Basic groups of microorganisms Physical conditions for Cultivation of microorganisms Bacterial Metabolism

7. Physical conditions for
Cultivation of microorganisms

10. Temperature
-microorganisms can grow over a broad range of temperature
*Growth rate – the number of of cell divisions per hour at the most
favorable temperatures
*Optimum growth temperature- the temperature at which a species
of microorganism grows most rapidly
Microorganisms may be divided into three groups on the basis of T in
which they grow best:
1. Psychrophiles- cold-loving microbes
- growth range of 0 to 20o C
- grow best at 15 to 20oC
- at ref T of 4 to 10oC, psychrophilic microbes
spoil food stored for prolonged periods
- bacteria, fungi, algae and protozoa found in
colder waters and soils such as the oceans and
the polar regions.

11. -most marine microorganisms belong to this group
- among the bacteria, many psychrophiles are members
of the genera Pseudomonas, Flavobacterium and
Alcaligenes
2. Mesophiles – moderate-temperature loving microbes
- the largest group
- most microbes are mesophiles
- growing best within a T range of 25 to 40oC
- grow in the lower part of the mesophilic range
(saprophytic bacteria, fungi, algae, & protozoa)
- grow in the upper part of the range (parasitic
microorganisms of humans and animals)
- those that are pathogenic for humans grow best
at about body T which is 37oC. (The elevated T of
a fever may inhibit the growth of some pathogens)
3. Thermophiles- heat-loving microbes
- grow at T from about 40 to 85oC
- they grow best between 50 and 60oC

12. -these microbes maybe found in volcanic areas, compost
heaps and hot springs
-most thermophilic microbes are procaryotes; no eucaryo-
tic cells are known to grow at T greater than 60oC
-several factors enable thermophiles like Bacillus
stearothermophilus to grow at elevated T:
1. their enzymes are produced more rapidly than the
enzymes of mesophiles so that those damaged by
high Ts are quickly replaced.
eg. Archaebacteria which able to grow above
boiling point of water:
Pyrodictium occultum –grow at 110oC
Pyrococcus woesi -104.8oC
Thermococcus celer- 103oC (these microorganisms
have been isolated from sediments near vents on
the ocean floor that spew forth superheated water)
2. ribosomes, membranes and various enzymes in ther-
mophiles fxn better at high T than at lower T.

13. The yellow coloring in the water at the Midway Geyser Basin in Yellowstone
National Park is caused by archaebacteria. Archaebacteria are known to survive
at extremely high temperatures like those produced from a geyser.

16. Gaseous Atmosphere
- some gases are used in cellular metabolism; others are may
have to be excluded from a culture because they are toxic to the cells.
-carbon dioxide and oxygen are the two principal gases that
affect the growth of microbial cells.
-on the basis of their response to gaseous oxygen, microorganisms
are divided into four physiological groups:
a. Aerobes
b. Facultative microorganisms
c. anaerobes
d.microaerophiles
Aerobic microorganisms
- microbes that normally require oxygen for growth
- can grow in a standard air atmosphere of 21% oxygen
- filamentous molds and the bacterial genera Mycobacterium and
Legionella are examples of aerobic microbes
-aerobes acquire more energy frm available nutrients than do
microbes that do not use oxygen

18. - Some aerobes may grow more slowly when O2 is limited so
care must be taken to provide adequate supply of this gas
(rarely a problem for microbes growing on the surface of a
solid medium but in a liquid medium, they rapidly use the O2
dissolved in the surface layer of medium.
To avoid this problem: liquid cultures of aerobic microbes are
sometime agitated on a mechanical shaker to increase the
supply of dissolved O2 and produce a larger cell crop within
a shorter incubation time.
Carbon dioxide
- some cells have specific needs in terms of CO2
-aerobic mammalian cells are best cultivated in an incubator
with a humid atmosphere and a continuous supply of 5% CO2
-some groups of microbes require elevated levels of CO2
eg. Neisseria gonorrhoeae –bacterium that causes
gonorrhea
- grows best in an 5 to 10% CO2
enriched atmosphere

19. Candle jar – an apparatus which is a special incubator that can supply
this atmosphere
How?: 1. after inoculated media are placed inside the jar
2. a candle is lit in the jar
3. lid is tightly closed
4. candle burns until there is no enough O2 to maintain
combustion(the jar atmosphere then contains
a reduced amount of free oxygen (about 17%
oxygen) and an increased carbon dioxide concn
(about 3.5%))
Facultative Microorganisms
- those that grow in an air atmosphere and can also grow
anaerobically
-they do not require O2 for growth, thou they may use it for
energy-yielding chemical reactions
-under anaerobic conditions, they obtain energy by metabolic
process called fermentation

20. -Members of the bacterial family Enterobacteriaceae, such as
Escherichia coli are facultative.
-many yeasts are facultative (e.g. Saccharomyces cerevisiae , the
common baker’s yeast)
Anaerobic microorganisms
-microbes that may be poisoned by O2
-can’t grow in an air atmosphere
- do not use oxygen for energy-yielding chemical reactions
- some anaerobes can tolerate low concn of O2 but strict
anaerobes are killed by brief exposure to the gas
-there is a wide range of O2 tolerance among anaerobes:
a. Highly oxygen-tolerant – Clostridium perfringens
b. Moderately tolerant – Clostridium tetani
c. Strict anaerobes – Methanobacterium and
Methanospirillum

21. -the toxicity of oxygen for strict anaerobes is due to certain molecules
produced during reactions involving oxygen. It produces superoxide
radicals (toxic and may cause damage to cells) which gives rise to hydro-
gen peroxide as an endproduct of superoxide breakdown by superoxide
dismutase (aerobically respiring cells are believed to survive only bec
they possess this enzyme which scavenges the superoxide radical).
*most obligate anaerobes lack superoxide dismutase thus cannot
tolerate the presence of free O2.
(1) O2- + O2- + 2H+ H2O2 + O2
(hydrogen peroxide)
(2) 2H2O2 2H2O + O2 (aerobic bacteria can dissipate this
by catalase or peroxidase but most
obligate anaerobes as well as aero-
tolerant lactic acid bacteria are un-
able to synthesize catalase)

31. Microaerophilic microorganisms
-like aerobes can use O2 for energy-yielding process
-unlike aerobes, can’t withstand the level of oxygen (21%)
present in an air atmosphere
-they usually grow best at oxygen levels bet 1 and 15%
- eg. Campylobacter jejuni (a bacterium that often causse diarrhea
in humans)
-requires low levels of oxygen when isolated from fecal
samples.

33. Oxygen Tolerance
Atmospheric
Type
Superoxide
Dismutase
Catalase
Aerobe
Facultative
Capnophile
(+) (+)
Aerotolerant
Anaerobe
Microaerophile
(+) (-)
Anaerobe (-) (-)

34. pH
- most pathogens grow best at neutral pH (pH 7) or
one that is slightly more alkaline (pH 7.4)
- pH minimum is about 4 with pH maximum for
growth is ph 11
- some species of Bacillus can grow at pH11 while
others are highly tolerant of acidic conditions.
- Thiobacillus species can grow at pH values as low as
0.5 (found in acidic drainage water from mines where
sulfur and iron present)

35. -food spoilage bacteria can’t grow at pH values of 3 to 4.
-molds and yeasts have a broader pH range than do bacteria
(their optimum pH for growth is about pH 5 to 6 (lower than
bacteria))
-optimal pH for growth of protozoa is generally bet 6.7 to
7.7
-algae (from pH 4 to 8.5 for optimal growth)

41. Reproduction in Procaryotic Microorganisms
-most bacteria multiply by asexual production which does not
involve sex cells (gametes)
- in most unicellular procaryotes, the mode of asexual reprodn is
transverse binary fission
-single cell divides into two daughter cells of approximately
equal size
-daughter cells may separate completely but in some
species they remain attached to form characteristic
pairs, clusters or chains

43. Bacterial multiplication by transverse binary fission
Parent cell
Cell elongation
Invagination of cell wall (septum) and
distribution of nuclear material
Formation of transverse cell wall (septum) and organized distribution of cellular
material into two cells
Separation into two identical daughter cells each capable of repeating this process

44. "Filamenting temperature-sensitive mutant Z".

47. e.g. Rhodopseudomonas acidophila
e.g. Nocardia species
e.g. Streptomyces and other
Actinomycetes
conidia

48. Growth of a Bacterial Culture
- follows an exponential or logarithmic increase in bacterial
numbers
1 2 4 8 16 32 ...
This increase may be expressed as a geometric progression:
1 21 22 23 24 ... . 2n
where n = refers to number of generations
Generation time = time interval required for each microbe to divide or
for the population in a culture to double.
- not all species have the same generation time
eg.E. coli – 12.5 min (generation time in a rich medium
Mycobacterium tuberculosis (13 to 15 hr)

51. Mathematical Expressions of Growth
N = 1 x 2n where N is the total, final population
Balanced growth – the exponential growth of a microbial culture
Phases of Growth

52. Growth Phase Growth Rate Characteristics
Lag Zero No increase in cell no.
Individual cells, increase in
size. Cells physiologically
active and synthesizing new
Enzymes to adapt to new
environment.
Exponential or Log Maximal and constant Condition of balanced
growth. Cells, most nearly
uniform in terms of
chemical composition and
metabolic and physiological
activities. Peak time of
physiological activity and
efficiency
Stationary Zero Accumulation of toxic
metabolic products and/or
exhaustion nutrients. Some
cells die while others grow
and divide.Number of viable
cells level off.
Table 5. Characteristics of Growth of a Unicellular Microbial Culture in each Phase of the Typical Growth
Curve

53. Death Negative Further accumulation of
inhibitory metabolic
products and depletion of
nutrients. Death rate
accelerates. Number of
viable cells decreases
exponentially. Depending
on species, a very few living
cells may persist into the
tail of the curve, forming
what may be called a
senescent phase. Typically
all cells normally die within
days to month

58. Synchronous Culture
-cells divide at the same time or synchronously
- a population can be synchronized by changing the physical
environment or the chemical composition of the medium
Continuous culture
-cultivation system which ensure continued new growth,
the culture volume and the cell concentration are both kept
constant by adding fresh, sterile medium at the same rate that used
cell containing medium is removed
-under these conditions, the rate at which new cells are
Produced in the culture vessel is balanced by the rate at which cells
Are being removed as part of the overflow from the vessel

62. Measurement of Population Growth
Method Examples of applications Manner in which growth is
expressed
Microscopic count Cell number in vaccines, milk and
cultures
Number of cells per ml
Electronic cell count (Same as for microscopic count) Number of cells per ml
Plate count Cell number in vaccines, milk,
cultures, soil, foods
Colony-forming units per ml or g
Membrane filter (Same as for plate count) Colony-forming unit per ml or g
Turbidity Microbiological assay, estimation
of cell mass in broth cultures or
other suspensions
Absorbance units
Nitrogen content Indirect measurement of cell
mass
mg nitrogen per ml
Dry weight (Same as for nitrogen content) mg cells per ml
Metabolic product Microbiological assays, indirect
measurement of metabolic
activity (growth)
Amount of product (e.g. Milli-
equivalents of acid) per ml
Table 5.1 Summary of Some Methods for Measuring

65. Serial Dilutions for Counting Bacteria
Dilution 1
1 :100
Dilution 2
1 :10,000
Dilution 3
1 1000,000
99 mL blank
99 mL blank
99 mL blank
Culture of bacteria
1 mL
1 mL
1 mL
1:100
1:1000
1:10000000
1:10000
1:1000000
1:100000
1 mL
1 mL
1 mL
0.1 mL
0.1 mL
0.1 mL
(10-2)
(10-3)
(10-4)
(10-5)
(10-6)
(10-7)

66. Bo = number of bacteria in 1 ml of original sample
D = Dilution factor (for a dilution of 1:10,000, D=10,000)
C = number of colonies counted
Then:
Bo = (D)(C)
Example:
If one plated out a dilution of 1:1000000 and after a
growth counted 78 colonies, what would be the number of
bacteria in the original culture?
Bo = (1000,000) (78)
= 7,800,000 per ml or 7.8 x 106

67. Similarly, one could blend 10g of a frozen meat pie into 90 ml of sterile water and then
dilute this for colony counts to determine the number of bacteria per gram of frozen
pie.
Dilution factor = Amount transferred + amount in bottle
(amount transferred) (amount plated)
For Example, if you transfer 0.1 ml into 99.9 ml of water and plate 0.1 ml,the
dilution factor will be:
0.1 + 99.9 = 10,000
(0.1) (0.1)
If 111 colonies grew from 0.1 ml of this dilution, the original culture contained
(111)(10,000) = 110,000 organisms per mililiter or 1.1 x 105

87. Maintenance of Stock Culture

94. Thank you for listening!