Transcript Lab 3

Lab 3 Goals and Objectives :
• Observe results:
1. From Ubiquity of Bacteria:
2. Of aseptic transfer
3. Of streak for isolation and analyze for how to improve
• Learn how to prepare specimen for staining
1.
2.
3.
Smear - A thin film of a microbes suspension on a slide
Dry the smear – To prevent cell lysis during fixing
Fix the smear - To attach the microbes to the slide
• Learn Simple Stain method - Use of a single basic dye (the
chromophore is a cation).
– Methylene blue, carbolfucsin, crystal violet
– The positive charged dye is attracted to the bacterial cytoplasm which
has a negative charge, and staining takes place.
– can see the size and shape of the bacteria
Exercise 7: Ubiquity of Bacteria: Observe results
Figure 7.1
Lab book pg 101
Go over shape and arrangement for bacteria (Exercise 7) (Sup. packet pg 56 )
• Exercise 9: Aseptic Technique: Check results of aseptic transfer
• Exercise 10: Pure Culture Techniques: Check results of streak for
isolation
Red = S. marcescens / Small yellow = M. luteus / Cream = E. coli
– Analyze for how to improve
– Repeat once using heavy growth from one plate (plate to plate)
Exercise 11: Smear preparation
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Slides are pre-cleaned
Set up fan and slide warmer on back counter for slide drying
Students use E. coli broth and slant from Exercise 9
Students make smears of interesting colonies from ubiquity plates
(Exercise 7)
No Cornebacterium (can’t see granules so why bother)
1.
Smear - A thin film of a solution of microbes on a slide
- 2 loops from a bacterial culture in liquid media – broth are placed
on a glass slide
- A small amount of bacterial colony from an agar plate is placed
in a droplet of water.
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Best smears will be barely visible to the naked eye when complete.
2. Air dried – prevent cell lysis
3.
Heat fixing – fixes the cells to the slides, kills most organisms and
prepares them for staining
- The slide is passed through a flame
- Slides can be kept in a box
- No need to be stained immediately.
Figure 11.1
Target circle
optional: spread
out wide to
make smear
as thin as possible
Water first always
with solid media
No big blobs!
Must be
COMPLETELY
dry!
Exercise 12: Simple Stain (Methylene blue)
– Stain slides from Exercise 11
1. Flood the smear prepared slide with a basic dye such as methylene blue for a
minute or so.
2. Wash of the dye gently with water
3. Blot slides with paper towel, no bibulous paper
– All stain remains in tray, not down sink
• In order to find the focal plane put a marker dot on the finished dry
slide, same side as the bacteria, before going to the scope and use the dot
to find your focus.
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• Label a slide box with your name on tape and store any slides you wish to
save. Blot oil off for storage. No methanol on slides (removes sample)
• !!!CLEAN YOUR SCOPE WELL!!!
Figure 12.1
paper towel
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Each pair needs for lab 3:
– 2 BHIA plates
– Pre-cleaned slides
– Loeffler’s Methylene Blue
– A tray
– Burner
– Loop
– Bottle with water
– Each person get and label slide box for stage of slides made (as desired)
Plates and tubes from last lab