Copyright © 2006 Pearson Education, Inc., publishing as Benjamin Cummings PowerPoint ® Lecture Slide Presentation prepared by Christine L. Case M I C R O B I O L O G Y a n i n t r o d u c t i o n ninth edition TORTORA  FUNKE  CASE 3 Staining Specimen

Copyright © 2006 Pearson Education, Inc., publishing as Benjamin Cummings  Observations of microorganisms are made with stained preparations  Staining: coloring microbes with a dye to emphasize certain structures  Smear: A thin film of material containing microbes on a slide.  A smear is usually fixed to attach the microbes to the slide and to kill the microbes.  Fixing is usually done by passing a smear slide over a bunsen burner Fixing Preparation of Specimens for Light Microscopy

Copyright © 2006 Pearson Education, Inc., publishing as Benjamin Cummings UN 3.3 Preparing Smears for Staining  Live or unstained cells have little contrast with the surrounding medium. However, researchers do make discoveries about cell behavior by observing live specimens.

Copyright © 2006 Pearson Education, Inc., publishing as Benjamin Cummings Simple Stains  Simple stain: Use of a single basic dye.  Primary purpose is to highlight the entire microorganism  To view cell shape and basic structures  A mordant may be used to intensify the stain  Purpose:  Increase absorption of stain  Coat certain structures (ie: flagella) to make them thicker and easier to see

Copyright © 2006 Pearson Education, Inc., publishing as Benjamin Cummings Differential Stains  React differently with different kinds of bacteria  Used to distinguish (differentiate) bacteria from each other  Two most common are Gram stain and acid-fast stain

Copyright © 2006 Pearson Education, Inc., publishing as Benjamin Cummings Gram Stain – Hans Christian Gram (1884)  Steps:  Heat fixed smear is covered in purple dye, usually crystal violet (called primary stain)  Purple dye is washed off and smear is covered with iodine (a mordant)  Iodine is washed off and slide is washed with alcohol acting as a decolorizing agent (removes purple from some species, but not all)  Alcohol is rinsed off and then stained with safranin (a red dye)

Copyright © 2006 Pearson Education, Inc., publishing as Benjamin Cummings Gram stain  Gram staining classifies bacteria as either gram positive or gram negative  Gram positive bacteria are those whose cytoplasm retains a dark purple color after the attempted decolorization with alcohol  Gram negative bacteria are those whose cytoplasm lose the dark purple color after decolorization  Often colorless after alcohol wash – why the safranin is used, to dye them red/pink

Copyright © 2006 Pearson Education, Inc., publishing as Benjamin Cummings Color of Gram–positive cells Color of Gram–negative cells Primary stain: Crystal violet Purple Mordant: Iodine Purple Decolorizing agent: Alcohol-acetone PurpleColorless Counterstain: Safranin PurpleRed Differential Stains: Gram Stain PLAY Animation: Microscopy

Copyright © 2006 Pearson Education, Inc., publishing as Benjamin Cummings Differential Stains: Gram Stain Figure 3.11b

Copyright © 2006 Pearson Education, Inc., publishing as Benjamin Cummings Why the purple and pink colors?  It all goes back to the cell walls  Gram positive bacteria have a thicker peptidoglycan cell wall  Gram negative bacteria contain a layer of lipopolysaccharide within their cell wall  Crystal violet cannot leave the peptidoglycan cell wall  The alcohol wash begins to break down the lipopolysaccharides allowing the crystal violet to leave gram negative cell walls

Copyright © 2006 Pearson Education, Inc., publishing as Benjamin Cummings Differential Stains: Acid-Fast Stain  Cells that retain a basic stain in the presence of acid-alcohol  Stain binds strongly only to bacteria with a waxy material in their cell walls  Ex: mycobacterium tuberculosis (causes tuberculosis) Figure 3.12 Above: Mycobacterium leprae (causes leprosy) in tissue have been stained red with an acid- fast stain. Non acid-fast cells are stained with methylene blue (counterstain)

Copyright © 2006 Pearson Education, Inc., publishing as Benjamin Cummings Shapes of bacteria  Staining allows us to view the various shapes of bacteria  Main shapes:  Coccus (spherical)  Bacillus (rod-shaped)  Spirillium (spiral)

Copyright © 2006 Pearson Education, Inc., publishing as Benjamin Cummings Arrangement  These 3 shapes are found in 2 main configurations:  Clumps are called “Staphylo” (pronounced STAH-fill-oh)  Chains are called “Strepto” (pronounced STREP-toh)

Copyright © 2006 Pearson Education, Inc., publishing as Benjamin Cummings Now let’s put it all together.. Clumps of Circles are called Staphylococcus Clumps of Rods are called Staphylobacillus Clumps of Spirals are called StaphylospirillumAND Chains of Circles are called Streptococcus Chains of Rods are called Streptobacillus Chains of Spirals are called Streptospirillum

Copyright © 2006 Pearson Education, Inc., publishing as Benjamin Cummings Staphylococcus Staphylobacillus Streptobacillus Streptococcus

Copyright © 2006 Pearson Education, Inc., publishing as Benjamin Cummings Staphylospirillum Staphylococcus Streptospirillum Streptobacillus

Copyright © 2006 Pearson Education, Inc., publishing as Benjamin Cummings Staphylospirillum Staphylobacillus Streptospirillum Streptococcus

Copyright © 2006 Pearson Education, Inc., publishing as Benjamin Cummings Staphylococcus StaphylobacillusStreptobacillus Streptococcus