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From the Virtual Microbiology Classroom on ScienceProfOnline.com
Image: Compound microscope objectives, T. Port

2. ** Please plug in your microincinerators **
Laboratory Exercise 1b How to Use a Compound Microscope Viewing a Simple Stain of Bacteria Under Oil Immersion
Aren’t They Cute?
** Please plug in your microincinerators **
From the Virtual Microbiology Classroom on ScienceProfOnline.com
Image: Simple stain of Staphylococcus, T. Port

3. Low Power Objective Lens
Has yellow band around it.
Magnifies objects 10x.
Q: What is the Total Magnification? ____ TM
Start with this lens when looking at a bacterial smear.
Q: What does the term parfocal mean?
From the Virtual Microbiology Classroom on ScienceProfOnline.com
Image: Microscope objective lenses, T. Port

4. Practice Safe Microscopy! Protect Your High-Dry Lens
Before putting any oil on your slide, make sure that the hi-dry, blue banded lens is covered up with a finger cot!
From the Virtual Microbiology Classroom on ScienceProfOnline.com
Image: Microscope objective lenses, T. Port

5. Oil Immersion Objective Lens
Has B&W band around it.
Magnifies objects 100x.
Q: What is the Total Magnification? ____ TM
Make sure that your bacterial smear is clearly in focus at 100xTM.
Put a drop of immersion oil directly on each of the three bacterial smears on your slide, then switch to the oil immersion lens.
From the Virtual Microbiology Classroom on ScienceProfOnline.com
Image: Microscope objective lenses, T. Port

6. Observing bacteria under oil immersion:
Don’t EVER use coarse focus when working with high dry or oil immersion.
Remember PARFOCAL!
Using oil immersion:
View bacteria with low power 100xTM (cant see much, but at least get them in your sights)
Protection for your high dry (blue band, 400xTM)
Drop of oil
Oil immersion lens
ONLY USE FINE FOCUS ADJUSTMENT!!!
When done, use lens paper to clean up your lens and the stage
From the Virtual Microbiology Classroom on ScienceProfOnline.com

7. When obtaining a bacterial sample from a tube or plate of media do so gently! The bacteria is growing as a thin film on top of the media! Don’t scrape so hard that you have pieces of agar in your sample!
If obtaining bacterial sample from slant tubes:
never pick up test tube by the cap.
do NOT set cap down on lab bench
flame neck of the test tube before & after
obtaining sample.
Image: E. coli growing on TSY agar in slant tube and in Petri dish, T. Port
From the Virtual Microbiology Classroom on ScienceProfOnline.com

8. Making a Bacterial Smear & Simple Stain1.
1. With a wax pencil, draw 3 dime-sized circles on a slide.
2. Flip slide over so wax is on the bottom.
3. Use DI water dropper to place a very small drop of water inside each circle.
* sterilize inoculation loop in microincinerator and let cool *
4. Using inoculation loop, obtain a small sample of Staphylococcus epidermidis from the source plate or tube, as instructor has demonstrated. Swirl loop in circle of water on left.
* IMPORTANT!!!: Very gently obtain sample of bacteria from the source plate or tube. The bacterial colonies are found growing on the surface of the TSY agar. DO NOT remove agar with your sample!!
5. Using inoculation loop, obtain a small sample of Escherichia coli (E. coli) from the source plate or tube. Swirl loop in circle of water in the middle.
6. Using inoculation loop, obtain a small sample of Bacillis subtilis from the source plate or tube. Swirl loop in circle of water on right.
7. Heat fix the slide on top of your microincinerator. Allow it to stay in the platform for 5 minutes after water has completely evaporated. This kills the bacteria and sticks it to the slide.
8. Stain the slide with crystal violet for 45 seconds then observe under oil immersion.
Draw circles with wax pencil 3.
Put a drop of water in each circle.
4 - 6
Inoculate circle of water with each of the following: 1. Staph epi 2. E. coli, 3. Bacillus subtilis. 8.
After heat fixing on microincinerator, stain with crystal violet, rinse, then look at with scope.
From the Virtual Microbiology Classroom on ScienceProfOnline.com

9. FYI: The example above is Staph.
Although you will be using 100xTM to get your specimen in focus, you will not be able to see individual bacteria until you use the 1000xTM oil immersion lens.
Bacterial smear at 1000xTM
(oil immersion)
Bacterial smear at 100xTM
FYI: The example above is Staph.
Remember, you want to use the microscope with a built in camera so that you can take pictures of the bacteria you see at 1000xTM.
From the Virtual Microbiology Classroom on ScienceProfOnline.com
Images: Both images Staphylococcus, by T. Port

10. Confused? Here are links to resources that further explain microscopy:
Microscopy Laboratory Main Page on the Virtual Microbiology Classroom of Science Prof Online.
Compound Microscope Parts and Use video from Science ProfOnline.
How to Make a Wet Mount of an Elodea Plant Cell video from ScienceProfOnline.
How to Make a Wet Mount of an Onion Epithelial Cell video from ScienceProfOnline.
How to Make a Wet Mount of a Cheek Cell video from ScienceProfOnline.
Microscope Mania crossword puzzle.
Viewing Bacteria Under Oil Immersion, SPO Lab Notes article.
How to Prepare a Microscope Slide of Bacteria, SPO Lab Notes article.
Prokaryotic Cell interactive diagram from Cells Alive website.
“Germs”. Music by Weird Al Yankovic. Video by RevLucio.
Smart Links
(You must be in PPT slideshow view to click on links.)
From the Virtual Microbiology Classroom on ScienceProfOnline.com

11. Are microbes intimidating you. Do yourself a favor
Are microbes intimidating you? Do yourself a favor. Use the… Virtual Microbiology Classroom (VMC) ! The VMC is full of resources to help you succeed, including:
practice test questions
review questions
study guides and learning objectives
You can access the VMC by going to the Science Prof Online website
Images: E. coli, Giant Microbes; Prokaryotic cell, Mariana Ruiz