Remember the two important rules:
2. Never use the coarse adjustment
with the 40x and 100x objectives.
Ex. 3: Staining Techniques Gram Stain, part 1
• Chromophores
• Basic vs. acidic dyes
Objectives
• Explain the value of staining microorganisms
• Prepare a specimen slide including air-drying and
heat-fixing
• Identify the most common shapes of bacteria
• Explain the difference between acidic and basic
stains
• Explain rational and procedure of Gram stain
• Perform and interpret Gram stain
• Recognize the differences between eu- and prokaryotic cells and estimate all cell sizes using microoculometer
Differential Stain
1. Primary stain (stains all cells on
slide)
2. Decolorizing step (removes stain
from certain types of cells)
3. Counterstain (stains the
decolorized cells)
Strongly advised
When doing any of the microbiology
labs:
1. make sure to carefully follow the procedure
outlined in the Materials and Methods
section
2. Pay attention to any additional advice given
by your instructor or technician
3. Always read the labs before
coming to class!
4. Have fun!
Lab Bench Organization
Prepare a Cheek Cell Smear
Allow smear to air
dry
Heat fix
Movie Clip on Heat Fixing
http://deimos.apple.com/WebObjects/Core.woa/Browsev2/laspositascollege.edu
Gram Stain is ….
…the most important bacterial stain!
Therefore:
Memorize steps as soon as possible
Gram Staining Reagents
Decolorizing
agent
Primary stain
Mordant
Counterstain
Gram Stain Mechanism
(slide from lecture)
• Crystal violet-iodine crystals form in cell.
• Gram-positive
– Alcohol dehydrates peptidoglycan
– CV-I crystals do not leave
• Gram-negative
– Alcohol dissolves outer membrane and leaves holes
in peptidoglycan.
– CV-I washes out
Staining
Blotting
Using the Oil Immersion Lens
When viewing the specimens with the microscope, estimate cell sizes
eyepiece micro-oculometer.
by using the
While in theory, each microscope should be calibrated separately, for
our purposes it will be good enough to use the following conversion
chart:
10 x objective lens:
1 ocular unit = 10 m
40 x objective lens:
1 ocular unit = 2.5 m
100 x objective lens: 1 ocular unit = 1 m
Remember the Trouble
Shooting
Trouble focusing on the object? Check the
following:
• Is the light adjusted properly?
• Was the object in focus under low
power?
• Is the oil touching the lens?
• Is the lens dirty?
Gram Stain Animations
ASM Microbe Library
MacGraw Hill Animation
YouTube presentation