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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

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

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

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

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

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