Lectures 9, 10

Question 1

How oxygen helpful and toxic?

3 reactions L9S3?

Answer 1

Oxygen needed to breathe
Oxygen generates free radicals that damage DNA, proteins, and lipids
Aerobically living organisms produce series of enzymes that detoxify fee radicals

Superoxide dismutase
Catalase
Peroxidase
L9S3

Question 2

What are the 5 test tubes for obligate Aerobes, facultative anaerobes, obligate anaerobes, aerotolerant anaerobes, microaerophiles?

Answer 2

L9S4
Obligate aerobe- needs oxygen to survive
Facultative anaearobe- can grow with or without oxygen but abundance of growth at top of oxygen
Obligate anaerobe- can’t live in oxygen
Aerotolerant anaerobe- growth throughout (oxygen doesn’t affect growth)
Microaerophile- mid range growth

Question 3

What is complex media and defined media?

Answer 3

Complex media- we don’t know every chemical in the media
Partially digested beef
Ground up dried yeast

Defined Media- every chemical in the media is known and quantified
Sugars, amino acids

Question 4

What are broths, plates, slants, and semi-solid deeps?

Pros and cons

Answer 4

Broths- grow large quantities of culture, difficult to check for purity
If it gets contaminated it can spread everywhere
Plate- readily score and separate colonies
Does not store well
Can separate colonies large surface area
Slants- mini plates for storage with smaller volume, difficult to check for purity
Semi-solid deeps- can score motility and oxygen requirements, also storage, difficult to check for purity
Less solidifying agent not fully solid or liquid

Question 5

What are the 3 playing methods?

Answer 5

Lawn
Single colonies
Growth
L9S9

Question 6

What is agar?

Answer 6

Carbohydrate from the cell walls of red algae
Melts at 85 Celsius solid to liquid
Solidifies at 40 Celsius liquid to solid
L9S10

Question 7

What is selective media, differential media, enrichment media?

Answer 7

Selective media- encourage the growth of some bacteria while preventing the growth of others (some things grow some don’t)
Differential media- produce different visual cues as to the type of organism growing (both grow just produce different effects)
Enrichment media- type of selective media used to allow proliferation or rare bacteria in a mixed population

Question 8

What is the MacConkey plate?

Answer 8

MacConkey Agar- selective and differential
Selects against gram + using bile salts and crystal violet (not for gram staining)
Contains lactose and pH indicators (fermenters turn purple, non fermenters stay creamy, acid diffusion colours plate)
Fermenters break down lactose
L9S13

Question 9

What is the Mannitol Salt Agar (MSA) plate?

Answer 9

Selective and differential
High salt ~10%
Contains mannitol (a sugar alcohol) that turns fermenters yellow and non fermenters red

Need halophiles bacteria to grow cause of high salt content

L9S14

Question 10

What is blood agar plates (BAP)?

What is alpha, beta, and gamma?

Answer 10

Enrichment and differential
5-10% sheep blood
Test for hemolytic activity (cells which can burst red blood cells, releases iron that the bacterial cells need)

Hemolysis may be enhanced in low oxygen environments

Alpha is discolouration in or under colony
Beta is clearing
Gamma is no-hemolysis
L9S15

Question 11

What is Saboraud Dextrose Agar (SAD)?

Answer 11

Similar to nutrient agar but with a high amount of glucose (2%)
Sugar and acidic pH selects for yeasts and molds
Yeasts and molds that can grow at higher sugar concentrations will be on this plate
L9S16

Question 12

What is the SIM Deep diagnostic tubes?

3 things they check for?

Answer 12

Differential x3

1. Sulfur reduction- of H2S is produced it will react with ammonium iron sulfate and turn black
2. Indole production- if tryptophan is metabolized it will produce indole, indole is detected by dropping Kovaks reagent on top after incubation (red=positive)
3. Motility- If cells grow away from stab they are motile

Question 13

What is the oxidase test?

Answer 13

Test for cytochrome 3 oxidase
- can the organism use oxygen to generate ATP?
Disks or strips are wetted, a colony is applied, and colour change is observed over a few minutes
Blue/purple- +
No change- -

Oxidase positive= reagent + cytochrome C = purple

Question 14

What is a dichotomous key?

Answer 14

Series of yes or no observations to quickly narrow the field of bacterial species to identify an unknown
Starts general then becomes more specific

Slide 4 example

Question 15

How do you perform several dichotomous experiments at once?

Answer 15

Use an enterotube II for oxidase (-) gram (-) bacilli (need to know those three things first)
Used to easily identify medically important bacteria by doing several metabolic tests at one time
Slide 5

Question 16

What are two additional identification methods? (DNA/antibody)

Answer 16

DNA testing- extract bacterial DNA and test using various procedures
Antibody testing- assaying if specific antibodies bind unknown cells to determine identity

Question 17

What is logarithmic growth in bacteria?

What are the 4 phases of a growth curve?

Answer 17

Exponential growth of bacteria makes a growth curve we must approach with log

Bacteria can divide from under an hour to days
Rapid division can cause growth curve
Slide 8

Lag phase
Log, or exponential growth, phase
Stationary phase
Death, or logarithmic decline, phase
Slide 9

Question 18

What are turbidity estimates?

Answer 18

Slide 11

Measuring light blockage by bacteria with a spectrophotometer

Question 19

What’s serial dilution counting?

Answer 19

Slides 12-13
Start with colony count
Divide by the volume plated to give cfu/mL of tube used
Multiply by volume of tube to get total cfu in tube
Divide by volume placed in that tube from the previous to give cfu/mL of previous tube

Question 20

What is the counting chamber?

Answer 20

Counts cells but not live cells
Use grid height width and depth and use cm to find volume
Slide 3 Lecture 11