Physical requirements for growth

1
Physical requirements for growth

• Prefixes and suffixes
• Bacteria are highly diverse in the types of
  conditions they can grow in.
• Optimal or required conditions implied by
  -phile meaning love
• Some bacteria prefer other conditions, but can
  tolerate extremes
• Suffix -tolerant
• Note the difference!

http//www.kodak.com/global/images/en/health/filmI
maging/thermometer.gif
2
When growing microbes..

• The physical/chemical conditions that are most
  important
• Presence or absence of oxygen
• Temperature range
• pH range
• Water activity (how wet)
• Note that by changing the conditions to make them
  unfavorable we can prevent bacterial growth.

3
Oxygen friend or foe?

• Early atmosphere of Earth had none
• First created by cyanobacteria using
  photosynthesis
• Iron everywhere rusted, then collected in
  atmosphere
• Strong oxidizing agent
• Reacts with certain organic molecules, produces
  free radicals and strong oxidizers
• Singlet oxygen, H2O2(peroxide), O3- (superoxide),
  and hydroxyl (OH-) radical.
• Free radicals are highly reactive chemicals that
  damage proteins, nucleic acids, and other cell
  molecules.

4
Protections of bacteria against oxygen

• Bacteria possess protective enzymes, catalase and
  superoxide dismutase.
• Catalase breaks down hydrogen peroxide into water
  and oxygen gas.
• Superoxide dismutase breaks superoxide down into
  peroxide and oxygen gas.
• Anaerobes missing one or both slow or no growth
  in the presence of oxygen.

Fe3 -SOD O2- ? Fe2 -SOD O2 Fe2 -SOD
O2- 2H ? Fe 3 -SOD H2O2
5
Relation to Oxygen

• Aerobes use oxygen in metabolism obligate.
• Microaerophiles require oxygen (also obligate),
  but in small amounts.
• Anaerobes grow without oxygen SEE NEXT

A aerobeB microaerophile

• Capnophiles require larger amounts of carbon
  dioxide than are found normally in air.

6
Anaerobes grow without O2

• Classifications vary, but our definitions
• Obligate (strict) anaerobes killed or inhibited
  by oxygen.
• Aerotolerant anaerobes do not use oxygen, but
  not killed by it.
• Facultative anaerobes can grow with or without
  oxygen

C could be facultative or aerotolerant.D
strict anaerobe
7
Effect of temperature

• Low temperature
• Enzymatic reactions too slow enzymes too stiff
• Lipid membranes no longer fluid
• High temperature
• Enzymes denature, lose shape and stop functioning
• Lipid membranes get too fluid, leak
• DNA denatures
• As temperature increases, reactions and growth
  rate speed up at max, critical enzymes denature.

8
Bacteria and temperature

• Bacteria have temperature ranges (grow between 2
  temperature extremes), and an optimal growth
  temperature. Both are used to classify bacteria.
• As temperature increases, so do metabolic rates.
• At high end of range, critical enzymes begin to
  denature, work slower. Growth rate drops off
  rapidly with small increase in temperature.

9
Classification of bacteria based on temperature
10
Terms related to temperature

• Special cases
• Psychrotrophs bacteria that grow at normal
  (mesophilic) temperatures (e.g. room temperature
  but can also grow in the refrigerator
  responsible for food spoilage.
• Thermoduric more to do with survival than
  growth bacteria that can withstand brief heat
  treatments.

11
pH Effects

• pH -logH
• Lowest 0 (very acid) highest 14 (very basic)
  Neutral is pH 7.
• Acidophiles/acidotolerant grow at low pH
• Alkalophiles/alkalotolerant grow at high pH
• Most bacteria prefer a neutral pH
• Many grow well from pH 6 to 8
• Some bacteria create their preferred conditions
• Lactobacillus creates low pH environment in vagina

12
Low water activityhalophiles, osmophiles, and
xerotolerant

• Water is critical for life remove some, and
  things cant grow. (food preservation jerky,
  etc.)
• Halophiles/halotolerant relationship to high
  salt.
• Marine bacteria archaea and really high salt.
• Osmophiles can stand hypertonic environments
  whether salt, sugar, or other dissolved solutes
• Fungi very good at this grandmas wax over
  jelly.
• Xerotolerant dry. Subject to desiccation. Fungi
  best
• Bread, dry rot of wood
• Survival of bacterial endospores.

13
Bacterial growth defined

• Since individual cells double in size, then
  divide into two, the meaningful increase is in
  the population size.
• Binary fission cell divides into two cells. No
  nucleus, so no mitosis.
• Cells do not always fully detach produce pairs,
  clusters, chains, tetrads, sarcina, etc.
• GROWTH increase in number of bacteria

14
Mathematics of bacterial growth

• Because bacteria double in number at regular
  intervals, they grow exponentially
• N N0 x 2n where N is the number of cells after
  n number of doublings and N0 is the starting
  number of cells.
• Thus, a graph of the Log of the number of
  bacteria vs. time is a straight line.

15
The Bacterial Growth Curve

• Bacteria provided with an abundant supply of
  nutrients will increase in number exponentially,
  but eventually run out of nutrients or poison
  themselves with waste products.

• Lag phase
• Exponential or
• Log phase
• Stationary phase
• Decline or Death
• phase.

4
3
2
1
16
Growth curve (continued)

• Lag phase growth lags cells are acclimating to
  the medium, creating ribosomes prior to rapid
  growth.
• Log phase cells doubling at regular intervals
  linear graph when x-axis is logarithmic.

• Stationary phase no net increase in cell
  numbers, some
• divide, some die. Cells preparing for
  survival.
• Decline phase highly variable, depends on type
  of bacteria and conditions. Death may be slow and
  exponential.

17
More about Growth

• The Growth curve is true under ideal conditions
  in reality, bacteria are subject to starvation,
  competition, and rapidly changing conditions.
• Generation time the length of time it takes for
  the population to double.

• Growth of bacteria is nonsynchronous, not every
  bacterium is dividing at the same time.
• Instead of stepwise curve, smooth curve

18
Exponential growth

• Balanced growth
• Numbers of bacteria are doubling at regular
  intervals.
• All components of bacteria are increasing in
  amount at the same rate
• 2x as many bacteria 2x as much protein, 2x as
  much peptidgolycan, 2x as much LPS, etc.
• During exponential growth, bacteria are not
  limited for any nutrients, i.e. they are not
  short of anything.

19
Measurement of cell numbers

• Direct methods cells actually counted.
• Petroff-Hausser counting chamber (right), 3D
  grid. Count the cells, multiply by a conversion
  factor.
• Dry a drop of cells of known volume, stain, then
  count.

20
Coulter Counter
Coulter-counter single-file cells detected by
change in electric current.
21
Counting cells with plates

• Viable plate count
• Relies on bacteria being alive, multiplying and
  forming colonies.
• Spread plate sample is spread on surface of
  agar.
• Pour plate sample is mixed with melted agar
  colonies form on surface and within agar.
• Colonies counted with a colony counter.

biology.clc.uc.edu/.../Meat_Milk/ Pour_Plate.htm
22
Filtration and plate counting

• Membrane filters are very thin with a defined
  pore size, e.g. 0.45 µm.
• Bacteria from a dilute sample are collected on a
  filter filter placed on agar plate, colonies
  counted.
• Used when concentration of bacteria is low.

http//dl.clackamas.cc.or.us/wqt111/coliform-8.jpg
http//www.who.int/docstore/water_sanitation_heal
th/labmanual/p25bs.jpg
23
Spectrophotometry

• Bacteria scatter light, making a turbid (cloudy)
  suspension.
• Turbidity is usually read on the Absorbance scale
• Not really absorbance, but Optical Density (OD)
• More bacteria, greater the turbidity (measured as
  OD)

Based on www.umr.edu/gbert/ color/spec/Aspec.html

24
More about Spectrophotometry

• Does NOT provide an actual number unless a
  calibration curve ( of bacteria vs. O.D.) is
  created.
• Indirect counting method
• Quick and convenient, shows relative change in
  the number of bacteria, useful for determining
  growth (increase in numbers).
• Does NOT distinguish between live and dead cells.
  To create a calibration curve, best to plot OD
  vs. number of cells determined with microscope
  (not plate count).

25
Biomass

• Measure the total mass of cells or amount of any
  component such as protein, PS, DNA, KDO.
• Especially when cells are doubling, the amounts
  of all the components of a cell are increasing
  at the same rate, so any could be measured.
• Not so in stationary phase.

In this example, total biomass increases
exponentially over time.
http//www.pubmedcentral.nih.gov/pagerender.fcgi?a
rtid242188pageindex10page