2. LECTURE OUTLINE
Reproduction & Growth
Requirements for Growth
Physical
Chemical
Measurement of Microbial Growth
Culture Media
Obtaining Pure Cultures
Preservation Methods
Parungao-Balolong 2011
Thursday, July 14, 2011
3. LECTURE OUTLINE
Reproduction & Growth
Requirements for Growth
Physical
Chemical
Measurement of Microbial Growth
Culture Media
Obtaining Pure Cultures
Preservation Methods
Parungao-Balolong 2011
Thursday, July 14, 2011
4. REPRODUCTION IN
PROKARYOTES
Binary fission
Budding
Conidiospores
(actinomycetes)
Fragmentation of
filaments
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Thursday, July 14, 2011
5. MICROBIAL GROWTH
Microbial growth =
increase in number of cells,
not cell size
Nutrients = substances used
in biosynthesis and energy
production (required for
microbial growth)
Environmental Factors =
temperature, oxygen levels,
osmotic concentration
Parungao-Balolong 2011
Thursday, July 14, 2011
6. GROWTH GROWTH
◦Increase in cellular
constituents
◦Leads to a rise in cell number
Budding, Binary Fission
For coenocytic
organisms
(multinucleate)
◦Growth results in
increased cell
size not number
Parungao-Balolong 2011
Thursday, July 14, 2011
7. MICROBIAL NUTRITION
Macroelements or Macronutrients
◦Carbon, oxygen, hydrogen, nitrogen, sulfur, phosphorus,
potassium, calcium, magnesium and iron
Trace elements or Micronutrients
◦Manganese, zinc, cobalt, molybdenum, nickel and copper
Parungao-Balolong 2011
Thursday, July 14, 2011
8. GROWTH FACTORS
BIOTIN PYRIDOXINE or VIT B6
◦Carboxylation (Leuconostoc) ◦Transamination (Lactobaci!us)
CYANOCOBALAMIN or VIT NIACIN
B12 ◦Precursor of NAD and NADP
◦Molecular rearrangements (Bruce!a)
(Euglena)
RIBOFLAVIN or VIT B2
FOLIC ACID ◦Precursor of FAD and FMN
◦One-carbon metabolism (Caulobacter)
(Enterococcus)
THIAMINE or VIT B1
PANTOTHENIC ACID
◦Aldehyde group transfer (Baci!us
◦Fatty acid metabolism (Proteus) anthracis)
Parungao-Balolong 2011
Thursday, July 14, 2011
9. MICROBIAL NUTRITION
CARBON SOURCES
Autotrophs CO2 sole or principal biosynthetic carbon source
Heterotrophs Reduced, preformed, organic molecules from
other organisms
ENERGY SOURCES
Phototrophs Light
Chemotrophs Oxidation of organic or inorganic compounds
HYDROGEN AND ELECTRON SOURCES
Lithotrophs Reduced inorganic molecules
Organotrophs Organic molecules
Parungao-Balolong 2011
Thursday, July 14, 2011
10. MICROBIAL NUTRITION
MAJOR NUTRITIONAL TYPES SOURCES OF ENERGY, REPRESENTATIVE
HYDROGEN/ELECTRONS AND MICROORGANISMS
CARBON
PHOTOLITHOTROPHIC Light energy Algae
AUTOTROPHY Inorganic hydrogen/electron Purple and green sulfur
donor bacteria
CO2 carbon source Blue-green algae
(cyanobacteria)
PHOTOORGANOTROPHIC Light energy Purple non-sulfur bacteria
HETEROTROPHY Organic hydrogen/electron Green non-sulfur bacteria
donor
Organic carbon source (CO2
may also be used)
Parungao-Balolong 2011
Thursday, July 14, 2011
11. MICROBIAL NUTRITION
MAJOR NUTRITIONAL TYPES SOURCES OF ENERGY, REPRESENTATIVE
HYDROGEN/ELECTRONS AND MICROORGANISMS
CARBON
CHEMOLITHOTROPHIC Chemical energy source Sulfur-oxidizing bacteria
AUTOTROPHY (inorganic) Hydrogen bacteria
Inorganic hydrogen/electron Nitrifying bacteria
donor Iron bacteria
CO2 carbon source
CHEMOORGANOTROPHIC Chemical energy source Protozoa
HETEROTROPHY (organic) Fungi
Organic hydrogen/electron Most non-photosynthetic
donor bacteria
Organic carbon source
Parungao-Balolong 2011
Thursday, July 14, 2011
12. THE GROWTH CURVE
Population growth is studied by analyzing the growth curve of
microorganisms
Growth of microorganisms reproducing by binary fission can be
plotted as the logarithm of cell number versus the incubation time
(Growth curve)
Parungao-Balolong 2011
Thursday, July 14, 2011
13. THE GROWTH CURVE
The Growth Curve can be obtained via a Batch Culture
◦Microorganisms are cultivated in a liquid medium
◦Grown as a closed system
◦Incubated in a closed culture vessel with a single batch of
medium
◦No fresh medium provided during incubation
◦Nutrient concentration decline and concentrations of
waste increase during the incubation period
Parungao-Balolong 2011
Thursday, July 14, 2011
14. THE LAG PHASE
No immediate increase in cell mass or cell number (Cell is synthesizing
new components)
The necessity of a lag phase:
◦Cells may be old and ATP, essential cofactors and ribosomes depleted
must be synthesized first before growth can begin
◦Medium maybe different from the one the microorganism was growing
previously
new enzymes would be needed to use different nutrients
◦Microorganisms have been injured and require time to recover
Cells retool, replicate their DNA, begin to increase in mass and finally
divide
Parungao-Balolong 2011
Thursday, July 14, 2011
15. THE LAG PHASE
LONG LAG PHASE
◦Inoculum is from an old culture
◦Inoculum is from a refrigerated
source
◦Inoculation into a chemically-
different medium
SHORT LAG PHASE (or even
absent)
◦Young, vigorously growing
exponential phase culture is
transferred to fresh medium of same
composition
Parungao-Balolong 2011
Thursday, July 14, 2011
16. EXPONENTIAL
/LOG PHASE
Microorganisms are growing and dividing at the maximal rate possible
given their genetic potential, nature of medium and conditions under
which they are growing
Rate of growth is constant
Microorganism doubling at regular intervals
The population is most uniform in terms of chemical and
physiological properties
Why the curve is smooth:
◦Because each individual divides at a slightly different moment
Parungao-Balolong 2011
Thursday, July 14, 2011
17. STATIONARY
PHASE
Population growth ceases and the growth curve becomes
horizontal (around 109 cells on the average)
Why enter the stationary phase:
◦Nutrient limitation (slow growth)
◦Oxygen limitation
◦Accumulation of toxic waste products
Parungao-Balolong 2011
Thursday, July 14, 2011
18. DEATH
PHASE
Detrimental environmental changes like nutrient
depletion and build up of toxic wastes lead to the decline
in the number of viable cells
Usually logarithmic (constant every hour)
DEATH: no growth and reproduction upon transfer to
new medium
Death rate may decrease after the population has been
drastically reduced due to resistant cells
Parungao-Balolong 2011
Thursday, July 14, 2011
19. LECTURE OUTLINE
Reproduction & Growth
Requirements for Growth
Physical
Chemical
Measurement of Microbial Growth
Culture Media
Obtaining Pure Cultures
Preservation Methods
Parungao-Balolong 2011
Thursday, July 14, 2011
20. Temperature
Minimum growth temperature REQUIREMENTS
Optimum growth temperature FOR GROWTH:
Maximum growth temperature PHYSICAL
Parungao-Balolong 2011
Thursday, July 14, 2011
21. INFLUENCE OF LIPID CONTENT
◦PSYCHROPHILY
HIGH CONTENT OF
UNSATURATED FATTY ACIDS
HELP MAINTAIN A SEMI-FLUID
MEMBRANE STATE AT LOW
TEMPERATURE
◦THERMOPHILY
PROTEINS OR ENZYMES =
INCREASED NUMBER OF SALT
BRIDGES (RESIST UNFOLDING
IN THE AQUEOUS MILIEU)
MEMBRANES = RICH IN
SATURATED FATTY ACIDS
(STABLE AT HIGH
TEMPERATURES)
Parungao-Balolong 2011
Thursday, July 14, 2011
22. TEMPERATURE RANGE
STENOTHERMAL
MICROBES
◦Narrow range
◦Neisseria gonorrhea
EURYTHERMAL
MICROBES
◦Wide range
◦Enterococcus faecalis
Parungao-Balolong 2011
Thursday, July 14, 2011
23. pH
Most bacteria grow between pH 6.5 and 7.5
Molds and yeasts grow between pH 5 and 6
Acidophiles grow in acidic environments
Parungao-Balolong 2011
Thursday, July 14, 2011
24. REQUIREMENTS FOR
GROWTH: PHYSICAL
Osmotic pressure
Hypertonic
environments,
increase salt or sugar,
cause plasmolysis
Extreme or obligate halophiles require high osmotic
pressure
Facultative halophiles tolerate high osmotic pressure
Parungao-Balolong 2011
Thursday, July 14, 2011
25. REQUIREMENTS FOR
GROWTH: PHYSICAL
WATER SOURCE BACTERIA FUNGI ALGAE
ACTIVITY
1.00 blood Most Gram none none
(pure water) negative and
non-halophiles
0.90 ham Most cocci and Fusarium, Mucor,
Bacillus Rhizopus
0.60 Chocolate none Saccharomyces rouxii none
0.55
(DNA
disordered)
Parungao-Balolong 2011
Thursday, July 14, 2011
26. REQUIREMENTS FOR
GROWTH: PHYSICAL
1atm
BAROTOLERANT
◦Increased pressure does adversely affect them but
not as much as it does non-tolerant bacteria
BAROPHILIC
◦Grow more rapidly at high pressures
TRIVIA: one barophile has been recovered from the
Mariana trench near the Philippines (10, 500m depth)
◦Can only grow at pressure greater than 400-500 atm
(at 2°C)
Parungao-Balolong 2011
Thursday, July 14, 2011
27. REQUIREMENTS FOR
GROWTH: CHEMICAL
Carbon
Structural organic
molecules, energy
source
Chemoheterotrophs use
organic carbon sources
Autotrophs use CO2
Parungao-Balolong 2011
Thursday, July 14, 2011
28. REQUIREMENTS FOR
GROWTH: CHEMICAL
Nitrogen
Trace elements
In amino acids and proteins
Inorganic elements required
Most bacteria decompose proteins
in small amounts
Some bacteria use NH4+ or NO3–
Usually as enzyme cofactors
A few bacteria use N2 in nitrogen fixation
Sulfur
In amino acids, thiamine and biotin
Most bacteria decompose proteins
Some bacteria use SO42– or H2S
Phosphorus
In DNA, RNA, ATP, and membranes
PO43– is a source of phosphorus
Parungao-Balolong 2011
Thursday, July 14, 2011
29. REQUIREMENTS FOR
GROWTH: CHEMICAL
Oxygen (O2)
Parungao-Balolong 2011
Thursday, July 14, 2011
30. REQUIREMENTS FOR
GROWTH: CHEMICAL
Singlet oxygen: O2 boosted to a higher-energy state
Superoxide free radicals: O2–
Peroxide anion: O22–
Hydroxyl radical (OH•)
Parungao-Balolong 2011
Thursday, July 14, 2011
31. LECTURE OUTLINE
Reproduction & Growth
Requirements for Growth
Physical
Chemical
Culture Media
Measurement of Microbial Growth
Obtaining Pure Cultures
Preservation Methods
Parungao-Balolong 2011
Thursday, July 14, 2011
32. Culture medium:
CULTURE Nutrients prepared
MEDIA
for microbial growth
Sterile: No living
microbes
Inoculum:
Introduction of
microbes into
medium
Culture: Microbes
growing in/on
culture medium
Parungao-Balolong 2011
Thursday, July 14, 2011
33. CULTURE MEDIA
TYPES: Chemically-Defined and Complex
Chemically defined media: Exact chemical composition is known
Complex media: Extracts and digests of yeasts, meat, or plants
Nutrient broth
Nutrient agar
Parungao-Balolong 2011
Thursday, July 14, 2011
34. RECALL: HISTORY OF
BEFORE AGAR GELATIN
◦Liquid medium ◦Frederick Loeffler
◦Meat extract
POTATO SLICES medium + gelatin
◦Robert Koch (1881) ◦But gelatin liquid at
◦Used boiled potato, room temperature
sliced
AGAR
◦Not all bacteria grew
well ◦Fannie Eilshemius
Hesse (1882)
◦Agar used for jams
and jelly
Parungao-Balolong 2011
Thursday, July 14, 2011
35. AGAR
Fannie, wife of Walther Hesse, was
working in Koch's laboratory as her
husband's technician and had
previously used agar to
Complex polysaccharide
Used as solidifying agent for culture
media in Petri plates, slants, and deeps
Generally not metabolized by
microbes
Liquefies at 100°C
Parungao-Balolong 2011
Thursday, July 14, 2011
36. ANAEROBIC CULTURE
METHODS
Reducing media
Contain chemicals (thioglycollate or oxyrase) that combine O2
Heated to drive off O2
Parungao-Balolong 2011
Thursday, July 14, 2011
37. ANAEROBIC CULTURE
METHODS
Parungao-Balolong 2011
Thursday, July 14, 2011
38. SELECTIVE MEDIA &
DIFFERENTIAL MEDIA
SELECTIVE: Suppress unwanted microbes and
encourage desired microbes.
DIFFERENTIAL
: Make it easy to
distinguish
colonies of
different
Parungao-Balolong 2011
Thursday, July 14, 2011
39. SELECTIVE MEDIA &
DIFFERENTIAL MEDIA
Parungao-Balolong 2011
Thursday, July 14, 2011
40. ENRICHMENT MEDIA
Encourages growth of desired microbe
used when the population of your target microbe is low
used when your target microbe is damaged
MRS = lactic acid bacteria Lactose Broth = enterics
Parungao-Balolong 2011
Thursday, July 14, 2011
41. LECTURE OUTLINE
Reproduction & Growth
Requirements for Growth
Physical
Chemical
Culture Media
Measurement of Microbial
Growth
Obtaining Pure Cultures
Preservation Methods
Parungao-Balolong 2011
Thursday, July 14, 2011
42. MATHEMATICS OF
GROWTH
GENERATION TIME
◦The time required for a
microbial population to double
in number
MEAN GROWTH RATE
CONSTANT(k)
◦The rate of microbial
population growth expressed in
terms of the number of
generations per unit time
MEAN GENERATION
TIME (g)
Parungao-Balolong 2011
Thursday, July 14, 2011
43. DO THE MATH...
If 100 cells growing for 5 hours produced
1,720,320 cells:
Parungao-Balolong 2011
Thursday, July 14, 2011
44. MATHEMATICS OF
GROWTH
N0 = initial population number
Nt = the population at time t
n = the number of generations in time t
Nt = N0 x 2n
To solve for n:
◦log Nt = log N0 + n ⋅ log 2
◦n = log Nt – log N0 = log Nt – log N0
'' ' log 2'' ' 0.301
Parungao-Balolong 2011
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45. SAMPLE
COMPUTATION
Parungao-Balolong 2011
Thursday, July 14, 2011
46. SAMPLE
COMPUTATION
Given an initial density of 4 x 104
Parungao-Balolong 2011
Thursday, July 14, 2011
47. SAMPLE
COMPUTATION
Given an initial density of 4 x 104
After 2 hours the cell density became 1 x 106
Parungao-Balolong 2011
Thursday, July 14, 2011
48. SAMPLE
COMPUTATION
Given an initial density of 4 x 104
After 2 hours the cell density became 1 x 106
Compute for the generation time
Parungao-Balolong 2011
Thursday, July 14, 2011
49. SAMPLE
COMPUTATION
Given an initial density of 4 x 104
After 2 hours the cell density became 1 x 106
Compute for the generation time
Solution:
Parungao-Balolong 2011
Thursday, July 14, 2011
50. SAMPLE
COMPUTATION
Given an initial density of 4 x 104
After 2 hours the cell density became 1 x 106
Compute for the generation time
Solution:
◦t = 2
Parungao-Balolong 2011
Thursday, July 14, 2011
51. SAMPLE
COMPUTATION
Given an initial density of 4 x 104
After 2 hours the cell density became 1 x 106
Compute for the generation time
Solution:
◦t = 2
◦n = log (1 x 106) – log (4 x 104)
Parungao-Balolong 2011
Thursday, July 14, 2011
52. SAMPLE
COMPUTATION
Given an initial density of 4 x 104
After 2 hours the cell density became 1 x 106
Compute for the generation time
Solution:
◦t = 2
◦n = log (1 x 106) – log (4 x 104)
'' ' ' 0.301
Parungao-Balolong 2011
Thursday, July 14, 2011
53. SAMPLE
COMPUTATION
Given an initial density of 4 x 104
After 2 hours the cell density became 1 x 106
Compute for the generation time
Solution:
◦t = 2
◦n = log (1 x 106) – log (4 x 104)
'' ' ' 0.301
◦n = 4.65
Parungao-Balolong 2011
Thursday, July 14, 2011
54. SAMPLE
COMPUTATION
Given an initial density of 4 x 104
After 2 hours the cell density became 1 x 106
Compute for the generation time
Solution:
◦t = 2
◦n = log (1 x 106) – log (4 x 104)
'' ' ' 0.301
◦n = 4.65
◦Generation time = 2/4.65 or 0.43 hoursParungao-Balolong 2011
(t/n)
Thursday, July 14, 2011
55. GENERATION TIME
MICROORGANISM TEMPERATURE (°C) GENERATION TIME
(hours)
Escherichia coli 40 0.35
Bacillus subtilis 40 0.43
Mycobacterium 37 12
tuberculosis
Euglena gracilis 25 10.9
Giardia lamblia 37 18
Sacharomyces 30 2
cerevisiae
Parungao-Balolong 2011
Thursday, July 14, 2011
56. DIRECT
MEASUREMENTS
Plate counts: Perform serial dilutions of a sample
Direct methods
Plate counts
Filtration
Direct microscopic count
Dry weight
Parungao-Balolong 2011
Thursday, July 14, 2011
57. DIRECT MEASUREMENTS:
Plate Count
Inoculate Petri
plates from serial
dilutions
Parungao-Balolong 2011
Thursday, July 14, 2011
58. DIRECT MEASUREMENTS:
Plate Count
After incubation, count colonies on plates that have
25-250 or 30-300 colonies
report as (CFUs)
Parungao-Balolong 2011
Thursday, July 14, 2011
62. INDIRECT
MEASUREMENTS: MPN
Multiple Tube
Fermentation Test as
measured in MPN or
Most probable Number
Count positive tubes and
compare to statistical
MPN table. Parungao-Balolong 2011
Thursday, July 14, 2011
63. LECTURE OUTLINE
Reproduction & Growth
Requirements for Growth
Physical
Chemical
Measurement of Microbial Growth
Culture Media
Obtaining Pure Cultures
Preservation Methods
Parungao-Balolong 2011
Thursday, July 14, 2011
64. PURE CULTURE
A pure culture contains only one species or strain.
A colony is a population of cells arising from a single cell
or spore or from a group of attached cells.
A colony is often called a colony-forming unit (CFU).
PURE Mixed
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65. OBTAINING PURE
CULTURE: Streak Plating
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66. OBTAINING PURE
CULTURE: Spread Plating
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Thursday, July 14, 2011
67. OBTAINING PURE
CULTURE: Pour Plating
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Thursday, July 14, 2011
68. OBTAINING PURE
CULTURE: Pour Plating
Parungao-Balolong 2011
Thursday, July 14, 2011
69. COLONY
CHARACTERISTICS
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70. OBTAINING PURE
CULTURE: The Essentials
Julius Richard Petri (1887)
Easy to use, stackable (saving space),
requirement for plating methods
Parungao-Balolong 2011
Thursday, July 14, 2011
71. POURING MEDIA ON
YOUR DISHES
Parungao-Balolong 2011
Thursday, July 14, 2011
72. LECTURE OUTLINE
Reproduction & Growth
Requirements for Growth
Physical
Chemical
Measurement of Microbial Growth
Culture Media
Obtaining Pure Cultures
Preservation Methods
Parungao-Balolong 2011
Thursday, July 14, 2011
73. PRESERVATION
METHODS: Long Term
Deep-freezing: –50°to –95°C
Lyophilization (freeze-drying): Frozen (–54° to –72°C) and
dehydrated in a vacuum
Parungao-Balolong 2011
Thursday, July 14, 2011