MICROBIOLOGY

BACTERIAL GROWTH

Instructor
Terry Wiseth

Northland Community & Technical College

 BACTERIAL GROWTH
Culture
Increase in the population of cells
Generation time
the time it takes to divide (double) is
called

2
 BINARY FISSION
division exactly in half
most common means of bacterial
reproduction
forming two equal size progeny
genetically identical offspring
cells divide in a geometric
progression doubling cell number

3
 BINARY
FISSION
Doubling time is
the unit of
measurement of
microbial growth
 CULTURE GROWTH
Growth of culture goes through four
phases with time
1) Lag phase
2) Log or Logarithmic
phase

3) Stationary phase

4) Death or Decline
phase

5
BACTERIAL GROWTH CURVE
 LAG PHASE
Organisms are adjusting to the
environment
little or no division Mouse click for lag
phase adjustment

synthesizing DNA, ribosomes and

enzymes
in order to
breakdown
nutrients, and to
be used for growth

7
 LOGARITHMIC PHASE
Division is at a constant rate
(generation time)
Cells are most susceptible to inhibitors

8
 STATIONARY PHASE
Dying and dividing organisms are at an
equilibrium
Death is due to reduced nutrients, pH
changes, toxic waste and reduced
oxygen
Cells are smaller and have fewer
ribosomes
In some cases cells do not die but they
are not multiplying

9
STATIONARY PHASE

10
 DEATH PHASE
The population is dying in a geometric
fashion so there are more deaths than
new cells
Deaths are due to
1) factors in stationary phase
2) lytic enzymes that are released
when bacteria lyse

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DEATH PHASE

12
 ENUMERATION OF BACTERIA
1) viable plate count
2) direct count
3) most probable number (MPN)

4
1 5 9
10
3 6 8
2

13
MEASURING MICROBIAL
GROWTH
DIRECT VS INDIRECT
VIABLE VS TOTAL
 BACTERIAL GROWTH
Culture
Increase in the population of cells
Generation time
the time it takes to divide (double) is
called

15
 DIRECT MEASUREMENTS
DIRECT MICROSCOPIC COUNT
PLATE COUNT
MOST PROBABLE NUMBER
 DIRECT COUNT
Dilutions of samples are observed
under a microscope
the number of bacterial cells from a
given volume of sample
are counted
dead cells are also
counted
automated particle
counters can be used

17
DIRECT COUNT
DIRECT COUNT
Cell density is determined
by:
1) Counting the number of
bacteria in one small
square
2) Multiplying by the dilution
factor of the sample

Dilution factor of 107

Number of bacteria in
square = 5
5 X 107 = 50,000,000
bacteria per ml
 PLATE COUNT
POUR PLATE
SERIAL DILUTIONS
COUNT PLATES BETWEEN 3O AND 300
COLONIES
ADVANTAGES DISADVANTAGES
EXTREMELY SAMPLING
SENSITIVE ERRORS
DOES NOT NEED TIME CONSUMING
COMPLICATED TEDIOUS
EQUIPMENT LARGE NUMBERS
MUST BE
COUNTED TO
REDUCE
SAMPLING ERROR
 VIABLE PLATE COUNT
Most common procedure for assessing
bacterial numbers
1) serial dilutions of a suspension of
bacteria are plated and incubated

22
 VIABLE PLATE COUNT
2) the number of colonies developing
are then counted
it is assumed that each colony
arises from an individual bacterial
cell

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 VIABLE PLATE COUNT
3) by counting the colonies and
taking into account the dilution
factors the concentration of bacteria
in original sample can be determined
4) only plates having between 30 and
300 colonies are used in the
calculations

See next slide for

bigger diagram

24
VIABLE PLATE COUNT
 VIABLE PLATE COUNT
5) multiply the number of colonies
times the dilution factor to find the
number of bacteria in the sample
Example
Plate count = 54

Dilution factor = 1:10,000 ml

Calculation

54 X 10,000 = 540,000 bacteria/ml

26
 VIABLE PLATE COUNT
TNTC
if the number of colonies is too great
(over 300) the sample is labeled TNTC
Too Numerous To Count
limitation of viable plate count
selective as to the bacterial types that
will grow given the incubation
temperature and nutrient type

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 VIABLE PLATE COUNT
Dilution factor of Click to
1/1,000 (10 -3) incubate

417 colonies
TNTC
 VIABLE PLATE COUNT
Dilution factor of Click to
1/1,000,000 (10 -6)
incubate

22 colonies Too few the

count is less
than 30
 VIABLE PLATE COUNT
Dilution factor of Click to
1/100,000 (10 -5) incubate

42 colonies

Calculate the number

of bacteria per ml
 VIABLE PLATE COUNT
Calculate:

42 colonies

dilution factor of 100,000

42 X 100,000 = ???

4,200,000 bacteria/ml

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 MOST PROBABLE NUMBER
A SINGLE LIVE
CELL CAN GIVE
RISE TO A TURBID
CULTURE
SERIAL
DILUTIONS
USES
STATISTICAL
TABLE
ADVANTAGES DISADVANTAGE
ALLOW COUNTS TIME
OF MICROBES CONSUMING
THAT ARE TEDIOUS
DIFFICULT TO
GROW ON SOLID
MEDIA
CAN BE USED TO
COUNT CELLS IN
MIXED LIQUID
CULTURE
 MOST PROBABLE NUMBER
Statistical method based on probability
theory
multiple serial dilutions are performed
to reach a point of extinction
dilution level at which no cells are
deposited

34
 MOST PROBABLE NUMBER
Criteria have been developed for
indicating whether a dilution tube has
bacteria present
the pattern of
positive and
negative results
are compared
with a table of
statistical
probabilities for
obtaining those
results
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MOST PROBABLE NUMBER
The pattern to
tubes that show
growth (brown) and
those that do not
(orange) are
compared with a
statistical table to
calculate MPN
 MOST PROBABLE NUMBER

MPN of the original sample is 170 per 100 mL

Fig. i7.6
 INDIRECT MEASUREMENTS
MEASURE PROPERTIES OF
POPULATION
TURBIDITY
DRY WEIGHT
METABOLIC ACTIVITY
 TURBIDITY

NUMBER IS
PROPORTIONAL TO
WEIGHT OF A SAMPLE
SPECTROPHOTOMET
ER
Turbidity
 USE OF THE
SPECTROPHOTOMETER
ESTIMATES MASS
OF DENSE
CULTURES
CHARTS GROWTH
COMPARED TO
STANDARD
GROWTH CURVE
ADVANTAGES| DISADAVANTAGES
RAPIDITY CAN BE USED ONLY ON
REPRODUCIBLE DENSE CULTURES
DOES NOT DISTINGUISH
BETWEEN LIVING AND
DEAD CELLS
CAN NOT BE USED ON
CELLS THAT AGGREGATE
NEED STANDARD CURVE
 DRY WEIGHT

CENTRIFUGATION OR
FILTRATION
DRYING IN OVEN AT
105 DEGREES C FOR
24 HOURS
Dry Weight
ADVANTAGES DISADVANTAGES

USED TO MAKE TIME CONSUMING

STANDARD TEDIOUS
CURVE FOR SAMPLE MUST
MEASURING CELL CONTAIN
MASS MORETHAN 10
ACCURATE MILLION CELLS
REPRODUCIBLE
 METABOLIC ACTIVITY
RATE OF
METABOLITE
PRODUCTION
UTILIZATION OF
SUBSTRATE
REDUCTION OF
DYES
Metabolic
Activity
ADVANTAGES DISADVANTAGES

CAN BE USED INDIRECT

WITH COMPLEX MEASURMENT
MEDIA SUCH AS DOES NOT GIVE
MILK OR SOIL AN ACCURATE
NO MEASUREMENT
INSTRUMENTS TIME CONSUMING
REQUIRED
 FACTORS INFLUENCING BACTERIAL
GROWTH
Rates of growth and death are greatly
influenced by environmental
parameters
each bacterial species has a specific
tolerance range for specific
environmental factors
outside this range of environmental
conditions bacteria lose their viability
ability to reproduce

50
 FACTORS INFLUENCING BACTERIAL
GROWTH
1) Nutrition
2) Temperature
3) Oxygen
4) Salinity
5) pH
6) Pressure
7) Radiation

51
 NUTRITION
Basic bacterial requirements
water
carbon
nitrogen
other

52
 WATER
Used to dissolve materials to be
transported across the cytoplasmic
membrane

53
 CARBON
required for the construction of all
organic molecules
autotrophs use inorganic carbon
(CO2) as their carbon source
heterotrophs use organic carbon

54
 NITROGEN
Obtained from
inorganic source
e.g. Nitrogen gas (N2), Nitrate
(NO3), Nitrite(NO2), and Ammonia
(NH3)
organic source
e.g. Proteins, broken down to
amino acids
Many organisms use nitrogen gas by
nitrogen fixation to produce ammonia

55
 OTHER NUTRIENTS
Required in small amounts are
Iron
Sulfur
Phosphorus

56
 TEMPERATURE
One of the most important factors
optimal growth temperature
temperature range at which the
highest rate of reproduction occurs
optimal growth temperature for human
pathogens ????

57
 TEMPERATURE
Microorganisms can be categorized
based on their optimal temperature
requirements
Psychrophiles
0 - 20 C
Mesophiles
20 - 40 C
Thermophiles
40 - 90 C
Most bacteria are mesophiles
especially pathogens that require 37 C

58
 BACTERIAL TEMPERATURE
REQUIREMENTS

100
Psychrophile Thermophile
% Max 50
Growth Mesophile

0
0 C 37 C 90 C
0 0 0

Variable
 BACTERIAL
TEMPERATURE
REQUIREMENTS
 TEMPERATURE
Psychrophiles
some will exist below 0 oC if liquid
water is available
oceans
refrigerators
freezers

Pigmented bacteria
in Antarctic ice

61
 TEMPERATURE
Mesophiles
most human flora
and pathogens

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 TEMPERATURE
Thermophiles
hot springs
effluents from laundromat
deep ocean thermal vents

63
 OXYGEN
Required for aerobic respiration and
energy production
Organisms are classified according to
their gaseous requirements
Obligate aerobes
Facultative anaerobes
Obligate anaerobes

64
 OXYGEN
Obligate aerobic
grow only when oxygen is available

Obligate anaerobic
grow in the absence of oxygen

Facultative anaerobe
require oxygen but exhibit maximal
growth rates at reduced oxygen
concentrations
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 OXYGEN

Obligate Facultative Obligate

Aerobe Anaerobe Anaerobe
 SALINITY
Halophiles
bacteria that specifically require
NaCl for growth
Moderates
grow best at 3% NaCl solution
many ocean dwelling bacteria
Extreme
grow well at NaCl concentrations of
greater than 15%
salt lakes, pickle barrels
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 SALINITY

Halophiles growing within salt lakes

often turn the water pink
this sometimes occurs in Great Salt
Lake, Utah

68
 SALINITY
Staphylococcus are salt tolerant up to
concentrations of 10% NaCl
grow on surface of skin

69
 BACTERIAL pH REQUIREMENTS

microbes have different optimum pH

requirements
ACIDOPHILES
some bacteria can grow in acid
substrates
NEUTROPHILES
most microbes prefer a pH near
neutrality
ALKALINOPHILES
microbes which can grow in very
alkaline substrates
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BACTERIAL pH REQUIREMENTS
 BACTERIAL pH
REQUIREMENTS
BACTERIAL pH REQUIREMENTS
 PRESSURE

Osmotic Pressure
exerted on the plasma membranes due
to solute concentrations of a solution
osmotolerant
bacteria able to grow in solutions
with a high solute concentration

74
 PRESSURE

Hydrostatic Pressure
exerted by the weight of water

barotolerant
bacteria able to grow at deep
ocean depths

75
 RADIATION
Photosynthetic microorganisms
require light at minimum levels of
intensity and proper wavelengths
exposure to light can cause the death of
some microorganisms
some bacteria will produce pigments
that protect them from exposure to
lethal effects of light

76
 RADIATION
Ultraviolet radiation is harmful to the
DNA of bacteria
causes abnormalities in cell growth
and division

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 END
BACTERIAL GROWTH

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 FILTRATION
PREPARATION
FOR OTHER
METHODS