LAB - Colonial & Gram Morphology, Culture Media

Question 1

where we can see a visual difference between different species

Answer 1

differential medium

Question 2

Inhibitory reagents such as dyes, chemicals or antimicrobials are added to make the medium …

Answer 2

selective

Question 3

This can be added for anaerobic medium

Answer 3

A reducing agent (e.g. cysteine or thioglycollate)

Question 4

“nutrient” broth

Answer 4

a simple fluid media designed to support the growth of most bacteria
with non-fastidious growth requirements. The constituents of the broth are peptone, beef extract, sodium chloride and distilled water

Question 5

what makes a nutrient broth a solid medium?

Answer 5

the addition of a complex polysaccharide AGAR

- extracted from seaweed, acting as a source of calcium, but it is not utilized by most bacteria

Question 6

BAP

Answer 6

• enriched medium, blood agar is prepared by adding sterile, defibrinated sheep blood to a sterile, molten nutrient agar base (TSB) cooled to approximately 50°C
• also considered a non-selective media,
• as well as differential, as it is used to differentiate different types of hemolysis
• to maximize recovery of fastidious bacteria, blood agar bases can be enriched by the addition of yeast extract
• vitamin K, hemin and cysteine (reducing agent) supplements may be added for isolation of strict anaerobes

Question 7

CHOC

Answer 7

• chocolate agar
• BAP that has been heated before plates are poured (BROWN)
• enriched & non-selective
• used for culture of fastidious organisms (such as Haemophilus & gonococci)
• growth is enhanced if supplemented w yeast extract (V factor + X factor); sheep blood inactivates NAD - will not support growth of Haemophilus

Question 8

Alternative method of supplying NAD on unheated blood agar

Answer 8

cross-streaking inoculated cultures with Staphylococcus aureus (“Staph. streak”)
- staphylococci lyse RBCs (beta hemolysis) and release NAD into the medium, allowing the Haemophilus to grow as “satellitic” colonies in the area adjacent to the staphylococci

Question 9

Colisin-Nalidixic Acid Agar (CNA)

Answer 9

selective medium used for the recovery of aerobic
and anaerobic Gram positive bacteria
- basically a BAP with antibiotics colisin and nalidixic acid, which inhibit Gram-negative bacteria

Question 10

MAC

Answer 10

• selective-differential medium; selective as it inhibits growth of gram-positive bacteria but allows growth of the Enterobacteriaceae and the non-fermentative gram-negative rods
• differentiates between “lactose fermenters” (pink colonies) and “non-lactose fermenters” (white or colorless colonies)

Question 11

this media exist for the selective and differential isolation of specific bacteria and yeasts

Answer 11

Chromogenic media

Question 12

grows best at cold temperatures (10-20°C)

Answer 12

psychrophile

Question 13

mesophile

Answer 13

– grows best at moderate temperatures (20-40°C)

Question 14

grows best at high temperatures (50-60°C)

Answer 14

thermophile

Question 15

obligate aerobes

Answer 15

require oxygen (at least 18% O2)

Question 16

grows with or without oxygen

Answer 16

facultative anaerobes

Question 17

capnophilic

Answer 17

grows better when atmosphere enriched with CO2 (5-10%, CO2, ~18% O2)

Question 18

require a reduced level of oxygen (5-6% O2)

Answer 18

microaerophilic

Question 19

humidophilic

Answer 19

require increased humidity (70-80%)

Question 20

Incomplete haemolysis of red blood cells with partial breakdown of haemoglobin.

Answer 20

alpha-hemolysis

- visible as a zone of partial clearing with green discoloration surrounding colonies

Question 21

beta-hemolysis

Answer 21

Complete lysis of RBC with complete breakdown of haemoglobin. There are distinct, totally clear zones around colonies

Question 22

There is no zone of hemolysis surrounding colonies, i.e., the bacteria are nonhemolytic. Normally this is not recorded as a significant finding.

Answer 22

gamma-hemolysis

Question 23

gv

Answer 23

gram variable

Question 24

pleo

Answer 24

pleomorphic

Question 25

basal nutrient media

Answer 25

designed to support growth of most organisms (non-fastidious growth requirements)

• various sugars + indicator added to make it differential
• inhibitory reagents such as dyes, chemicals, or antimicrobials added to make selective
• may be enriched with blood, serum, yeast for growth of fastidious bacteria
• reducing agent (eg. cysteine or thioglycollate) for anaerobic medium

Question 26

“nutrient” media

Answer 26

• designed to support growth of most organisms (non-fastidious)
• peptone, beef extract, sodium chloride, and distilled water
• addition of complex polysaccharide AGAR, it becomes solid or semi-solid (from seaweed); acts as a source of calcium but not used by most bacteria

Question 27

Sheep Blood Agar (BAP)

Answer 27

• enriched medium
• sterile, defibrinated sheep blood to a sterile, molten nutrient agar base (TSB) cooled to approximately 50 C (5% v/v)
• non-selective media, as well as differential = differentiate different types of hemolysis
• maximize recovery of fastidious bacteria = add yeast extract
• reducing agents: vit K, hemin, and cysteine to isolate strict anaerobes

Question 28

Chocolate agar (CA/CHOC/CAP)

Answer 28

• BAP that has been heated before plates are poured
• enriched and non-selective
• used for culture of fastidious organisms (Haemophilus and gonococci)
• growth enhanced w yeast
• X factor (hemin) and V factor (NAD)
• sheep enzyme has an enzyme that inactivares NAD so unheated sheep blood will not support the growth of H. influenzae (requires NAD)

Question 29

Alternative method of supplying NAD on unheated blood agar

Answer 29

is by cross-streaking inoculated cultures with S. aureus (“staph streak”
- staph lyse RBCs = beta
and release NAD into medium
= Haemophilus grow as satellitic colonies in area adjacent to staph

Question 30

these media can include antibiotics and can be used to isolate drug-resistant bacteria

Answer 30

Chromogenic media

- MRSA, VRE, etc.

Question 31

key features of bacterial colonies that serve as important criteria for identification

Answer 31

• size
• shape
• elevation
• pigmentation
• surface texture
• opacity

Question 32

these are used to detect single colonies from mixed cultures in order to obtain pure cultures

Answer 32

sterile wires

NOT loops

Question 33

colonies are examined for these characteristics

Answer 33

• surrounding zone of hemolysis

- surface morphology including colour, opacity, and size