Microlab quiz 4 SG

What is the purpose of staining bacterial cells?

allow bacteria cells and their structures to become visible when viewed microscopically

What information can be gained by staining bacterial cells?

the cell shape, arrangement, and presence of cellular structures

What is basic dyes?

Positively charged ions that are attracted to the negative charge of the bacteria cell

what is acidic dyes?

have negatively charge ions that are attracted to the glass or surrounding medium and stain specific cellular structures

Give an example of basic dyes

- methylene blue

- crystal violet

- safranin

- malachite green

Give an example of acidic dyes

nigrosine?

What are the steps in prepping a smear

1. label a clean slide

2. flame the loop, let the loop cool, obtain one loop of water to slide

3. flame the loop, let the loop cool, flame the top of the test tube, and obtain a small amount

4. reflame the top of the test tube and the loop

5. air dry the slide

6. heat fix the slide by passing it through the flame 2-3 times. The smear should be facing up and away from the flame

Define smear

the aseptic addition of a bacterium to a clean, glass slide

Define heat fixing

for bacteria to adhere to slide and therefore prevent the specimen from washing away during the staining procedure

Know the steps in a simple stain

1. prepare a bacterial smear

2. add a basic dye (methylene blue) to the smear for 1 minute

3. rinse with water

4. blot dry using bibulous paper

5. view under the microscope using scanning, low, high, and oil objective lenses

6. draw the results (in color) on the results table

What is the significance of a thin smear

What is the disadvantage of having a thick smear?

Different shapes of bacteria

bacillus (rod-shaped), coccus ( spherical- shaped) and spirillum (corkscrew)

What are the different cellular arrangements

diplo (pairs), strepto (chains), staphylo (grape- like clusters), and tetrads (fours)

What are the steps in the gram stain

1. make a smear

2. add the primary stain, crystal violet, let it sit on the smear for 1 minute

3. rinse with water

4. add iodine (mordant) and let it sit on the smear for one minute

5. decolorize with ethyl alcohol for 15 seconds (or until the slide runs clear)

6. rinse with water

7. add the counterstain stain, safanin, and let it sit on the smear for one minute

8. rinse with water

9. blot dry using bibulous paper and viewing using oil immersion lens

10. draw your results (in color) on the results table

What's the purpose of a gram stain

is to separate bacteria into two categories: gram positive and gram negative based on the cell wall composition

Know mordant in gram stain

iodine

Know decolorizer in the gram stain

ethyl alcohol

Know the primary stain in the gram stain

crystal violet

Know the counterstain in the gram stain

safranin

Know the cell wall structure of a gram positive bacterium and its color after the staining process

Know the cell wall structure of a gram negative bacterium and its color after the staining process

What are the steps In the negative stain?

1. add 1-2 drops of nigrosine dye to one end of a clean slide

2. use a toothpick to scrape between your teeth and add the scraping to the nigrosine

3. using 2nd slide, spread the suspension over the entire slide

4. air dry and view

Purpose of negative stain

Uses the acidic dye nigrosine to determine the presence or absence of a bacteria capsule

Define capsule

polysaccharide structures that surround the cell wall and prevent phagocytosis

What acidic dyes bind to the negative stain?

Nigrosine

steps of acid fast stain

1. Make a smear

2. Add the primary stain, carbolfuchsin to the smear and microwave (in glass dish) for 30 seconds

3. Cool, decolorize with acid alcohol for 15 -20 seconds

4. Rinse with water

5. Add the counterstain, methylene blue, to the smear and let it sit for 30-45 seconds

6. Rinse with water

7. Blot dry and view using the oil immersion

8. Draw the results (in color) on the results table

purpose of acid fast stain

To determine the presence of mycolic acid and identify bacteria belonging to the genus mycobacterium

Know the mordant in acid fast stain

Heat

Know the decolorizer in acid fast stain

Acid alcohol

Know the primary stain of acid stain

Carbolfuchsin

Know the counterstain in the acid fast stain

Methylene

Know the result and be able to explain the results for acid fast stain

Acid fast positive- bright red

Acid fast negative- blue

Be able to give an example of an acid fast stain positive bacterium

Know the steps in the endospore stain

1. make a smear

2. add the primary stain, malachite stain, to the smear and microwave for 30 seconds in glass Petri dish

3. cool, rinse with water

4. add the counterstain, safranin, to the smear and let it sit for 30-45 seconds

5. rinse with water

6. blot dry and view using oil immersion objective lens

Understand the purpose of the endospore stain

distinguish between spore-forming and non-spore-forming bacteria?

Define spore

a cell surrounded by a protective covering

Define vegetative cell

metabolically active, replicating cell

which bacteria can produce spores

Bacillus and Clostridium

Know when bacteria produce spores

when the cells become stressed due to lack of nutrients, extreme heat, and exposure to uv light

what is the mordant in the endospore stain

heat

what is the decolorizer in the endospore stain

water

what is the primary stain in the endospore stain

malachite green

what's the counterstain in the endospore stain

safranin

Know the results (color) and be able to explain the results for the endospore stain

result of the endospore stain is spores appear green and the vegetative cells appear red