Preparing a Bacterial Smear

1
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From the Virtual Microbiology Classroom on
ScienceProfOnline.com
Image Compound microscope objectives, T. Port
2
Laboratory Exercise 1bHow to Use a Compound
Microscope Viewing a Simple Stain of Bacteria
Under Oil Immersion
Arent They Cute?
Please plug in your microincinerators
Image Simple stain of Staphylococcus, T. Port
From the Virtual Microbiology Classroom on
ScienceProfOnline.com
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?

Image Microscope objective lenses, T. Port
From the Virtual Microbiology Classroom on
ScienceProfOnline.com
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!

Image Microscope objective lenses, T. Port
From the Virtual Microbiology Classroom on
ScienceProfOnline.com
5
Oil Immersion Objective Lens

• Has BW 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.

Image Microscope objective lenses, T. Port
From the Virtual Microbiology Classroom on
ScienceProfOnline.com
6
Observing bacteria under oil immersion

• Dont 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! Dont
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 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.
• 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!!
• sterilize inoculation loop in microincinerator
  and let cool
• 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.

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
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.
Images Both images Staphylococcus, by T. Port
From the Virtual Microbiology Classroom on
ScienceProfOnline.com
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.

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. 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