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Lab 11, 12, 13
| Question | Answer |
|---|---|
| What is the most frequently used staining technique in microbiology? | Gram Stain |
| What is usually the first step in identification of an isolated unknown organism? | Gram stain |
| The Gram stain is a Differential stain, what does this mean? | It differentiates the gram negative and gram positive cells by color |
| What does the response to this stain (Positive or Negative) depend on? | The composition of the cell wall |
| How is the cell wall of a gram POSITIVE cell made up? | made up of a very THICK layer of Peptidoglycan |
| What is Peptidoglycan? | Long fibrous network of NAM(N-acetylmuramic acid) and NAG (N-acetyl glucosamine) crossed linked by short peptide chains |
| How is the cell wall of a gram NEGATIVE cell made up? | made up of very THIN layer of Peptidoglycan surrounded by an outer membrane containing lipopolysaccharide (LPS) |
| What does the Gram stain technique require the use of? | 1.dyes 2.Stains 3.Mordant 4.decolorizer |
| What would be used as PRIMARY STAIN? | Crystal violet |
| What would be used as the MORDANT? | Grams Iodine |
| What does the mordant do? | Fixes the color into the cell or intensifys color ( iodine binds to RNA and crystal violet in cell, creating a large color complex ) |
| What is used for DECOLORIZATION? | Acetone Alchohol |
| What is Acetone Alchohol? | A solvent/denaturant that creates large holes in the gram negative cells outer lipopolysaccharide cell wall and washes out the iodine crystal violet complex |
| How will the cells appear AFTER the decolorization step? | Gram positive cells will appear purple Gram negative cells will appear colorless |
| Final step of gram stain is Safranin, what is this used for? | Counterstain |
| How will the cells appear after the Safranin (Counterstain) is applied and rinsed off? | Gram positive cells will appear purple Gram Negative cells will appear pink |
| What are the 4 Steps in Gram Staining? | 1.Crystal violet 2.Grams Iodine 3.Alcohol 4.Safranin (red dye) |
| What happens when gram positive cells die? | The cell walls break down and no longer retain the purple crystal violet iodine complex |
| True or False, When older gram positive cultures are gram stained the result can be ambiguous with BOTH pink and purple cells being present? | TRUE |
| When the organism is gram positive you are just seeing old culture in which not all of the cells are still alive, True or False? | TRUE |
| What organisms are seen (on slants or plates) during gram staining? | 1.Pseudomonas aeruginosa 2.Staphylococcus aureus |
| What type of cells eventually form Endospores? | Vegetative cells (actively metabolizing) of members of genera bacillus (aerobe) and Clostridim (anaerobe) begin the process of sporulation and eventually form endespores |
| What is an Endospore? | A survival structure formed within the bacterial cell wall that allows the cell to survive until environmental conditions improve |
| What does the Endospore contain? | 1.Copy of the organisms chromosome 2.a little cytoplasm 3.small amount of cell machinery |
| What is the Spore coat composed of? | Keratin, protein, calcium, dipicolinic acid, peptidoglycan |
| At what point do Vegetative cells begin the process of sporulation? | When stressed by factors as nutrient limitation, temperature changes, and waste accumilation |
| What process begins with the replication of bacterial chromosome and the spatial separation of the chromosomes I different regions within the cells cytoplasm? | Sporulation |
| How is separation of the chromosome accomplished? | by the formation of a wall or septum within the cell |
| The region of one side of the septum that will eventually become the endospore? | forespore |
| The region on the other side of the septum is metabolically active and produces and deposits compounds necessary for completion of the spore coat layers, what is the name of this side? | Sporangium |
| What does a "free spore" imply? | A mature endospore whose cell wall has broken down and released the endospore |
| What would you call an endospore that developes at the end of a cell? | Terminal endospore |
| What would you call an endospore that forms in the middle of the cell? | Central endospore |
| What do you call an endospore that is located in between the center and the end of the cell? | Subterminal endospore |
| True or False, the Endospore stain is classified as a Differential or Structural stain? | TRUE |
| What is the Primary stain for Endospore staining? | Malachite green |
| True or False, with Endospore staining, the heat fixed smears are steamed over boiling water with the malachite green? | TRUE |
| When the endospore smears are decolorized, they are counterstained, what is the counter stain used? | Safranin (to dye the vegetative cells) |
| What color will endospores stain to? | Endospores will be green to blue green Vegetative cells will appear red |
| How long must slides be heated with steaming water bath for the staining method to be effective? | at least 5 minutes |
| What organisms are used during the endospore stain? | -Bacillus spp (24 hr culture) -Bacillus spp (1 week culture) |
| What does Acid Fast staining differentiate? | differentiates between acid fast and non acid fast bacteria |
| What does the acid fast genre include? | - Mycobacterium and some members of Nocardia (mycobacterium include causative agents of tuberculosis TB, and leprosy) |
| What causes Acid Fast cells to have a waxy cell wall? | Mycolic acid in the cell walls |
| What does the Mycolic Acid in the cell walls cause? | The organisms to be resistant to desiccation and disinfectants |
| What staining method must be used for acid fast cells? | Ziehl Neelson method and the Kinyoun method |
| What does the Ziehl Neelson method use to facilitate stainig the cell? | Steaming water bath |
| Which method is sometimes called the cold method? | Kinyoun method, because it does not require steam |
| What is the Primary stain for Acid Fast staining? | Carbol Fuchsin |
| During Acid fast staining, what are the smears decolorized with? | Acid Alcohol |
| What are smears that retain color AFTER the decolorization called? | Acid Fast- bright red fuscha color remains |
| What is the Counterstain that is used in Acid Fast staining? | Methylene Blue |
| After the counterstain, the non acid fast cells will appear what color? | Blue |
| The steps in the Kinyoun method are ALMOST identical to the steps in the Ziehl Neeson method, what is the difference? | The Kinyoun method does not require slide to be steamed, |
| Why does the Kinyoun method not require steaming of the slide? | Because the primary stain reagent contains higher concentrations of phenol and basic fuchsin that in Ziehl Neelson reagent allowing staing of cells at benchtop temperatures |
| What organisms are used for the Ziehl Neelson method? | -Mycobacterium smegmatis -Staphylococcus aureus -Patient F. culture |
Created by:
Melissa nicole