Bacteriology

The Study of Bacteria

Topics

Bacterial Ultrastructures
Biofilms
Bacterial Metabolism
Bacterial Sporolation
Bacterial Reproduction and Growth Kinetics
Bacterial Cultivation
Environmental Factors of Growth
Detection, Identification and Characterization

Bacteriology

Biofilms

Definition

A biofilm is any group of microorganisms in which cells stick

to each other on a surface.
These adherent cells are frequently embedded within a selfproduced matrix of extracellular polymeric substance (EPS).
Factors
which may include cellular recognition of specific or non-specific
attachment sites on a surface
nutritional cues
in some cases, by exposure of planktonic cells to sub-inhibitory
concentrations of antibiotics.
When a cell switches to the biofilm mode of growth, it
undergoes a phenotypic shift in behavior in which large suites of
genes are differentially regulated.

Biofilm Formation

There are five stages of biofilm

development (see illustration at
right):

Initial attachment:
Irreversible attachment:
Maturation I:
Maturation II:
Dispersion:

Topics

Bacterial Ultrastructures
Biofilms
Bacterial Metabolism
Bacterial Sporolation
Bacterial Reproduction and Growth Kinetics
Bacterial Cultivation
Environmental Factors of Growth
Detection, Identification and Characterization

Bacteriology

Microbial Growth

Microbial Growth

Increase in cell number

Binary Fission

Conditions for
bacterial
growth

There are five main conditions for bacterial

growth
Temperature
Moisture
Time
Ph level
Oxygen

Temperature

Bacteria grow best at 37C which is body

temperature. They reproduce quickly between
3C and 63C which is the danger zone. Cook
chilled food should be stored at 0C to 3C.
To control temperature food should be
Cooled to below 3C
Heated to above 63C

Key temperatures

Above 121C all bacteria and spores are killed

72C temperature of reheated food
63C Food should be heated above this
temperature
3-63C danger zone for bacterial growth
-18C temperature of freezer
-22 temperature to freeze food.

Temperature

Psychrophiles
Mesophiles
Thermophiles

Psychrophiles: Cold
Loving

True Psychrophiles
sensitive to temperature lower than 20C
Optimum temp at 15C

Psychotrophs
Between 20 30C
Responsible for most low temperature food
spoilage

Mesophiles Middle
Loving

Most pathogens and common spoilage

organisms
25 40C
Optimum Tem: 37C
Adapted to live in the bodies of animals

Thermophiles: Heat
Loving

50 60C
Cannot grow below 45C
Adapted to live in sunlit soil, compost piles,
and hot springs
Extreme Thermophiles: Archaebacteria

Temperature

Moisture

Bacteria like moist

conditions. Many foods
contain liquid
Controls for moisture
Dehydration-removing the
water like in dried milk
High sugar contentmakes less water
available like in jam
Salt removes water by
osmosis like in bacon.
Freezing turns water into
solids

Time

Bacteria multiply
rapidly. One bacterium
can become one
million in less than
seven hours.
To control bacteria
multiplying you should
Eat food as soon after
it is made
Cool quickly and store
in a fridge or freezer.

PH Level

Bacteria grow best in a

neutral PH between 6.6
and 7.5. They cannot
survive below ph 4.5.
Ph levels can be
controlled by acidity
regulators which keep
food below ph4.5.
Vinegar has a ph 3.5
and is used to preserve
foods like onions

PH Levels

Acidophiles
Neutrophiles
Alkaliphiles

Acidophiles

0.1 5.4
Lactobacillus

Neutrophiles

5.4 8.5
Includes most human pathogens

Alkaliphiles

7 12 or higher
Vibrio cholerae and Alkaligenes faecalis pH 9
Agrobacterium pH 12

Oxygen

Some bacteria but not

all need oxygen to
reproduce.
To prevent bacteria
getting oxygen
manufacturers use
vacuum pack foods of
a system known as
MAP where the oxygen
is removed and
replaced with a less
active gas.

Oxygen
Requirements

Obligate Aerobes require oxygen to live

Pseudomonas

Facultative Anaerobes
Can use oxygen, but can grow in its absence
E. coli, Staphylococcus, yeasts, many intestinal
bacteria

Obligate Anaerobes
Cannot use oxygen and are harmed by the
presence of Oxygen
Clostridium

Growth in thioglycollate broth reveals oxygen

preferences

Oxygen gradient

Obligate
aerobes

Faultative
anaerobes

Obligate
anaerobes

Aerotolerant
anaerobes

Microaerophiles

Resazurin
dye is red
in the
presence of
oxygen

Thyoglycollate binds molecular oxygen, reducing it and removing it:

R-SH + O2 R-SO2

Anaerobic and Low O2 Culture

Methods

Candle jar

Brewer or anaerobic jar

CO2 packet

Osmotic Pressure

80 90% water
Hypertonic Solutions used to control
spoilage and microbial growth
Hypotonic Solution microbe may lyse or
burst if cell wall is weak

Osmotic Pressure

Halophiles
Moderate to large salt concentrations
Ocean Bacteria (3.5% salt)

Extreme or Obligate Halophiles

20 30%
Bacteria in the Dead Sea

Facultative Halophiles
Do not require high salt concentrations for
growth
But tolerate 2% or more

Chemical
Requirements

Carbon
Nitrogen, Sulfur, and Phosphorous

Carbon

Makes up 50% of dry weight of cell

Chemoheterotrops carbon from their energy
Source: lipids, proteins, and carbohydrates

Chemoautotrophs and Photoautotrops

Carbon from Carbon dioxide

Nitrogen, Sulfur, and

Phosphorous

Nitrogen

14% of dry cell weight

Protein
Ammonium
Nitrogen Gas
Nitrates

Sulfure

Used to form proteins and some vitamins

Sources
Protein
Hydrogen Sulfide
Sulfates

Phosphorous

Source: Mainly inorganic phosphate salts and

buffers

Other elements

Calcium
Required for the synthesis in Gram positive
bacteria

Trace Elements
Used as enzyme cofactors
Commonly found in tap water

Iron
Copper
Molybdenum
Zinc

Topics

Bacterial Ultrastructures
Biofilms
Bacterial Metabolism
Bacterial Sporolation
Bacterial Reproduction and Growth Kinetics
Bacterial Cultivation
Environmental Factors of Growth
Detection, Identification and Characterization

Bacteriology

Bacterial Reproduction

Bacterial
Reproduction

Introduction

Bacteria can reproduce in one of two ways:

1. Asexually
2.Sexually

Asexual
Binary
Fission:
Reproduction

one parent involved

offspring are identical to parent & each other
Advantages:
1. simple: only 1 parent
2. offspring are fully formed (no maturation
needed)
3. very fast (20 min. in ideal conditions)
after 24 hrs:
1 bacterium
2x106 kg of
cells
(enough to cover the earth)

Asexual Reproduction

Disadvantages:
no genetic variety
one unfavourable environmental condition
can wipe out whole population

Asexual
Reproduction
Binary Fission:

Sexual Reproduction

two parents involved

offspring are genetically different
to parents & to each other
Advantages:
genetic variety i.e. some are able to adapt to
unfavourable conditions (ex: antibiotic resistance)
Disadvantages:
1. more complex: slower because must find a
compatible partner

2. no new individuals produced

(i.e. no increase in population)

Bacterial Conjugation

donor recipient
cell (+)
cell ( - )

plasmi
d

plasmid copies itself

passes through pili
(cytoplasmic bridge) into
recipient cell
cells separate with both
cells containing the
plasmid

pil
i

Mechanisms That Produce Variation

If binary fission produces clonal offspring, why are

bacteria so genetically diverse???
Two factors contribute to genetic diversity among and
within bacterial species
Mutation
Recombination

Mutation

A mutation is a change in the DNA of a gene, ultimately

leading to genetic diversity

Mutations can be spontaneous or caused by mutagens

Spontaneous - Errors during DNA replication
Mutagens - Chemical or physical factors that damage DNA

Spontaneous mutations are extremely rare, occurring on

average only once in 10 million cell divisions, per gene
Because bacteria divide rapidly & exponentially, mutation is
a relevant factor generating genetic diversity

Genetic Recombination

In sexually reproducing organisms this is the main way

The combining of DNA from two sources

genetic variation is produced

In eukaryotes, it involves the sexual processes of meiosis and

fertilization
In prokaryotes three other processes are used transformation,
transduction, and conjugation (= bacterial sex)
Results in horizontal gene transfer the transfer of genetic
material within a generation, instead of from one generation to
the next a major force in the long-term evolution of bacteria

Conjugation
The direct transfer of genetic material between two bacteria
cells that are temporarily joined
DNA transfer is one-way, from male to female
The donor (male) uses an appendage called the sex pilus that forms a
cytoplasmic mating bridge
DNA gets transferred via this bridge in the form of a plasmid
The plasmid encodes the ability to mate as well as other traits such as
antibiotic resistance

Transformation

The alteration of a bacterial cells genotype and phenotype

by the uptake of naked, foreign DNA from the surrounding
environment
Many bacteria possess cell surface proteins that facilitate
transformation in natural populations
E. coli is used in biotechnology applications of genetic
recombination (genetic engineering)
Cells are cultured in high CaCl2 to become competent
Cells are then transformed with human genes that code for proteins
such as insulin or growth hormone that are needed in large amounts

Transduction
Phages (viruses that infect
bacteria) carry bacterial
genes from one host cell to
another as a result of
mistakes in the phage
reproductive cycle
In the process called
generalized transduction,
this transfer is random
Figure 18.16!

Bacterial Populations Evolve

Rapidly

Natural selection operates on genetic (heritable) variation, such

as is generated readily by mutation in bacteria

A mutation that confers a reproductive advantage increases in

frequency in subsequent generations, and eventually becomes
fixed in the population
Bacteria reproduce quickly and therefore have a short
generation time relative to most other organisms
The rapid evolution of antibiotic resistance in bacteria is a
medically important example of natural selection at work

Bacteriology

Growth Curve

Growth Kinetics

Growth patterns and kinetics in batch culture

- growth phases
In batch culture:
- lag phase
- logrithmic or exponential growth phase
- deceleration phase
- stationary phase
- death phase

Typical growth curve for a bacterial population

Batch Growth Kinetics

Lag phase

A period of adaptation for the cells to

their new environment
New enzymes are synthesized.
A slight increase in cell mass and volume, but no
increase in cell number
Prolonged by low inoculum volume, poor inoculum
condition (high % of dead cells), age of inoculum, nutrientpoor medium
Multiple lag phases: (diauxic growth) medium contains
more than one carbon source

Diauxic growth

Typical growth curve for a bacterial population

Typical growth curve for a bacterial population

Batch Growth
Kinetics

Exponential growth phase

In this phase, the cells have adjusted to their
new environment and multiply rapidly
(exponentially)
Balanced growth all components of a cell grow
at the same rate.
Growth rate is independent of nutrient
concentration, as nutrients are in excess.

The slope net is constant.

Typical growth curve for a bacterial population

Typical growth curve for a bacterial population

Batch Growth Kinetics

Deceleration growth phase

Very short phase, during which growth
decelerates due to either:
Depletion of one or more essential
nutrients
The accumulation of toxic by-products of
growth (e.g. Ethanol in yeast
fermentations)
Period of unbalanced growth: Cells
undergo internal restructuring to increase

Typical growth curve for a bacterial population

Batch Growth Kinetics

Stationary Phase:

With the exhaustion of nutrients (S0) and

build-up of waste and secondary metabolic
products
The growth rate equals the death rate.
There is no net growth in the organism
population.
Cells may have active metabolism to produce
secondary metabolites.
Primary metabolites are growth-related: ethanol
by S. cerevisae.
Secondary metabolites are non-growth-related:
antibiotics, pigments.

Kinetic Pattern of Growth and

Product Formation

Growth-associatedMixed-growth-associated
Non growth-associated

Batch Growth Kinetics

Death Phase:
The living organism population decreases with
time, due to a lack of nutrients and toxic
metabolic by-products.
The rate of death usually follows:

dN
'
kd N
dt
'
k d is the first - order death rate constant.

Growth Curve for Bacteria (Logarithmic Plot)

Figure 6.14

Phases of Growth

Lag
Adapt to nutrients

Log
Active growth

Stationary
Death = Growth rate

Death
Nutrients consumed
pH too low (why?)

Optimize curves in production

Chapter 6

Bacteriology

Culture Media

Culture medium

is the mixture of various nutrients that is

suitable for the growth of microorganisms.

Types of Culture Media

based on the function and chemical components
based on the physical state

71

Based on the function and the chemical components:

Basic Medium
--contains the basic nutrients for the most bacterial growth;
--the base of other kind of media.
--e.g. broth.

Nutrient Medium/Enriched Medium

Additional or special nutrients (e.g., serum, growth
factors, trace elements) are added to support some
fastidious bacterial growth.
e.g. blood agar.

72

 Selective Medium
the medium that can prevent the certain bacterial
growth while permitting others.
e.g. SS agar

Differential Medium
Some special substrates and indicators are added into the
media in order to produce a visual differentiation
when several bacteria grow on the same kind of medium.
e.g. EMB agar (Eosin-methylene blue agar).

73

E.coli on EMB

S.dysenteriae on EMB

74


Citrate slant
Double sugar iron slant

75

Anaerobic Medium
a medium for the cultivation of certain anaerobes. The
medium contains reducing agent, such as non-saturation
fatty acid.

76

Based on the physical state

Liquid medium:

Without agar.
for the proliferation of bacteria.

Solid medium:
1.5-2.5% agar.
for the isolation and identification of bacteria
e.g., slant, Petri dishes/plates.

Semisolid medium:
0.3-0.5% agar.
for the observation of bacterial motility and preservation of
bacteria.

77

Bacterial growth patterns

In liquid medium:
Superficial growth;
Turbidity/diffuse;
Precipitate growing;
(sediment)

In solid medium:
Confluent growth / Smear;
Colony:
a cluster of microorganisms growing
on a solid medium. It is directly visible
and arises from a single cell.

78

79

 In semi-solid medium:
Only grow along the line of inoculation
Grow diffusely

80

81

Selective Media
Goal: To chemically (or
physically) suppress
unwanted microbes
and encourage desired
microbes.

MSA

Mannitol salt agar : selective for halophiles

with 7% salt (osmotic challenge) and
differential for mannitol fermenters: good for
skin bacterial cultures.
EMB Agar: kills gram positives with eosin
and methylene blue, selective for gram
negatives. Differential for lactose fermenters.
Good for growing enterics.
McConkey Agar: supresses gram positives
with crystal violet and bile salts; also
differential for

EMB

MA

Figure 6.9b, c

Differential Media
Distinguish between different species based on a
metabolic ability.

Blood agar
(sheeps blood)
reveals if
hemolytic

Mannitol salt agar

contains the pH sensitive
dye phenol red (yellow
when acidic)

Se

Sa

Figure 6.9a

Enrichment Media

Encourages growth of desired microbe by providing

special growth conditions or added growth factors

Thioglycollate

Anaerobic or
Brewer Jar

Lysed red blood cells provide

unique nutrients in
blood/chocolate agar

Glucose Salts Agar (enriches

for microbes that can growth
only on glucose and some
inorganic nutrients

Pure Cultures Used To Study Characteristics Of A Particular Species

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)

Bacteriology

Bacterial Enumeration

Estimating Bacterial Numbers by

Indirect methods
Direct Measures

Plate counts of viable bacterial forming colonies

Counting low viable bacterial numbers by filtration
Counting viable bacteria with Most Probable
Number
Counting bacteria per ml in direct microscopy

Indirect Measures
Turbidity/Absorbance with a spectrophotometer
Metabolic activity tracking conversion of colored
molecules
Dry weight by weighing a set volume and knowing
weight of one cell

Plate Assays: Spread Plate or Pour Plate

Methods
After incubation, count colonies on plates that have
30-300 colonies (CFUs)

The dilution in a
particular tube =
ml of fluid added
to tube/total
volume after
addition; e.g.
1ml/(9ml + 1ml) =
1/10 = 10-2

Figure 6.15

Direct Measurements of
Microbial Growth

Figure 6.19

Direct Measurements of Microbial Growth

Filtration: Good for measuring very dilute samples of bacteria

Figure 6.17a, b

Direct Measurements of Microbial Growth

Multiple tube
MPN test
Count positive
tubes and
compare to
statistical MPN
table
Produces a
range of
concentrations

Figure 6.18b

Estimating Bacterial Numbers by Indirect

methods
Direct Measures

Plate counts of viable bacterial forming colonies

Counting low viable bacterial numbers by filtration
Counting viable bacteria with Most Probable
Number
Counting bacteria per ml in direct microscopy

Indirect Measures
Turbidity/Absorbance with a spectrophotometer
Metabolic activity tracking conversion of colored
molecules/enyzme assay
Dry weight by weighing a set volume and knowing
weight of one cell

Estimating Bacterial Numbers by Indirect

Methods
Turbidity

Figure 620

Metabolic Conversion/Enzyme Assay

1 bacterium produces 4.6 x 10

12

NADH/sec/cell

under idea growth conditions.

In a 1 ml sample of growing cells, 5.2 x 1023
NADH/sec/ml are produced per second (as revealed
by a color-based assay of NADH on the sample)
Therefore, (4.6 x 1012 NADH/sec/ml) x (5.2 x 1023
NADH/sec/cell) = 2.3 x 1024 cells/ml

Determining dry mass of a fixed

volume

An E. coli cell has a dry mass of

about 7.0 x 10-19 mg.
A 1 ml sample with a dry mass of
2 mg therefore has:
2 mg/ml x 1 cell/7 x 10-19 mg
= 2.8 x 1020 cells/ml

Microbial Growth

Physical Requirements of Microbes

Chemical Requirements

Carbon source in medium

Nitrogen, sulfur, phosphorous, trace elements
Oxygen requirements

Obligate aerobes, anaerobes, facultative anaerobes

Free radical oxygen (O2-) and H2O2 dangers; superoxide dismutase and
catalase = aerobes

Culture Media for Microbes

Temperature (optimal enzyme operation)

Psychrophiles, mesophiles, thermophiles

pH (optimal enzyme operation)

Using buffers in media

Molds & yeasts versus bacteria

Chemically defined vs. complex media

Anaerobes: reducing media/Brewer jar
Other: animals, eggs, tissue culture, CO2
Media types

Selective, Differential, Enrichment

Bacterial Population Growth

Growth Curve: Lag, Log, Stationary, Death

Quantifying Growth

Topics

Bacterial Ultrastructures
Biofilms
Bacterial Metabolism
Bacterial Sporolation
Bacterial Reproduction and Growth Kinetics
Bacterial Cultivation
Environmental Factors of Growth
Detection, Identification and Characterization