Topic 4-L2 - Culturing Microbes

Question 1

Half of that weight of a microbial cell is

Answer 1

protein, ~ 25% is nucleic acid (mostly

ribosomes!), much of the rest is the cell envelope

Question 2

Microbes also require lower

Answer 2

abundance molecules (e.g. enzymatic cofactors)

Question 3

Although a lower % of cell material,
availability of these low-abundance
nutrients can dictate

Answer 3

growth of a microbe

Question 4

CHONPS

Answer 4

Key elements to build core macromolecules by cells (protein, DNA)

Question 5

Mg2+ plays key roles in stabilizing

Answer 5

negative charges in membranes, nucleic acids – also used by enzymes

Question 6

Many organic “micronutrients” & trace metals are required a very low
amounts

Microbes that require these (often scarce) nutrients often evolve highly

Answer 6

efficient mechanisms for their uptake

Question 7

Essential cations and anions for cells

Answer 7

Na, Mg,K,Ca,Cl

Question 8

Growth media or culture media (medium, singular) can be highly variable depending on the

Answer 8

microbe…and for a given microbe

Question 9

Defined media:

Answer 9

Media prepared by adding precise/known quantities of chemicals to water. Know the exact composition.

Question 10

Complex media:

Answer 10

Contain extracts or digested organic material with an unknown composition. E.g. yeast extract, casein (milk protein) digests

Question 11

Defined medium has the advantage that

Answer 11

you know what you’re

working with.

Question 12

Complex media advantage is that they are very

Answer 12

common – cheaper, easier, work for a broad(er) array of different microbes

Question 13

Other microbes can require a great number of growth factors – things like vitamins, amino acids, purines/pyrimidines, etc.

Answer 13

• Many organisms have an obligate symbiotic lifestyle
• Organisms that live in nutrient-rich environments (such as many lactic
  acid bacteria)

Question 14

Some microbes can make most or all of the organic molecules they need

Answer 14

• Many microbes that live in nutrient-poor environments

- Certain “flexible” microbes that adapt to many different environments
such as E. coli

Question 15

Auxotrophy –

Answer 15

inability to produce a molecule you need for your growth. (Prototrohpy is opposite - can produce that molecule)

Question 16

E.coli can grow in a complex media contains

Answer 16

Peptone and yeast extract

Question 17

Selective media:

Answer 17

Used to isolate a limited range of microbes (often bacteria) – this could be a single species. Often a combination of positive (nutrients few organisms can grow on) and negative (substances that kill most microbes) selection.

Question 18

Differential media:

Answer 18

Contain some sort of an indicator (e.g. a dye that changes colour) when particular organisms are present

Question 19

Enrichment culture:

Answer 19

Similar idea to selective media…but less selective and richer medium. Promotes growth – increase numbers from isolates to make it easier to isolate a particular microbe. Usually somewhat selective.

Question 20

Solid plates useful for

Answer 20

isolating single colonies –(ideally) originates from a single cell that grows to large numbers & can be readily seen

• identify morphology, contamination, start pure cultures

Question 21

Liquid cultures (e.g. used in experiments) should generally be started using

Answer 21

isolated single cultures

streaked on agar plates

Question 22

All media is selective?

Answer 22

Yes

Question 23

The great plate count anomaly

Answer 23

When “natural” samples are plated on generic growth plates (meant to be “non-selective”)…99.9% of microbes don’t grow.

Question 24

We cannot culture the vast majority of microbes? Why or why not

Answer 24

NO we cant, cuz of syntrophy

Question 25

syntrophy

Answer 25

(microbes feeding off one

another – microbes require other community members to grow)

Question 26

Counting microbial cell numbers:

Answer 26

1. Direct count: counted using a microscope
2. Viable plate counts: colonies counted
3. Turbidimetric: Absorbance of light by a microbe growing in a uliquid culture is measured in a spectrophotometer
4. Other indirect methods: O2 consumption, CO2 production,metabolic activity. Quantitative PCR to determine the number of
   “genome equivalents”.

Question 27

Direct – microscopic counts

Answer 27

Known volume, gridded microscope.

• doesn’t require growth (advantage)
• prone to inaccuracies, requires staining. Can confuse microbe with debris.

Question 28

Viable plate counts

Answer 28

• reliable and common
• need to known how to grow microbe
• assumes colonies emerge from single cell. (Problem when clumping/aggregating)
• assume ALL cells grow to form colonies (some cells are viable but aren’t culturable)

Question 29

Turbidity measurements

Answer 29

• microbes scatter light, amount of light scattered proportional to liquid sample. (Common and reliable)
• correlation b/w optical density and cell #
• only for single isolated pure cultures in liquid sample

Question 30

A significant advantage of turbidity measurements is that they are not

Answer 30

labor intensive and growth can be continuously or regularly measured over time

Question 31

Disadvantage for turbidity measurements

• Limited useful range:

Answer 31

At low low cell numbers, insufficient light scattering not detected. High enough cell numbers saturate signal (no longer linear)

Question 32

Microbial “growth”

Answer 32

refers to increase in population size – cell division resulting in multiplying in numbers

Question 33

Generation time (doubling time):

Answer 33

The amount of time it takes how long it takes for one cell to become two (cells to double in numbers & mass). Varies greatly depending on microbe & growth conditions

Question 34

Batch cultures -

Answer 34

cultures in a fixed volume in a closed container like a flask or a test tube.

Question 35

Continuous cultures –

Answer 35

cultures within systems where waste product are being removed and new media fed in

Question 36

Growth phases of batch culture

Answer 36

• Lag phase : adjustment phase
• exponential phase
• stationary phase: food limited, waste accum.
• decline phase

Question 37

Cell numbers double at some regular interval – this interval can vary greatly depending on

Answer 37

microbe/growth conditions

Question 38

Generation time formula

Answer 38

Generation time = (growth time) / (number of generations)

• g = t/n

Question 39

Growth during exponential phase

Answer 39

Total number of cells at any time (N t) will be equal to the starting number of cells (N0) multiplied by 2n, where n is the number of generations

• Nt = N0 x 2n

Question 40

Exponential growth can lead to very large numbers of microbes accumulating in a short time

In the real world, cells

Answer 40

rarely grow exponentially at such as fast rate…but it can happen.

Question 41

Continuous culture

Answer 41

Cell can be grown indefinitely in the lab if we can remove waste products (and cells) and add fresh media

Question 42

Chemostat (continuous culture device) can be used to

Answer 42

grow cells at a steady state – culture volume, number of cells, nutrient-waste status all kept constant.

• Cells grow at same rate that they are removed
• useful for industrial/experimental applications

Question 43

Dilution rate -

Answer 43

volume always added & removed at same rate - controls growth rate in cont. culture

• (To maintain same number of cells…if you replace the full volume every 2 hours, cells must double every 2 hours)

Question 44

Nutrient availability in the medium controls how

Answer 44

many cells you’ll have (density of culture)

More nutrients – equilibrium will be established with more cells/ml – greater yield

Question 45

planktonic growth

Answer 45

(free-living organisms in liquid)

Question 46

sessile growth

Answer 46

(growth attached to a surface)

Question 47

Growth on surface can develop into

biofilms –

Answer 47

cells encased in polysaccharide matrix attached to surface

Question 48

In many cases, bio films are complex

Answer 48

communities with significant
differentiation (cells with different
properties in different layers of biofilm)

Question 49

Biofilm formation can start with a

Answer 49

planktonic cells attaching to a surface

via appendages such as pili, fimbriae or even the flagellum

Question 50

After attachment to surface, the next steps for bio films formation is

Answer 50

• colonization
• development
• disperse

Question 51

Colonization begins and the cells

Answer 51

grow (multiply) and produce extracellular polysaccharides

Question 52

During development, cells change their biological program - express unique

Answer 52

combinations/amounts of genes to facilitate this biofilm lifestyle. Different cells (e.g. surface exposed vs. lower layers), different programs.

Question 53

disperse –

Answer 53

resume planktonic state. Form a new biofilm elsewhere?