1. Copyright © 2010 Pearson Education, Inc. Bell Ringer  Why is C. diff a difficult microbe to treat in an infected hospital patient?

2. Copyright © 2010 Pearson Education, Inc. Objectives  Students will describe and perform the Gram stain technique on two different species of bacteria.  Why is this important?  It is always the first step done in order to identify an unknown pathogen  Graded on  Gram stain results  Morphology  Arrangement  Relative Size

3. Copyright © 2010 Pearson Education, Inc. Differential Stains  Used to distinguish between bacteria  Gram stain  Gram positive = purple  Gram negative = pink

4. Copyright © 2010 Pearson Education, Inc. Differential Stain  3 steps to a differential stain  1) Primary stain  Colorize cells  2)Decolorizing agent  Has the ability to remove the primary stain  3) Counterstain  Contrasting color to the primary stain  If the primary stain was removed, the cell absorbs the counterstain

5. Copyright © 2010 Pearson Education, Inc. Gram Stain  Based on differences in the bacterial cell walls  Both gram-positive and gram-negative have peptidoglycan  Repeating disaccharides attached by polypeptides  Surrounds and protects cell

6. Copyright © 2010 Pearson Education, Inc. Gram-Positive vs. Gram-Negative Cells  Gram-positive  Many layers of peptidoglycan  Teichoic acid spanning the peptidoglycan layer  Gram-negative  One or a few layers of peptidoglycan  Outer membrane consists of lipopolysaccharide (LPS)  Resists enzymes, detergents, and heavy metals, antibiotics  Harder to kill

7. Copyright © 2010 Pearson Education, Inc.

8. Gram Stain  Utilizes four reagents  1) Primary stain – Crystal Violet (purple)  Stains all of the cells purple  2) Mordant – Gram’s Iodine  Increases cells affinity for primary stain  Binds primary stain and forms (CV-I) complex  Intensify color of stain

9. Copyright © 2010 Pearson Education, Inc. Gram Stain  3) Decolorizing agent – Acid alcohol (Ethanol)  Acts to dehydrate proteins and dissolve lipids  Gram-negative cells - alcohol dissolves outer lipid layer  CV-I complex becomes easy to remove from thin peptidoglycan layer  Cells become colorless and unstained  Thicker peptidoglycan layer in gram-positive cells traps CV-I complex  Alcohol dehydrates pore causing thick peptidoglycan layer to trap stain

10. Copyright © 2010 Pearson Education, Inc. Gram Stain  4) Counterstain – Safranin (pink)  Decolorized, gram-negative, cells stain pink  Gram-positive cells retain purple stain

11. Copyright © 2010 Pearson Education, Inc. Color of Gram-positive cells Color of Gram-negative cells Primary stain: Crystal violet Mordant: Iodine Decolorizing agent: Alcohol-acetone Counterstain: Safranin Gram Stain Purple Clear Pink

12. Copyright © 2010 Pearson Education, Inc. Common Errors  Old culture  Do not want to exceed 24 hours  G+ appear pink or mixed  Thick smear  Thick smears trap the primary stain  Cannot decolorize properly  Over-decolorize  Most important step  Alcohol can remove the primary stain from G+ cells  G+ appear pink

13. Copyright © 2010 Pearson Education, Inc. While You Are Waiting  1) Complete the questions in the lab report  2) P. 68 in textbook  Describe acid-fast, endospore, flagella, and negative staining procedures  Include  Explain what the stain is used for  Describe what a positive and negative cell look like  Explain the various stains used in the procedure