week 2: microbial growth and enumeration

Question 1

ways to grow bacteria in the lab

Answer 1

1. Liquid cultures
   A) Batch culture (tube, flask etc. with a finite volume of media)
   B) Continuous culture
2. Semi-solid media
   Media containing agar or other solidifying agents, usually in petri plates

Question 2

binary fission and equation

Answer 2

bacteria division during exponential growth

* 2n where n=number of divisions Ex: 10 x 2n where 10=starting number of bacteria and n=number of divisions

Question 3

batch/liquid culture phases

Answer 3

• Lag phase: bacteria preparing to growth
  
  • Log/Exponential phase: growing at max rate
  • Stationary phase: cells stop growing, turning on stress responses to help retain viability
  • Death phase: cells die with a half-life, no more nutrients

Question 4

determination of generation time (g)

Answer 4

• Time it takes for cells to double in number
  
  • Learn how to read log graphs
    G depends on growth conditions and genetics, depends on media used

Question 5

continuous culture

Answer 5

In a continuous culture, all cells in a population achieve a steady state, allows detailed study of bacterial physiology
○ Can maintain log phase state, constant number of bacteria over time
○ As bacteria divides, the waste and excess is removed as nutrients are added
○ If bacteria is not dividing, culture is being diluted over time from to continuous flow of media added
* The chemostat ensure logarithmic growth by constantly adding and removing equal amounts of culture media

Question 6

counting bacteria

Answer 6

• Bacteria must be liquid to count them (ex: solid samples must be suspended in liquid or solid surfaces must be sampled with a swab and the swab dipped in liquid
  
  • Expressed in terms of
    ○ Number of bacteria/mL of liquid
    or
    ○ Number of bacteria/g of solid
    or
    ○ Number of bacteria/surface area

Question 7

enumeration methods

Answer 7

1. Viable plate counting
2. Optical density measurements
3. Hemocytometer
   • For each, must know
     ○ Direct or indirect method?
     ○ Specific requirements?
     ○ Advantages and disadvantages?
     What are you measuring?

Question 8

pour plate and spread plate methods

Answer 8

Pour plate method
1. Pipette bacterial sample onto petri dish
2. Pour nutrient agar
3. Swirl to mix
4. Incubate and colonies grow on agar surface and subsurface

Spread plate method
1. Pipette bacterial sample onto surface of agar plate
2. Spread sample evenly over surface
3. Colonies grow on surface

Both counts are expressed as CFU/mL
Both don’t always give same number of counts

Question 9

optical density measurements

Answer 9

Optical density measurements

* Spectrophotometers measure photons of light after passing through sample 
* Need standard curve to convert OD readings to actual number of cells 
* OD increases with increase of [cells]

Question 10

direct microscopic count

Answer 10

Direct microscopic count

* Microorganisms can be counted directly by placing dilutions on a special microscope slide containing gride that hold a known volume of liquid
1. Slide with shallow wells hold sample
2. Coverslip placed over slide
3. Bacterial suspension is added to wells, seeps under coverslip to fill the shallow space of known volume over the grid Bacterial cells in each square are counted under a microsco