Shopping List (per student)

Shopping List (per student)
• 2 clean slides
• Lens paper
• Bibulous paper
• Clothespin
• Wax pencil
• Coverslips
• Inoculating loop (in drawer)
• Marker (in drawer)
• Bacti-cinerators “on”
• Last lab’s experiments (incubator)
• Lab manual

Transferring Bacteria
• Pick up small amount of bacteria on
loop & transfer it to new slant

• Streak Plate
• Swab 1st quadrant
with Staph aureus
Fig. 3.3
• Rotate & streak 2nd
quadrant
• Rotate & streak 3rd
quadrant
• Rotate & streak 4th
quadrant

• Streak Plate
• Draw “T” on plate
• Swab 1st quadrant
with Staph aureus
• Sterilize loop
• Streak 2nd
quadrant
• Sterilize loop
• Streak 3rd quadrant

Bacterial colony shapes

Smear Preparation
Exercise 4

Preparing a Smear (Exercise 4)
• Smear = thin layer of cells
attached to slide for staining
• 1) Draw circle on slide
• 2) Heat loop (5 seconds)
• 3) Resuspend culture
• 4) Add 4 loopfuls to slide
• 5) Spread over circle on slide
• 6) Heat loop
• 7) Allow slide to air dry
• 8) Heat fix over Bacti-
Cinerator (5 seconds)

Assignment: Smear Preparation
• Prepare smears using the following cultures:
• 1) EC = E. coli
• 2) SA = S. aureus
• 3) BM = Bacillus megaterium

Gram Staining
Exercise 5

• Bacteria have slight negative charge
• Basic dyes (+ cations): used for positive staining (attract)
» Crystal violet, Methylene blue, basic fuchsin
• Acidic dyes (- anions): Used for negative staining (repel)
» India ink, nigrosin, Eosin, Acid fuchsin

Gram Stain
• Gram stain = stain that divides bacteria into 2 groups (+ or -)
• Based on chemical differences in cell wall = peptidoglycan

Microscopy
Activity 6

Bacteria: Size & Organization
Smallest living organisms
range from 0.2-10 µm
Chlamydia (STD)
Rickettsia (Rocky Mtn Spotted Fever)
0.2-2.0 microns
intracellular
Prokaryotic organization
Nucleoid = chromosome
with no membrane
Few & internal organelles
*Ribosomes = protein factories

Effect of magnification

Pathway of light

compound light microscope

Microscope Parts & Function
Part
Function
Main switch
Light intensity knob
Eyepiece
Field of view
Interpupillary distance adjustment
Diopter adjustment ring
Condenser
Aperture iris diaphragm knob
Stage
Specimen holder
Y-axis knob
x-axis knob
Revolving nosepiece
Objectives: 4x,10x,40x,100x oil
Coarse focus adjustment knob
Fine focus adjustment knob
Controls power (on/off) to the scope
Adjusts the light intensity
Contains viewing lenses (10x)
Circular illuminated area seen through the eyepieces
Adjusts to match distance between student’s eyes
Adjusts focuses for student’s individual eyesight
Channels light through specimen and objective lens
Opens/closes amount of light passing through the specimen
Resting surface for the specimen slide
Holds specimen slide in place on stage
Front and back movement of the specimen holder
Right and left movement of the specimen holder
Rotates (black ridged band) and positions objective lenses
Allows magnification of specimen
Locates specimen and focuses under 10X objective lens
Focuses specimen under 40x and 100x oil objective lenses

Calculating Total Magnification
Total magnification = objective x ocular
Lens Used
Lens Magnification
Total Magnification
10x objective lens
40x objective lens
100x objective lens with oil

How to Use Microscope
1. Place slide on stage & center over light
2. Place 4x objective in place
3. Light intensity dial turned to 3
4. Bring condenser all the way up
5. Slowly use coarse adjustment knob to lower stage
6. Use fine adjustment know to “fine focus”
7. Adjust contrast user diaphragm lever under stage
8. Focus at 4x, 10x, 40x
9. Add oil on TOP of coverslip & focus at 100x
ONLY USE COARSE FOCUS KNOB AT 4X!!!!

Microscope Operation
1. Turn on microscope
2. Adjust brightness: light intensity knob = 3
3. Rotate 4x objective into place
4. Insert slide & center over light: X-axis & Y-axis knobs
5. Move stage all the way UP
6. Use coarse adjustment to lower stage until specimen appears
7. Use fine adjustment knob to sharp focus
8. Adjust light intensity using the iris diaphragm lever
Lower magnifications low light
Higher magnifications high light
9. Adjust distance between eyepieces to match your eyes
10. Look through right eye & use fine adjustment knob to sharp focus
11. Look through left eye & turn diopter adjustment ring to focus
12. Fine focus at 10x & 40x

Oil immersion lens

Oil Immersion
1. Focus with 40X lens.
2. Rotate nosepiece until 40X & 100X objectives straddle
slide
3. Add drop of oil on TOP OF COVERSLIP
4. Rotate oil immersion lens (100x) into place
5. Fine focus only!
6. Clean objectives with lens paper & windex when
finished!

Bacteria cell shapes photos

Contrast
• There is no contrast in cells
» few pigments
» few molecules that absorb light
• Can supply contrast by staining

Types of stains

3 views of a Paramecium
Bright-field
Dark-field
Phasecontrast

Gram Staining

•1) Bacillus = Rod shaped. (pl. bacilli)
•(diplobacilli, streptobacilli)
•2) Coccus = Round shaped. (pl. cocci)
•(diplococci, streptococci, staphylococci)
•3)Spiral = Spiral shaped
•(spirilla, vibrio, spirochete)

Bacteria may appear as single
cells or in groups:
• These terms describe typical bacteria
groupings:
• 1) diplo = paired cells
• 2) strepto = long chains
• 3) staphylo = grape-like clusters

Key to Slides
1. E. coli = Gram-negative (rods)
2. S. aureus = Gram-positive (cocci clusters)
3. E. coli = Gram-negative
S. aureus = Gram-positive

Table 4.6a

Table 4.6b