LAB 2 - COLONIAL AND GRAM MORPHOLOGY, CULTURE MEDIA

Question 1

A plate is growing white, opaque colonies, non-hemolytic on a BAP. The sample was taken from
a patient with a UTI. What organism is of most concern? Are there any additional tests that
must be done? If so, what are they and what are the expected results?
a. What would you report if it is the organism of concern?

Answer 1

• Staphylococcus saprophyticus
• Gram stain (g+c, clps) → catalase (+) → coagulase (-) → DNAse → (-) novobiocin (R)

Question 2

What makes a media differential? What makes a media selective? What makes a media
enriched?

Answer 2

Selective media: Inhibitory reagents (dyes, chemicals, antimicrobials) inhibits growth of certain bacteria

Enriched media: Serum, blood, yeast added to grow fastidious bacteria

Question 3

BAP:
1. Components
(include inhibitory
agents, indicators,
etc.)
2. Function of
major
components
3. Type of media (i.e..
Selective,
differential,
enriched, etc.)
4. Examples of
organisms that
grow on this
media

Answer 3

1. Defibrinated sheep blood (5%) on TSB agar
2. Bacterial growth
3. Enriched, non-selective, differential (hemolysis)
4. Staphylococcus, streptococcus, almost everything…

TSB - Tryptic Soy Broth

Question 4

CAP:
1. Components
(include inhibitory
agents, indicators,
etc.)
2. Function of
major
components
3. Type of media (i.e..
Selective,
differential,
enriched, etc.)
4. Examples of
organisms that
grow on this
media

Answer 4

1. Heated sheep blood (5%) on TSB agar, yeast extract, hemin (X factor), NAD (V factor)
2. Growth of fastidious bacteria; heated BAP destroys NADase and releases hemoglobin for growth
3. Enriched, non-selective, differential (hemolysis)
4. Haemophilus influenzae

TSB - Tryptic Soy Broth
NAD - Nicotinamide adenine dinucleotide

Question 5

Colisin-Nalidixic Acid
Agar (CNA):
1. Components
(include inhibitory
agents, indicators,
etc.)
2. Function of
major
components
3. Type of media (i.e..
Selective,
differential,
enriched, etc.)
4. Examples of
organisms that
grow on this
media

Answer 5

1. Antibiotics (colisin+nalidixic acid), dyes chemical inhibitors added to nutrient/ enriched media
2. Inhibits growth of normal flora
3. Selective
4. Gram positive bacteria

Question 6

MAC:
1. Components
(include inhibitory
agents, indicators,
etc.)
2. Function of
major
components
3. Type of media (i.e..
Selective,
differential,
enriched, etc.)
4. Examples of
organisms that
grow on this
media

Answer 6

1-2. Peptone, proteone peptone, NaCl, lactose (substrate), neutral red (indicator), crystal violet (inhibits gram+ve), bile salts
3.
- Selective: inhibits growth of gram+ve bacteria; allows growth for Enterobacteriaceae and non-fermentative bacilli
- Differential: lactose fermenters (pk) from non-lactose fermenters (wh)
4. Enterobacteriaceae

Question 7

What is an advantage of using MAC for Proteus sp.?

Answer 7

Inhibits Proteus sp. from spreading over the plate and other colonies

Question 8

Explain alpha, beta and gamma hemolysis? What is happening to the red cells?

Answer 8

Alpha: incomplete hemolysis with partial breakdown of hemoglobin; partial clearing with green discoloration around colonies

Beta: complete lysis of RBC and breakdown of hemoglobin; distinct clearing around colonies

Gamma: no hemolysis

Question 9

Why is it good practice to avoid streaking too close to the edge of a plate?

Answer 9

There are contaminants near the edge of the plate