Microbiology: Observing Bacteria Laboratory -1 Name ________________________________

Date ___________________

Prelab: Part 1 – Introduction to the microscope- please read through this handout and label the picture on the next page before starting the lab Care and safety:

§ Carry microscopes with 2 hands, holding the arm and the base. § Clean lenses with lens paper before and after use. § Keep microscopes away from the edge of the lab bench. Keep the bench area around the

microscope clear of unnecessary clutter. § Remove the slide, wipe the stage and clean the lens before putting the microscope away. § Always unplug scope by grasping the plug not the wire!

Getting to know the microscope Please label the picture with the correct term that corresponds to that part of the microscope: (arm, diaphragm control, objective lenses, base, fine adjustment, coarse adjustment, ocular lens, light source, stage.) Note the label on each lens, both ocular and objective. How is the overall magnification power determined? How does increased magnification affect field of vision? Which objective focuses closest to the slide when it is in focus?

Hints for use: § Be sure the bottom of the slide is clean and dry. § Place the object to be viewed in the center of the slide. § Center the slide itself of the stage and clip it into position. § Start with low power (4x – aka: scanning objective)

o Put slide on stage and clip it into place with stage clips. o Raise the stage as high as it will go without the slide touching the objective lens. o Looking through the eyepiece, use the coarse adjustment to slowly raise the objective

until you have good focus. o Finish focusing using the fine adjustment knob. o Adjust the diaphragm for best viewing. o Complete all tasks at low power - then…

§ To view under medium-high (40x) power:

o Adjust the slide so the object you want to view is in the center of the field under low power.

o Switch to the larger, medium power objective. (Make sure the lens does not touch the slide cover!)

o Focus with the fine adjustment only. Do not use the coarse adjustment! o Adjust the diaphragm for the best contrast. o Repeat for high power.

• To view under 100X power:

o Higher magnification requires more light o turn the objective and place a drop of immersion oil on the slide o snap the 100X objective in place

§ If you can’t see your sample, check the following:

o Is the objective clicked into position? o Is the diaphragm clicked into position? o Is the light turned on?

Part II. How big are bacteria? Observe the prepared slide of yeast/blood/bacteria under 100X power. Which Kingdom does each of these cells belong? What type of cell are they (prokaryotic vs. eukaryotic)? Draw to scale an example of each cell below. Note on drawing your field of vision to scale (determining the size of the specimen):

Any object observed under a microscope can be described in the word small, but precisely how small is critical information to many scientists. A millimeter, 1/1,000 of a meter is typically used to measure small objects, but is far too large a unit for microscopic objects. Rather, scientists use the unit of a micrometer 1,000 times smaller than the millimeter. The Greek letter µ is used to symbolize micro, 1/1,000,000 of a meter.

1. Using the 10x objective, place a clear plastic ruler on the stage and focus on the mm marks. Record how many millimeters fit across the diameter of your field of view. Record this # ____________mm = ___________um.

2. Use the following formula to calculate your field of vision.

3. Once you’ve determined your field of view, you can get a rough estimate of the size of your organism by figuring out how many of those organisms could fit across the field of view and then dividing by that number. For example: under low power you determine the field of view is 1.5mm = 1,500um.

1,500um x 10x/400x = 37.5um is your field of view under high power. If you estimate 20 organisms can fit across the diameter of your field of view, then the size of your organism is around 1.9um!

Label the items in your drawing. Kingdom Cell type (prok or euk) Drawing Yeast Blood Bacteria

Total Magnification ______Field of view________

What was the largest organism you observed? the smallest? How many polio viruses (25nm) could fit across the diameter of an E. coli bacteria (assume the diameter of E. coli is 1.5um)? Show your calculations. Using the chart below, as a reference for size, could your microscope be used to view a polio virus? Explain.

Part IV Oil Immersion Explain why oil is needed for observing bacteria at high power (use the diagram below as a reference). Use terms such as resolution, index of refraction and magnification in your description.

Explanation: What would occur if water were used in place of immersion oil? Part III Observations of microbes using light microscopy and oil immersion.

Protozoa: a.______________________ b. ___________________________

     

   

   Magnification_________           Magnification_________  Size  of  object  _________           Size  of  object  _________   Bacterial Shapes: From the prepared slides of Mixed Types of Bacteria, draw and label the 3 examples of the major shapes. What multimeric form (streptococci, staphlobacilli, etc..) did you observe, if any? a.________________________ b. __________________________ c. __________________________

Magnification_________            Size  of  object  _________              Fungi  and/or  Archaea:  a.______________________ b. ___________________________

Magnification_________           Magnification_________