Transcript Preparing a Bacterial Smear & Viewing Under Oil Immersion

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From the Virtual Microbiology Classroom on ScienceProfOnline.com
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Image: Compound microscope objectives, T. Port
Laboratory
Exercise 1b
How to Use a
Compound Microscope
Viewing a Simple Stain of
Bacteria Under Oil Immersion
** Please plug in your microincinerators **
From the Virtual Microbiology Classroom on ScienceProfOnline.com
Image: Simple stain of Staphylococcus, T. Port
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
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
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
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
–
Protection for your high dry (blue band,
–
–
–
Drop of oil
Oil immersion lens
ONLY USE FINE FOCUS
ADJUSTMENT!!!
When done, use lens paper to clean up
your lens and the stage
–
(cant see much, but at least get them in your sights)
400xTM)
From the Virtual Microbiology Classroom on ScienceProfOnline.com
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.
From the Virtual Microbiology Classroom on ScienceProfOnline.com
Image: E. coli growing on TSY agar in
slant tube and in Petri dish, T. Port
Making a Bacterial Smear & Simple Stain
1.
1. With a wax pencil, draw 3 dime-sized circles on a slide.
2. Flip slide over so wax is on the bottom.
Draw circles with wax pencil
3. Use DI water dropper to place a very small drop of water
inside each circle.
3.
* 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.
Put a drop of water in each circle.
* 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!!
* sterilize inoculation loop in microincinerator and let cool *
4-6
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.
* sterilize inoculation loop in microincinerator and let cool *
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.
* sterilize inoculation loop in microincinerator and let cool *
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.
From the Virtual Microbiology Classroom on ScienceProfOnline.com
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.
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
100xTM
Bacterial smear at
1000xTM
(oil immersion)
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
Confused?
Here are links to resources that further explain microscopy:
•
Microscopy Laboratory Main Page on the Virtual Microbiology Classroom of
•
Compound Microscope Parts and Use
•
How to Make a Wet Mount of an Elodea Plant Cell
•
How to Make a Wet Mount of an Onion Epithelial Cell
•
How to Make a Wet Mount of a Cheek Cell
•
Microscope Mania
•
Viewing Bacteria Under Oil Immersion,
•
How to Prepare a Microscope Slide of Bacteria, SPO Lab Notes
•
Prokaryotic Cell
•
“Germs”.
Science Prof Online.
video from Science ProfOnline.
ScienceProfOnline.
ScienceProfOnline.
video from
video from
video from ScienceProfOnline.
crossword puzzle.
SPO Lab Notes article.
article.
interactive diagram from Cells Alive website.
Music by Weird Al Yankovic. Video by RevLucio.
(You must be in PPT slideshow view to click on links.)
From the Virtual Microbiology Classroom on ScienceProfOnline.com
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
www.ScienceProfOnline.com
Images: E. coli, Giant Microbes; Prokaryotic cell, Mariana Ruiz