Quiz 2

Question 1

gram stain stains

Answer 1

• Primary stain: crystal violet
• Mordant: Gram’s iodine
• Decolorizing agent: ethanol (or isopropanol)
• Counter-stain: safranin

Question 2

gram +

Answer 2

thick peptidoglycan layer of cell wall, stains purple by crystal violet

Question 3

gram -

Answer 3

thin peptidoglycan layer, stains pink from the safranin

Question 4

gram’s iodine

Answer 4

forms a complex with the peptidoglycan so that the stain sticks to the cell

Question 5

endospores purpose

Answer 5

• encapsulate genetic material (DNA) and essential cell
  components
• Enable bacteria to survive unfavorable environmental conditions
   Starvation
   Extreme heat/cold
   Desiccation
   Toxic chemicals/antibiotics/radiation

Question 6

endospore walls

Answer 6

Thick walled, dehydrated form of bacterial cell

Question 7

what happens to endospores once favorable conditions return

Answer 7

the endospores can germinate to
form vegetative cells (a regularly dividing cell)

Question 8

endospore-producing species

Answer 8

• clostridium
  
  • causes botulism (FBI paralyzes by interfering with nerve signaling)
• bacillus anthracis
  
  • causes antrax routes of
    entry include breathing in spores, ingestion, or skin contact

Question 9

endospore staining

Answer 9

primary stain: malachite green (15 mins) for endospores
counterstain: safranin for vegetative cells

Question 10

mycobacterium species

Answer 10

Special cell wall composition – presence of mycolic acids
- Immunoevasion – mycolic acid allows Mycobacterium to grow inside of macrophages (an inhospitable environment for most other microbes)
- Resistance to antibiotics
- Resistance to chemical damage, dehydration
- Virulence factor in Tuberculosis
- Waxy mycolic acid layer makes the mycobacteria slow growing and difficult to identify
- Mycobacteria can be recovered from clinical specimens through a
process called digestion, which releases the bacteria from fluids and cells, followed by decontamination to remove other normal microbes.
* no relationship to fungi

Question 11

what stain is used to identify mycobacterium species

Answer 11

acid-fast stain = KF (positive red) methylene blue (negative)

Question 12

why did we do a streak plate

Answer 12

to see different populations of bacteria after dilution

Question 13

T-streak plate steps

Answer 13

1. Pick up culture
2. Inoculate in Zone 1
3. Flame loop
4. Streak through Zone 1
   into Zone 2
5. Flame loop
6. Streak through Zone 2
   into Zone 3

Question 14

microbes related to hand washing

Answer 14

• Transient bacteria: contaminants; may be present for a finite time (weeks, months); may be pathogenic
• Resident bacteria: permanent residents of normal
  human microbiome; usually, not pathogenic
  
  • Otherwise known as your ”normal flora”

Question 15

common skin microbiota

Answer 15

• Corynebacterium diphtheriae
• Staphylococcus aureus
• Micrococcus luteus
• Staphylococcus epidermidis
• Pityrosporum ovale

Question 16

micropipettors

Answer 16

P1000 and P200

Question 17

P1000

Answer 17

• Used for volumes between 200-1000 μL
• Volume indicator should read: 0|9|0 (= 900 μL)
• Use larger blue tips

Question 18

P200

Answer 18

• Used for volumes between 20-200 μL
• Volume indicator should read 1|0|0 (= 100 μL)
• Use smaller yellow tips

Question 19

using a micropipette

Answer 19

1. Depress plunger to FIRST STOP before you place it into the sample
2. Slowly return the plunger to the up position to suck up the liquid (take care not to suck up any
   air – this will make your volume inaccurate!)
3. Release liquid by depressing plunger all the way
   down to the SECOND STOP
4. CHANGE TIPS AFTER EVERY TRANSFER!!!
   
   • Bacteria on inside of tip will skew dilution

Question 20

serial dilutions

Answer 20

• Method used to efficiently calculate the number of microbes in a
  sample
• Original sample is too concentrated to count directly (if we put it
  on a plate, a lawn of bacteria would grow: entire plate covered)
• Lower and lower concentrations of sample are made by serially
  diluting the sample
• Dilutions will thin out the lawn until we can see single colonies, which we
  can physically count
• Today’s Serial dilution = 1 part original solution + 9 parts
  diluent

Question 21

diluent

Answer 21

a fluid used to dilute the concentrated
sample (e.g. water)

Question 22

Aliquot

Answer 22

A smaller volume withdrawn from a total sample volume

Question 23

Dilution factor

Answer 23

• Fraction by which your original
  The sample concentration is diluted
• amount of bacteria divided by the total volume

Question 24

final dilution

Answer 24

product of dilution factors at every step in the series (If you make three consecutive 1/10 dilutions your final dilution is 1/10 x 3= 1/1,000)

Question 25

CFU

Answer 25

colony-forming units

Question 26

kilo

Answer 26

1000 = 10^3

Question 27

Base unit

Answer 27

(1) = 10^0

Question 28

milli

Answer 28

0.001 (1/1,000) = 10^-3

Question 29

micro

Answer 29

0.000001 (1/1,000,000) = 10^-6

Question 30

nano

Answer 30

0.00000001 (1/1,000,000,000) = 10^-9

Question 31

100 microliters =

Answer 31

0.1 mL

Question 32

CFU/mL calculation

Answer 32

1. find dilution factor 2. multiple dilution factor by # of times the procedure was done 3. Multiply that by the amount plated (in mL!!!!) 4. multiply amount plated by number of colonies