Laboratory Experiment 1: Examination of Natural Microbes

Many students taking microbiology for the first time feel that they are thoroughly familiar with the
use of the microscope from their previous biology courses. In microbiology, however, you are dealing with
such minute forms that it is essential to use good microscope technique. To observe microbes clearly you
will especially need proper, focused illumination, a good selection of objectives and patience. In this
experiment we will attempt to duplicate the observations Antoni van Leeuwenhoek made with his simple
microscopes more than three hundred years ago....that is to observe the major types of microbes: bacteria,
algae, fungi and protozoa. We, however, have a definite advantage: the modern microscope. Think about
that if you were in fact, Leeuwenhoek, would you have recognized these things as alive?

A hay infusion is an excellent way to provide microbes at a high density during any time of the year.
This process allows you to follow the development of microbes as they become visible over time.

Objectives:
1. Observe and identify some major types of microbes from a pond water.
2. Culture microbes by making a hay infusion.
3. Diagram the observed, over time, microbes such as amoebas, protozoa, rotifers, insect larvae, algae
and bacteria.
4. Appreciate the importance of microbes in the ecological system.
Materials:
Pond water with clumps of algae & other Hay
suspended material Milk
Jar, such as a mayonnaise or jam jar Corn syrup
pipettes or dropper Petroleum jelly (Vaseline)
slides and square coverslips Toothpicks
Antibacterial soap for cleanup
Procedure:
Laboratory 1.A. : Microbes in Pond Water

1. Take a clean glass slide and place two drops of pond water that contain visible clumps of algae or
suspended material. DO NOT USE clear water. Gently place a coverslip over the material and flatten with
your finger if necessary. Wipe up any excess liquid with a Kimwipe or towel.
2. Observe first with the 4X lens to center visible clumps and get the microscope in close focus. Switch to
the 10X lens and attempt to distinguish the various large forms of microbes: algae and protozoa. When
you have located one, switch to the 40X lens and examine in close detail. Measure the cell dimensions
with the ocular micrometer and diagram at least two algae and two protozoa.
3. Attempt to discern cell walls, internal organelles such as vacuoles, chloroplasts, nuclei, and flagella if
flagellated. Attempt to tell the difference between the cyanobacteria and eukaryotic algae. Diagram any
features you can identify.
4. Attempt to see bacteria. Bacteria are the size of mitochondria. You will need to use the fine adjustment:
going up and down slowly to see bacteria pop into focus and then out. They should appear clearly
outlined when in the plane of focus and dark-blurred as they move out of the plane of focus. You should
be able to see rods, cocci and filaments. Which forms are nonmotile? motile?
Questions:
1. What types of major microbes did you observe for each sample?
2. Diagram at least two algae, protozoa, and any bacteria you see.
Making a Hay Infusion

1. Put the pond water (500 ml) into a jar and add pieces of cut hay. Cover the jar loosely and partially.
2. Add 1-2 drops of milk. The water will turn slightly turbid. The milk is food for the bacteria and they will
start to reproduce. The ciliates feed on the bacterial and will also reproduce.
3. Take a picture (Using the ProScope USB microscope) of the contents of the jar every two days.
4. Save the pictures in a folder entitled “Hay Infusion.” Make sure the date for each observation is also
recorded.
5. Let the jar stand for several days, protected from direct sunlight as this may result in overheating and
the heat will reduce the oxygen concentration. Do make sure that the beaker receives sufficient light,
though. Photosynthetic algae present in the pond water will produce oxygen.
6. Keep adding 1-2 drops of milk when the turbidity disappears. Bubble some air through the water at
regular intervals.
7. After about a week, the hay infusion population will peak. Using a pipette, get small water samples from
the top of the water, the bottom of the jar, and near the floating debris in the middle. Using samples
from all of these areas will give you the best chance of getting different types of microbes for viewing.

Laboratory 1.B. : Microbes in Hay Infusion (After 1 week)

1. Make a slide of several different water samples to compare (top, middle, bottom). If the microbes move
too quickly to take pictures of them, add a drop of corn syrup to each sample.
2. Take pictures of each slide using the various powers of magnification. Try to observe each sample both
before and after adding the corn syrup.
3. Draw and label any organisms you find in the water samples. Use reference materials to identify the
organisms. Also use the photographs from the ProScope USB microscope to help identify each microbe.
Compare and contrast the pictures to determine likenesses and differences in the various microbes.
Describe any differences you observe over time.

Questions:
1. What changes did you notice each day in the hay infusion project?
2. Which water sample produced the most microbes? Why do you think this sample produced the most
microbes?
3. How many different microbes were you able to isolate and identify?
4. Which lens magnification made the microbes appear to be the clearest?

(Extension Activity: Continue to feed the hay infusion by adding more pond water and hay each week. Using
the microscope, make new slides from the water samples every four to five days. Record any changes or
additional microbes you detect.)

Conclusion:

(Please use separate sheet for your answers)