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What is the medical application for Flourescence Microscope?
- Rapid ID of bacterial antigens in tissue smears, sections, and fluids
- Rapid ID of many disease-causing organisms
This microscopy is based on the principal of removal of incident illumination by selective absorpion. What does this mean?
Flourescence Microscopy: light that has been absorbed by the specimen and re-emitted at an altered wavelength, which is filtered, removing ineffective wavelengths.
What are the functions of the following:
1. exciter filter
2. barrier filter
3. heat filter
4. mercury vapor arc lamp
- Exciter filter: removes long wavelengths
- Barrier filter: blocks ultraviolet radiation but allows visible light through
- Heat filter:
When is flourescence microscopy used in clinical laboratory? How is this done?
- Can mark and view antibodies, good for diagnosing diseases
- Checking antibodies that stick to a mitochondria and checking those antibodies to see if they light up
T or F: Positive staining is good for viewing yeasts and capsules.
False: negative stanining
This staining is achieved by mixing bacteria with a negative acidic stain. The negative stain will repel with the negatively charged bacterial wall.
T or F: You heat fix a negative stain
T or F: you use oil immersion for flourescence microscopy
T or F: You use a thin smear technique when preparing a slide for a negative stain.
When Is negative staining used? (3 reasons)
- 1. If you don'nt want to heat fix a specimen
- 2. To see capsules (see different contrast than capsule stain exercise)
- 3. Fast process
Which stains can be used for negative staining?
Any stain (acidic) that has a negative charge so it can be repelled by the cell wall.
When do you use simple staining over negative staining?
While negative staining is satisfactory for simple observations, more specific stains are necessary if bacterial detail is to be observed.
What dyes are used in Negative staining?
Acidic dyes: India Ink
What dyes are used in Positive staining? (4)
Basic dyes: crystal violet, methylene blue, safranin, malachite green
This term refers to use of a single stain or dye to create a contrast between the bacteria and the background
When is simple staining often employed?
When info is needed about cell shape, size and arrangement
What are the steps of making a negative stain? (4 steps)
- 1. Apply a small amount of bacteria to slide
- 2. Add Acidic dye and mix
- 3. Make a spread of the mixture evenly and thinly
- 4. Air dry and use oil immersion
T or F: you heat fix in a positive stain
In positive staining, basic dyes (crystal violet, carbolfuchsin, or methylene blue) bind to bacterial cells by ____ interactions.
Crystal violet, carbolfuchsin, and methylene blue have what kind of charge when interacting with bacterial cell walls?
Positive: binds to negatively charged molecules like nucleic acids, proteins, and surfaces of prokaryotic cells.
What is the procedure for making a positive stain? (5 steps)
- 1. Prepare a smear of bacteria on a slide and air dry
- 2. Heat fix
- 3. Place on staining rack and stain with of 3 stains:
- - methylene blue for 1 min.
- - carbolfuschin for 10 secs
- - crystal violet for 30 secs
- 4. Wash stain with water and let dry
- 5. Use oil immersion.
What are the 2 purposes of heat fixing?
- 1. Kills bacteria
- 2. Sticks them to slide
What is the benefit of simple staining?
To see cell shape, size and arrangements in an easy manner
Are basic dyes or acidic dyes used in positive staining? Why?
Basic: they have a positive charge and attract to thhe negatively charged cell wall
Name 3 basic stains
- 1. Crystal violet
- 2. Carbolfuschin
- 3. Methylene blue
This differential staining is the most useful and widely used stain in bacteriology.
T or F: Gram negative bacteria retain the color of the primary dye, and negative lose the primary dye when washed in a decolorizing solution.
False: Gram-positive bacteria retain the color of the primary dye, negative take on the color of the second dye.
What is the primary dye in gram staining?
What does the iodine solution function as and what is its purpose?
Mordant: it increases the interaction between the bacterial cell and the dye that causes the dye to form large complexes in the peptidoglycan meshwork of the bacterial wall
After the primary stain and iodine is applied, what is used to decolorize the primary stain? What happens to the gram positive and negative stains?
- 95% ethanol or isoproponaol is used:
- - Gram positive bacteria retain the crystal violet-iodine
- - gram negative lose theirs and become colorless
What is used as the counterstain in gram staining? What is the action?
Safranin stains the colorless, gram-negative bacteria pin but does not alter the dark purple color of gram-positive bacteria.
T or F: gram-positive cultures will always remain in that state.
False: may turn gram negative if they get too old.
What does it mean when a bacteria is gram variable?
When some cells in the same culture will be gram positive and some gram negative.
How can gram-stain results be confirmed?
- By placing a drop of 10% KOH on a slide and mixing with lòopful of bacteria
- Wait 30 secs, then pull loop slowly through suspension, away from slide.
- Positive organisms will remain fluid, negative will produce a mucoid string.
What is the process of making a gram stain?
- 1. Add sample to slide, air dry then heat fix.
- 2. Place on staining rack, and smear with crystal violet for 30 secs, then rinse with water
- 3. Cover with iodine mordant for 1 min then rinse with water
- 4. Decolorize with ethanol drop by drop until on a slant, until only clear liquid is running. Should be about 5 secs. Rinse with water
- 5. Counterstain with safranin for 60 secs, then rinse with water.
- 6. Let dry and examine under immersion oil.
If you heat fix an organism, do you need to apply a cover slip?
Using the typical stains from lab, what color will gram positive and negative stains be?
- Negative: pink
- Positive: blue to purple
Explain what is happening to the cell's wall in each step during gram staining:
Step 1 - Crystal violet
Step 2 - Iodine (mordant)
Step 3 - Alcohol
Step 4 - Safranin
- Step 1: cells stain purple, both negative and positive will be affixed with the dye
- Step 2: Cells remain purple. In gram positive, the dye complex is trapped in the wall. Gram negative has no effect of iodine.
- Step 3: Gram positive cells remain purple, and negative become colorless. This happens because the outer membrane is weakened, and dye flows out of the wall
- Step 4: Gram positive remains purple, gram negative appears red. In positive, red dye is masked by violet. Negative, red dye stains the colorless cell
What is the difference between a simple and differential stain?
- Simple: Throw it on, all organisms look similiar
- Differential: can see if cells are gram + or -
This step in gram staining is the most crucial and most likely to cause poor results
Why can't older cultures be used for gram staining?
Their cell walls break down and can't hold onto stains
What part of the bacterial cell is most involved with gram staining? Why?
Peptidoglycan Layer: It holds the crystal violet
These bacteria are IDed in the medical field using acid-fast staining.
Genus mycobacterium: M. Leprae (leprosy) and M. Tuberculosis
If a bacteria does not readily stain with simple stains, what is another solution? What does it mean when a bacteria is "acid-fast?"
- When a bacteria is not easily stained with simple stains, it can be heated with carbolfuchsin, driving the stain into the cell wall.
- Acid-fast refers to once the bacteria has taken up the carbolfuchsin, they are not easily decolorized by acid-alcohol.
What makes a bacteria acid-fast?
It is due to the high lipid content in the cell wall of the microorganism.
How are cells IDed as acid-fast or non-acid-fast?
When microorganisms are decolorized by acid-alcohol, acid-fast ones will retain their color and appear red. Non-acid-fast will appear blue due to the counterstaining with the methylene blue.
What is the purpose of the heat during the acid-fast staining procedure?
Heat drives dye into the cell
What is the function of the counterstain in the acid-fast staining procedure?
Stains things that primary stain didn't take up. It IDs the non-acid-fast stains that were decolorized.
Are acid-fast bacteria gram positive or negative?
Gram + due to thick peptidoglycan layer and mycolic acid.
The absence of this makes a bacteria non-fast-fast.
Why is gram stain not an adequate substitute for an acid-fast stain?
Acid-fast bacteria will not hold the purple violet due to the myolic acid lipid
T or F: Endospores do not stain easily, but do not resist decolorization.
False: They do not stain easily, but once stained, STRONGLY resist declorization.
What are endostores stained with? What is done to help the stain penetrate?
Malachite green: Heat
What is used to counterstain the rest of the cell once the endospore is stained with malachite green? What color is the counterstain?
Safranin.: light red
How is heat added to endospore staining? How is the malachite green added?
- By placing the slide on top of a boiling water bath (tin can filled with water)
- A paper towel, the size of the slide, is placed on top of slide and malachite green is soaked onto the paper towel.
Where are endospores located within the vegetative cells?
Internally: this is a genetic trait to help determine the organism
Name 2 disease-causing bacteria that produce endospores