Transcript Staining
Bell Ringer • Identify the two pictures below with Gram reaction, morphology, and arrangement 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 – Gram stain – Acid-fast stain – Endospore stain • Special Stains – Negative (capsule) stain – Flagellar stain © 2012 Pearson Education Inc. Figure 4.16 Simple stains-overview 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. 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 underdecolorization © 2012 Pearson Education Inc. 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: Smear is decolorized; Gram-positive cells remain purple, but Gram-negative cells are now colorless. Result: Gram-positive cells remain purple, Gram-negative cells are 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. 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. Staining • Special Stains – Negative (capsule) – Acidic dyes – – – – Repelled by bacteria Stain environment Eosin and nigrosin Capsules © 2012 Pearson Education Inc. Bacterium Capsule Background stain 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. 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. Identification of Microorganisms • Identifying Characteristics – Physical characteristics – Morphology (shape) – Coccus and Bacillus – Physical appearance of bacterial colonies © 2012 Pearson Education Inc. 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. Figure 4.23 Two biochemical tests for identifying bacteria-overview Gas bubble Acid with gas Inverted tubes to trap gas Acid with no gas Inert Hydrogen sulfide produced No hydrogen sulfide Figure 4.24 One tool for the rapid identification of bacteria, the automated MicroScan system Wells 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. Figure 4.25 An agglutination test, one type of serological test-overview Negative result Positive result Negative result Positive result 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. Figure 4.26 Phage typing Bacterial lawn Plaques 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. 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. Figure 4.27 Use of a dichotomous taxonomic key-overview Gram-positive cells? No Yes Gram-positive bacteria Rod-shaped cells? No Yes Can tolerate oxygen? Cocci and pleomorphic bacteria No Yes Ferments lactose? Obligate anaerobes No Yes Non-lactosefermenters Can use citric acid (citrate) as sole carbon source? No Yes Produces gas from glucose? No Shigella Produces hydrogen sulfide gas? No Yes Escherichia Yes Produces acetoin? No Citrobacter Salmonella Yes Enterobacter