Remember the two important rules:

1. Only use oil on 100x objective lens. Never get it onto any of the other objectives!

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 pro-karyotic cells and estimate all cell sizes using micro-oculometer

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 advisedWhen 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

Primary stain Mordant

Decolorizing agent

Counterstain

Gram Staining Reagents

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

by using the eyepiece micro-oculometer. 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 YouTube presentation

MacGraw Hill Animation

THE END