Ch. 9 -Microbial Growth

What is binary fission?

• Asexual reproduction; common mechanism for bacteria to divide

Where does DNA replication in binary fission begin?

• Ori: origin of replication

What does the second part of binary fission involve?

• Formation of division septum: cleavage furrow

What does the last part of binary fission involve?

• Cell separation; may remain in pear shape

What protein orchestrates DNA replication in binary fission?

• FtsZ

What is doubling time?

• Amount of time it takes for cell to divide (time for population to double)

What formula is used to calculate the number of cells over multiple generations?

• Nn = N0 × 2^n

Start with 1 cell, how many cells after 3 generations?

• 1 × 2³ = 8 cells

What are the 4 phases of the growth curve?

1. Lag phase

2. Log phase

3. Stationary phase

4. Death or decline phase

What occurs in the lag phase?

1. No increase in number of bacteria (bacteria assessing nutrients, if it can grow)

What occurs in the log (exponential) phase?

• Exponential increase in number of bacteria (fastest, consuming all nutrients)

What occurs in the stationary phase?

• Plateau phase in number of bacteria (rate of cell division = death)

nutrients limited; begin ot run out

What occurs in death or decline phase?

• Exponential decrease in number of bacteria

What are persistors?

• Survivors of death phase; with slow metabolic rate (mycolic acid)

increase antibiotic resistance

What is a chemostat?

• Equipment that brings in new media and removes waste

What two methods can you use to measure bacterial growth?

1. Direct cell count

2. Indirect cell count

For a direct cell count, what must be done?

• Must dilute orginal sample

What are the 3 types of direct cell counts?

1. Petroff- Hausser chamber

2. Coulter counter

3. Viable plate count

What must be done in a Petroff Hausser chamber? Can you tell if bacteria is alive or dead?

• Place a drop of bacteria and specific coverslip to separate into chambers, count directly and find average

Bacteria alive or dead

What is done in a coulter counter? Can you tell if bacteria is alive or dead?

• Uses electrode and introduces bacteria to observe electrical difference

Bacteria alive or dead

What is done in a viable plate count? What is each colony called?

• Counting number of colonies on place (pour/spread)

Colony Forming Unit (CFU)

What is the range goal of CFU for serial dilution?

• 30-300 CFU

How do you start a serial dilution?

1. Start by extracting 1 mL from original culture

What is the second step to a serial dilution?

2. Add 1.0 mL to 9.0 mL (1 to 10 ratio)

What is the last step to a serial dilution?

3. Repeat 3 more times to reach 50 CFU

What are the dilutions in multiples of?

• In multiples of 10

For what concentration is the spread or pour plate method used?

• Sample too concentrated; requires serial dilutions

What are the steps to the pour plate?

1. Bacterial sample mixed with warm agar

2. Sample is poured in plate

3. Plate swirled around; allowed solidify

4. Plate incubated until colonies grow

What are the steps to spread plate?

1. Sample poured onto solid medium

2. Spread over surface

3. Plate incubated over medium

For what concentration is the most probable number used?

• Very dilute; small concentration sample

What does the most probable number method estimate?

• Estimate viable microbes

What is the first step of the most probable number method?

1. Separate original sample into 3 tubes; 10 mL, 1 mL, 0.1 mL

What is the second step of the most probable number method?

2. Count amount of tubes that showed growth; 5: 2: 0 (code)

What is the code from the most probable number method used for?

• Use code; compare to a chart to find amount of bacteria in 100 mL sample

What is an example of the most probable number

• Coliforms in pond water

What are the two methods for indirect cell counts?

1. Spectrophotometer

2. Measuring dry weight

What is the function of spectrophotometer?

• Shine light through sample

What is turbidity?

• Cloudiness, not much light

What is transmittance? When is it high?

• Amount of light passing through sample

Not turbid

What is absorbance? When is this high?

• Amount of light blocked or refracted by sample

Turbid

What is the disadvantage of the spectrophotometer method?

• Bacteria could be alive (viable) or dead

How does the measuring dry weight work?

• Centrifuge cells out and place them in weigh boat