Bell Ringer Identify the two pictures below with Gram reaction, morphology, and arrangement

1) Complete the questions in the lab report and finish Gram staining In The Next 30 Minutes… 1) Complete the questions in the lab report and finish Gram staining Collect 2) Work on handout P. 68 in textbook Describe acid-fast, endospore, flagella, and negative staining procedures Homework if not complete You will have a 15 point quiz at the end of class covering the material taught today

Staining Simple Stains Differential Stains Special Stains Gram stain Acid-fast stain Endospore stain Special Stains Negative (capsule) stain Flagellar stain © 2012 Pearson Education Inc. 3

Figure 4.16 Simple stains-overview

Use more than one dye so that cells can be distinguished Staining Differential Stains Use more than one dye so that cells can be distinguished Gram stain Acid-fast stain Endospore stain © 2012 Pearson Education Inc. 5

Staining Differential Stains Gram stain Gram (+) & Gram (-) Primary stain- crystal violet Mordant- iodine Decolorizing agent- acid alcohol Counterstain- safranin Young cells Thin smear Avoid over over-decolorization and under-decolorization © 2012 Pearson Education Inc. 6

Figure 4.17 The Gram staining procedure-overview Slide is flooded with crystal violet for 1 min, then rinsed with water. Slide is flooded with iodine for 1 min, then rinsed with water. Result: All cells are stained purple. Result: Iodine acts as a mordant; all cells remain purple. Slide is flooded with solution of ethanol and acetone for 10–30 sec, then rinsed with water. Slide is flooded with safranin for 1 min, then rinsed with water and blotted dry. Result: Gram-positive cells remain purple, Gram-negative cells are pink. Result: Smear is decolorized; Gram-positive cells remain purple, but Gram-negative cells are now colorless. 7

Why do G+ cells appear purple? Why do Gram – cells appear pink? Cold Calling Why do G+ cells appear purple? Why do Gram – cells appear pink? What would result if you did not decolorize during the gram stain? How would the cells appear? Why would a G+ cell appear purple and pink after Gram staining?

Staining Differential Stains Acid-fast stain Mycobacterium & Nocardia M. tuberculosis and M. leprae Do not gram stain due to waxy cell walls Appear red or pink Carbolfuchsin Acid-alcohol Methylene Blue © 2012 Pearson Education Inc. 9

Acid-Fast Stain Why isn’t the Gram stain used to stain Mycobacterium?

Staining Differential Stains Endospore stain Bacillus & Clostridium Stain green Malachite green and steam Water Safranin © 2012 Pearson Education Inc. 11

Staining Special Stains Negative (capsule) Acidic dyes Repelled by bacteria Stain environment Eosin and nigrosin Capsules Bacterium Capsule Background stain © 2012 Pearson Education Inc. 12

Cold Calling How come negative stains do not dye the bacterial cells?

Staining Flagellar stain Mordant binds to flagella and change their color to increase contrast © 2012 Pearson Education Inc. 14

Quiz 15 Points 17 Questions C. diff and Staining

Identification of Microorganisms Identifying Characteristics Physical characteristics Biochemical tests Serological tests Phage typing Analysis of nucleic acids © 2012 Pearson Education Inc. 16

Identification of Microorganisms Identifying Characteristics Physical characteristics Morphology (shape) Coccus and Bacillus Physical appearance of bacterial colonies © 2012 Pearson Education Inc. 17

Identification of Microorganisms Identifying Characteristics Biochemical tests Utilize or produce certain chemicals Ferment various carbohydrates Produce hydrogen sulfide gas as a waste product Used in labs to identify pathogens Color change indicates metabolic reaction The pattern of colors correlates to a specific pathogen © 2012 Pearson Education Inc. 18

Figure 4.23 Two biochemical tests for identifying bacteria-overview Gas bubble Inverted tubes to trap gas Hydrogen sulfide produced No hydrogen sulfide Acid with gas Acid with no gas Inert 19

Figure 4.24 One tool for the rapid identification of bacteria, the automated MicroScan system Wells 20

Identification of Microorganisms Identifying Characteristics Serological tests The study of serum (liquid portion of blood) Antigen antibody reactions Antibodies- part of immune system that bind specifically to a antigen Agglutination test- antiserum mixed with target antigens Clumping occurs if antigens as present © 2012 Pearson Education Inc. 21

Negative result Positive result Negative result Positive result Figure 4.25 An agglutination test, one type of serological test-overview Negative result Positive result Negative result Positive result 22

Identification of Microorganisms Identifying Characteristics Phage typing Bacteriophages (phages)- viruses that infect bacteria Phages are specific to their host One bacterial strain is susceptible while another is not Bacteria is lawned on media Drops of different phage solutions are placed on plate Plaque- clear zones on bacterial lawn © 2012 Pearson Education Inc. 23

Figure 4.26 Phage typing Bacterial lawn Plaques 24

Identification of Microorganisms Identifying Characteristics Analysis of nucleic acids Sequencing of nucleic acids Polymerase chain reaction (PCR) Examine G + C ratios 20%-80% in prokaryotes © 2012 Pearson Education Inc. 25

Classification and Identification of Microorganisms Taxonomic Keys Dichotomous keys Series of paired statements where only one of two “either/or” choices applies to any particular organism Key directs user to another pair of statements, or provides name of organism © 2012 Pearson Education Inc. 26

Figure 4.27 Use of a dichotomous taxonomic key-overview Gram-positive cells? No Yes Rod-shaped cells? Gram-positive bacteria No Yes Cocci and pleomorphic bacteria Can tolerate oxygen? No Yes Obligate anaerobes Ferments lactose? No Yes Can use citric acid (citrate) as sole carbon source? Non-lactose- fermenters No Yes Produces gas from glucose? Produces hydrogen sulfide gas? No Yes No Yes Produces acetoin? Shigella Escherichia Salmonella No Yes Citrobacter Enterobacter 27