Lab 3

Question 1

What are the main differences between bacterial cells (prokaryotic cells) and eukaryotic cells?

Answer 1

Bacterial cells are much small and simpler, and they don’t have a nucleus or organelles

Question 2

What are the (3) categories of cell shapes?

Answer 2

1) Cocci, coccus(pl); 2) bacilli, bacillus (pl); 3) spirilla (spirillum (pl)

Question 3

Describe the cocci bacterial shape

Answer 3

Spherical or generally round shaped

Question 4

Describe the bacilli bacterial shape

Answer 4

Rod shaped

Question 5

Describe the spirilla bacterial shape

Answer 5

Curve shaped

Question 6

What are the (2) categories of variations in bacterial cell shapes?

Answer 6

1) Vibrios; 2) spirochetes

Question 7

What are vibrios?

Answer 7

Vibrios are an arc-shaped variation of bacilli bacteria

Question 8

What are spirochetes?

Answer 8

Spirochetes are a spiral shaped variation of spirilla bacteria

Question 9

What is the general dilution equation?

Answer 9

V1D1 = V2D2 [where V1 = volume of the first sample (or stock); V2 = final voume (diluent + V1); D1 = Dilution of the first sample (=1 if it’s the stock); D2 = final dilution]

Question 10

What is the mathematical representation of the original cell formula (OCD)?

Answer 10

OCD = CFU / (v x D) [where CFU = # or colony forming units; v = volume plated; D = total dilution]

Question 11

What is a chromogen?

Answer 11

Substance that can be readily converted into a dye or other colored compound

Question 12

What are the two parts of a chromogen?

Answer 12

1) Chromophore (color solute); 2) Auxochrome (charged solute)

Question 13

Describe the differences between positve and negative cell walls

Answer 13

Positive cell walls are very porous and allow the stain to readily leak into the plasma membrane; negative cell walls have more lipid which resists the leaking of stain into the plasma membrane (alcohol is required to help this process by extracting the lipid, therefore making the cell wall more porous - alcohol dehydration oppositely makes postive cell walls less porous, therefore trapping the dye in the cells)

Question 14

What are the two categories of stains?

Answer 14

1) Simple; 2) differential

Question 15

Which type of stain is a gram stain?

Answer 15

Differential

Question 16

What are the three basic stains used?

Answer 16

1) Crystal violet; 2) saffranin; 3) methylene blue

Question 17

What are we looking for when using a gram stain?

Answer 17

We are looking at the difference in cell walls, whether they are positive or negative

Question 18

What color will a positive cell wall turn in a gram stain?

Answer 18

Purple

Question 19

What color will a negative cell wall turn in a gram stain?

Answer 19

Pink

Question 20

What are the basic steps of a gram stain?

Answer 20

1) Prepare a smear of the sample; 2) stain the smear using the gram technique; 3) view under a microscope using oil immersion lens

Question 21

Describe the gram staining technique

Answer 21

1) Place drop of water on slide; 2) collect sample using aseptic technique; 3) spread and mix water and sample in a circular motion; 4) allow it to air dry completely; 5) heat fix it; 6) flood smear with crystal violet, let sit for 60-seconds, and then rinse with water; 7) flood smear with iodine, let sit for 60-seconds, and then rinse with water; 8) decolorize with alchol until the fluid runs clear (10-seconds), and then rinse with water; 9) counter stain with safranin, let sit for 60-seconds, and then rinse with water; 10) blot dry

Question 22

What are (5) categories of bacterial cell arrangement?

Answer 22

1) Diplo; 2) strepto; 3) tetrad; 4) sarcina; 5) staphylo

Question 23

Describe the diplo arrangement

Answer 23

Pair of cells

Question 24

Describe the strepto arrangement

Answer 24

Chain of cells

Question 25

Describe the tetrad arrangement

Answer 25

Group of 4 cells

Question 26

Describe the sarcina arrangement

Answer 26

Group of 8 cells

Question 27

Describe the staphylo arrangement

Answer 27

Irregular cluser of cells

Question 28

What is the purpose of staining cells?

Answer 28

Cells are stained to make them more visible under the microscope; the cytoplasm in cells is transparent and other organelles are dffcult to see

Question 29

What three cell properties does staining help determine?

Answer 29

1) Cell morphology; 2) size; 3) arrangement

Question 30

What is a solution?

Answer 30

Homogenous mixture of two or more substances (may be in solid, liquid, or gas form)

Question 31

What is a solvent?

Answer 31

A substance, usually a liquid, capable of dissolving another substance

Question 32

What is a solute?

Answer 32

A substance dissolved in another substance

Question 33

Why does staining work (from a chemical standpoint)?

Answer 33

Because stains have a postively charged chromophore which forms an ionic bond with the negatively charged bacterial cell

Question 34

What is a mordant?

Answer 34

It is a preservative

Question 35

What cell arrangements can cocci display?

Answer 35

All

Question 36

What cell arrangements can bacilli display?

Answer 36

Strepto (most typical), diplo, staphylo

Question 37

What cell arrangements can spririlli display?

Answer 37

Single (although staphylo can be found with heavy handed technique)

Question 38

Procedure for preparing smear

Answer 38

1) Drop of water on slide; 2) collect sample using aseptic technique; 3) spread and mix with water in circular motion (about the size of quarter); 4) allow to air dry completely; 5) heat fix

Question 39

What makes a good smear?

Answer 39

1) Appropriate thickness to view individual cells (not heavy handed); 2) can withstand repeated washing during staining; 3) will retain original cell morphology after fixation and staining

Question 40

Differences between simple and differential stains

Answer 40

SIMPLE: only 1 color, and only shows morphology, arrangement, and size; DIFFERENTIAL: contrasting colors, and also indicates type of cell wall

Question 41

What role does alcohol play in the gram staining process?

Answer 41

Decolorizer

Question 42

Why are bacterial cells (or any cells) easily colored by basic stains?

Answer 42

Because they have negative (-) cell walls and the stain has a positive (+) charge