1.0 Introduction to Bacteriology

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Microbial Physiology

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Cell organelles

Both contains: DNA Plasma membrane Cytosome Ribosome

Eukaryotic Cell Contain a membrane-bound nucleus and numerous

membrane-enclosed organelles Mitochondria, lysosomes, golgi apparatus- not found

in prokaryotes Animal, plants, fungi More complex Found in many different forms

Nucleus Contains most of the genetic material (DNA) of the

cell Additional DNA: mitochondria and (if present)

chloroplasts Nuclear DNA is complexed with proteins to form

chromatin organized as a number of linear chromosomes

Genetic control of the cell is carried out by the production of RNA in the nucleus (the

process of transcription)

Subsequent transfer of this RNA to a ribosome in thecytoplasm, where protein synthesis is directed (theprocess of translation)

The resulting proteins carry out cell functions Also located in the nucleus is the nucleolus or

nucleoli where ribosomes are assembled

Nucleus is bounded by a nuclear envelope a double membrane perforated with pores connected to the rough endoplasmic reticulum

membrane system

INTRODUCTION TO BACTERIOLOGY


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Introduction toIntroduction toIntroduction toIntroduction to MicrobiologyMicrobiologyMicrobiologyMicrobiology

Cytoskeleton Consists of microtubules, intermedimicrofilaments

Maintain cell shape and anchor org Causes cell movement Frequently undergoes assembly an

according to cellular needs

Ribosome Site of protein synthesis in the cell Each ribosome consists of

a large subunit and a small sub each contains rRNA (ribosomal

ribosomal proteins In protein synthesis

the mRNA (messenger RNA) mribosome

amino acids attached to tRNAsare brought to the ribosome

Amino acids are joined to produce Ribosomes exist free in the cytopla

the endoplasmic reticulum (ER) Free ribosomes synthesize the

function in the cytosol Bound ribosomes make protei

distributed by the membrane sthose which are secreted from

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anelles

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oves through the

(transfer RNAs)

he proteinm and bound to

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ns that areystems, includingthe cell.

Plasma Membrane Phospholipid bila

encloses every liv Blocks uncon

soluble mate Various proteins

bilayer penetrate Responsible for t

membrane1. controlling th

and out of th

2. mediating thstimuli (signa3. interacting w

Mitochondria Site of cellular re

generates Ausing oxygen

Primary function:triphosphate) froand Pi (inorganic

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er with embedded proteinsing celltrolled movements of water-rials into or out of the cellmbedded in the phospholipidinto and through the bilayere specific functions of the plasma

e flow of nutrients and ions intoe cells

e response of a cell to externall transduction)ith bordering cells

pirationP from substrates in reactions

synthesize ATP (adenosineADP (adenosine diphosphate)

phosphate).


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Prokaryotic cell

Fundamentally different in their internalorganization from eukaryotic cells

lack a nucleus and membranous organelles The genetic material (DNA) is localized to a region

called the nucleoid has no surrounding membrane Consists of a single circular molecule of loosely

organized DNA lacking a nuclear membrane andmitotic apparatus The cell contains large numbers of ribosomes that

are used for protein synthesis Periphery of the cell is the plasma membrane Outside the plasma membrane of most prokaryotes

is a fairly rigid wall which gives the organism itsshape

Plasmid Contains a variety of genes for antibiotic

resistance and toxins Extrachromosomal double stranded circular

DNA molecules that are capable of replicating

independently of the bacterial chromosome Carry genes for the following functions:

Antibiotic resistance Resistance to heavy metals such as mercury Resistance to ultraviolet light Pili Exotoxins including several enterotoxins

The walls of bacteria consist of peptidoglycans Murein, mucopeptide Gives rigid support Protects against osmotic pressure Site of action of penicillins and cephalosporins

Some bacteria have flagella and/or pili For locomotion To pull two cells in close contact Facilitate the transfer of genetic material

Bacteria are held by peptidoglycan, protectingagainst osmotic rupture

Gram-positive bacteria: substantial layer 50 to 100molecules thickness

Gram-negative bacteria: only 1-2 molecules thick Outer lipopolysaccharide layer is present on top

of peptidoglycan

Bacterial structures (outside the cell wall):Flagella

Long whiplike appendages used for chemotaxis Process of moving the bacteria toward nutrients

and other attractantsCapsule

Protects against phagocytosis Gelatinous layer covering the entire bacterium Important mainly for 4 reasons:


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Determinant of virulence of many bacteria sinceit limits the ability of phagocytes to engulf thebacteria

Specific identification of an organism Capsular polysaccharides are used as antigens in

certain vaccines for they are capable of elicitingprotective antibodies

May play a role in the adherence of bacteria tohuman tissue

Pilus or fimbria Mediates attachment to cell surfaces Sex pilus mediates attachment of 2 bacterias during

conjugation Hairlike filaments that extend from the cell surface Shorter and straighter than flagella Found mainly on gram negative bacterias

Spore Provides resistance to dehydration, heat and

chemicals Formed in response to adverse conditions Has no metabolic activity and can remain dormant

for years As a result of their resistance to heat, sterilization

cannot be achieved by boilingGlycocalyx (slime layer)

Mediates adherence to surfaceGram Stain

developed in 1884 by Hans Christian Gram characterizes bacteria based on the structural

characteristics of their cell walls

thick layers of peptidoglycan in the "Gram-positive" cell wall stain purple

thin "Gram-negative" cell wall appears pink By combining morphology and Gram-staining, most

bacteria can be classified as belonging to one of fourgroups

Gram-positive cocci or bacilli Gram-negative cocci or bacilli

Useful in: Identification of many bacteria Influencing the choice of many antibiotic

because in general, gram (+) bacteria are moresuspceptible to penicillin G than gram (-)

Acid-Fast Cell Wall In addition to peptidoglycan, the acid-fast cell wall of

Mycobacterium contains a large amount of glycolipids, mycolic acids

Porins required to transport small hydrophilic

molecules thru the outer membrane of the acid-fast cell wall

Because of its unique cell wall, it is stained by the acid-fast procedure will resist decolorization with acid-alcohol and

stain red, the color of the initial stain,

carbolfuchsin

Common Features of Prokaryotic and Eukaryotic Cells DNA, the genetic material contained in one or more

chromosomes and located in a nonmembrane boundnucleoid region in prokaryotes and a membrane-bound nucleus in eukaryotes

Plasma membrane , a phospholipid bilayer withproteins that separates the cell from the surroundingenvironment and functions as a selective barrier forthe import and export of materials

Cytoplasm , the rest of the material of the cell withinthe plasma membrane, excluding the nucleoidregion or nucleus, that consists of a fluid portioncalled the cytosol and the organelles and otherparticulates suspended in it

Ribosomes , the organelles on which proteinsynthesis takes place

Classification of Bacteria

seeks to describe the diversity of bacterial species bynaming and grouping organisms based on similarities

Basis of Classification cell structure cellular metabolism (differences in cell

components such as DNA, fatty acids, pigments) Serologic reactivity (antigens) Animal pathogenicity Antibiotic sensitivity

Bacterial Morphology


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Difference between Gram (+) and Gram (-)

Gram Positive Gra2 layers:

1. inner cytoplasmicmembrane

2. outer thickpeptidoglycan

layer (60-100%)

3 layers:1. inner cy

membrane2. thin pe

(5-10%)3. outer m

lipopoly

Low lipid content High lipid cNO endotoxin (except Listeriamonocytogenes)

Endotoxin (

No periplasmic membrane PeriplasmicNO porin channel Porin channVulnerable to lyzozyme andpenicillin attack

Resistant topenicillin at

6 gram-positive organisms that cause diseas 2 gram-positive cocci

Streptococcus Staphylococcus

4 gram-positive bacilli Bacillus Clostridium Corynebacterium Listeria

Rest of other bacterial organisms a

Gram Positive Bacteria: Purple/ Blue

Produce

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acterias

Negative

toplasmic

tidoglycan layer

embrane withsaccharide (LPS)

ntentPS) lipid A

spaceellyzozyme andacke in humans

e gram-negative

Gram positive bacilli (ex.

Gram positive cocci (Ex. SGram Negative Bacteria:

Gram negative diplococci (

sp ores

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acillus anthracis)

aphylococcus aureus)ink/ Red

ex. Neisseria )


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Gram negative rods (ex. Escherichia coli )Gram Staining

1. Crystal Violet Dye Stains all cells blue/purple

2. Iodine solution

Mordant Form crystal violet-iodine complex3. Organic Solvent: acetone or ethanol

Gram negative colorless Gram positive remain blue Solvent extracts the blue-dye complex from the lipid

rich, thin-walled gram negative4. Red dye Safranin

Stains decolorized gram (-) red or pink Gram (+) remains blue If step 2 is omitted and Grams iodine is not added,

gram (-) bacteria stain blue rather than pink. This isbecause the organic solvent removes the crystal

violet-iodine complex but not the crystal violetalone. Gram (+) bacteria also stain blue when iodineis not added.

A. ENCOURAGING GROWTH OF MICROORGANISMS INVITRO

Bacterial Growth Increase in the number of microorganisms

Bacterial Colony Mound of bacteria containing millions of cells

Culture Medium Nutrient material prepared for the growth of

microorganisms in the laboratory Inoculum

Microbes introduced into a culture medium toinitiate their growth

Culture Microbes that grow and multiply in or on culture

mediumInoculation of Culture Media

Inoculation of a liquid medium

Involves adding a portion of the specimen to themedium

Inoculation of a solid or plated medium Involves the use of a sterile inoculating loop to

apply a portion of the specimen to the surfaceof a medium

Sterile technique

When it is necessary to exclude all microorganismsfrom a particular area

Incubation Used in culturing human pathogens Incubator

Chamber that contains the appropriateatmosphere, moisture level and temperature

Bacterial Population Count Number of bacterial cells in a millilitre of liquid or in

a gram of solid material To determine the degree of bacterial contamination

in drinking water, food, etc.

MICROBIAL PHYSIOLOGY


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E.g. viable plate count- determines the number of viable bacteria in a liquid sample such as milk, water,ground food diluted in water or a broth culture

Bacterial Population Growth Curve Shows the growth of a bacterial population over

time 4 Phases Lag Phase

Time for bacteria to absorb nutrients, synthesizeenzymes and prepare for cell division

Little or no cell division during this phase Log Phase

Logarithmic growth phase or exponentialgrowth phase

Time when growth rate is greatest Period when microorganisms are particularly

susceptible to adverse conditions (e.g. radiation,

antimicrobial drugs) Stationary Phase

Nutrient exhaustion Waste product accumulation Harmful pH changes slow rate of cell division No. of bacteria dividing=No. of bacteria dying

Death Phase Logarithmic decline phase No. of cell deaths > No. of new cells formed

B. INHIBITING THE GROWTH OF MICROORGANISMSSterilization

Complete destruction of all microorganismsDisinfection

Destruction or removal of pathogens from nonlivingobjects by physical or chemical methods

Sanitization Reduction of microbial populations to levels

considered safe by public health standardsSepsis

Presence of pathogens in blood or tissuesAntisepsis

The absence of pathogens

Chemical Agents Disinfectants

Chemicals used to disinfect inanimate objects Antiseptics

Solutions used to disinfect living tissues Microbiostatic Agents Chemicals or drugs used to inhibit the growth

and reproduction of microorganisms Bacteriostatic Agents

Drugs which inhibit the growth adreproduction of bacteria by interfering withbacterial protein production, DNAreplication or other aspects of bacterialcellular metabolism

Bactericidal Agents Any substance that kills bacteria ideally and

nothing else either disinfectants, antiseptics or

antibioticsPhysical Methods

Heat Cold Dessication Radiation Ultrasonic waves Filtration Gaseous atmosphere

Broad-spectrum Antibiotics Active against several types of microorganisms

Acts against both gram (+) and gram (-) bacteria E.g. tetracyclines are active against many gram (-)rods, chlamydiae, mycoplasmas and rickettsiae

Narrow-spectrum Antibiotics Active against only specific families of bacteria E.g. vancomycin primarily used against certain gram

(+) cocci5 Sites of Action of Antimicrobials

Cell Wall synthesis Cell membrane function Nucleic acid synthesis or DNA replication Protein synthesis Synthesis of essential metabolites

Bacteria are held by peptidoglycan, protectingagainst osmotic ruptureGrowth Inhibitors of Pathogens


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Gram-positive bacteria: substantial layer 50 to 100molecules thickness

Gram-negative bacteria: only 1-2 molecules thick Outer lipopolysaccharide layer is present on

top of peptidoglycan

Mechanism of Action of Anti-Microbial AgentsInhibition of Cell Wall Synthesis

Can inhibit peptidoglycan synthesis in 2 differentmanners :

a. Inhibit synthesis of the linear strands (e.g.vancomycin)

b. Inhibit cross-linking of the strands (e.g. Beta-lactams- penicillins and cephalosporins)

Disrupt Cell Membrane Function Phospholipids of gram (-) bacteria Sterols of eukaryotic fungi ergosterol

Inhibit Nucleic Acid Synthesis Quinolones inhibit DNA gyrase

Prevents relaxation of supercoiled DNA duringreplication

Rifampicins inhibits bacterial RNA polymerase mRNA synthesis

Inhibit Protein Synthesis Targets of antimicrobial agents that inhibit protein

synthesis:o 1. 30S subunit (small subunit)

a. Aminoglycosides andStreptomycin bind irreversibly tothe 30S subunit and preventformation of the initiation complex

- stop protein synthesiso 2. 50S subunit (large subunit)

a. Erythromycin andChloramphenicol bind reversibly to50S - inhibit formation of growingpolypeptide chain

Inhibit Folic Acid Synthesis Two antimicrobial agents that inhibit folic acid

synthesis: Sulphonamide- sulfa drug Trimethoprim

Symbiosis Close and long-term relationships between different

biological speciesNeutralism

relationship between individuals of different specieswhere both individuals derive a benefit.

Commensalism Relationship between two living organisms whereone benefits and the other is not significantlyharmed or helped.

MutualismParasitism

Relationship is one in which one member of theassociation benefits while the other is harmed

Synergism

Role of Indigenous Flora Denotes the population of microorganisms that

inhabit the skin and mucous membranes of healthypersons

Roles: Plays role in maintaining health normal function

(ex. GIT flora synthesizes Vit.K and aids inabsorption of nutrients)

Prevents colonization of pathogens and diseasethru bacterial inteference

Bacterial Interference Microorganisms compete for receptors or binding

sites on host cells

Competition for nutrients Mutual inhibition by metabolic or toxic products

MICROBIAL ECOLOGY

NORMAL MICROFLORA


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When do normal flora become pathogenic? Normal flora may produce disease when introduce

into the bloodstream or tissues Streptococcus viridans (resident of upper

respiratory tract)

Large #s are introduced into the bloodstreamfollowing tooth extraction or tonsillectomy

May settle on deformed or prosthetic valvesinfective endocarditis

Predominant normal flora at various sitesSkin

Staphylococcus epidermidis Corynebacterium Proprionibacterium Micrococcus Yeasts

Conjunctivae Staphylococcus epidermidis

Nose and nasopharynx Staphylococcus epidermidis Staphylococcus aureus Streptococcus species

Mouth and Oropharynx S. epidermidis Non-group A streptococci Streptococcus pneumoniae Streptococcus mitis Stretococcus salivarius Neisseria Haemophilus

Veillonella Bacteroides Fusobacterium Treponema Lactobacillus Yeasts

Small intestine Lactobacillus Streptococcus species Enterococcus Veillonella Actinomycetes Yeasts

Large intestine Bacteroides Clostridium Fusobacterium Eubacterium Bifidobacterium Lactobacillus Peptostreptococcus Enterococcus Enterobacteriaceae

Gut Corynebacterium

Alpha-hemolytic & Nonhemolytic streptococci Staphylococcus epidermidis

Enterococcus Lactobacillus Mycobacterium smegmatis Enterobacteriaceae Bacteroides

Fusobacterium

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1.0 Introduction to Bacteriology