1. 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
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

2. Exercise 7: Ubiquity of Bacteria: Observe results
Figure 7.1
Lab book pg 101

3. Go over shape and arrangement for bacteria (Exercise 7) (Sup
Go over shape and arrangement for bacteria (Exercise 7) (Sup. packet pg 56 )

4. 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)

5. Exercise 11: Smear preparation
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)
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.
Best smears will be barely visible to the naked eye when complete.
Air dried – prevent cell lysis
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.

6. Figure 11.1
Water first always
with solid media
Target circle
optional: spread
out wide to
make smear
as thin as possible
No big blobs!
Must be
COMPLETELY
dry!

7. Exercise 12: Simple Stain (Methylene blue)
Stain slides from Exercise 11
Flood the smear prepared slide with a basic dye such as methylene blue for a minute or so.
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.
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!!!

8. Figure 12.1
paper towel

9. Burner Loop Bottle with water 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