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Micro Lab Part 1
Lab 4 Gram Staining/Acid Fast Staining
| Question | Answer |
|---|---|
| The Gram Stain is an example of what type of stain? | Differential Stain |
| What does the Gram Stain differentiate? | Gram Positive and Gram Negative bacteria |
| List the five steps of Gram stain in order; | 1)Heat fixed cells2)Crystal Violet (Primary Stain) 20 seconds3)Gram's Iodine (mordant) for 1 minute4) Ethyl Alcohol (decolorizing) 10-20 seconds5) Safranin (1 min) |
| Bacteria with a high lipid content contain what material that affects the staining properties of certain bacteria? | mycolic acid |
| List the steps of acid-fast staining; | 1)Carbolfuchsin and stain for 5 min (on heat)2)Wash w/water then decolorize w/acid-alchohol for 1 min., then rinse w/water. 3) Counterstain w/methylene blue for 30 sec. rinse to remove excess and blot dry w/bibuous paper. Examine under oil immersion |
| What is the function of a mordant? | A mordant causes the primary stain to adhere better or be taken up by the cell so that it is not removed during the de-colorizing step |
| For differential staining, how does a counterstain differ from a primary stain? | The counterstain must be a different color than the primary stain to aid in differentiaion |
| How do gram positive and gram negative bacteria differ in cellular structure? How does this contribute to their differential staining properties? | A gram + cell wall (thick) retains the crystal-violet iodine complex better in teh presence of decolorizer as compared to gram - cell, which has a thin cell wall |
| Which is the most critical step in the Gram-Stain procedure? Why? If this procedure is cone incorrectly, how might that affect the final results? | The decolorizer step because it is the step in which the cells become differentiated (Gram+ are purple and Gram- are colorless)Too much & Gram+ lose primary & stain pink. Too little & Gran- will not lose primary & will remain purple. |
| How does culture age affect the results of a Gram-stain? | Old cultures of gram+ cells may not retain stain as well as younger cultures and could give false negatives |
| How does culture age affect the results of a spore stain? | Old cultures of spore formers like bacillus are ideal bec under the conditions of nutrient depletion, sporulation is more likely to occur |
| Why must smear thickness be considered before performing gram stain? | Smears that are too thick will not stain properly bec the stains may not penetrate lower layers & thick clumps of cells may not easily decolorize |
| What color are bacterial endospores after a Gram-stain is performed? What does this tell you about the physical properties of endospores? | Basic dyes do not penetrate spores so gram staining will result in colorless spores. This indicates that spores are very resistant structures. |
| Bacillus anthracis, the causative agent of anthrax, is an endospore-former. Why does this trait enhance its capabilities as a biological weapon? | They are ideal because they are easily produced, can be dispersed in the air & are environmentally stable. |
| What makes Mycobacterium particularly resistant to staining? How are the bacteria in this genus grouped in terms of Gram classification? | It has a peptidoglycan layer filled w/mycolic acids that make the wall waxy & impenetrable to stains. Classified w/gram+ cell bec of cell wall thickness & genetic similarities |
| How do you think the acid-fast nature of Mycobacterium contributes to its virulence? | The waxy cell protects it against phagocytosis & some antibiotics while in the host (eg lungs for TB) so the pathogen has greater opportunity to cause disease. |
| The color of Staphylococcus aureus before primary stain is added | colorless |
| Pseudomonas aeuruginosa after primary stain is added | purple |
| Bacillus megaterium after the mordant is added | purple |
| Staphylococcus aureus cells after the decolorizer is used | purple |
| Moraxella (Branhamella) catarrhalis after the decolorizer is used | no color |
| Bacillus megaterium after the counterstain is added | purple |
| Pseudomonas aeruginosa after the counterstain is added | pink |
| List the four staining techniques of gram-stain and the cell type and color after completion of stain | Crystal Violet; iodine; alcohol-acetone, safarin results in Gram + Purple and Gram - Pink |
| List the four staining techniques of Spore Stain and the cell type and color after complettion of stain | Malachite green, heat, water, safranin. Sporangium=Green and Begetative cell=Pink |
| List the four staining techniques of Acid-Fast Stain and the cell type and color after | Carbolfuchsin, heat, acid-alcohol, methylene blue. Results; Acid fast bacteria=RED and non-acid fast bacteria= Blue |
| A bacterial cell wall is composed of; | peptidoglycan |
| The exosporium or endospore coast is composed of | proteins |
| Endospores are produced by bacteria in the genus | Bacillus and Mycobacterium |
| Acid fast staining is useful for identifying he causative agent of | leprosy and tuberculosis |
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