Medical MicrobiologyMedical Microbiology

Prof. Dr. Jie YAN (严杰 )Department of Medical

Microbiology and Parasitology

E-mail: med_bp@zju.edu.cn

School of Medicine Zhejiang University

Introduction to medical microbiologyIntroduction to medical microbiology

•The word “microbe” comes from the Greek words mikros, meaning small life. So microbes / microorganisms are small living things that are too small to be seen by naked eye.

•Microorganisms were probably the first organisms to appear on the earth.

•However, these organisms were not seen until about 3 centuries ago when lenses powerful enough to make them visible were made.

•Viruses, bacteria, fungi, protozoa and some algae are all in this category.

Microbes / MicroorganismsMicrobes / Microorganisms

•The distribution of microorganisms is universal in nature including air, soil, water, animals and human body.

DistributionDistribution

•There is a close relationship between microorganisms and human beings.

• Beneficial activities: Most microbes are benefit to human beings, some are necessary (nitrogen and arbon cycles).

• Harmful activities: Only a small portion of microbes cause human diseases, which called pathogenic microbes.

Relationship with human beingsRelationship with human beings

• Medical microbiology is a branch of Microbilogy to study biological character, pathogenicity and immunoty, laboratory diagnosis, and prevention and control of pathogenic microbes.

Medical MicrobiologyMedical Microbiology

Microbes in nitrogen cycleMicrobes in nitrogen cycle

• The prokaryotic cell, in contrast to the eukaryotic cell, has no nuclear

membranes, mitochondria, endoplasmic reticulum, Golgi body,

phagosomes and lysosomes.

Prokaryotes / EukaryotesProkaryotes / Eukaryotes

• Prokaryotes generally possess only a single circular chromosome,

which is bound to a specific site on the cell membrane - the mesosome. 

• Prokaryotic ribosomes are 70S (30S and 50S subunits) in size, whereas

eukaryotic ribosomes are larger (80S, 40S and 60S subunits).

•According to organizational structure, microbes can be divided into three types:

Prokaryotes (Eubacteria and Archaebacteria)

Eukaryotes (fungi, Protozoa, algae)

Acellular entities (viruses)

Classification of microbes Classification of microbes

•Eubacteria include Bacteria, Chlamydiae, Mycoplasmas,

Richettiae, Spirochetes, and Actinomycetes. Some of them cause

human diseases.

VirusesViruses

•Viruses are obligate parasites totally dependent on their host cells for replication.

•Viruses are very small particles and have no basic cell

structure. A simplest virus consists of one core and one

protein coat (capsid). The core composed with a nucleic acid

molecule, either DNA or RNA.

•Fungi is a kind of eukaryotic cells. So they have various

organelles, for examples, nuclear membranes, mitochondria,

endoplasmic reticulum, Golgi body, phagosomes and lysosomes.

FungiFungi

•The numerous emerging and re-emerging infectious diseases

such as AIDS, SARS, avian influenza, tuberculosis, viral

hepatitis and so on.

New challenge in medical microbiologyNew challenge in medical microbiology

Morphology and Structure of BacteriaMorphology and Structure of Bacteria

BacteriologyBacteriology

Size of bacteriaSize of bacteria

• Unit for measurement of bacteria is micrometer (μm)

• On the average, bacteria are 2-8 μm in length and 0.2-2.0 μm

in diameter. Exceptions include some spiral shaped bacteria

that can reach 4- 500 μm.

10001000

Shape of bacteriaShape of bacteria

• Spherical (Cocci, sing. Coccus )

• Rods (Bacilli, sing. Bacillus)

• Spiral (Spiral bacteria) vibrio spirillum helicobacterium

Spherical bacteriaSpherical bacteria Different arrangements depending

on the plane of division

DiplococciDiplococci: Pair of cells divide in one plane

StreptococciStreptococci: Chain of cells formed by dividing in one plane several times

TetradTetrad: Divide in two planes

SarcinaeSarcinae: Divide in three planes

StaphylococciStaphylococci: Divide in many planes and remain together as a cluster

• Considerable variation in length and diameter: 0.5-1 μm in width and 2-5 μm in length.

• Most of rod-shaped bacteria are single arrangement.

Rod-shaped bacteriaRod-shaped bacteria

Diplobacilli: Bacilli that remain in pairs after they divide.

Streptobacilli: Bacilli that remain in chains after they divide.

Coccobacilli: A short Bacilli that nearly looks like a cocci.

Divided into:

Vibrio: comma shaped

Spirillum: helical

Spiral-shaped bacteriaSpiral-shaped bacteria

Structure of Bacteria

• bacterial structures may be defined:

Cell envelope

Plasmids

Flagella

Pili

Capsules

Spores

Cell envelope

Plasmids

Flagella

Pili

Capsules

Sspores

Important bacterial structuresImportant bacterial structures

Cell envelopeCell envelope

•Bacterial envelope is divided into cell membrane and cell wall

(Gram positive) plus an outer membrane (Gram-negative).

Gram-positive cocciGram-positive cocci Gram-negative bacilliGram-negative bacilli

(Gram-staining method)(Gram-staining method)

•Cell wall consists of peptidoglycan layer and attached structures.

Gram-positive Gram-negative

Cell wallCell wall: general component-peptidoglycan: general component-peptidoglycan

Peptidoglycan

•glycan backbone: N-acetyl

muramic acid and N-acetyl

glucosamine are alternatively

linked by -1,4 linkage.

•4-peptide side chain: links to N-acetyl muramic acid.

•Penicillin can block the linkage between peptide side chain and bridges to kill gram-positive bacteria.

•peptide bridge: links side chains (gram-negative bacteria have no peptide bridges).

Cell wall: Cell wall: characteristits of gram-positive bacteriacharacteristits of gram-positive bacteria

•There are some special components such as teichoic acids, the major superficial antigen of gram-positive bacteria .

•Peptidoglycan layer is thick (15-50 layers).

Cell wall: Cell wall: characteristits of gram-negative bacteriacharacteristits of gram-negative bacteria

•Peptidoglycan layer is thin (1-2 layers).

•There is outer membrane located in outside of peptidoglycan layer but no any teichoic acids.

Outer membraneOuter membrane

•Outer membrane of a gram-negative bacterium is composed of

phospholipids, membrane proteins and lipopolysaccharide (LPS)

Lipopolysaccharide (LPS)Lipopolysaccharide (LPS)

• O antigen is a polysaccharide

to act as the somatic antigen of

gram-negative bacteria.

•Core polysaccharide links O

antigen with lipid A.

•lipid A decides toxicity.

•LPS is also called endotoxin (poisonous to mammal cells).

•LPS has 3 regions: an external O antigen, a middle core, and an inner lipid A.

• Maintaining bacterial shape.

• Resistance to osmotic pressure

• Providing a platform for surface appendages such as flagella and pili.

• Providing a pathogenic function to adhere host cells (For gram-positive bacteria, the major adhesin is teichoic

acids. For Gram-negative bacteria, the major adhesin is pili and some of outer mambrane proteins).

• Playing an essential role in bacterial division

• Participating bacterial material exchange

• Containing major antigens.

Cell wall: Cell wall: function

• When bacteria are treated with 1) enzymes (e.g. lysozyme) with

cell wall hydrolytic activity or 2) antibiotics inhibiting

peptidoglycan synthesis, wall-less bacteria are generated which

is called L-forms of bacteria.

• L-forms of bacteria can of bacteria can cause chronic infections.

• L-forms of bacteria are difficult to cultivate and usually

require a medium with a right osmotic strength.

• It is resistant to antibiotics (e,g. penicillin) and difficulty to

detect (e.g. absence of O antigen).

Wall-less forms of bacteriaWall-less forms of bacteria

Electron micrograph of Staphylococcus Electron micrograph of Staphylococcus

A: L-form; B: wild typeA: L-form; B: wild type

Cell envelope

Plasmids

Flagella

Pili

Capsules

Spores

Important bacterial structuresImportant bacterial structures

PlasmidsPlasmids

•Plasmids are small, circular / line,

extra-chromosomal double-stranded

DNA.

•Usually present in multiple copies and

are capable of self-replication.

•Often code for pathogenic factors and

antibiotic resistant factors. Are not

essential for bacterial survival.

Cell envelope

Plasmids

Flagella

Pili

Capsules

Spores)

Important bacterial structuresImportant bacterial structures

Flagella: general descriptionFlagella: general description

• Flagellum is composed of flagellin and provide motility.

•It extends from cell envelope and projects as a long strand.

•Flagellum is slender that can not be seen by light microscopy unless a special stain is applied.

Flagella: structureFlagella: structure

• Basal body:

a structure to insert into cell envelope.

• Flagellin is an antigen (H antigens).

Flagella: function: function

•Motility of bacteria: move towards foodstuffs or away from toxic materials.

•Identification of bacteria: According to the mobility and antigenicity

of flagellin (H antigen).

•Possible pathogencity: chemotaxis to the suitable sites in hosts

for colonization.

Cell envelope

Plasmids

Flagella

Pili

Capsules

Spores

Important bacterial structuresImportant bacterial structures

PiliPili

•Pili are hair-like strands of bacteria.

•They are shorter and thinner than flagella, only visible under

electron microscope.

•Pilus is composed of special protein called pilin.

•Two types can be distinguished:

Ordinary pili

•Shorter, thinner, numerous for a bacterium

•Relative to bacterial adhesion (adhering to host cells)

•Contribute to virulence of some pathogenic bacteria

Sex pili

•Longer, coarser, only 1-4 for a bacterium

•Relative to bacterial conjugation (a pattern of bacterial genetic material exchanges)

•The recent data revealed the sex pili of some bacteria has the ability to adhere host cells.

PiliPili

Ordinary pili

Sex pili

Donor bacterium

Recipient

Electron graph of piliElectron graph of pili

Cell envelope

Plasmids

Flagella

Pili

Capsules

Spores

Important bacterial structuresImportant bacterial structures

•Capsule is a structure surrounding outside of cell envelope.

•Usually, slime layer is thinner than capsule.

•They are usually demonstrated by the negative staining or

“capsule stain” which gives color to the background.

Capsules and slime layers

•They are usually composed of polysaccharide. However, in

some certain bacilli, they are composed of polypeptide.

• They are not essential to bacterial viability.

• Some strains within a bacetrial species can produce a capsule,

whereas the others can not.

•Capsules are often lost during in vitro culture.

•The capsules contribute to invasiveness (virulence) of bacteria

by protecting them from phagocytosis by phagocytes.

Capsules and slime layers

Cell envelope

Plasmids

Flagella

Pili

Capsules

Spores

Important bacterial structuresImportant bacterial structures

SporesSpores

• Under adverse conditions, such as nutrient / water depletion,

some bacteria form a thick wall inside the cytoplasmic

membrane leading to a resting stage known as spores.

•Spores contribute to bacterial resistance.

SporesSpores

•One spore-forming bacterium can only produce one spore

which has no propagation ability.

•One spore germinates into one vegetative bacterial cell which

can propagate / multiplication. •Spore can be seen after staining with dyes. Sometimes, it can

also be seen as a colorless area by using conventional bacterial

staining methods.

•Spores are commonly found gram-positive bacilli.

•Different sizes, shapes and positions of spores will help us to

identify spore-forming bacteria.

Structure of sporesStructure of spores

Core spore wall /core

Cortex Coat Exosporium

Classification of bacteriaClassification of bacteria

•Taxonomic termsTaxonomic terms: :

Family: a group of related genera.Family: a group of related genera.

Genus: a group of related species.Genus: a group of related species.

Species: a group of related strains.Species: a group of related strains.

Type: sets of strains within a species (e.g. biotypes, Type: sets of strains within a species (e.g. biotypes, serotypes).serotypes).

Strain: Strain: one line or a single isolateone line or a single isolate of a particular species. of a particular species.•The basic taxonomic group is The basic taxonomic group is speciesspecies. .

strain strain type type species species genusgenus familyfamily

O157:H7O157:H7 Coli Coli Escherichia Enterobacteriaceae Escherichia Enterobacteriaceae

Classification of bacteriaClassification of bacteria

StaphylococcusStaphylococcus aureusaureus

S. aureusS. aureus

Genus Genus species species

金黄色葡萄球菌金黄色葡萄球菌

species genusspecies genus

Summary Summary

Structure of bacteria include essential structures of cell

wall,

cell membrane, cytoplasm, and nuclear material (nucleoid).

Some bacteria also have one or more of the particular

structures of capsule, flagella, pili, endospores. Structure of cell wall, cell wall structural differences

between Gram-positive and Gram-negative bacteria, concept

of plasmid, and functions of bacterial particular structures are

the most important contents, because of their close association

with bacterial pathogenesis.

Growth, Propagation and Metabolismof Growth, Propagation and Metabolismof BacteriaBacteria

• Autotroph: can synthesize organic substances using CO2 as carbon source and N2 or NH3 as nitrogen source. The energy comes from oxidation of inorganic substances.

• Heterotroph: use different organic substances, such as proteins, saccharides and lipids, as the nutrient substances or materials and energy source.

▲Saprophyte: dead bodies of animals and plants, or decomposed foods.

▲Parasite: living hosts (animals and/or human). Nearly all the pathogens are parasites.

Nutrtion types of bacteriaNutrtion types of bacteria

• Water: mediator for biological responses.

• Carbon source

• Nitrogen source

• Inorganic salts: have many functions to act as a component

of organic substance as well as to maintain enzymatic

activity and osmotic pressure and pH, etc.

• Growth factors: vitamins, some special amino acids,

hemoglobin and coenzyme I or II (blood, serum) .

Nutrient substances of bacteriaNutrient substances of bacteria

• Nutrient substances

• pH: 7.2-7.4 for microbial pathogens.

• Temperature: 37ºC for microbial pathogens.

• Gas: O2

Conditions of bacterial growth and propagationConditions of bacterial growth and propagation

▲Obligate aerobe: needs O2 during growth and propagation.

▲Microaerophilic bacteria: 5% O2.

▲Facultative anaerobe: grow and propagate in aerobic or anaerobic enviroment.

▲Obligate anaerobe: has no special enzymes (e.g. SOD and catalase) to deal with ROS such as O 2 and H2O2 produced in metabolism.

¯

• Growth and propagation of a bacterial individual: binary

fission (2n), a process in which a parent cell splits into two

daughter cells with approximately equal size.

Bacterial growth and propagationBacterial growth and propagation

a. Bacterial cell first can been seen to enlarge or elongate.

a

b. Followed by formation of transverse membrane and new cell wall.

b

c. The new membrane and cell wall grow inward from the outer layers.

c

d. The cell divided into the two daughter cells.

d

Bacterial growth and propagationBacterial growth and propagation

• Generation time: under optimal conditions, the average time

required for a population of bacteria to double in number.

20-30 min for most of bacteria (e.g. E. coli).

• Colony: a bacterial cluster from propagation of a bacterium.

▲Obtain a pure bacterial species.

▲Often used for bacterial counting.

• Growth and propagation of a bacterial population:

Bacterial growth and propagationBacterial growth and propagation

Growth curve

OD600

Time

Lag Phase

Log Phase

Stationary Phase

Death Phase

Bacterial growth and propagationBacterial growth and propagation

• Phenomena of bacterial growth in liquid medium

ii ii ii iii iii

Broth (a common liquid medium)

cultures can exhibit: (i) forming

cloudiness in broth (growth with

uniform turbid pattern), or (ii)

forming a ring at the top of broth

(growth with suspension pattern),

or (iii) forming sediment at the

bottom of broth (growth with

sedimentary pattern).

• Pyrogen: cause fever (LPS of G- bacteria and glycopeptide

or glycolipid of G+ bacteria).

• Toxins: exotoxins and endotoxin (LPS).

• Invasive enzymes: e.g. collagenase (invasion and spreading)

and coagulase (resist phagocytosis of macrophages).

• Others: pigment, vitamine, antibiotic, bacteriocin (细菌素 ).

Constructive metabolism of bacteriaConstructive metabolism of bacteria

• Carbohydrate Fermentation Tests

DestructiveDestructive metabolism of bacteriametabolism of bacteria

Positive: yellow color oryellow color with gas bubbleNegative: red color and no gas bubble

For identification of bacteria !

DestructiveDestructive metabolism of bacteriametabolism of bacteria

Methyl Red (MR) Testhydrolyse pyruvate (丙酮酸 )

Voges-Proskauer (VP) Test hydrolyse pyruvate (丙酮酸 ) → diacetyl (二乙酰 )

DestructiveDestructive metabolism of bacteriametabolism of bacteria

DestructiveDestructive metabolism of bacteriametabolism of bacteria

The citrate test utilizes Simmon's citrate media to determine if a bacterium can grow utilizing citrate as its sole carbon and energy source.

Citrate Utilization Test

Growth of bacteria in the media leads to development of a Prussian blue color (positive citrate).

DestructiveDestructive metabolism of bacteriametabolism of bacteria

Indole Test

hydrolyse tryptophan to produce indole

DestructiveDestructive metabolism of bacteriametabolism of bacteria

To determine the ability of a bacterium to produce hydrogen sulfide (H2S) by enzymatic reaction on amino acids such as cysteine, cystineand methionine.

Positive result: The hydrogen sulfide combines with ferrous sulfide (Fe2S) in the triple sugar iron (TSI) agar to form a black to dark insoluble precipitate.

Hydrogen Sulfide (H2S ) Formation Test

DestructiveDestructive metabolism of bacteriametabolism of bacteria

Urease Test

Principle:Principle: The hydrolysis of urea by urease The hydrolysis of urea by urease

produces ammonia and carbon dioxide. The produces ammonia and carbon dioxide. The

formation of ammonia alkalinizes the formation of ammonia alkalinizes the

medium, and the pH is detected by the color medium, and the pH is detected by the color

change from light orange to pink-red.change from light orange to pink-red.

Positive result:Positive result: pink-red color pink-red color

Negative result:Negative result: light orange light orange

Death of MicroorganismsDeath of Microorganisms

Disinfection & Sterilization

Concept and DefinitionConcept and Definition

Sterilization:Sterilization: A physical or chemical process to kill all microbial

life including spores.

Disinfection:Disinfection: A physical or chemical process to kill vegetative microbes, but not kill spores.

Bacteriostasis:Bacteriostasis: A physical or chemical process to inhibit

bacterial growth / propagation in vitro and in vivo.

Antisepsis:Antisepsis: A physical or chemical process to inhibit bacterial

growth / propagation in vitro, but not kill bacteria.

Asepsis:Asepsis: a state of being free of living microbes.

Antimicrobial agentsAntimicrobial agents

▲ Physical Agents: Heat, Radiation, Filtration,

Low Temperature and Desiccation (Dry)

▲ Disinfectants and Antiseptics

PhysicalPhysical antimicrobial agents: Heatantimicrobial agents: Heat

▲ A temperature of A temperature of 100 100 ºCºC ( (boilingboiling) usually for ) usually for 2-52-5 minmin will kill will kill all vegetativeall vegetative formsforms but not kill spores. but not kill spores.

▲ A temperature of A temperature of 121 121 ºCºC for for 15-20 min15-20 min will kill will kill all microorganisms including sporesall microorganisms including spores ((autoclaveautoclave). ).

▲ Hot air sterilizationHot air sterilization by hot air ovens, heating at by hot air ovens, heating at 160 ºC160 ºC for for 2 h2 h,,

PhysicalPhysical antimicrobial agents: Ultraviolet Rayantimicrobial agents: Ultraviolet Ray

▲ Microbial killing effect of sun light is due in large part to the action of ultraviolet light.

▲ Activity of ultraviolet (UV) ray depends on: i) Length of exposure: 30 min; ii) Wavelength of UV ray: 260 nm - 270 nm

thymine-thymine dimmers

within the one DNA strand

will block base pairing and

DNA replication.

Summary Summary

The most important contents in this lecture are displayed as

the followings:1) Bacteria growth curve, especially the characteristics and application of

log phase and maximum stationary phase.

2) Concepts of sterilization, disinfection and asepsis, and the temperature

and time to kill bacteria including spores when using autoclaving and

hot air sterilization.

3) The microbicidal mechanism and application limits of UV radiation.

4) The types (names) of bacteria based on the difference of O2

requirement in growth.

5) Concepts of bacterial colony, pyrogen and invasive enzymes.