MEDICAL MICROBIOLOGY

Course for medical students

Associate Professor L. Yocheva, Ph.D.

Department of Biology, Medical Genetics and

Microbiology
Medical Faculty
SU “St. Kliment Ohridski”

Email: [email protected]
 Lecture 3
Bacterial Physiology.
Metabolism and Growth

1. Chemical composition, nutrition, enzymes.

2. Metabolism - catabolic and anabolic reactions of
bacteria.
3. Growth and reproduction.
4. Bacterial cultivation - principles and requirements.
 Key words

• Heterotrophic bacteria
• Metabolism
• Respiration, fermentation
• Enzymes
• Growth curve, generation time
• Bacterial colony
• Aerobes, facultative anaerobes, strict
anaerobes, microaerophiles
• Psychrophiles, mesophiles
 Chemical Composition

Bacteria, like other cells, contain approximately 70-

90% water
Dry substance of the cell:
• Carbon - 40-50%
• Hydrogen - 10-20%
• Nitrogen, oxygen, phosphorous - at 10%
• Sulphur - 5%
• In smaller amounts: sodium, potassium, calcium,
magnesium, zinc, iron ions.
• All these elements must be obtained from the
environment
Organic Components of the Bacterial Cell

• Proteins - 50-55%
• Nucleic acids - DNA (3%) and RNA (21%)
• Carbohydrates - 12-28%
• Lipids - 9%
• Pigments
Nutritional Requirements of the Bacterial
Cells – Organic Macro Nutrients

• Holophytic nutrition – with the entire

surface of the cell
• Organic nutrients are required in different
amounts by different species of bacteria
(fastidious and non fastidious).
• Carbohydrates are used for many
biosynthetic pathways.
• Amino acids are absolutely essential to many
bacteria.
 Nutritional Requirements of the Cells-
Trace Elements

• Microbes require very small amounts

of other chemical elements, such as
iron, copper, molybdenum and zinc.

• These are referred to as trace

elements.

• Most are essential for activity of

certain enzymes, usually as cofactors.
 Nutritional Requirements of Iron

• Bacteria secrete small molecules that bind

iron (siderophores)

• They internalize iron in the bacterial cell

• The human host also has iron transport

proteins (e.g. transferrin, lactoferrin)

• Competition between bacteria and infected

host for utilization of iron exist
 Nutritional Requirements –
Growth Factors

• All fastidious bacteria are dependent on

growth factors

• Vitamins - usually make up all or part of the

enzyme cofactors

• Purine and pyrimidine bases for nucleic

acid synthesis

• Amino acids for protein synthesis

 Auxotrophic and Prototrophic Bacteria

• Prototrophic bacteria – they are able to

synthesize their required growth factors

• Auxotrophic – they are unable to synthesize

particular organic compound required for its
growth

• Such bacteria need additional imports of

specific compounds (growth factors)
 Grouping of Bacteria according to
Used Source
• ENERGY:
˗ phototrophs use light
˗ chemotrophs use chemical compounds

• ELECTRONS:
˗ lithotrophs oxidize inorganic compounds
˗ organotrophs oxidize organic compounds

• CARBON:
˗ autotrophs use CO2
˗ heterotrophs use organic compounds
 Nutritional Type of Medical Bacteria

• "Medical" bacteria derive its energy and

carbon from organic compounds
• The vast majority of medically
important bacteria are
chemoorganotrophs. They are also
heterotrophs
• Some bacteria like Pseudomonas spp. are
chemolithotrophs
 Transport of Nutrients

Nutrient molecules can cross selectively

permeable plasma membranes through one
of the following three major mechanisms:

• Passive transport or simple diffusion

• Facilitated diffusion
• Active transport
 Passive Transport through the
Membrane

• Transport without expenditure of energy

• In the direction of the concentration gradient

• Types
˗ Simple diffusion (water, CO2, some ions) –
using nonspecific porins
˗ Facilitated diffusion (glycerol) – transport
of substances via a specific carrier
(protein), located into the cytoplasmic
membrane
Facilitated Diffusion
 Active Transport through the Membrane
• Dependent on energy
• Against the concentration gradient
• Specific transport permeases
• Types of active transport:
˗ Primary active transport - dependent on binding proteins;
uses chemical energy (ATP)
˗ Secondary active transport (ion transport) - uses energy
from the proton motive force; characteristic of aerobic
organisms
˗ Group translocation - the substance is changed
chemically; transfer of phosphate to the transported sugar
• Significance
▪ Many enzymes or nutritional substances are concentrated in
the cell by this way.
▪ Active transport is essential for cell growth.
Transport of Nutrients
Uniport, Symport and Antiport
 Catabolism and Anabolism

• The process of breakdown of various organic

substrates in order to obtain usable energy in
the form of ATP is known as catabolism

• Anabolism is a process, in which the energy

obtained in catabolic reactions is used for
synthesis of the bacterial components

• These two processes are referred to as

metabolism. Metabolism is the total of all
chemical reactions occurring in the cell
 Bacterial Enzymes
• Bacterial enzymes take part in both
processes: catabolism and anabolism
• Specific bacterial enzymes hydrolyze large
molecules in the external environment to smaller
molecules. They are transported across the cell
membranes into the cytoplasm by active or
passive transport
• Some enzyme are specific for synthesis
of different bacterial substances
• Important virulence factors
• Role in the identification of bacteria by
biochemical tests
 Classification of Bacterial Enzymes
• According to their mechanisms of action:
– Oxidoreductases, transferases, hydrolases,
lyases, ligases, isomerases
• According to their presence in the cell:
– Constitutive (produced independent of
internal or external stimuli)
– Inducible (an enzyme produced by a cell in
response to the accumulation or addition of a
some nutrient substances)
• According to their localization in the cell:
– Exo-, ecto- and intracellular enzymes
 Energy Metabolism of the Bacterial
Cells. Metabolism of Glucose

• Glycolysis is a most common biochemical

pathway for breaking down glucose into energy.
• In this process the glucose is converted to
one common, universal product - pyruvic
acid.
• Bacteria can produce energy from glucose by
different ways:
1. Fermentation and anaerobic respiration (both
of which occur in the absence of oxygen)
2. Aerobic respiration
• Only aerobic respiration can completely
convert glucose to CO2 and H2O plus energy.
 Fermentative Metabolism

• Organic substances serve as a donors and

acceptors of electrons

• Pyruvic acid is reduced to form organic

acids or alcohols

• The result of glycolysis: from 1 molecule

glucose - two molecules ATP and the
production of fermentation end-products –
acids and /or alcohol
 Examples of Fermentation End Products

• Lactic acid fermentation (L. acidophphilus)

• Mixed acid fermentation (E. coli )
• Propionic acid fermentation used by
Propionibacterium
• Ethanol production in alcohol fermentation,
etc.

• N.B. Fermentation pathways are useful as

tools in biochemical identification tests.
 Respiratory Metabolism

• Aerobic respiration
• Pyruvate from glycolysis is converted to
H2O and CO2 via Acetylcoenzime A and
the tricarboxylic acid cycle.
• It is multistep chain process and final electron
acceptor is O2.
• Complete respiration results in 38 mol ATP
produced for 1 mol of glucose, a much higher
energy yield than that of fermentation.
• Used with the aerobic and facultative anaerobic
bacteria.
 Respiratory Metabolism

• Anaerobic respiration
• Bacteria use inorganic substances as
terminal electron acceptors in place of
oxygen.
• Acceptors can be molecules such as nitrate
or sulfate.
• Anaerobic respiration can be used as
alternative to aerobic respiration in some
facultative bacteria, but is obligatory in some
anaerobes.
 Physical Conditions Required for
Growth

• Culture media are needed to grow

microorganisms in the laboratory and to
carry out microbial identification.

• A wide variety of media is available for

these purposes.

• Different physical conditions are required

for bacterial growth as temperature,
specific atmosphere, pH gradient, etc.
 Growth Atmosphere
• Obligate aerobes
˗ Completely dependent on aerobic respiration;
called strict aerobes
˗ N.B. Possess enzymes (superoxide dismutase,
catalase and peroxidase) for detoxification of
oxygen radicals
˗ Example - Pseudomonas aeruginosa,
Mycobacterium tuberculosis
• Obligate anaerobes
˗ Deprived of superoxide dismutase, catalase
and peroxidase
˗ Extremely sensitive to O2, grow only in an
anaerobic environment - strict anaerobes
˗ Example - genus Clostridium
 Growth Atmosphere (cont.)
• Aerotolerant anaerobes: Do not use O2 for
their metabolism, but do not generate toxic
oxygen radicals; grow better without O2.

• Microaerophilic bacteria: A microorganism that

requires oxygen to survive, but in lower levels
than are present in the atmosphere; typically 2-
10% O2 is needed; Campylobacter spp.

• Capnophiles: Capnophiles require an

elevated concentration of carbon dioxide
(e.g. 10% CO2); Neisseria spp.
- Many microaerophiles are also capnophiles
(Helicobacter pylori)
 Growth Atmosphere (cont.)

• Facultative anaerobes
˗ Facultative anaerobes can grow with
or without oxygen
˗ Receive energy in anaerobic and aerobic
way, but prefer aerobic

• Most pathogenic bacteria are

facultative anaerobes
 Growth Atmosphere

a) Aerobic
b) Anaerobic
c) Facultative
d) Microaerophilic
e) Aerotolerant
anaerobes
 Growth Temperature
• Basic temperatures – minimal, optimal
and maximal

• Psychrophiles (cold-loving). Grow best at

low temperature, opt 10-15 ℃. Can grow at
0-8 ℃; at normal refrigeration temperature
(4 ℃)

• Mesophiles – most pathogenic bacteria,

grow within a range between 20-40 ℃

• Thermophiles (heat-loving). Grow best at

high temperature, e.g. >60 ℃.
Thermophilic and Hyperthermophilic Bacteria

• Thermophilic and hyper thermophilic bacteria

produce heat-stable macromolecules such as
Taq polymerase

• It is used in the nucleic acid amplification steps of

the polymerase chain reaction (PCR)
 Requirements of Bacteria to pH and Salt
Concentration

• Acidophilic bacteria - grow well at pH below

neutral
• Bacteria growing at pH around neutral values
7.2-7.4 - predominant among medically
important bacteria
• Alkalophilic bacteria - grow well at alkaline pH

• Requirements for salt concentration - 0.5-0.8%

NaCl
• Halophilic bacteria - also grow at high
concentrations of 6-15% NaCl
 Bacterial Growth and Replication

• Bacterial growth – irreversible increase in the

cell biomass and size
• Bacterial replication - above a critical volume,
the bacterial cell divides by binary fission into
two identical daughter cells
• Generation time (doubling time) - the time required
for one cell to produce two cells, e.g. E. coli time is
20 min, but Mycobacterium time is 24 hrs.

• N.B. In laboratory, culture results are available after

some time: for E. coli the next day, while several weeks
are required for culture of M. tuberculosis
 Cell Division
• Amitotic fission – binary fission
• Replication of the bacterial chromosome is a start
for division
• The cell elongates
• A dividing septum is formed in the middle of the
cell from two layers peptidoglycan
• Special transpeptidases and other еnzymes are
involved in the process
• A split follows
• As a result of division, the number of bacteria
grows exponentially
Cell Division
Cell Division
 Bacterial Cultivation in Laboratory

Bacterial growth can be observed in two forms:

1. By the development of colonies on solid

nutrient media. Colony is the macroscopic
product derived from the division of one
parent cell.

2. By the transformation of a clear broth medium

to a turbid suspension of 107-109 cells/ml
 Growth Phases in Broth Culture

Periodical culture in Closed system

• Lag phase – initiation of growth, rapid synthesis
of ingredients, the cell number is constant
• Exponential (Log) phase – the bacterial cells
divide, increasing the cell number exponentially
with time
• Stationary phase – the number of newly
formed cells is equal to that of the dead once.
• Decline phase – a final phase with the death of
cells because of nutrient exhaustion and
accumulation of metabolic end-products
Growth Phases in Broth Culture in
Closed System
 Growth Phases in Broth Culture

• Continuous culture in Open system

• Nutrients continually being supplied to the
medium.
• Bacterial growth is sustained in a logarithmic
phase.
• Used in industrial microbiology for the
production of different bioproducts.
 Bacterial Culture Properties
1. Bacterial colony morphology on solid medium
form (S-, R- and M-form), elevation, margin,
surface, transparence, pigmentation, etc.
2. Growth in a liquid medium – broth culture
pellicle, turbidity, sediment, pigmentation
3. Motility indicators
4. Hemolysis - on blood agar it appears as
-hemolysis (a partial hemolysis) – is indicated by
green discoloration around the bacterial colony
-hemolysis (a complete hemolysis) - sharply
defined clear colorless zone of hemolysis
surrounding colonies
γ-hemolysis – without hemolysis
Bacterial Growth in Broth Media
 Bacterial Growth on Solid Media
Agar plate
An agar plate is a Petri dish that
contains a growth medium
solidified with agar, used to
culture microorganisms.

An agar culture of E. coli -

colonies
A colony is defined as a visible
mass of microorganisms all
originating from a single mother
cell, therefore a colony
constitutes a clone of bacteria
all genetically alike
 Agar (Solid) Media

Blood agar

Chocolate agar
Different Types of Hemolysis on
Blood Agar

A)- beta, B) - alfa , C) - no hemolysis

 Different Types of Colonies

M-colonies Prevotella melaninogenica

Mucoid-Type Colonies of an
Encapsulated Bacillus species
 Production of Pigment

Serratia marcescens
Pseudomonas aeruginosa

Serratia rubidaea
S-colonies

Shigella flexneri
 R-colonies

Shigella sonnei
 Cultivation EXCEPTIONS

• Some bacteria can not be cultivated,

e.g. Treponema pallidum.

• Cultivation of others is difficult and slow

(fastidious bacteria) e.g. Borrelia
burgdorferi.
 Methods of Bacterial Enumeration
Determining the total number of cells - viable and dead
• Turbidimetric method: determines the reduction in the
intensity of light as it passes through a bacterial liquid
suspension. The detector is placed in line with the
source of light.
• Nephelometry is the same, but it measures the
scattered light. The detector is set at an angle to the
light beam.
• Both methods are based on the fact that the intensity
of light can be approximately related to the
concentration of suspended bacteria.

• Optical standard – the turbidity of bacterial

suspension is visually compared to that in a series of
standard tubes.
Nephelometry and Turbidimetry
 McFarland Standard

The turbidity of each standard corresponds to a certain

number of bacterial cells per 1 ml volume(CFU/ml) and
optical density (OD).

McFarland Standard №1 = approximately 3X108 cfu/ml.

 Methods of Bacterial Enumeration

Determination of the viable cells number

• Estimation the number of viable cells through
dilutions of the sample and inoculation of the
bacteria on the plate. After incubation the
number of the colonies is counted.
• Method is called Determination of microbial
number
• The result is expressed as colony forming units
per ml (CFU/ml)
• This is a commonly used method.
Determination of Microbial Number