lab 4-6

Question 1

staining

Answer 1

enhances contrast & resolution; reveals details
of cellular morphology & arrangement

Question 2

basic stain (crystal violet)

Answer 2

• negative stain
• cell is stained
• positively charged attracts negative charged cells

Question 3

acidic stain (nigrosin)

Answer 3

• simple stain (only one staining reagent)
• background is stained
• negatively charges

Question 4

negative stain

Answer 4

• Determine morphology &
  cellular arrangement (shapes and size)
  – Typically for cells sensitive
  to heat‐fixation during
  smear preparation
  – Minimal distortion of cells
  (no heat‐fixation involved)

Question 5

differential staining

Answer 5

• use more than one staining reagent to differentiate cellular structures
• allows for detection of differences between bacteria and serves as an aid in identifying bacteria.
• gram, acid-fast, spore, and capsule stain
• primary stain, counterstain, and often (though not always) a decolorizing agent.

Question 6

gram stain

Answer 6

• most common differential stain; differentiates many bacterial types on basis of cell wall differences
• initial step in identification of unknown bacteria.

Question 7

Gram positive cell

Answer 7

• retention of primary stain and resists decolorization
• thicker peptidoglycan and cross linkage

Question 8

gram negative cell

Answer 8

• loss of primary stain from decolorization with alcohol
• less peptidoglycan, higher lipid content

Question 9

endospores

Answer 9

• dormant form of a bacterium that is formed under conditions of nutrient starvation and/or other physical, metabolic stresses.
• highly resistant to heat, desiccation, and radiation
• can be visualized without staining the spore

Question 10

two groups of spore-formers

Answer 10

1.) bacillus = soil microbes, aerobic, facultative anaerobes
2.) clostridium = anaerobic

Question 10

spore stain

Answer 10

• uses heat to allow the dye (malachite green) to penetrate
  the spore; safranin is used as a counterstain. Microscopy reveals green‐colored spores and red vegetative cells.

Question 10

spore gram/simple stain

Answer 10

coloration of vegetative portion of the cell, while the spores appear colorless

1.) heat-fixed emulsion (up to 4)
2.) cover smear with paper and apply malachite green stain
3.) steam for 5 minutes and rinse with water
4.) counterstain with safarin for 1 minute and rinse with water

Question 11

acid fast stain (kinyoun method)

Answer 11

• has mycolic acids (lipids in cell wall)
• carbolfuchsin solution (the primary stain), whose chemical nature allows it to penetrate the waxy acid‐fast cell wall mycolic acids; acid‐fast bacteria resist decolorization by acid‐alcohol.
• mycobacterium

Question 12

non-acid fast

Answer 12

bacteria must be counterstained (with brilliant green) in order to be seen.

Question 13

capsules

Answer 13

allows bacteria to avoid host defenses

Question 14

capsule stain

Answer 14

• combination of a negative stain and simple stain; a
  heat‐fixed smear IS NOT prepared.
  1.) add congo red stain to slide and add organisms
  2.) push the spreader slide to the other end
  3.) air dry
  4.) add maneval stain for 1 minute and rinse with water

Question 15

selective media

Answer 15

contains chemical components that inhibit certain bacteria,
favors the growth of others

Question 16

differential media

Answer 16

can visualize metabolic differences between bacterial types.

Question 17

PEA

Answer 17

• ONLY selective to cultivate gram positive organisms
• phenylethyl alcohol reduces growth of gram negative organisms

Question 18

MSA

Answer 18

• differential and selective
• differentiates mannitol fermenters (yellow) and non-fermenters
• high salt favors staphylococcus sp.

Question 19

CNA with sheep blood

Answer 19

• selective for just CNA, but selective and differential with sheep blood
• promotes isolation of gram-positive bacteria by suppressing growth of gram-negative with COLISITIN and NALIDIXIC ACID

Question 20

MA (macconkey agar)

Answer 20

• differential and selective
• inhibits gram positives (via bile salts); more gram negatives
• distinguishes between lactose (purple) & non‐lactose (colorless) fermenters.

Question 21

EMB (Eosin methylene blue agar)

Answer 21

• differential and selective
• dyes eosin and methyl‐ene blue inhibit growth of Gram-positive organisms.
• lactose fermenters=purple
• weak lactose fermenters=pinkish-purple
• non-lactose fermenters=colorless
• strong acid= metallic green sheen (e. coli)

Question 22

HEK (hektoen enteric agar)

Answer 22

• selective and differential
• inhibitors prevent growth of gram positive bacteria
• lactose fermentation and H2S production aids in identification of organism
• salmonella=black colonies
• shigella=translucent green colonies
  - both are non-lactose fermenters
• lactose fermenters (coliforms)=yellow colonies

Question 23

nutrient agar

Answer 23

• used as a control as it supports the growth of various types of bacteria and contains many nutrients necessary for growth