Lab Exam I

Question 1

Definition of Blood Agar Plate

Answer 1

Differential medium (RBC from sheep) is used to observe hemolytic activity of bacteria. Most commonly used to differentiate Streptococcus pneumoniae (alpha hemolysis) and Streptococcus pyogenes (beta hemolysis).

Question 2

alpha, beta, and gamma hemolysis

Answer 2

alpha hemolysis - partial lysis, brown or green
beta hemolysis - complete lysis, clearing
gamma hemolysis - no hemolysis, no change

Question 3

Procedure for smear preparation

Answer 3

1. Place small drop of water on slide.
2. Spread small amount of culture.
3. Air dry.
4. Heat fix by passing slide over flame 3 times.
5. Circle smear with wax pencil.

Question 4

Purposes of Quadrant Streak Plate Technique

Answer 4

• obtain pure culture
• determine if culture was pure or mixed
• determine colony morphology

Question 5

What is the purpose of streaking a plate in 4 quadrants?

Answer 5

Streaking the plate in 4 quadrants dilutes the culture, leaving bacteria further and further apart. Isolated colonies arise from a single cell.

Question 6

Label of each plate

Answer 6

1. Name
2. Specimen ID
3. Medium
4. Incubation conditions
5. Date

Question 7

Terms used to describe colony morphology

Answer 7

• Shape (circular, irregular)
• Surface (dry, moist, rough, wrinkled)
• Margin (smooth, wavy, lobed, branched)
• Elevation (flat, raised, convex, raised in center)
• Opacity (transparent, opaque, transluscent, iridescent)
• Pigmentation (white, tan, cream red, yellow)
• Other
  
  • agar color change
  • isolated colonies, swarmer

Question 8

Purpose of TSA Slant

Answer 8

Used to grow and maintain culture. Not for isolating colonies. Advantages of Slants include:

• easier to maintain sterility
• less desiccation for long-term storage
• less media used

Question 9

Definition of Gram stain

Answer 9

Used to determine if bacteria is Gram positive (purple) or Gram negative (pink)

Question 10

Procedure for Gram staining

Answer 10

• Prepare smear
• Soak in Crystal Violet for 1 minute; rinse with water
• Soak in Gram Iodine for 1 minute; rinse with water
• Decolorize with EtOH until runs clear; rinse with water
• Soak in Safranin for 1-3 minutes; rinse with water

Question 11

Define Kirby Bauer/Antibiotic Sensitivity Test

Answer 11

Purpose is to demonstrate the difference between broad and narrow spectrum antibiotics, using Gram-negative bacterium (E. coli) and Gram-positive bacterium (Staphylococcus epidermidis).

Question 12

Describe how Kirby Bauer Test is interpreted

Answer 12

• Zone of inhibition around each antibiotic disk measured in mm and compared against chart of interpretive standards.
• Broad spectrum antibiotics will be effective against both Gram-positive and Gram-negative bacteria.
• Narrow spectrum antibiotics will be effective only against a subset of bacteria.

Question 13

Define MacConkey Agar (MAC)

Answer 13

A Selective and Differential medium used to detect and differentiate Gram-negative enteric bacteria based on ability to ferment lactose. E. coli is lactose positive. Salmonella, Shigella are lactose negative.

Question 14

Selective component of MAC

Answer 14

• Crystal violet selects for Gram-positive

- Bile salts select for enteric

Question 15

Differential component of MAC and indicator

Answer 15

• Lactose

- Neutral red pH indicator turns red when pH <6.8, remains colorless when pH >6.8

Question 16

Define Mannitol Salt Agar (MSA)

Answer 16

A Selective and Differential medium used for isolation and differentiation of pathogenic Staphylococci, principally Staph. epidermidis and Staph. aureus.

Question 17

Selective component of MSA

Answer 17

• 7.5% NaCl selects for Staphylococci capable of growing in high salt concentration

Question 18

Differential component of MSA

Answer 18

• Mannitol
• Phenol red
  
  • pH < 6.8 yellow
  • pH 7.4-8.4 red
  • pH > 8.4 pink
• Staph. aureus (pathogenic) capable of fermenting mannitol, producing acid, which turns the pH indicator yellow.

Question 19

Define Catalase Test

Answer 19

• Detects the presence of catalase, and enzyme found in aerobic and facultative anaerobic bacteria.
• Superoxide dismutase converts superoxide radicals into H2O2 and O2. Catalase converts H202 into H2O and O2.
• Differentiate between Staphylococcus (catalase positive) and Streptococcus (catalase negative).

Question 20

Define KOH Test

Answer 20

A nonstain alternative to the Gram stain test. If Gram-negative bacteria is mixed with KOH solution, the KOH crosses the thin cell wall and lyses the cells. Chromosomal material is released into the emulsion. The DNA unwinds into a viscous, stringy material. Gram positive bacteria remain unchanged.

Question 21

Define Snyder Agar

Answer 21

Selective and differential agar typically used to determine the relative risk of an individual developing dental caries (due to Lactobacilli). Agar is green.

Question 22

Selective component of Snyder Agar

Answer 22

pH 4.8

Question 23

Differential component of Snyder Agar

Answer 23

• Glucose
• Lactobacillus ferments glucose to produce lactic acid.
• The green agar turns the agar yellow faster in the presence of lactobacilli.

Question 24

Procedure for conducting Snyder Agar Test

Answer 24

1. Collect saliva in small cup.
2. Obtain molten Snyder agar from hot water bath. Do not allow to let cool.
3. Pipette o.2mL of saliva into tube.
4. Vortex tube.
5. Incubate at 37C
   yellow in 24 hours –> high susceptibility to dental caries
   yellow in 48 hours –> moderate susceptibility
   yellow in 72 hours –> slight susceptibility

Question 25

Define Thioglycolate Broth

Answer 25

Used to determine oxygen requirement for bacteria. An indicator is added to detect presence of free oxygen. Observing location of growth in the column will indicate oxygen requirement.