Growth and Culturing

of Bacteria
 Growth and Cell Division
• Microbial Growth Defined: Growth is an orderly increase in
the quantity of cellular constituents.
• It depends upon the ability of the cell to form new protoplasm
from nutrients available in the environment.

1. Mother or parent cell doubles in size

In most bacteria, growth involves increase in cell mass and number
of ribosomes, duplication of the bacterial chromosome, synthesis of
new cell wall and plasma membrane, partitioning of the two
chromosomes, septum formation, and cell division.

2. Divides into two daughter cells

Microbial growth is defined as the increase in the number of

cells, which occurs by cell division.

This asexual process of reproduction is called binary fission.

 Cell Division
• Binary fission (equal cell division): A cell
duplicates its components and divides
into two cells

• Septum: A partition that grows between

two daughter cells and they separate at
this location

• Budding (unequal cell division): A small,

new cell develops from surface of
exisiting cell and subsequently separates
from parent cell
Binary Fission
Thin section of the bacterium Staphylococcus,
undergoing binary fission
Budding in Yeast
 Some Methods used to measure bacterial growth

Method Application Comments

Enumeration of bacteria in milk Cannot distinguish living from
Direct microscopic count
or cellular vaccines nonliving cells
Enumeration of bacteria in milk,
Viable cell count (colony Very sensitive if plating
foods, soil, water, laboratory
counts) conditions are optimal
cultures, etc.
Estimations of large numbers of Fast and nondestructive, but
Turbidity measurement bacteria in clear liquid media cannot detect cell densities less
and broths than 107 cells per ml
Measurement of total N or Measurement of total cell yield only practical application is in
protein from very dense cultures the research laboratory
Measurement of Biochemical Requires a fixed standard to
activity e.g. O2 uptake CO2 Microbiological assays relate chemical activity to cell
production, ATP production, etc. mass and/or cell numbers
Measurement of dry weight or probably more sensitive than
Measurement of total cell yield
wet weight of cells or volume of total N or total protein
in cultures
cells after centrifugation measurements
 Phases of Growth
• Consider a population of organisms
introduced into a fresh, nutrient medium

• Such organisms display four major

phases of growth
1. The lag phase
2. The logarithmic phase
3. The stationary phase
4. The death phase
 The Lag Phase
• Organisms do not increase significantly in
number

• Replication of DNA

• They are metabolically active

• Grow in size, synthesize enzymes, and

incorporate molecules from medium

• Produce large quantities of energy in the form

of ATP
 The Log Phase
• Organisms have adapted to a growth
medium

• Growth occurs at an exponential (log) rate

• The organisms divide at their most rapid

rate

• a regular, genetically determined interval

(generation time)
Synchronous growth: A
hypothetical situation in which
the number of cells in a culture
would increase in a stair-step
pattern, dividing together at the
same rate

Nonsynchronous growth: A
natural situation in which an
actual culture has cell dividing at
one rate and other cells dividing
at a slightly slower rate
 Growth Rate and Generation Time
•Bacterial growth rates during the phase of exponential growth, under
standard nutritional conditions (culture medium, temperature, pH, etc.),
define the bacterium's generation time.

•Generation times for bacteria vary from about 12 minutes to 24 hours

or more. The generation time for E. coli in the laboratory is 15-20
minutes, but in the intestinal tract, the coliform's generation time is
estimated to be 12-24 hours. For most known bacteria that can be
cultured, generation times range from about 15 minutes to 1 hour.

•Symbionts such as Rhizobium tend to have longer generation times.

Many lithotrophs, such as the nitrifying bacteria, also have long
generation times.

•Some bacteria that are pathogens, such as Mycobacterium

tuberculosis and Treponema pallidum, have especially long generation
times, and this is thought to be an advantage in their virulence.
Generation times for some common bacteria under optimal conditions of growth.

Generation Time
Bacterium Medium
(minutes)
Escherichia coli Glucose-salts 17
Bacillus megaterium Sucrose-salts 25
Streptococcus lactis Milk 26
Streptococcus lactis Lactose broth 48
Staphylococcus aureus Heart infusion broth 27-30
Lactobacillus acidophilus Milk 66-87

Rhizobium japonicum Mannitol-salts-yeast extract 344-461

Mycobacterium tuberculosis Synthetic (LJ medium) 792-932

Treponema pallidum Rabbit testes 1980
 Calculation of Generation Time

When growing exponentially by binary fission, the increase in a bacterial

population is by geometric progression. If we start with one cell, when it divides,
there are 2 cells in the first generation, 4 cells in the second generation, 8 cells in
the third generation, and so on. The generation time is the time interval required
for the cells (or population) to divide.

G (generation time) = t (time, in minutes or hours)/ n (number of generations)

G=t/n
G = generation time (time for the cells to divide)
t = time interval in hours or minutes
B = number of bacteria at the beginning of a time interval
b = number of bacteria at the end of the time interval
n = number of generations (number of times the cell population doubles during
the time interval)
b = B x 2n (This equation is an expression of growth by binary fission)
 What is the generation time of a bacterial population that
increases from 10,000 cells to 10,000,000 cells in four hours
of growth?
Solve for n:

Log b = log B + n log2

n = log b – log B
log2

n = log b – log B
0.301

n = 3.3 log b / B
G= t______
3.3 log b / B
G=t/n
G= 240 minutes
Solve for G 3.3 log 107/104

G= t____ G = 240 minutes

3.3 log b / B 3.3 x 3

G = 24 minutes
Microbes growing continuously in a chemostat
• Stationary Phase:

1. Cell division decreases to a point that new cells are

produced at same rate as old cell die.

2. The number of live cells stays constant.

3. (up to 109 cells/ml)

One bacterium in 11 hours can generate 109 cells

• Decline (Death) Phase:

1. Condition in the medium become less and less

supportive of cell division
2. Cell lose their ability to divide and thus die
3. Number of live cells decreases at a logarithmic rate
 Serial Dilution and Standard Plate
Counts
• Standard plate count: One method of
measuring bacterial growth

• Agar plate: A petri dish containing a

nutrient medium solidified with agar

• Serial dilutions are used to dilute the

original bacterial culture before you
transfer known volume of culture onto
agar plate
Serial Dilution
 Calculation of the number of bacteria per milliliter of culture
using serial dilution

Pour plate: made by

first adding 1.0ml of
diluted culture to 9ml of
molten agar

Spread plate: made by

adding 0.1ml of diluted
culture to surface of
solid medium
Counting colonies using a bacterial colony counter
Bacterial colonies viewed through the magnifying
glass against a colony-counting grid
Countable number of colonies
(30 to 300 per plate)

Which of these plates would be the

correct one to count? Why?
 Direct Microscopic Counts

• Another way to measure bacterial growth

• Petroff-Hausser counting chamber

• Bacterial suspension is introduced onto chamber with

a calibrated pipette

• Microorganisms are counted in specific calibrated

areas

• Number per unit volume is calculated using an

appropriate formula
The Petroff-Hausser Counting Chamber
 Most Probable Number (MPN)
• Method to estimate number of cells

• Used when samples contain too few

organisms to give reliable measures of
population size by standard plate count

• Series of progressively greater dilutions

• Typical MPN test consists of five tubes of

each of three volumes (e.g. 10, 1, and 0.1ml)
A MPN test: those tubes in which gas bubbles are
visible (labeled +) contain organisms

Used only when the bacterial

cells are very few in number, so
that they can’t be counted by
plating method.

The number of organisms in the

original culture is estimated
from a most probable number
table.

Here in this case the MPN is 50/

dL
 Positive carbohydrate fermentation test

CO2

+ Gas/+Acid + Acid - No Acid or Gas

Turbidity, or a cloudy appearance, is an indicator of
bacterial growth in urine in the tube on the left
A Spectrophotometer: This instrument can be used to
measure bacterial growth by determining the degree of
light transmission through the culture
Factors Affecting Bacterial Growth
• The kinds of organisms found in a given
environment and the rates at which they grow can
be influenced by a variety of factors, both physical
and biochemical

• Physical factors include: pH, temperature, oxygen

concentration, moisture, hydrostatic pressure,
osmotic pressure, and radiation

• Nutritional factors include: availability of carbon,

nitrogen, sulfur, phosphorus, trace elements and,
in some cases, vitamins
 pH
• Optimum pH: the pH at which the
microorganism grows best (e.g. pH 7)

• According to their tolerance for

acidity/alkalinity, bacteria are classified
as:

1. Acidophiles (acid-loving): grow best at pH 0.1-5.4

2. Neutrophiles: grow best at pH 5.4 to 8.0
3. Alkaliphiles (base-loving): grow best at pH 7.0-11.5
 Temperature
• Obligate: organism must have specified
environmental condition

• Facultative: organism is able to adjust to and

tolerate environmental condition, but can also live
in other conditions

• According to their growth temperature range,

bacteria can be classified as:
1. psychrophiles: 15-20oC
2. Mesophiles: 25-40oC
3. Thermophiles: 50-60oC
Thermophiles: Thermophilic sulfur bacteria can live and
grow in the runoff waters from such geysers despite the near-
boiling temperatures
Growth rates of psychrophilic, mesophilic, and thermophilic
bacteria
 Oxygen
• Aerobes: require oxygen to grow
• Obligate aerobes: must have free oxygen for aerobic
respiration (e.g. Pseudomonas)
• Anaerobes: do not require oxygen to grow
• Obligate anaerobes: killed by free oxygen (e.g. Bacteroides)
• Microaerophiles: grow best in presence of small amount of
free oxygen
• Capnophiles: carbon-dioxide loving organisms that grow
best under conditions of low oxygen
• Facultative anaerobes: carry on aerobic metabolism when
oxygen is present, but shift to anaerobic metabolism when
oxygen is absent
• Aerotolerant anaerobes: can survive in the presence of
oxygen but do not use it in their metabolism
Patterns of Oxygen Use
 Hydrostatic Pressure

• Water in oceans and lakes exerts pressure

exerted by standing water, in proportion to
its depth

• Pressure doubles with every 10 meter

increase in depth

• Barophiles: bacteria that live at high

pressures, but die if left in laboratory at
standard atmospheric pressure
 Osmotic Pressure

• Environments that contain dissolved

substances exert osmotic pressure, and
pressure can exceed that exerted by
dissolved substances in cells

• Hyperosmotic environments: cells lose water

and undergo plasmolysis (shrinking of cell)

• Hypoosmotic environment: cells gain water

and swell and burst
 Halophiles
• Salt-loving organisms which require moderate to
large quantities of salt (sodium chloride)

• Membrane transport systems actively transport

sodium ions out of cells and concentrate
potassium ions inside

• Why do halophiles require sodium?

1. Cells need sodium to maintain a high intracellular
potassium concentration for enzymatic function
2. Cells need sodium to maintain the integrity of their
cell walls
Responses to Salt
 The Great Salt Lake in Utah

•Bacteria found here are

Gram-negative and they
lack cell wall peptidoglycan.

•They are insensitive to

most of the antibiotics
used.

•Obligate aerobes.

•Unlike green plants,

pigments they use for light-
dependent ATP synthesis
are red-orange carotinoids
and red-purple
Water here is 10 times saltier than the ocean
bacterioruberins and
bacteriorhodopsins.
 Nutritional Factors

1. Carbon sources
2. Nitrogen sources
3. Sulfur and phosphorus
4. Trace elements (e.g. copper, iron,
zinc, and cobalt)
5. Vitamins (e.g. folic acid, vitamin B-
12, vitamin K)
A USDA scientist working
on his microbial brew – a
mix of some 80 ingredients
to support growth of
nutritionally fastidious
spiroplasmas.
Fastidious means
organisms have special
nutritional requirements
that can be difficult to meet
in the laboratories.
Such as Nisseria gonorrhoae,
mycobacterial lapre, Chlamydial spp.
 Spiroplasma spp.
responsible for hundreds of crop and animal diseases
 Locations of Enzymes
• Exoenzymes: production of enzymes that are
released through cell or plasma membrane. These
are mainly hydrolases, they add water as they split
large molecules. Namely, Amylase, cellulase,
sucrase, maltase, lactase, lipases, proteases,
caesinase, gelatinase etc.

• Extracellular enzymes: usually produced by gram-

positive rods, which act in the medium around the
organism.

• Periplasmic enzymes: usually produced by gram-

negative organisms, which act in the periplasmic
space
 Sporulation
• The formation of endospores, occurs in
Bacillus, Clostridium and a few other gram-
positive genera

• Protective or survival mechanism, not a

means of reproduction

• As endospore formation begins, DNA is

replicated and forms a long, compact, axial
nucleoid
• Core (living part of endospore): most of cell’s RNA and some
cytoplasmic protein molecules gather around DNA

• Dipicolinic acid: contained in the core along with calcium ions

• Endospore septum: grows around the core, enclosing it in a

double thickness of membrane

• Cortex: laminated layer forms when peptidoglycan is released

into space between endospore septum membranes

• Spore coat: keratin-like protein, impervious to chemicals is laid

down around the cortex

• Exosporium: found in some endospores, a lipid-protein

membrane formed outside the coat
Vegetative and Sporulation Cycles in Bacteria capable
of Sporulation
 Germination

• A spore returns to its vegetative

state, occurs in three stages:

1. Activation
2. Germination proper
3. Outgrowth
Bacterial endospores in two Clostridium species
•Things to remember

•Grow in liquid culture or on solid agar plates -

can isolate pure culture by streaking

•Culture - bacterial population in liquid

•Inoculum - starting source of culture

 Culturing Bacteria
• Culturing of bacteria in the laboratory
presents two problems:

1. A pure culture of a single species is

needed to study an organism’s
characteristics
2. A medium must be found that will
support growth of the desired organism

• Pure culture: a culture that contains

only a single species of organism
The Streak Plate Method uses agar plates to
prepare pure cultures
A Streak Plate of Serratia marcescens. Note the greatly
reduced numbers of growth /colonies in each successive region
Various colony morphologies

Top view

Lateral view
 Types of Culture Media
• Natural Media: In nature, many species of
microorganisms grow together in oceans, lakes,
and soil and on living or dead organic matter

• Synthetic medium: A medium prepared in the

laboratory from material of precise or reasonably
well-defined composition

• Complex medium: contains reasonably familiar

material but varies slightly in chemical
composition from batch to batch (e.g. peptone, a
product of enzyme digestion of proteins)
 Commonly Used Media
• Yeast Extract

• Casein Hydrolysate

• Serum

• Blood agar

• Chocolate agar
 Selective, Differential, and Enrichment
Media
• Selective medium: encourages growth of some
organisms but suppresses growth of others
(e.g. antibiotics)

• Differential medium: contains a constituent that

causes an observable change (e.g. MacConkey agar)

• Enrichment medium: contains special nutrients that

allow growth of a particular organism that might not
otherwise be present in sufficient numbers to allow it
to be isolated and identified
 Rich vs minimal media

Minimal - no organics other than C source; have Na, K, Mg, Ca, Fe,
NH4, Cl, PO4, SO4

Selective media - identify auxotrophs and antibiotic resistant

bacteria

Color-indicator plates - identify which sugars can be used as C

source

MacConkey agar - contains sugar and pH sensitive dye (red in

low pH, white in high pH) If add lactose, Lac+ cells grow, ferment
lactose, decrease pH and stain colony red. Lac- cells grow, use amino
acids as C source, breakdown products include ammonia which
increases pH, decolorizes dye and colony is white.
Three species of Candida can be differentiated in mixed
culture when grown on CHROMagar Candida plates
Identification of urinary tract pathogens with
differential media (CHROMagar)
Candle Jar culture of anaerobes and microaerophiles
To culture obligate
anaerobes, all molecular
oxygen must be removed
and kept out of medium.
Agar plates are
incubated in sealed jars
containing chemical
substances that remove
oxygen and generate
carbon dioxide or water
Anaerobic Transfer
 Preserved Cultures
• To avoid risk of contamination and to reduce
mutation rate, stock culture organisms should
be kept in a preserved culture, a culture in
which organisms are maintained in a dormant
state
1. Lyophilization
2. Frozen at -70oC
3. Refrigeration

• Reference culture (type culture): a preserved

culture that maintains the organisms with
characteristics as originally defined
 Methods of Performing Multiple Diagnostic
Tests

1. The Enterotube System-

used to identify enteric pathogens or
organisms cause intestinal diseases such as
typhoid, paratyphoid fevers, shigellosis,
gastroenteritis and some kind of food
poisoning.

1. The Analytical Profile Index (API) System

The Enterotube Multitest System
Sum for E. coli 36601 and sum for Klebsiella pneumonae is 34363
The API System: Various species of Enteobacteriaceae are shown
here with differences in reactions that enable them to be identified
Cocci Bacilli/Coccobacilli
Neisseria Aerobic
+ -

Growth on SBA
+ -
Bacteroides
Oxidase
+ -
Haemophilus Legionella
Glucose fermented Lactose Fermented
+ - + -
Vibrio Brucella Escherichia Salmonella
Bordetella Klebsiella Shigella
Campylobacter Proteus
Pseudomonas Yersinia
Sterilization
and
Disinfection
Sterilization and
Disinfection
• Disinfectant: Typically chemical agents
that are applied to inanimate objects

• Antiseptics: Typically chemical agents

that are applied to living tissues
• Sterilization: The killing or removal of
all living cells, viable spores, viruses in a
material or on an object

• Sterility: there are no living organisms

in or on an object

• Disinfection: The reduction of the

number of pathogenic microorganisms to
the point that they pose no danger of
disease