The role of microbiology in the dentist’s medical practice. The history of the

microbiology development.Classification and morphology of the

bacteria. Physiology of microorganisms. Growth and reproduction of the bacteria.

Chair of Microbiology, Virology, and Immunology

Lecturer Prof. S.I. Klymnyuk

The role of microbiology in the dentist’s medical practice.

The history of the microbiology development.

Classification and morphology of the bacteria.

Microbiology is a science, which study most shallow living creatures - microorganisms.

Before inventing of microscope humanity was in dark about their existence. But during the centuries people could make use of processes vital activity of microbes for its needs. They could prepare a koumiss, alcohol, wine, vinegar, bread, and other products. During many centuries the nature of fermentations remained incomprehensible.

Brueghel: The Triumph of Death (1560)

Microbiology learns morphology, physiology, genetics and microorganisms systematization, their ecology and the other life forms.

Specific Classes of MicroorganismsSpecific Classes of Microorganisms Algae

Protozoa Fungi (yeasts and molds)

Bacteria Rickettsiae

Viruses Prions

The Microorganisms are extraordinarily widely spread in nature. They literally ubiquitous forward us from birth to our death. Daily, hourly we eat up thousands and thousands of microbes together with air, water, food. On our skin, in mouth and nasal cavities, on mucous membranes and in bowels enormous amount of microorganisms live and act. Many of them are found in earth cortex and in the air, and in the ocean’s, sea’s, river’s water, on all of latitudes, mainlands and continents.

For the first time term “microbe" was offered by French scientist Sh. Sedillot in 1878. It derives from Greek “microbe", that means briefly living, or most shallow living creature. Science, which learns the microorganisms, was named by E. Duclaux microbiology. For short development period this science accumulated great factual material. The separate microbiological branches such as bacteriology, mycology, protistology, virology quickly appeared.

Comparative sizes of Bacteria

Periods of microbiology development

• Morphologic

• Physiologic

• Prophylactic

Development of microbiological science was interlinked with art of glass and diamonds grinding. This brought to creation of the first microscope by Hans and Zacharian Jansen in Holland in 1590.

The discovery of microorganisms is associated with the name of Antony van Leeuwenhoek (1632-1723).

In 1683 Leeuwenhoek described the basic bacterium forms. His scientific supervisions Leeuwenhoek described in special letters and sent off them to the London Royal Scientific Society. He sent away about 300 letters. The Leeuwenhoek’s letters brought on enormous surprise among English scientists. They opened a fantastic world of invisible creatures. He named them “living animals" (animalcula viva) and in one of letter wrote: “In my mouth there are more animacula viva, than peoples in all United Kingdom".

These wonderful discovery of Dutch naturalist were the embryo, with which science of bacteria developed. Namely from these times starts the so-called morphological period in microbiology history (XVII middle of age). It is also called micrographycal period, as the study of microorganism came only to description of their dimensions and forms. Biological properties and their significances for man still a long time remained incomprehensible.

However, using the primitive microscopes of that time it was difficult to determine the difference between separate bacteria species. Even celebrated founder of scientific systematization of all of living organisms Karl Linney renounced to classify the bacteria. He gave them general name “chaos".

In the second half of XIX century microbiology strongly affirms as independent science. Namely these sciences were fruitful soil, onwhich Pasteur's talent evinced.

He studied wine "illness“, fermentation, made Pasteurization method, offered to grow microbes on artificial nutrient media, he proved, that on definite cultivation conditions the pathogenic bacteria lose its virulence, made vaccine against anthrax, rabies.

Physiological period has began

Not less important are scientific works of celebrated German scientist R. Koch.

He performed classic researches on etiology of anthrax, opened tuberculosis bacilli, cholera vibrio, proposed to isolate pure bacterial cultures on solid nutrient media (gelatin, potatoes), developed the preparations staining methods by aniline dye-stuffs, method of hanging drop for examination of bacteria motility, offered apparatus for sterilization

The Patriarch of world and Ukrainian microbiology - I. Metchnikov

He studied inflammation pathology, phagocytosis, bases about antagonism of bacteria.

From all microbes-antagonists I.Metchnikov preferred the lactic bacteria. On their base he offered three medical preparations -sour clotted milk, yogurt and lactobacillin.

Now they are called by eubiotics. Classic Metchnikov's researches defined a prophylactic period in microbiology history.

In 1892 D. Ivanovskiy described an virus of mosaic tobacco illness – new class of infectious agents

Microorganisms constitute a very antique group of living organisms which appeared on the Earth's surface almost 3000 million years ago.

There are natural and artificial classifications system.

Bergey's Manual of Determinative Bacteriology - the "bible" of bacterial taxonomy.

Classifying Bacteria

• Bergey’s Manual of Systematic Bacteriology– Classifies bacteria via evolutionary or genetic

relationships.

• Bergey’s Manual of Determinative Bacteriology– Classifies bacteria by cell wall composition,

morphology, biochemical tests, differential staining, etc.

The Three-Domain System

Figure 10.1

Prokaryotes

Procaryotae Kingdom has 4 Divisions according to the structure of cell wall and Gram staining:

Gracilicutes (gracilis - thin, cutis - skin) – Gram-negative bacteria,

Firmicutes (firmus - firm) – Gram-positive bacteria,

Tenericutes (tener – soft, tender) – microbes without cell wall,

Mendosicutes (mendosus - mistaket) – microbes with atipical peptidoglican

35 of the major groups of bacteria are distinguished primarily on morphological characteristics, namely: cell shapes (rods, cocci, curved, or filament forming); spore production; staining reactions; motility.

Other groups are defined based on their metabolism, on combinations of morphological and physiological characteristics.

Some of the Major Groups of Bacteria in Bergey's Manual

Spirochetes

Very slender rods that are helically coiled around a central axial filament; includes the bacteria that cause syphilis and Lyme disease

Gram-positive cocci

Bacteria that have a cell wall structure that results in their staining blue-purple by the Gram stain procedure and that are spherical; include the streptococci and

staphylococci Endospore-forming

rods and cocci Bacteria that form heat-resistant bodies called endospores within their cells; include the bacteria that cause gas gangrene, botulism,

tetanus, and anthrax

Species is population of microbes, which have the only source of origin, common genotype, and during the present stage of evolution are characterized by similar morphological, biochemical, physiological and other signs

If deviations from the typical species properties are found on examination of the isolated bacteria, then culture is considered a subspecies.

Infrasubspecies subdivisions

serovar (antigenic properties)

morphovar (morphological properties)

chemovar (chemical properties)

biovar (biochemical or physiological properties)

pathovar (pathogenic properties)

phagovar (relation to phages)

The term clone was applied to designate a group of individuals arising from one cell

Population is an elementary evolutional unit (structure) of a definite species

The term strain designates a microbial culture obtained from the different sources or from one source but in different time

Taxonomic Grades

Rank Example

Kingdom Procaryotae

Division Gracilicutis

Class Scotobacteria

Order Spirochaetales

Family Leptospiraceae

Genus Leptospira

Species L. interrogans

Bacterial Nomenclature

• Binomial naming system– Two word naming system

• First word is genus name– Always capitalized

• Escherichia• Second word is species name

– Not capitalized• coli

• When writing full name genus usually abbreviated– E. coli

• Full name always italicized– Or underlined

Morphological Classification of Bacteria

Bacteria (Gk. bakterion - small staff) are unicellular organisms lacking chlorophyll.

Morphologically, bacteria possess four main forms:

spherical (cocci)

rod-shaped (bacteria, bacilli, and clostridia)

spiral-shaped (vibrios, spirilla and spirochaetes)

thread-shaped (non-pathogenic)

Cocci groupings

Coccus

Diplococcus

Streptococcus

Tetrad

Sarcinae

Staphylococcus

Cocci (Gk. kokkos berry). These forms of bacteria are spherical, ellipsoidal, bean-shaped, and lanceolate. Cocci are subdivided into six groups according to cell arrangement, cell division and biological properties

Micrococci (Micrococcus). The cells are arranged singly or irregularly. They are saprophytes, and live in water and in air ( M. roseus, M. luteus, etc.).

Diplococci (Gk. diplos double) divide in one plane and remain attached in pairs. These include: Meningococcus (causative agent of epidemic cerebrospinal meningitis, and gonococcus, causative agent of gonorrhoea and blennorrhoea) Pneumococcus (causative agents of pneumonia)

Streptococci (Gk. streptos curved, kokkos berry) divide in one plane and are arranged in chains of different length. Some streptococci are pathogenic for humans and are responsible for various diseases.

Tetracocci (Gk. tetra four) divide in two planes at right angles to one another and form groups of fours. They very rarely produce diseases in humans.

Staphylococci (Gk. staphyle cluster of grapes) divide in several planes resulting in irregular bunches of cells, sometimes resembling clusters of grapes. Some species of Staphylococci cause diseases in man and animals

Sarcinae (L. sarcio to tie) divide in three planes at right angles to one another and resemble packets of 8, 16 or more cells. They are frequently found in the air. Virulent species have not been encountered

Rods. Rod-shaped forms are subdivided into:

bacteria,

bacilli,

clostridia

Bacteria include those microorganisms which, as a rule, do not produce spores (colibacillus, and organisms responsible for enteric fever, paratyphoids, dysentery, diphtheria, tuberculosis, etc.).

Bacilli and clostridia include organisms the majority of which produce spores (hay bacillus, bacilli responsible for anthrax, tetanus, anaerobic infections, etc.)

According to their arrangement, cylindrical forms can be subdivided into three groups: monobacteria monobacilli

E. coli Y. pestis

C. tetani

C. botulinum

diplobacteria diplobacilli

K. pneumoniae

streptobacteria streptobacilli

Haemophilus ducreyi

(chancroid)

Bacillus anthracis

(anthrax)

Spiral-shaped bacteria

Vibriones (L. vibrio to vibrate) are cells which resemble a comma in appearance. Typical representatives of this group are Vibrio cholerae, the causative agent of cholera, and aquatic vibriones which are widely distributed in fresh water reservoirs.

Spirilla (L. spira coil) are coiled forms of bacteria exhibiting twists with one or more turns. Only one pathogenic species is known {Spirillum minus} which is responsible for a disease in humans transmitted through the bite of rats and other rodents (rat-bite fever, sodoku)

Spirochaetes (L. spira curve, Gk. chaite cock, mane) differ from bacteria in structure with a corkscrew spiral shape

Borrelia. Their cells have large, obtuse-angled, irregular spirals, the number of which varies from 3 to 10. Pathogenic for man are the causative agents of relapsing fever transmitted by lice (Borrelia hispanica), and by ticks (Borrelia persica, etc.). These stain blue-violet with the Romanowsky-Giemsa stain

Leptospira (Gk. leplos thin, speira coil) are characterized by very thin cell structure. The leptospirae form 12 to 18 coils wound close to each other, shaping small primary spirals. The organisms have two paired axial filaments attached at opposite ends (basal bodies) of the cell and directed toward each other.

Leptospira interrogans which is pathogenic for animals and man cause leptospirosis

Treponema (Gk. trepein turn, nema thread) exhibits thin, flexible cells with 6-14 twists. The micro-organisms do not appear to have a visible axial filament or an axial crest when viewed under the microscope

A typical representative is the causative agent of syphilis Treponema pallidum

Properties of prokaryotes and eukaryotes

Prokaryotes Eukaryotes

The nucleoid has no membrane separating it from the cytoplasm

Karyoplasm is separated from the cytoplasm by membrane

Chromosome is a one ball of double twisted DNA threads. Mitosis is absent

Chromosome is more than one, There is a mitosis

DNA of cytoplasm are represented in plasmids

DNA of cytoplasm are represented in organelles

There aren’t cytoplasmic organelle which is surrounded by membrane

There are cytoplasmic organelle which is surrounded by membrane

The respiratory system is localized in cytoplasmic membrane

The respiratory system is localized mitochondrion

There are ribosome 70S in cytoplasm

There are ribosome 80S in cytoplasm

Peptidoglycan are included in cell’s wall (Murein)

Peptidoglycan aren’t included in cell’s wall

The structure of procaryotes

Nucleus. The prokaryotic nucleus can be seen with the light microscope in stained material. It is Feulgen-positive, indicating the presence of DNA. Histonelike proteins have recently been discovered in bacteria and presumably play a role similar to that of histones in eukaryotic chromatin

The DNA is seen to be a single, continuous, "giant" circular molecule with a molecular weight of approximately 3 X 109. The unfolded nuclear DNA would be about 1-3 mm long (compared with an average length of 1 to 2 µm for bacterial cells)

Plasmids: R, Col, Hly, Ent, Sal

Plasmids small circular, double-stranded DNA free or integrated into the chromosome duplicated and passed on to offspring not essential to bacterial growth & metabolism may encode antibiotic resistance, tolerance to toxic metals, enzymes & toxins used in genetic engineering- readily manipulated & transferred from cell to cell There may be several different plasmids in one cell and the numbers of each may vary from only one to 100s in a cell

Prokaryotic RibosomeProkaryotic Ribosome

A ribosome (70 S) is a combination of RNA and protein, and is the site for protein synthesis Composed of large (50S) and small (30S) subunits S = Svedverg unit, measures molecular size

The 80S ribosomes of eukaryotes are made up of 40S and 60S subunits.

• Storage granules– Metachromatic

granules – Polysaccharide

granules– Lipid inclusions– Sulfur granules– Carboxyzomes – Magnetosomes

• Gas vesicles

Inclusions, granules

Volutin granules

Corynebacterium diphtheriae

Loeffler's technique Neisser's staining

Composted of A. The cytoplasmic membrane

To act as a physical barrier btw cytoplasm and environments and selectively controls the movement of substaces into and out of the cell“Semipermeable”

B. Cell wallThe rigid layer that protect the fragile cytoplasmic membrane from rupturingTo maintains cell’s shape

C. Capsule or slime layer (glycocalyx)

Cell Envelope

Cell membrane

PeripheralMembraneProtein

IntegralMembraneProtein

PeripheralMembraneProtein

Phospholipid

Bacterial plasma membrane are composed of 40 percent phospholipid and 60 percent protein. The phospholipids are amphoteric molecules with a polar hydrophilic glycerol "head" attached via an ester bond to two nonpolar hydrophobic fatty acid tails, which naturally form a bilayer in aqueous environments. Dispersed within the bilayer are various structural and enzymatic proteins which carry out most membrane functions.

Mesosome

The predominant functions of bacterial membranes are:

1. Osmotic or permeability barrier;

2. Location of transport systems for specific solutes (nutrients and ions);

3. Energy generating functions, involving respiratory and photosynthetic electron transport systems, establishment of proton motive force, and transmembranous, ATP-synthesizing ATPase;

4. Synthesis of membrane lipids (including lipopolysaccharide in Gram-negative cells);

5. Synthesis of murein (cell wall peptidoglycan);

6. Assembly and secretion of extracytoplasmic proteins;

7. Coordination of DNA replication and segregation with septum formation and cell division;

8. Chemotaxis (both motility per se and sensing functions);

9. Location of specialized enzyme system.

• Unique chemical structure– Distinguishes Gram positive from Gram-negative– bacteria and archaea bacterial species

• Rigidity of cell wall is due to peptidoglycan (PTG) – Compound found only in bacteria – Archaea –psudomurein or other sugars, proteins,

glycoproteins

• Many antimicrobial interfere with synthesis of PTG

• Penicillin; Lysozyme

Cell wall

• Basic structure of peptidoglycan– Alternating series of two

subunits• N-acetylglucosamin (NAG)• N-acetylmuramic acid (NAM)

– Joined subunits form glycan chain

• Glycan chains held together by string of four amino acids

– Tetrapeptide chain:L-ala-D-glu-DAP-D-ala L-ala-D-glu-Lys-D-ala

• Interpeptide bridge

Structure of peptidoglycan

Structures associated with gram-positive and gram-negative cell walls.

Differences of cell wall structure in Gram-positive and Gram negative cells

L FormsL Forms

Glycocalyx

CapsuleProtects bacteria from phagocytic cells

Slime layerEnable attachment and aggregation of bacterial cells

Capsules Most prokaryotes contain some sort of a polysaccharide layer outside of the cell wall polymer

Only capsule of B. anthracis consist of polypeptide (polyglutamic acid)

CapsuleCapsule

The capsule is covalently

bound to the cell wall.

Associated with virulence in bacteria.

Example:

Streptococcus pneumoniae

Slime LayerSlime Layer

The slime layer is loosely bound to the cell.

Carbohydrate rich material enhances adherence of cells on surfaces

Example:Streptococcus mutans and “plaque formation”

Biofilms Biofilms

The slime layer is associated with cell aggregation and the formation of biofilms

Example:Staphylococcus epidermidis biofilms on catheter tips

•Adhesion•Avoidance of immune response•Protection from dehydration •Protection of bacterial cells from engulfment by protozoa or white blood cells (phagocytes), or from attack by antimicrobial agents of plant or animal origin. •They provide virulent properties of bacteria (S. pneumoniae, B. anthracis)

General capsule function

Flagella

• 3 parts– filament – long, thin,

helical structure composed of proteins

– hook- curved sheath– basal body – stack of

rings firmly anchored in cell wall

• rotates 360o

• 1-2 or many distributed over entire cell

• functions in motility

Flagellar arrangements

1. Monotrichous – single flagellum at one end (cholera vibrio, blue pus bacillus),

2. Lophotrichous – small bunches arising from one end of cell (blue-green milk bacillus,Alcaligenes faecalis)

3. Amphitrichous – flagella at both ends of cell (Spirillum volutans),

4. Peritrichous – flagella dispersed over surface of cell, slowest E. coli, salmonellae of enteric fever and paratyphoids A and B

Bacterial MotilityBacterial Motility

The rotation of the flagella enables bacteria to be motile.

Flagella are important for:

Motility (dispersal)

Antigenic determinant

Number and location species specific

Pili and FimbriaePili and Fimbriae• Short, hair-like structures on the surfaces of procaryotic cells • Proteinaceuse filaments (~20 nm in diameter)• Very common in Gram-negative bacteria

• Functions:– Adherence to surface/ substrates: teeth, tissues– Involved in transfer of genetic information btw cells– Have nothing to do with bacterial movement (Except the twitching move

ment of Pseudomonas)

Fimbriae are smaller than flagella and are important for attachment

Bacterial endospores• Bacterial spores are often called “endospore” (since they are

formed within the vegetative cell)• Produced in response to nutrient limitation or extreme environments• Highly resistant• Highly dehydrated (15% water)• Metabolically inactive• Stable for years• Not reproductive • Functions: to survive under an extreme growth conditions such as

high temperature, drought, etc.

Bacillus, Clostridium, Sporolactobacillus, Thermoactinomyces, Sporosarcina, Desulfotomaculum species sporulate

Spore

Spores

• Key compositions:– Dipicolinic acid (DPA)– Calcium (Ca2+)

• Structure– Core / Cytoplasm– Plasma membrane– Core wall/ spore wall– Cortex – Spore coat– Exosporium

Endospores

The sporulation process begins when nutritional conditions become unfavorable, depletion of the nitrogen or carbon source (or both) being the most significant factor. Sporulation involves the production of many new structures, enzymes, and metabolites along with the disappearance of many vegetative cell components.

Spores are located: 1) Centrally (B. anthracis);

2) Terminally (С. tetani);

3) Subterminally (C. botulinum, C. perfringens)

The spores of certain bacilli are capable of withstanding boiling and high concentrations of disinfectants. They are killed in an autoclave exposed to saturated steam, at a temperature of 115-125 C, and also at a temperature of 150-170 C in a Pasteur hot-air oven.

Physiology of microorganisms. Growth and reproduction of the

bacteria

Metabolism refers to all the biochemical reactions that occur in a cell or organism.

The study of bacterial metabolism focuses on the chemical diversity of substrate oxidations and dissimilation reactions (reactions by which substrate molecules are broken down), which normally function in bacteria to generate energy.

Chemical composition of bacteria

Protein 55 %

Total RNA 20.5 %

DNA 3.1 %

Phospholipid 9.1 %

  Lipopolysaccharide  3.4 %

Murein 2.5 %

Inorganic ions  1.0 %

Bacterial cell consists of:

Water – 70-90 % Dry weight – 10-30 %

Proteins – 55 %, 2,35 million of molecules, 1850 different types of molecules

RNA – 20,5 %, 250000 molecules, 660 different types of molecules

DNA – 3,1 %, 2 molecules

Lipids – 9 %, 22 million of molecules

Lipopolysaccharides –3,4 %, 1,5 million of molecules

Peptidoglycan – 1 molecule

Microbial metabolism1. Catabolism (Dissimilation)

- Pathways that breakdown

organic substrates

(carbohydrates, lipids, &

proteins) to yield metabolic

energy

for growth and maintenance.

2. Anabolism (Assimilation)

- Assimilatory pathways for

the formation of key

intermediates and then to

end products (cellular

components).

4. Intermediary metabolism -

Integrate two processes

Pyruvate: universal intermediate

Aerobic respiration

Fermentation

Glycolysis (EMP pathway)

Substrate-level phosphorylation

Catabolism

The bacterial cell is a highly specialized energy transformer. Chemical energy generated by substrate oxidations is conserved by formation of high-energy compounds such as adenosine diphosphate (ADP) and adenosine triphosphate (ATP) or compounds containing the thioester bond

O

║

(R –C ~ S – R), such as acetyl ~ S-coenzyme A

Another form of energy - transmembrane potential - ΔμН+

Chemiosmosis• Production of ATP in Electron

Transport• Electrochemical Gradient

Formed between membranes• H+ (Protons) generated from

NADH• Electrical Force (+) & pH Force

(Acid)• Gradient formed• ATPase enzyme that channels

H+ from High to Low concentration– 3 ATP/NADH– 2 ATP/NADH

Fermentation: metabolic process in which the final electron acceptor is an organic compound.

Sources of metabolic energyRespiration: chemical reduction of an electron acceptor through a specific series of electron carriers in the membrane. The electron acceptor is commonly O2, but CO2, SO4

2-, and NO3- are employed by some microorganisms.

Photosynthesis: similar to respiration except that the reductant and oxidant are created by light energy. Respiration can provide photosynthetic organisms with energy in the absence of light.

Substrate-level phosphorylation

The Krebs cycle intermediate compounds serve as precursor molecules (building blocks) for the energy-requiring biosynthesis of complex organic compounds in bacteria. Degradation reactions that simultaneously produce energy and generate precursor molecules for the biosynthesis of new cellular constituents are called amphibolic.

Energy RequirementsOxidation of organic compounds - Chemotrophs

Sunlight - Phototrophs

Carbon source

- Autotrophs (lithotrophs): use CO2 as the C source

Photosynthetic autotrophs: use light energy

Chemolithotrophs: use inorganics

- Heterotrophs (organotrophs): use organic carbon (eg.

glucose) for growth.

- Clinical Labs classify bacteria by the carbon sources

(eg. Lactose) & the end products (eg. Ethanol,…).

Nitrogen source

Ammonium (NH4+) is used as the sole N source by most

microorganisms. Ammonium could be produced from N2 by

nitrogen fixation, or from reduction of nitrate (NO3-)and

nitrite (NO2).

Metabolic Requirements

Physiologic types of bacterial existence

Carbon Source

Energy Source

Oxidation of organic compounds - Chemotrophs

Sunlight - Phototrophs

Organic - Heterotrophs Inorganic - Autotrophs

Electrone donor

Inorganic - Lithotrophs Оrganic -Organotrophs

Chemoorganoheterotrophic bacteria

Sulfur source

A component of several coenzymes and amino acids.

Most microorganisms can use sulfate (SO42-) as the S source.

Phosphorus source

- A component of ATP, nucleic acids, coenzymes,

phospholipids, teichoic acid, capsular polysaccharides; also is

required for signal transduction.

- Phosphate (PO43-) is usually used as the P source.

Metabolic Requirements

Mineral source- Required for enzyme function.- For most microorganisms, it is necessary to provide sources

of K+, Mg2+, Ca2+, Fe2+, Na+ and Cl-.

- Many other minerals (eg., Mn2+, Mo2+, Co2+, Cu2+ and Zn2+)

can be provided in tap water or as contaminants of other

medium ingredients.- Uptake of Fe is facilitated by production of siderophores

(Iron-chelating compound, eg. Enterobactin).

Growth factors: organic compounds (e.g., amino acids, sugars, nucleotides, vitamines) a cell must contain in order to grow but which it is unable to synthesize. Purines and pyrimidines: required for synthesis of nucleic acids (DNA and RNA);Amino acids: required for the synthesis of proteins; Vitamins: needed as coenzymes and functional groups of certain enzymes.

Transport systems

The proteins that mediate the passage of solutes through membranes are referred to as transport systems, carrier proteins, porters, and permeases. Transport systems operate by one of three transport processes.

In a uniport process, a solute passes through the membrane unidirectionally. In symport processes (cotransport) two solutes must be transported in the same direction at the same time; in antiport processes (exchange diffusion), one solute is transported in one direction simultaneously as a second solute is transported in the opposite direction.

Transport systems

Diffusion systems

• passive diffusion

• facilitated diffusion

• ion-driven transport

• binding protein dependent transport

• group translocation

• Membrane is selectively permeable– Few molecules pass through freely– Movement involves both active and passive

processes

Passive processes – no energy (ATP) required – Along gradient – simple diffusion, facilitated diffusion,

osmosis

• Simple diffusion

• Facilitated diffusion

Can reduce concentration gradient Can reduce concentration gradient but can’t create onebut can’t create one

Osmosis• Osmotic pressure

Active processes• energy (ATP)

required– Active transport– Group

translocation

Facilitated diffusion

Active transport

Transport systems

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 ????

• Microorganisms can be categorized based on their optimal temperature requirements– Psychrophiles

• 0 - 20 ºC– Mesophiles

• 20 - 40 ºC– Thermophiles

• 40 - 90 ºC• Most bacteria are mesophiles

especially pathogens that require 37 ºC

TEMPERATURE

BACTERIAL TEMPERATURE REQUIREMENTS

Variable

100

50

0

0 0C

% Max

Growth

37 0C 90 0C

Psychrophile

Mesophile

Thermophile

Effects of Temperature on Growth

Mesophiles10o-50o

Thermophiles70o-110o

BC YangFor lecture only

• PsychrophilesPsychrophiles– some will exist below 0 oC if liquid water is

available• oceans• refrigerators• freezers

TEMPERATURE

Pigmented bacteria in Antarctic ice

• MesophilesMesophiles– most human flora

and pathogens

TEMPERATURE

• ThermophilesThermophiles– hot springs– effluents from

laundromat– deep ocean thermal

vents

TEMPERATURE

Respiration in Bacteria

Obligate Aerobe

Microaerophile

Obligate Anaerobe

Facultative Anaerobe (Facultative Aerobe)

Aerotolerant Anaerobe

Capneic bacteria

124

Categories of Oxygen Requirement

Aerobe – utilizes oxygen and can detoxify it obligate aerobe - cannot grow without oxygen

(Mycobacterium tuberculosis, Micrococcus spp., Bacillus spp., Pseudomonas spp.

facultative anaerobe – utilizes oxygen but can also grow in its absence (Echericihia spp., Salmonella spp., Sta[phylococcus spp.)

microaerophylic – requires only a small amount of oxygen (Helycobacter spp., Lactobacillus spp.)

125

Categories of Oxygen Requirement

Anaerobe – does not utilize oxygen

• obligate anaerobe - lacks the enzymes to detoxify oxygen so cannot survive in an oxygen environment (Clostridium spp., Bacteroides spp.)

• aerotolerance anaerobes – do no utilize oxygen but can survive and grow in its presence (Streptococcus pyogenes)

126

Carbon Dioxide RequirementAll microbes require some carbon dioxide in

their metabolism.

• capneic – grows best at higher CO2 tensions than normally present in the atmosphere (Brucella abortus)

OXYGENObligate Aerobe

Facultative Anaerobe

Obligate Anaerobe

Four Toxic Forms of Oxygen

Toxic Oxygen Forms Are formed:

Singlet oxygenduring photosynthesis as molecular oxygen with electrons are boosted to higher energy state

Superoxide radicalsduring incomplete reduction of oxygen in aerobic and anaerobic respiration

Peroxide anionduring reactions that neutralizes superoxide radicals

Hydroxyl radicalfrom ionizing radiation and from incomplete reduction of hydrogen peroxide

Four Toxic Forms of Oxygen

Toxic Oxygen Forms Are neutralized by:

Singlet oxygencarotenoids that remove the excess energy of singlet oxygen

Superoxide radicalssuperoxide dismutases, enzymes that detoxify them

Peroxide anioncatalase or peroxidase, enzymes that detoxify peroxide anion

Hydroxyl radicalcatalase, peroxidase, and antioxidants such as vitamins C and E that protect against toxic oxygen products

Enzymes and Their Role in Metabolism

Enzymes, organic catalysts of a highly molecular structure, are produced by the living cell. They are of a protein nature, are strictly specific in action, and play an important part in the metabolism of micro-organisms. Their specificity is associated with active centres formed by a group of amino acids.

Some enzymes are excreted by the cell into the environment (exoenzymes) for breaking down complex colloid nutrient materials while other enzymes are contained inside the cell (endoenzymes).

Bacterial enzymes are subdivided into some groups:

1. Hydrolases which catalyse the breakdown of the link between the carbon and nitrogen atoms, between the oxygen and sulphur atoms, binding one molecule of water (esterases. glucosidases, proteases. amilases, nucleases, etc.).

2. Transferases perform catalysis by transferring certain radicals from one molecule to another (transglucosidases, transacylases. transaminases).

3. Oxidative enzymes (oxyreductases) which catalyse the oxidation-reduction processes (oxidases, dehydrogenases, peroxidases, catalases).

4. Isomerases and racemases play an important part in carbohydrate metabolism. Rearrangement atoms of a molecule.

5. Lyases (remove chemical groups from molecules without adding water).

6. Lygases (join two molecules together and usually require energy from ATP).

Enzymes

Significance of the enzymes

With the help of amylase produced by mould fungi starch is saccharified and this is employed in beer making, industrial alcohol production and bread making. Proteinases produced by microbes are used for removing the hair from hides, tanning hides, liquefying the gelatinous layer from films during regeneration, and for dry cleaning.

Fibrinolysin produced by streptococci dissolves the thrombi in human blood vessels. Enzymes which hydrolyse cellulose aid in an easier assimilation of rough fodder.

Due to the application of microbial enzymes, the medical industry has been able to obtain alkaloids, polysaccharides, and steroids (hydrocortisone, prednisone, prednisolone. etc.).

Bacteria play an important role in the treatment of caouichouc, coffee, cocoa, and tobacco.

Enzymes permit some species of microorganisms to assimilate methane. butane, and other hydrocarbons, and to synthesize complex organic compounds from them.

With the help of the enzymatic ability of yeasts in special-type industrial installations protein-vitamin concentrates (PVC) can be obtained from waste products of petroleum (paraffin’s).

Metabolism Results in Reproduction

• Microbial growth – an increase in a population of microbes rather than an increase in size of an individual

• Result of microbial growth is discrete colony – an aggregation of cells arising from single parent cell

• Reproduction results in growth

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

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

BACTERIAL GROWTH CURVE

LAG PHASE• Organisms are adjusting to the

environment – little or no division

• synthesizing DNA, ribosomes and enzymes – in order to

breakdown nutrients, and to be used for growth

Mouse click for lag phase adjustment

LOGARITHMIC PHASE• Division is at a constant rate

(generation timegeneration time)

• Cells are most susceptible to inhibitors

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

STATIONARY PHASE

DEATH PHASE

in 37oC, pH 5.1 ; in 45oC, pH 6.2In bioreactors

BC YangFor lecture only

ENUMERATION OF BACTERIA

• 1) viable plate count

• 2) direct count

• 3) most probable number (MPN)1

23

45

6

7

8

9

10

VIABLE PLATE COUNT

• Most common procedure for assessing bacterial numbers– 1) serial dilutions of a suspension of bacteria

are plated and incubated

– 2) the number of colonies developing are then counted

• it is assumed that each colony arises from an individual bacterial cell

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

VIABLE PLATE COUNT

See next slide for bigger diagram

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

• CalculationCalculation

– 54 X 10,000 = 540,000 bacteria/ml

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

VIABLE PLATE COUNT

VIABLE PLATE COUNT

“TNTC”417 colonies

Dilution factor of 1/1,000 (10 -3)

Click to incubate

VIABLE PLATE COUNT

22 coloniesToo few the count

is less than 30

Click to incubate

Dilution factor of 1/1,000,000 (10 -6)

VIABLE PLATE COUNT

42 colonies

Dilution factor of 1/100,000 (10 -5)

Calculate the number of bacteria

per ml

Click to incubate

• Calculate:

– 42 colonies

– dilution factor of 100,000

• 42 X 100,000 = ???

• 4,200,000 bacteria/ml

VIABLE PLATE COUNT

Nutrient media

• Ordinary (simple) media • Special media (serum agar, serum broth, coagulated

serum, pota toes, blood agar, blood broth, etc.).• Elective media • Enriched media • Differential diagnostic media: (1) proteolytic action; • (2) fermentation of carbohydrates (Hiss media); • (3) haemolytic activity (blood agar); • (4) reductive activity of micro-organisms; • (5) media containing substances assimilated only by

certain microbes.

Biochemical properties

Colonies

Colonies

Colonies

Pure Cultures Isolation

Isolated colonies obtaining

Important Point: