Microbial Nutrition

1
Lecture 5

• Microbial Nutrition Growth
• Chapter 7
• Foundations in Microbiology

2
Microorganism must Eat!!!

• Macronutrients
• Required in large amounts
• Molecules that contain C, H, S O
• Protein, carbohydrates lipids
• Micronutrients
• Required in trace (very low) amounts
• Mn, Zn, Ni, plus many others
• These must be supplied in the growth medium!!!

3
Organic / Inorganic

• Organic
• Contain C
• Usually products of living cells
• CH4, glucose, starch, lipids, proteins, nucleic
  acids
• Inorganic
• No C (except CO2)
• Metals salts
• O2, CO2, H2O, PO4, MgSO4, FeSO4, NH4Cl

4
92 naturally occurring elements 25 are essential
for life 6 major elements make up 96 of the
mass of most living organisms O Oxygen (for
almost all organic comps) P Phosphorus (DNA,
RNA and ATP (energy)), membranes
(phospholipids) C Carbon (for all organic
compds) S Sulfur (for proteins) H -
Hydrogen (for almost all organic comps) N
Nitrogen (for proteins, DNA, RNA)
5
Chemical Composition of E. coliDry weight

• Carbon 50
• Oxygen 20
• Nitrogen 14
• Hydrogen 8
• Phosphorus 3
• Sulfur 1
• Potassium 1
• Sodium 1
• Calcium 0.5
• Magnesium 0.5

Water is 70 of total weight
Table 7.2 page 189
6
Chemical Composition of E. coliDry weight

• Protein 50
• RNA 20
• DNA 3
• Carbohydrates 10

7
Growth Media Recipes

• What do we need?
• Carbon Source Glucose
• Nitrogen Source NH4Cl, NaNO3, or protein
• Sulfur Source Na2SO4 or protein
• Phosphorus K2HPO4 and/or KH2PO4 (also acts as
  buffer-resists change in pH of medium as cells
  grow)
• Trace Metal Solution Contains Fe, Mg, Mn, Ni,
  Cu, Co, K and others
• Vitamin solution (if necessary)

8
Parasitic Lifestyle

• Live in or on a host
• Derive nutrition from their host
• Pathogens
• Inclined to cause disease or even death
• Obligate parasite
• Unable to grow outside of a living host
• Facultative parasite
• Able to grow outside a living host
• Obligate intracellular parasite
• Spend all or part of their life cycle within a
  host cell

9
Saprophytic Lifestyle

• Saprophytic
• Decomposer
• Dead organic matter
• Obligate saprobes
• Strictly on dead organic matter
• Facultative Parasite
• Can infect a living host under certain
  circumstances
• Opportunistic pathogen

10
Temperature and Growth

• Minimum temperature
• - Lowest temperature an organism can grow
• Maximum temperature
• - Highest temperature an organism can grow
• Optimal temperature
• Fastest growth rate metabolism
• Lowest Doubling Time

11
(No Transcript)
12
Low Temperature

• Psychrophile
• Optimal temp lt 15ºC and capable of growth at OºC
• Obligate psychrophiles generally can not grow
  above 20ºC
• Psychrotrophs or facultative psychrophiles
• Grow slowly at low temp but have an optimum temp
  gt 20ºC

13
Intermediate Temperature

• Mesophiles
• Optimal growth temperature 20º to 40ºC
• Most human pathogens (human body temperature
  37ºC)
• Some mesophiles can with stand short exposure to
  high temperatures (this is why pour plates
  work!!!)

14
High Temperature

• Optimal temp gt 40ºC
• Moderate thermophiles
• Optimal growth temperature between
• 40 and 80ºC
• Hyperthermophiles
• Optimal growth temperature gt 80ºC
• Hot springs or deep sea thermal vents

15
Oxygen (O2) and Growth

• Aerobe or aerobic organism
• Use O2 can eliminate H2O2
• Obligate aerobe
• Cannot live without O2
• Facultative aerobes
• Capable of growth in the absence of O2
• Microaerophile
• Requires O2 but at low concentration

16
Anaerobic

• Anaerobe
• Does not require O2
• Strict or Obligate anaerobe
• Cannot tolerate any O2
• Aerotolerant anaerobes
• Do not utilize O2 but survive in its presence
• Capnophiles (usually anerobic or microaerophilic)
• Prefer a high CO2 ( means
  concentration)

17
(No Transcript)
18
Use of Oxygen (O2)

• Required for aerobic respiration
• C6H12O6 O2 ? CO2 H2O ATP
• Glucose ? Glycolysis ? Krebs Cycle ? Respiratory
  Chain
• O2 is the final electron acceptor (celled
  terminal electron acceptor)
• Generates a lot of ATP
• Anaerobic Respiration
• NO3-, SO4-2 or CO3-2 used as terminal electron
  acceptor
• Generates less ATP per amount of terminal
  electron acceptor
• Respiratory chain represents a series of proteins
  usually in the cell membrane of prokaryotes that
  are electron carriers that ultimate drop off
  electrons to the terminal electron acceptor

19
Fermentation

• Anaerobic process
• Pyruvate ? ethanol
• Yeast and bacteria
• Pyruvate ? lactic acid
• Bacteria
• Pyruvate ? acetic acid
• Bacteria
• Electron donor and acceptor
• are organic compounds
• No electron transport chain
• Less energy than from respiration

20
O2 is EXTREMELY Reactive

• Build up of O2 in the cell can be deadly
• Destructive by-products of O2
• Superoxide ion
• O2-
• Peroxides
• H2O2
• Hydroxyl ion
• OH-
• There are enzymes that detoxify these products

21
Superoxide dismutase

• O2- O2- 2H ? H2O2 O2
• H2O2 H2O2 ? 2H2O O2

Catalase
22
pH and growth

• Most bacteria are neutrophilic and their optimal
  growth pH is between pH 6 and 8
• Acidophiles optimal pH lt 3
• Obligate acidophile
• optimal growth pH between 0-1 and cant grow at
  7
• Alkalinophiles (alkaliphile)
• Optimal growth at high pH (gt 8)
• Obligate grows at pH gt 10 but cant at 7
• Note Fungi are much more tolerant of acidic pH
  and the optimum growth pH for many is around 5

23
Osmotic Pressure (Water Activity)

• Water Activity (aw) For pure water aw 1.000
  Affects growth strongly and selects for
  particular organisms
• - Human blood 0.995
• - Bread 0.950
• - Maple Syrup 0.900
• - Salt Lakes, Salted Fish 0.750
• - Cereals, Dried Fruit 0.700

24
Osmotic Pressure (Water Activity)

• Water Activity (aw) For pure water aw 1.000
• Halophiles a type of extremophile
• Osmophile
• Hypertonic / hypersaline environments
• Salt lakes, salt ponds
• Obligate halophile
• Optimal growth 25 NaCl but requires at least
  9 NaCl
• Facultative halophiles
• Resistant to salt but dont normally reside in
  high salt environments Staphylococcus aureus (8
  salt)

25
(No Transcript)
26
(No Transcript)
27
Ecological Associations

• Symbiotic
• Two organisms live in close association
• Required by one or both individuals
• Mutualism
• Mutually beneficial relationship
• Commensalism
• One species derives benefit without harming the
  other
• Parasitism
• One species derives benefit and the other is
  harmed

28
Other Associations

• Synergism
• Interrelationship
• 2 or more free-living organisms
• Benefits all but is not necessary for survival
• Antagonism
• One species secretes a substances that inhibits
  or destroys another species
• Antibiotics
• Antimicrobial proteins

29
Cell Division Endospores

• Binary fission (budding) vs. Sporulation
• Binary fission and budding are forms of
  reproduction
• Sporulation is, in most cases, not a form of
  reproduction but is used for survival of the cell
  under harsh conditions. There are some exceptions
• Note Endospores are found only in Gram-positive
  bacteria
• Implicated in disease examples Bacillus
  anthracis, Clostridium botulinum, Clostridum
  tetani

30
Binary Fission
31
Binary / Transverse Fission and Budding

• One cell becomes two
• Division plane forms across the width of the cell
• Parent cell enlarges
• Replication of the chromosome
• Transverse septum
• Continuous

32
Sporulation

• Dormant bodies
• Resting structure of some Gram
• Bacillus, Clostridium Sporoscarcina
• Vegetative cycle
• Endospore cycle
• Unfavorable environmental conditions
• Heat, irradiation, desiccation, disinfectants
• Thick impervious cortex
• Long lived
• 250 mya spore

33
Protein filaments migrate from the middle of the
cell to opposite poles Two rings form near each
pole Production of a spore only occurs at one
pole Forespore or prespore forms Spore matures
within the mother cell cell lyses and pore is
feed Spore withstands extreme environmental
conditions
34
Other Spore Formers

• Epulopiscium spp.
• Unusually large, bacterial symbiont of the
  intestinal tract of marine surgeon fish
• Surgeon fish are herbivores detritivorous
• Some strains of Epulopiscium do not reproduce
  vegetatively
• Viviparity

35
Viviparity
36
(No Transcript)
37
Estimating the Number of Bacterial Cells in a
Sample (e.g., water, food, soil)

• Main method is the plate count method which we
  will go over herewill count only live cells in a
  sample (Viable count) but not all live cells may
  form colonies
• Other methods are microscopic methods but there
  are limitations cells could be live or dead
  unless a vital stain is used and flow
  cytometry which is expensive

38
Plate Count Method

• Dilute a sample with sterile saline until the
  microbial cells can be counted accurately
• A broad range of dilutions is used since the
  exact number of bacteria is not known
• Plates should have between 30 and 300 colonies
• Fewer than 30 colonies is not statistically
  accurate
• Too Few To Count (TFTC)
• More than 300 colonies is simply to difficult to
  count
• Colonies are too close together
• Too Numerous To Count (TNTC)

39
Colony Forming Units (cfu)

• Each viable cell will develop into a colony
• There are two assumptions
• Each colony arose from one cell
• Cant be certain that two cells close together
  produced one colony
• Random sample from the population
• Statistically accurate

40
Calculate Colony Forming Units

• Count the bacterial colonies on the plates the
  have between 30 300 colonies
• Divide the number of colonies by the dilution
  factor
• There are 130 colonies on the 10-6 dilution
• 130 / 10-6 1.3 x 108 or 130,000,000
• Report cfu / mL or cfu / gram

41
Large number of cells
0.1
0.01
0.1
0.01
0.01
10-7
10-8
10-6
10-2
10-4
42

• The 1st plate has TNTC
• The 3rd plate has TFTC
• The 2nd plate has 21 colonies and will be used
  for the calculations
• The concentration of the cells is
• 21 / 0.1 mL 210 cells / mL
• 210 X 107 2.1 X 109

43
Population Growth
44
Measurement of Microbial Population

• Viable plate count method
• Counting colonies on agar medium
• Spectrophotometric analysis
• Measure of turbidity

45
Growth Curve
46
Growth Curve

1. Lag phase flat period of adjustment,
   enlargement little growth
2. Exponential growth phase a period of maximum
   growth will continue as long as cells have
   adequate nutrients a favorable environment
3. Stationary phase rate of cell growth equals
   rate of cell death cause by depleted nutrients
   O2, excretion of organic acids pollutants
4. Death phase as limiting factors intensify,
   cells die exponentially in their own wastes

47
Spectrophotometer

• Useful laboratory tool - inexpensive
• Electronically compares the amount of light
  transmitted through a sample with that
  transmitted through a blank.
• The ratio of the amount of light transmitted
  through a sample to that transmitted through a
  blank is called the transmittance

48
Spectrophotometer
light transmitted through a sample light
transmitted through the blank
t
T t x 100
49
(No Transcript)
50
Absorbance

• A -log10(t)
• A Optical Density or O.D.
• O.D.400 nm or A400 nm

51
(No Transcript)