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Micro Lab Part 1
Lab 5 Negative Staining/Capsular Staining/Spore Staining
| Question | Answer |
|---|---|
| Negative stains are ____________ and thus have a ___________ ___________ chromophore | acidic, negatively charged |
| Negative stains; cells appear as | transparent objects against a dark backround |
| Examples of negative stains are; | india ink and nigrosin |
| Negative staining is useful for determining | cell dimensions because heat fixation is not performed so no shrinkage of cells occurs; it is also useful for observing spirochaetes (thin cells) |
| Describe the four steps of a Negative staining technique using the spreader slide | 1) Organisms are dispersed into a sm drop of nigrosin or inidia ink.2) Spreader slide is moved toward drop until it connects and spreads along edge3) Spreader slide is then pushed to the left dragging the suspension over bottom of the slide; air dry |
| Some bacterial cells are surrounded by an extracellular slime layer called; | a capsule or glycocalyx |
| What does the capsule or glycocalyx do? | Prevents WBC's from engulfing and destroying bacterial pathogen, enabling the organism to invade or attach to surfaces |
| Steps of capsular staining (combing simple stain and negative stain) | 1)2 loopfuls of organism mixed w/india ink2)Ink spread over slide & air dried3)Heat fix 4) Stain w/crystal violet for 1min5) Wash off w/water and blot w/bibuous paper |
| A basic dye is utilized to stain bacterial cells in what techniques? | Simple and capsular |
| A stain that does not penetrate cells is used to color the backround in what technique? | Negative and capsule |
| Which technique is useful in observing spirochetes? | Negative |
| Which technique is useful in observing the glycocalyx of certain bacterial species | Capsule |
| In which technique are cells mixed with a stain before they are smeared on the slide? | Negative, capsule |
| In which tecnique is heat fixation of the slide not recommmended? | Negative, gentle heat for capsule |
| In which technique is water used to remove excess stain from the slide | Simple and capsule |
| What causes a stain to adhere to bacterial cells? Why are all colored dyes not necessarily useful for simple staining? | Basic dyes, which carry a (+)chg will adhere to neg charged cell surface (phospholipids) bec of chg attraction, acidic dyes will not adhere |
| Why are encapsulated strains of Streptococcus pneumoniae much more likely to cause disease than strains that do not produce a capsule? | They are protected against phagocytosis in the host & r more likely to cause infection than unencapsulated strains |
| For the simple stain procedure, one can use: | crystal violet, methylene blue |
| For the negative stain procedure, one can use; | india ink, nigrosin |
| Before heat fixation, a wet smear of bacterial cells on a slide must first be | air dried |
| Bacterial capsules can consist of; | peptidoglycan and polypeptides w/unique amino acids |
| What contributes to the resistance of endospores? | Exosporium (protein coat) |
| What procedure is used to stain endospores? | Spore staining w/ the schaeffer-fulton method |
| Procedure of Schaeffer-Fulton spore stain method | 1) saturate slide w/malachite green; steam over water for 5 minutes (add stain if it boils off)3)Counterstain w/safranin for 20 sec4)Rinse w/water and blot dry w/bibulous paper2) cool slide & rinse w/water for 30 sec |
| What does a negative stain look like? | Pink backround with clear bacterial cells |
| What does a capsule stain look like? | Bright pink backround with clear cells that are encapsulated with blue |
| What does an endospore stain look like? | light backround with bluegreen/red cells |
| What does a gram stain look like? | Gram positive are purple and gram negative are pink |
| What does acid-fast stain look like? | light backround with blue bacteria...some are bright pink |
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