat wood, but they don’t have the enzymes they need to digest it. They rely on microbes such as Trichonympha (a protist) and bacteria to break down the cellulose in wood into glucose that the termite can use for energy. The relationship is mutually beneficial - the termite chews on the wood and breaks it down into tiny particles. Then those particles are broken down in the termite gut by the microbes into a form of food (glucose) that everyone can use. How do baby termites get “inoculated” with these microbes they need to digest food? They eat fluid that comes out the posterior of the parent termite (proctodeal trophallaxis) which is rich in the microbes they need.

Part II - Environmental Plates

Microbes are all around us - on the surface of your cell phone, the bottom of your shoes, door handles, the soil, etc. We are also covered in microbes both inside and outside of our bodies. In fact, all of life is so dependent on the microbes in our environment that it would be an apocalypse if they all suddenly disappeared!

Today we take a quick and imperfect sampling of some microbes on and around us. Why imperfect? We will be using nutrient agar to culture microbes that we capture on a sterile cotton swab. However most organisms won’t grow on nutrient agar, so we will only see the ones that do. It’s still lots of fun, and we’ll look at the organisms we do grow under the microscope in Lab #4.

Materials

Per Class

• Prepared slides of Amoeba, Euglena, and Paramecium
• Living cultures of Amoeba, Euglena, and Paramecium
• Termites
• Pond samples
• Transfer pipets
• Forceps

Per Student

• Clean glass slides and coverslips
• Compound microscope
• Lens paper
• 2 agar plates
• 4 cotton or calcium alginate swabs

Experiment

Observe Living Protozoa

1. Obtain a microscope and place it on the bench. Check to be sure the stage is all the way down and the scanning objective is in place.
2. Your instructor will show you how to prepare wet mounts of the protozoan specimens. Sketch what you see in the data sheet or lab notebook.
3. Use forceps to transfer a termite into a drop of water on a clean slide. Add a coverslip, and use the blunt end of the forceps or your finger to squash the termite. Mash the intestine to release Trichonympha and other protozoa and bacteria.
4. Examine drops of pond water samples. Sketch and describe the organisms you find.
5. When you are finished observing specimens, clean the objective lenses with lens paper. Return all lenses and adjustments to their storage positions before putting the microscope away.

Prepare Environmental/Body Parts Plates

1. Choose 2 different environments to investigate. Use a sharpie to draw a line at the bottom of each agar plate to divide it into two halves, and label each half with the environment you plan to sample (Figure \(\PageIndex{2}\)).
2. If you are collecting from a dry surface, dip the cotton swab in sterile water (if available), and then swab the surface.
3. Open the lid of the agar plate, and light streak back and forth across the agar in the labeled region. Discard the swab.
4. Choose 2 body parts to investigate. Any body parts are acceptable except vaginas and anuses, with a goal not to inadvertently grow up any of the more significant pathogens.
5. Label the bottom of the plates with your first initial and last name, and the date. Incubate at 37^oC for 48 hours or at 30^oC for 5 days.

Figure \(\PageIndex{2}\): Diagram of how to prepare your environmental and body part plates

Data

Sketch the protozoa that you view under the microscope. Indicate the total magnification for each sketch, and label any structural characteristics you can identify (nucleus, cell membrane, flagella, pseudopods, etc.).

Questions

1. What features did the cells you observed have in common? How were they different?
2. If you observed the same organism on a prepared slide and a wet mount, how did the images compare?