Microbial Nutrition , full description on microbial biology and its nutition . various types of growth media with all necessary examples.

1. Microbial
Nutrition

2. Feast or famine: normal is what’s
normal for you: Oligotrophs vs.
copiotrophs
 Oligo means few; oligotrophs are adapted to life in
environments where nutrients are scarce
 For example, rivers, other clean water systems.
 Copio means abundant, as in “copious”
 The more nutrients, the better.
 Medically important bacteria are copiotrophs.
 Grow rapidly and easily in the lab.
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3. Culture Medium
 Defined vs. Complex
 Defined has known amounts of known chemicals.
 Complex: hydrolysates, extracts, etc.
 Exact chemical composition is not known.
 Selective and differential
 Selective media limits the growth of unwanted microbes or allows growth of
desired ones.
 Differential media enables “differentiation” between different microbes.
 A medium can be both.
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4. Defined Medium for Cytophagas/Flexibacters
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Component grams
K2HPO4 0.10
KH2PO4 0.05
MgCl2 0.36
NaHCO3 0.05
{CaCl2 1 ml*
{BaCl2.2H2O
Na acetate 0.01
FeCl.7H2O 0.2 ml*
RNA 0.10
alanine 0.15
arginine 0.20
aspartic acid 0.30
glutamic acid 0.55
glycine 0.02
histidine 0.20
isoleucine 0.30
leucine 0.20
lysine 0.40
phenylalanine 0.30
proline 0.50
serine 0.30
threonine 0.50
valine 0.30

5. 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!
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http://www.kodak.com/global/images/en/health/filmImaging/thermometer.gif

6. Oxygen: friend or foe?
 Early atmosphere of Earth had none
 First created by cyanobacteria using photosynthesis
 Oxygen gas rusted iron in Earth’s crust, then excess 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.
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7. 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.
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Fe3+ -SOD + O2
- → Fe2+ -SOD + O2
Fe2+ -SOD + O2
- + 2H+ → Fe 3+ -SOD + H2O2

8. Relation to Oxygen
 Aerobes: use oxygen in metabolism;
obligate.
 Microaerophiles: require oxygen
(also obligate), but in small
amounts.
 Anaerobes: grow without
oxygen; SEE NEXT
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•Capnophiles: require larger amounts of carbon dioxide
than are found normally in air.
A: aerobe
B: microaerophile

9. 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
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C: could be facultative
or aerotolerant.
D: strict anaerobe

10. 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.
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11. 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.
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12. Classification of bacteria based on
temperature
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13. 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.
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14. pH Effects
 pH = -log[H+]
 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
 What is pH of human blood?
 Some bacteria create their preferred conditions
 Lactobacillus creates low pH environment in vagina
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15. Low water activity: halophiles,
osmophiles, and xerotolerant
 Water is critical for life; remove some, and things can’t
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; grandma’s wax over jelly.
 Xerotolerant: dry. Subject to desiccation. Fungi best
 Bread, dry rot of wood
 Survival of bacterial endospores.
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16. Nutritional Requirements
 Nutrients are substances required for biosynthesis of
macromolecules, energy production and growth
 macroelements (macronutrients)
 required in relatively large amounts
 C, H, N, O, P, S and K, Mg, Fe and Ca
 micronutrients (trace elements)
 Mn, Zn, Co, Mo, Ni, and Cu
 required in trace amounts, used as cofactors by enzymes
 often supplied in water or in media components

17. Mystery Behind Life
 Molecules of life are formed through reductive pathway that
requires:
 Source of electrons and protons
 Source of energy
 A carbon source at oxidized state
 Process:
 energy is used to release electrons from an inroganic/organic source
 Transfer onto a carbon containing molecule
 The reduced form of carbon is used to build new macrmolecular
derivatives

18. Growth Factors
 Are organic compounds
 essential cell components (or their precursors) that the cell
cannot synthesize
 must be supplied by environment if cell is to survive and
reproduce

19. Classes of growth factors
 amino acids
 needed for protein synthesis
 purines and pyrimidines
 needed for nucleic acid synthesis
 vitamins
 function as coenzymes

20. Nutritional classification of
Microorganisms
 Nutritional classes :
 Based on carbon source:
 autotrophs
 use carbon dioxide as their sole or principal carbon source
 heterotrophs
 use organic molecules as carbon sources which often also serve
as energy source
 Based on energy source
 phototrophs use light
 chemotrophs obtain energy from oxidation of chemical compounds
 Based on electron source
 lithotrophs use reduced inorganic substances
 organotrophs obtain electrons from organic compounds

21. Nutritional Resource Management
 Depending on how carbon, energy and electron sources are used
microroganisms can be divided into five nutritional categories:
(auto/hetero) (photo/chemo) (litho/organo)

22. Uptake of Nutrients by the Cell
 Some nutrients enter by passive diffusion. (Membranes are
permeable for them)
 Most nutrients enter by:
 facilitated diffusion
 active transport
 group translocation

23. Passive Diffusion (simple diffusion)
 molecules move from region of higher concentration to lower
concentration because of random thermal agitation
 is not energy dependent
 H2O, O2 and CO2 often move across membranes this way

24. Passive diffusion is restricted
Substance Rate of intake
Water 100.00
Glycerol 0.1
Tryptophan 0.001
Glucose 0.001
Chloride ion 0.000001
Sodium ions 0.0000001

25. Facilitated Diffusion
 similar to passive diffusion e.g.,
 movement of molecules is not energy dependent
 direction of movement is from high concentration to low
concentration
 concentration gradient impacts rate of uptake

26. Facilitated Diffusion …
 differs from passive diffusion
 uses carrier molecules (transporters, eg. permeases)
 smaller concentration gradient is required for significant
uptake of molecules
 effectively transports glycerol, sugars, and amino acids
 more prominent in eucaryotic cells than in procaryotic cells

27.  rate of facilitated
diffusion increases
more rapidly
at a lower
concentration
 diffusion rate
reaches a plateau
when carrier becomes
saturated
 carrier saturation
effect not seen in PD
Passive and Facilitated Diffusion

28. Note conformational change of carrier
Facilitated diffusion…
Examples
 Members of major intrinsic
proteins (MIP) that form porin
 Aquaporin: channels to
transport water
 glycerol transport channel

29. Active Transport
Bacteria use active transport to accumulate scarce sources of
nutrients from their natural habitat
 energy-dependent process
 ATP or proton motive force used
 moves molecules against the concentration gradient
 concentrates molecules inside cells
 involves carrier proteins
 carrier saturation effect is observed at high solute concentrations

30. ABC transporters
 ATP-Binding Cassette transporters
 observed in bacteria, archaea, and
eucaryotes
 Transports sugars like arabinose,
galactose, ribose etc
 Cargo delivery by porins (OmpF) to
periplasmic space where:
 Solute binds to a specific binding
protein (SBP) that delivers it to the
transporter
 Transporter conformation changes
 ATP binds to transporter subunits in
lumen side
 Upon ATP hydrolysis the solute is
transferred into the cytoplasm

31. Active Transport using proton gradient
 PMF instead of ATP can be
indirectly utilized to
transport sugars into
bacterial cells
 Sugars can be transported
by a symporter that is driven
by Na+ gradient outside the
cell
 Na+ gradient itself is
generated through H+
gradient coupled antiporter
that pumps the Na+ to the
periplasmic space

32. Coupled transport: Symport and
antiport
 Na-Sugar symporter
 Na/Ca antiporter
 Na increases in cytoplasm
 How to balance?
 Coupled to Na/K pump

33. Group Translocation
 chemically modifies molecules as it is brought into cells
 PEP sugar phosphotransferase system (PTS):
 best known system, transports a variety of sugars
 while phosphorylating them using phosphoenolpyruvate (PEP) as
the phosphate donor
 Found among the member of enterobacteriacae, clostridium,
staphylococcus, and lactic acid bacteria

34. Active transport by group translocation
 energy-dependent
process: PEP
 Phosphate is
carried via E1, HPr
to cyotosolic
protein IIA
 IIB receives P and
passes to a sugar
molecule that has
been transported
into the cell via IIC
protein

35. Iron Uptake
 ferric iron is very insoluble so
uptake is difficult
 Microorganisms chelate Fe3+
using,
Hydroxamates
 Form complexes with ferric
ion
 complex is then transported
into cell

36. Iron Uptake
 ferric iron is very insoluble so
uptake is difficult
 Microorganisms chelate Fe3+
using,
Siderophores, eg.
enterochelin
 Pass through OM via FepA
 FebB (a SBP), delivers to
ABC (FepG,FepD, FepC)
delivery to cytoplasm
 Reductio to Fe2+

37. Culture Media
 most contain all the nutrients required by the
organism for growth
 classification
 chemical constituents from which they are made
 physical nature
 function

38. Types of Culture Media
Physical Nature Composition Application
Liquid
Defined
(synthetic) Supportive
Semi-solid Complex Enriched
Solid Differential
Selective

39. Defined or Synthetic Media
 all components and
their concentrations
are known

40. Complex Media
 contain some
ingredients of
unknown
composition and/or
concentration

41. Some media components
 peptones
 protein hydrolysates prepared by partial digestion of various
protein sources
 extracts
 aqueous extracts, usually of beef or yeast
 agar
 sulfated polysaccharide used to solidify liquid media

42. Functional Type of Media
 supportive or general purpose media
 support the growth of many microorganisms
 e.g., tryptic soy agar, Nutrient broth, Luria Bertani
 enriched media
 general purpose media supplemented
by blood or other special nutrients
 e.g., blood agar

43. Types of media…
Selective media
 favor the growth of some microorganisms and
inhibit growth of others
 e.g., MacConkey agar
selects for gram-negative bacteria

44. Types of media…
Differential media
 distinguish between different groups
of microorganisms based on their
biological characteristics
 e.g., blood agar
 hemolytic versus nonhemolytic
bacteria
 e.g., MacConkey agar
 lactose fermenters versus
nonfermenters
E. coli
S. enterica

45. Techniques: Isolation of Pure
Cultures
Pure culture
Isogenic population of cells
arising from a single cell
Isolation techniques
 spread plate
 streak plate
 pour plate

46. The Spread Plate and Streak Plate
 involve spreading a mixture of cells
on an agar surface so that
individual cells are well separated
from each other
 each cell can reproduce to form a
separate colony (visible growth or
cluster of microorganisms)

47. Streak plate technique
 using a sterile loop transfer cells from solid or broth culture onto
an agar plate
 streaking lines are made with an intermittent
flaming the loop
 Cells are diluted on the streak lines and
separated as individual cells
 Each cell grows and forms a colony after
proper incubation
Click for animation

48.  dispense cells onto
medium in a Petri dish
 sterilize spreader by
dipping into 70% alcohol
followed by flaming
 spread cells across surface
 incubate plate
Spread plate technique

49. Pour plate technique
 sample is diluted
several times, eg
10-fold dilution
series
 diluted samples
are mixed with
liquid agar
 mixture of cells
and agar are
poured into
sterile culture
dishes
 What is the cfu/ml
of culture?

50. Calculation of bacterial cell
concentration
 Question: plating of triplicate 100 ul from 10-7 dilution of
an actively growing E.coli culture produced 37, 42 and
44 isolated colonies on nutrient agar plates following
ovenight incubation at 37⁰C. Calculate the number of
the colony forming units per milliliter of the original
culture.
Answer: 4.1x 109 cfu/ml