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Micro Lab 3 & 4
| Question | Answer |
|---|---|
| Bacterial Staining Theory | Bacterial dyes that are BASIC STAINS and most bacterial cells have a negatively charged surface |
| Basic Stains | Positively charged stains |
| Procedure for bacterial smears (1) | Clean slides |
| Procedure for bacterial smears (2) | Label slides |
| Procedure for bacterial smears (3) | Prepare smear |
| Procedure for bacterial smears (4) | heat fix slides |
| Purpose of Heat Fix | Kills bacterial cells, sticks cells to slide surface, provides better stain penetration |
| Simple staining procedure (1) | Stain, with Crystal Violet, flooding slide for 60 seconds |
| Simple staining procedure (2) | Rinse slide |
| Simple staining procedure (3) | Blot dry with bibulous paper |
| Simple staining procedure (4) | Observe and make representative drawing ; measure size |
| Why are thick/dense smears less likely to provide a good smear preparation for microscopic evaluation? | Dense smears diminish the amount of light that can pass through, making it difficult to visualize the morphology of single cells |
| Why should you be careful not to overheat the smear during the heat-fixing process? | Overheating the slide can distort the morphology of the bacteria |
| Can simple staining techniques be used to identify more than the morphological characteristics of microorganisms? | Yes, simple stain shows not only morphology (cell shape and arrangement) but also cell size-which is not a part of cell morphology. |
| Will failing to heat-fix E.coli to a slide differ from correctly preparing a slide? | Slightly. E.coli would still stain but not as well as if it had been heat fixed. |
| Lab 3 microbes used: | Staphylococcus epidermis, Bacillus subtilis and my pure culture |
| Lab 4 microbes used: | Staphylococcus epidermis, Escherichia coli and my pure culture |
| Bacterial smear | bacterial sample is spread as a thin film on a clean microscope slide |
| Gram Stain Procedure (1) | Primary stain: Crystal Violet and let stand for 60 seconds, then rinse with distilled water |
| Gram Stain Procedure (2) | Mordant: Gram's iodine and let stand for 60 seconds, then rinse with distilled water |
| Gram Stain Procedure (3) | Decolorizing Agent: 95% alcohol, hold slide at 45 degree angle & drop by drop until almost clear |
| Gram Stain Procedure (4) | Counterstain: Safranin for 60 seconds, then rinse with distilled water |
| Safranin | simple stain, turns G- orange-pink |
| Gram Stain Procedure (5) | Blot dry with bibulous paper |
| G+ cell stain color | Violet |
| G- cell stain color | orange-pink |
| What are the advantages of differential staining procedures over the simple staining technique? | Differential staining allows one to differentiate G+ from G- cells, whereas simple staining only shows cell size and morphology. |
| Purpose of Primary stain in a differential staining procedure? | Impart its color to all cells |
| Purpose of Counterstain in a differential staining procedure? | If primary stain is washed out, the counterstain is absorbed |
| Purpose of Decolorizing agent in a differential staining procedure? | It may or may not remove the primary stain from the entire cell or only from certain cell structures. |
| Purpose of Mordant in a differential staining procedure? | It increases the cells affinity for stain |
| Why is it essential that the primary stain and the counterstain be of contrasting colors? | If the colors didn't have contrast, then it would be difficult to differentiate between G+ and G- cells |
| Which is the most crucial step in the performance of the Gram staining procedures? | The decolorization step. |
| Over-decolorization | Will result in a loss of primary stain, causing G+ to appear G- |
| Under-decolorization | Will remove the CVI (crystal-violet-iodine) complex, causing G- to appear G+ |
Created by:
kissteele