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Staining
Microscope adjustment for stained cells
Bright-field microscopy
how big should the smear in preparing a smear on a slide?
1-1.5 cm in diameter
Result from not heat fixing enough?
Loss of cells during staining (you will see no cells)
What happens if you heat fix too much?
Cells will be distorted and useless for viewing
How long to wait for crystal violet in simple stain?
30 seconds
What if you wash a slide with too much water after applying crystal violet?
it could wash the cells away
What if you rub the smear with bibulous paper?
You can wipe away the bacteria you want to observe
steps for simple stain
1. make smear and let air dry
2. heat fix
3. add crystal violet stain for 30 s
4. rinse with water, blot with bibulous paper
5. let dry and observe
2. heat fix
3. add crystal violet stain for 30 s
4. rinse with water, blot with bibulous paper
5. let dry and observe
Gram-positive cells
very thick peptidoglycan cell wall around a single cell membrane. Stain purple
Gram-negative cells
much thinner peptidoglycan cell wall and more compelx cell boundary system with two membranes. Stain pink.
purpose of iodine in gram staining
mordant: it helps bind the crystal violet stain to the peptidoglycan wall
Purpose of alcohol in gram staining
it differentially decolorizes cells. The very thick peptidoglycan of Gram-positive cells prevents loss of color, whereas in Gram-negative bacteria the crystal violet is more easily removed.
Purpose of safranin
counterstain, it lightly colors Gram-negative cells pink so they can also be observed.
Affect of age on cells
Gram staining should be done on cultures incubated for 24-48 hrs because old cells have thinner peptidoglycan layer of Gram-positive cells.
What happens if you overexpose gram stain to alcohol?
It will decolorize Gram-positive cells too much
steps to gram staining
1. prepare smear, dry and heat fix
2. stain with crystal violet for 30 s and rinse with water
3. add iodine for 20 s, rinse with alcohol and then water
4. stain with safranin for 1 min, wash with water
5. blot and observe
2. stain with crystal violet for 30 s and rinse with water
3. add iodine for 20 s, rinse with alcohol and then water
4. stain with safranin for 1 min, wash with water
5. blot and observe
Three features that we observe in a flagella stain:
number of flagella, location relative to the cell and the arrangement of flagella relative to each other
three most common types of bacterial flagella arrangements
monotrichous, lophotrichous and peritrichous
only one flagellum present
monotrichous
a bunch of flagella at one or both ends
lophotrichous
Flagella dispersed all over the cell
peritrichous
Ryu stain
use for flagella staining, stains bacteria and their flagella purple
How does Ryu stain work? What does it consist of?
Uses crystal violet in an alcoholic solution as primary stain. Upon evaporation a precipate is left around the flagella increasing its thickness. The stain also contains tannic acid and aluminum potassium phosphate as mordants, and phenol as antifungal agent
Steps of Flagella Staining
1. touch loop with culture on it to a drop of water on a slide and cover with a slip. DON'T MIX
2. after about 5-10 min apply a drop of Ryu stain to the edge of the cover slip, the stain will flow under the cover slip by capillarity and mix witht the cell suspension. Let sit for 2-3 min
3. use oil immersion to examine cells
2. after about 5-10 min apply a drop of Ryu stain to the edge of the cover slip, the stain will flow under the cover slip by capillarity and mix witht the cell suspension. Let sit for 2-3 min
3. use oil immersion to examine cells
A cellular capsule is composed of
mucoid polysaccharides or polypeptides that repel most stains
What type of stain is a capsule stain?
negative stain- the cell and glass are stained but the capsule is not
Do you heat fix in capsule staining? Why or why not?
No because heat will destroy the capsule
India ink
used for capsule staining- it colors the background glass so that capsules can be observed negatively
procedure for capusle staining
1. place one loopful of india ink at one end of the micrscope slide
2. mix one loopful of sterile saline water with the ink
3. aseptically transfer and mix a small amount of bacteria
4. take a second slide and hold at a 45° angle, touch the liquid at the end of the slide
5. without raising the slide, slowly and gently pull the top slide back to spread the stain
6. discard top slide
7. don't heat fix, allow slide to thoroughly air dry
8. flood with crystal violet for 30 s
9. rinse with tap water
10. allow to air dry, DON'T BLOT
11. observe using oil immersion
2. mix one loopful of sterile saline water with the ink
3. aseptically transfer and mix a small amount of bacteria
4. take a second slide and hold at a 45° angle, touch the liquid at the end of the slide
5. without raising the slide, slowly and gently pull the top slide back to spread the stain
6. discard top slide
7. don't heat fix, allow slide to thoroughly air dry
8. flood with crystal violet for 30 s
9. rinse with tap water
10. allow to air dry, DON'T BLOT
11. observe using oil immersion
Common placements of endospores are
terminal (end of cell), central (middle of cell) or subterminal (between the middle and the end)
What is the appearance of cell and spore after an endospore stain?
cell is pink (from safranin counterstain) and spore is green-blue (from malachite green)
malachite green
used for endospore staining
why do you have to stain a spore differently?
They are resistant ot staining due to a thick wall so you have to heat to drive the stain into the spore
Endospore staining procedure
1. Wear gloves malachite green will stain everything, it is toxic and won't wash out
2. Prepare a smear, air dry and heat fix
3. Prepare a large boat from tin foil and place on preheated hot plate. Put the slide in the boat.
4. Cover smear with malachite green stain. Place a small piece of filter paper on the stain
5. Adjust heat to prevent boiling but want it to be steaming-hot
6. Don't allow stain to dry out continue to add more stain
7. Remove slide from hot plate after 5 min
8. Cool and rinse with water
9. add safranin for 1 min, wash with water, blot, air dry and observe
2. Prepare a smear, air dry and heat fix
3. Prepare a large boat from tin foil and place on preheated hot plate. Put the slide in the boat.
4. Cover smear with malachite green stain. Place a small piece of filter paper on the stain
5. Adjust heat to prevent boiling but want it to be steaming-hot
6. Don't allow stain to dry out continue to add more stain
7. Remove slide from hot plate after 5 min
8. Cool and rinse with water
9. add safranin for 1 min, wash with water, blot, air dry and observe