Microbial Growth & Methods of Measuring Growth

Question 1

List

Kinds of Cell Division

3

Answer 1

1. Binary fission
2. Budding
3. Biofilm

Question 2

List

Examples of fast growers & their generation times

3

Answer 2

1. E. coli (15-18 mins)
2. S. aureus (27-30 mins)
3. Bacillus subtilis (26 mins)

Question 3

List

Examples of slow growers & their generation times

2

Answer 3

1. Leptospira (6-8 hr)
2. Mycobacterium (18-24 hr)

Question 4

Differentiate

Closed Culture System vs Open Culture System

Answer 4

Closed
* fixed amount of nutrient at the start
* leave for 18-24 hours
* no way to get rid of excess waste

Open
* keep adding fresh medium
* remove waste/excess
* mix for aeration

Question 5

Identify

What is used to detect turbidity?

Answer 5

spectrophotometer

Question 6

Define

viable count

Bonus: Where can you find it?

Answer 6

• count of live cells
• found in plates w/ media

Question 7

List

Growth Phases

4

Answer 7

1. Lag Phase
2. Logarithmic/Exponential Phase
3. Stationary Phase
4. Death Phase

Question 8

Identify

Plots different stages of (bacterial) life

Answer 8

Bacterial Growth Curve

Question 9

phase with metabolic activity, but no growth

Answer 9

Lag Phase

Question 10

List

What is the duration of the Lag Phase dependent on?

2

Answer 10

1. Nutrients available
2. Species

Question 11

• phase where there is a lot of growth
• ↑ cell population, ↑ rate
• generation time of organism can be determined

Answer 11

Logarithmic/Exponential Phase

Question 12

Fill in the blank

To stay in the Logarithmic Phase, use an ____ culture system. This system is also called ____.

Answer 12

open, chemostat

Question 13

Main components of chemostat

3

Answer 13

1. inflow of new medium & air
2. mixing of medium (aeration)
3. outflow of waste

Question 14

Factors that govern cell density

2

Answer 14

1. Dilution rate
2. Concentration of limiting nutrient

Question 15

Why would you want to stay in the Logarithmic Phase?

Answer 15

For extraction purposes; you keep producing more cells & can extract more in a shorter amt of time

Question 16

Phase where

• Replication Rate = Death Rate
• Limited Nutrients
• ↑ Microbial waste

Answer 16

Stationary Phase

Question 17

During the stationary phase, the environment becomes ____, with a [higher/lower] pH and a [higher/lower] oxygen level.

Answer 17

toxic, higher, lower

Question 18

Phase with

• little to no nutrients
• ↑ toxic waste
• Endospores form

Answer 18

Death Phase

Question 19

What happens to cells during the death phase?
Why do they do that?

Answer 19

Involution: they change shape

• ex. rod → spherical
• to limit cell’s nutrient use

Question 20

Why do endospores form during the Death Phase?

Answer 20

Endospores are how cells defend themselves in a toxic environment.

Question 21

Identify

• process that helps reduce bacterial load
• series of tubes with a diluent

Answer 21

Serial Dilution

Question 22

List

Methods of Measuring Bacterial Growth

7

Answer 22

1. Serial Dilutions
2. Viable Cell Count
3. Direct Cell Count
4. Most Probable Number Technique
5. Most Probable Count
6. Filtration
7. Spectophotomoetry

Question 23

Why do we do serial dilation?

Answer 23

If you don’t, your sample might have too many colonies –> won’t be visible when trying to count them

Question 24

Kinds of diluent

Answer 24

1. Normal Saline Solution (NSS): 0.85% saline solution
2. Phosphate Buffer Solution (PBS)

Question 25

What happens when using a diluent & how do you use it?

Answer 25

* Use it within 10-15 mins * **Non-nutritious**: cells don't grow, but they also don't die

Question 26

# Explain Serial Dilution Process

Answer 26

1. E. coli (1 mL) in Test Tube 1 (9 mL diluent) 2. Vortex (mix) 3. Transfer 1 mL TT1 in TT 2 with 9 mL diluent 4. Vortex 5. Transfer 1 mL TT 2 in TT 2 with 9 mL diluent 6. Repeat until 30-300 colonies 7. Plate counting

Question 27

Why should the colony count be 30-300 when plate counting? What do you declare when the colony count is outside that range?

Answer 27

[< 30] - TFTC (Too Few to Count) * statistically insignificant [> 300] - TNTC (Too Numerous to Count) * won't see individual colonies, too many layers

Question 28

# Fill in the Blank The Most Probable Number Technique is used for samples with a _[high/low]_ amount of bacteria

Answer 28

low

Question 29

# Identify when a microorganism thrives in environments with small amounts of oxygen

Answer 29

microaerophilic

Question 30

# Differentiate Spread-plate method vs Pour-plate method

Answer 30

Spread-Plate * sample is spread on agar → surface colonies Pour-plate * sample is added first., then medium is added & mixed → colonies grow in agar & on top of agar

Question 31

where sample is placed for direct cell count

Answer 31

Petroff-Hauser counting chamber / haemocytometer

Question 32

# Explain The Great Plate Count Anomaly

Answer 32

* **no. from direct cell counts > no. from viable plate counts** * Direct cell count: counts both live and dead cells * Viable plate count: “forgets” non-culturable cells (impossible for all kinds of cells to grow in 1 specific condition)

Question 33

What are solutions to the Great Plate Count Anomaly?

Answer 33

1. Use more more selective/general medium (to target less organisms) 2. Vary culture conditions 3. Alter experiment

Question 34

Technique usually used on water (bottled water), food, air samples

Answer 34

Most Probable Number Technique

Question 35

What are the bases for positive reults in MPNT? | 2

Answer 35

1. turbidity 2. gas production

Question 36

Describe the setup used in the Most Probable Number Technique.

Answer 36

1) x sets of test tubes, with x tubes per set * Set 1: 10 mL of sample * Set 2: 1 mL of sample * Set 3: 0.1 mL of sample 2) Incubate tubes (37ºC, 24 hr) 3) Compare the number of positive test tubes with what is on MPN table

Question 37

# T/F [Bonus: If false, what is wrong?] MPN Technique is usually part of a bigger experiment.

Answer 37

T

Question 38

What are phases of the "bigger experiment" that uses the MPN Technique? Why do we do each phase?

Answer 38

1. Presumptive Phase - to observe gas production 2. Confirmatory Phase - to confirm whether or not the colonies are fecal and/or harmful 3. Completed Phase - identifies actual organism

Question 39

What other item is used in the presumptive phase, and why do we use it?

Answer 39

Durham tube * hard to observe gas production without it * a bubble can be found inside it

Question 40

Incubation Conditions for Presumptive phase & Confirmatory Phase

Answer 40

Presumptive: 37℃ for 24 hours Confirmatory: 45 ℃ for 24 hours

Question 41

# T/F Getting a negative result in the Presumptive Phase requires you to proceed to the Confirmatory Phase

Answer 41

F | you are not required

Question 42

How long does the Confirmatory Phase take? What kind of results do you need to proceed to Completed Phase?

Answer 42

2 weeks positive tubes (in terms of turbidity)

Question 43

What broth/agar is used in each phase of the MPN Technique?

Answer 43

1. Lauryl Sulfate Tryptone Broth (LSTB) - Presumptive phase 2. Brilliant Green Lactose Bile Broth (BGLB) - Confirmatory Phase 3. Eosine Methyl Blue (EMB) Agar - Completed Phase

Question 44

What is the size of the filter in a membrane filter? Why do we use that size

Answer 44

0.5 micrometers, so that the target organisms pass through the filter

Question 45

# Explain Filtration Method

Answer 45

1. Use membrane filter 2. Place filter on agar 3. Leave for 24 hours

Question 46

measures amount of light unscattered aka light absorbed

Answer 46

Optical Density (OD)

Question 47

# Fill in the blank [↑/↓] light absorbed = [↑/↓] OD | Spectrophotometry

Answer 47

↑ light absorbed = ↑ OD

Question 48

[↑/↓] turbidity = [↑/↓] OD

Answer 48

↑ turbidity = ↑ OD

Question 49

What are the advantages & disadvantages of Spectrophotometry?

Answer 49

Advantage: fast (~10 mins) Disadvantage: counts dead cells