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Devised By Paul Ehrlich
 
Devised By Paul Ehrlich
 
Used for bacteria resistant to Gram's stain ie: M. Tuberculosis and Nocardia species
 
Used for bacteria resistant to Gram's stain ie: M. Tuberculosis and Nocardia species
 +
These two species are not gram (+) or gram (-)
 +
They are resistant to gram's stain they have a thin peptidoglycan layer with external arabinogalactan layers which are esterified into glycolipids (mycolic acids). This gives the cell wall a waxy appearance.
  
 
====Ziehl-Neelsen staining (hot method)====
 
====Ziehl-Neelsen staining (hot method)====

Revision as of 02:34, 11 March 2008

Medical Microbiology

Study of microscopic organisms includes

  • Bacteriology Bacteria simplest smallest single celled free living organisms
  • Virology viruses - non cellular parasitic, not living organisms
  • Mycology fungi - microscopic (molds and yeast), macroscopic (mushrooms and puff balls), unicellular and multicellular
  • Protozoology - protozoa, single celled
  • Phycology/Algology - algae - simple aquatic organisms ie seaweeds

Koch's postulates

  1. The same microbe is always associated witha specific disease
  2. this microbe can be recovered and grown in pure culture
  3. The pure culture must cause disease in an experimental animal
  4. The original microbe must be recovered from t he experimental disease

Examples of Problems:

  • Mycobacterium leprae - requres animal host
  • Neisseria gonorrhoeae - only human host
  • Opportunistic pathogens - immunocompromised host

Use of Molecular Postulates

  • Criteria a for determining cause of infections using molecular techniques
  1. Virulence factor: gene or gene product must be found in pathogenic stains but not in non-pathogenic stains
  2. When a virulence factor is introduced into a non-pathogenic strain it should become a pathogenic strain
  3. Genes for virulence must be expressied during the course of disease
  4. Antibodies specific for the virulence gene products should be protective


Classifications of Microogranisms

Organisms:

  • Bacteria, Archaeabacteria and Eukaryotes.
  • Infectious Agents: Viruses, viroids, prions

Bacterial and fungal classification using taxonomy

  • Kingdom, Phylum, Class, Order, Family, Genus, Species
  • Order has suffix of ales, Family has suffix of aceae
  • Biovar - variant strain that differs physiologically or biochemically
  • Serovar - sub division of species
  • Strain - a genetic varient or subtype

Viral Taxonomy

  • Order suffix virales
  • Family suffix viridae
  • Subfamily suffix virinae
  • Genus suffix virus
  • Species individual virus

Bacterial Morphology

Size

Bacteria range in size between .2 micrometers and 10 micrometers , usually around 1-2 micrometeres in diameter (Mycoplasma species range from .15 -.3 micrometers in diameter Although bacteria have small size, the importance of this is their large surface area to volume ratio. Prokaryotes do not require complex nutrient uptake and transportation mechanisms (use passive diffusion)

Bacterial Shapes

  1. Spherical - Coccoid (Coccus (singular), cocci (plural)
  2. Cylindrical - Rod (Bacillus (singular), Bacilli (plural)
  3. Curved, Helical Twisted, Spiral Vibrio (refers to vibratory motility)
  4. Square Cuboid (not infectious)

Cell Arrangement

  • Bacteria can be arranged singularly, in pairs, in chains or in clusters
  • Specific Terminology is used for coccoidal Bacterial arrangements
    • Single Single
    • Pairs: Diplococci Streptococcus pneumoniae
    • Chains Streptococci Streptococcus pyogenes
    • Clusters Staphylococci All Staphlococcus species
    • Tetrads Example: Sarcina species
    • These terms do not exsist for bacillus
  • Coccobacillus - a very short bacillus, a term indicating an intermediate appearance of a bacilli and coccus
  • Pleomorphic - cannot retain proper shape or proper stain. Could be very old or very young bacteria

Staining Techniques

Grams staining

Invented in 1884 by Hans Christian gram. This is the most important staining technique for identifying and classifying bacteria.
Utilizes the ability of bacteria to retain crystal violet-iodine complex after exposure to organic solvent.
Note: Never carried out on a throat swab or fecal location.
Steps:

  1. Flood with Crystal violet (primary stain)
  2. Flood with Gram's iodine (mordant)
  3. Drop wise add decoloriser (acetone or alcohol) *critical step*
  4. Immediately rinse with water
  5. Flood with safranin (counter stain)

Gram Positive Bacteria stain Blue/Purple Gram Negative Bacteria stain Red

Utility of Gram Stains

  1. Determine the adequacy of the specimen for culture
  2. Make a presumptive etiologic diagnosis and early clinical decision
  3. Suggest a need for non-routine laboratory procedure
  4. Help make accurate interpretation of culture results
  5. Provide better insight into the current infection

Acid Fast Staining

Devised By Paul Ehrlich Used for bacteria resistant to Gram's stain ie: M. Tuberculosis and Nocardia species These two species are not gram (+) or gram (-) They are resistant to gram's stain they have a thin peptidoglycan layer with external arabinogalactan layers which are esterified into glycolipids (mycolic acids). This gives the cell wall a waxy appearance.

Ziehl-Neelsen staining (hot method)

  1. Flood with hot basic carbolfuchsin; decolorize with acid-alkali
  2. Counterstain with methylene blue or malachite green
  3. Acid fast bacteria - Red/Pink; Non Acid-fast bacteria - Blue/Green

Kinyoun Stain (cold method)

Same as Ziegl-neelsen method but does not require heat

Fluorochrome stain

(auramine-rhodamine)

  • Primary stain is a fluorescent dye and the counterstain is an oxidizing agent (Potassium permanganate)
  • The bacteria would be yellowish/green against black background

Bacterial Ultrastructure

Flagellum

Flagella (plural form)

General features
  • Extends outwards from within the cell
  • Provides motility...but some bacteria are motile but lack flagella
  • May have an essential role for colonization (contributes to virulence)
  • Not essential for bacterial survival
  • All coccoidal cells lack flagella
Flagellum arrangement

Polar (at one or both ends/poles) or Lateral(situated around the entire surface)

Composition of flagellum

Composed of 3 parts

Helical filaments
  • long & thin ~20nm dia by 1-7 micrometers in length
  • Composed of protein - flagellin, antigenic termed the H (Haugch antigen). This H-antigen is useful for identifcation of bacteria
    • <math>H^+</math> possess flagella (motile)
    • <math>H^-</math> lack flagella (non-motile)
Hook
  • a short curved structure which anchors the filament into the basal body
Basal Body
  • Contains rod and 1-2 sets of double plates/rings.
  • All flagella contain two rings (S & M) for rotation located in the cytoplasmic membrane.
  • Gram negative flagella possess additional 2 rings (L & P) located in outer membrane of cell wall which stabilize the filament)
    • 2 rings Gram positive bacteria
    • 4 rings Gram negative bacteria
Movement
  • Taxis - involuntary movement of organism in response to a stimulus
  • Magnetotaxis - magnetic field
  • Phototaxis - light
  • Thermotaxis - heat
Chemotaxis
  • response to chemical stimulus (nutrients or growth inhibitors within the environment
  • Chemicals are recognized by protein receptors located in the cytoplasmic membrane
Recognition of response
  • + response- organism swims from low concentration to high concentration (up the concentration gradient)
  • - response - organism swims down the concentration gradient
  • Non response- random walk
Mechanism
  • Rotation of basal body plates in the clockwise or counterclockwise direction
  • ccw rotation - flagella sweeps around cell in common axis which results in a forward motion
  • cw rotation - Reversal of basal plates which causes the flagella to stop rotating and results in a tumbling motion to allow for a change in direction
  • random movement indicates absence of concentration gradient
  • movement during the presence of an attractant gradient resulting in a reduced tumbling frequency.

Additional outer appendages

  • Play no role in motility
  • Hollow , helical 9-10 nm diameter, thinner, shorter and more numerous than flagella.
  • Composed of PILIN protein (antigenic, can be used for classification & identification)
Pilus/Pili
  • F-Pilus (Fertility/sex)
  • A bacterium may possess a single pilus or up to 10
  • Found only in Gram negative bacteria
  • Function: transfer of genetic material between bacterial cells
Fimbria
  • known as common attachment pili (type I pili) or Colonization factor antigens (CFA)
  • enterotoxigenic E.coli
  • Mostly gram negative bactera and some gram positive bacteria
  • Function: adhesion of bacteria to surfaces, ie: tissues helps bacteria to establish colonization at a particular site (virulence)

Glycocalyx

  • Known as the capsule or slime layer
  • External polysaccharide
  • Exception: B.anthracis has a polypeptide glycocalyx
  • non vital
  • not all bacteria possess a glycocalyx
  • shows some degree of organization
Slime layer
  • poor organization, weakly attached to cell wall and easily removed.
  • ie: Coagulase Negative Staphylococcus epidermis
  • Slime produced allows attachment to medical plastic implants leading to biofilm formation
Capsule
  • organized, tightly adhered to cell wall harder to remove
  • known as the K antigen (or M antigen in Streptococcus pyogenes or Vi antigen in Salmonella
Function
  1. Adherence in order to form colonies with other species members and to stick to surfaces
  2. Antigenic activity for id(detect K antigen)
  3. Antiphagocytosis - resists phagocytosis
  4. Prevention of neutrophil killing of engulfed bacteria by limmiting access of the lysosome contents into the interior of the bacterial cell
  5. Prevention of Polymorphonucleur (PMN) Leukocyte migration to site of infection- substance released from capsule paralyzes PMN leukocyte
  6. Toxicity to host cell
  7. Protection of bacterial cell against - oxygen toxicity (anaerobes), desiccation & nutrient loss
Quelling Reaction

a swelling reaction that detects the presence of a capsule add antiserum and capsulated bacteria -> results in swelling use specific antigens (K/M/Vi) for serotyping

Cell Wall

  • surrounds all eubacteria except mycoplasma species
  • important bacterial characteristics
  • structure and function distinctive
  • composed of peptidoglycan (murein) backbone-contains L&d amino acid isomers .This is unique to bacteria
  • composed of a diasaccharide of NAG (N-actylemuramic acid) and NAM (Nacetylglucosamine) by a Beta 1-4 glycosidic bonds
  • Provides rigidity and strength
  • prevents osmotic lysis
  • 3rd aaof one tetrapeptide to the 4th aa of a second tetra peptide on NAM on adjacent glycan backbones
  • This provides rigidity and strenthg and prevents osmotic lysis

Gram negative E coli Gram positive Ecoli 3rd aa: diaminopmelic acid 3rd aa: L-lysine direct lipprotein cross linkage Pentaglycine cross linkage few cross linkages Greater cross linkage > 90o open mesh framework A tight framework

Gram Positive Bacterial cell Wall
  • Simple composition of thick peptidoglucan layer (50-60% of weight)
  • Teichoic and lipoteichoic acids are only found in gram positive bacteria
  • these are highly anionic so they sequester cations
  • bind proteins, act as adhesive sites(adhesins) for viruses
  • are antigenic; used for id and classification
  • Cell wall may contain CH20 and proteins depending upon species
  • (M,T,R proteins of group A streptococcus, protein A of staphlyococcus aureus)
Roles of lipoteichoic acid during disease
  • dermal necrosis (schwartzman rxn)
  • induction of cell mitosis at site of infection
  • stimulate specific immunity
  • adhesion to the human cell
  • complement activation
  • induction of hypersensitivity (anaphylaxis)
Why do gram positive bacteria stain purple
  • crystal violet pierces cell wall and sits above cytoplasmic layer
  • gram's iodine passes peptidoglycan layer and sits on the cytoplasmic layer to help cell retain crystal violet
  • add decolorizer to remove the excess stain
  • peptidoglycan is hydrated
  • add organic solvent -> causess peptidoglycan to become dehydrated -> crystal violet is trapped in periplasmic space
Gram negative bacteria
  • complex composition of thin peptidoglycan layer comprimising 5-10% of bacterial cell dry weight
  • outer membrane present attached to peptidoglycan layer by lipoproteins known as braun proteins
  • membrane contains porins (protein channels) as well as lipopolysaccharide which extends outward
Lipopolysaccharide LPS structure
  • entire structure is known as endotoxin -
  • Lipid A anchors LPS to outer membrane (toxic)
  • Core polysaccharide portion external to lipid A
  • Terminal polysaccharide - repeating subunits known as O-antigen
  • provides bacterial serotypes used forclassification and identification ecoli O157:H7
=Effects of Endotoxin=
  • fever
  • hemorrhagic necrosis (schwartzman reaction )
  • disseminated intravascular coagulation
  • Production of TNF
  • Activate alternative complement pathway
  • stimulate bone marrow cell prolferation
  • enhance the immune and limulus lysate reaction
  • can lead to septic shock
Lipoligosaccharide (LOS)

Some gram negative bacteria ( Bordetella pertusis , neisseria meningitidis and camplyobacter jejuni have LPS without the O polysaccharide but with an extended core region these have a LOS Lipoligosaccharide comprises of 2 components -> no terminal repeating subunits

  1. Lipid A (toxic
  2. core oligosaccharide external to lipid A
  • virulence factor : LOS levels correlate with release of IL-1, IL-6 and TNFalpha -imp in meningococcemia and meningoccal meningitis


Gram negative bacteria stains pink/red because the thin layer of peptidoglycan, can't dehydrate much to trap crystal violet, also the crosslinkages allows gram negatives ....outer membrane layer

acid fast stain

mycobacterium and nocardia are not gram+ or gram - they are resistant to gram's stain they have a thin peptidoglycan layer with external arabinogalactan layers which are esterified into glycolipids (mycolic acids). This give the cell wall a waxy cappearance.


Chemical agents on bacterial cell walls

effects of lysozyme

produced by humans in saliva,mucus, blood sweat and tears cleaves the beta 1-4 bonds between nam and nag destroys all or part of the cell wall result -> wall degraded, w/ wall support .5 sucrose) cell will not lyse)

-spheroplast: mostly gram negative -> portion of cell wall remains
-protoplast: mostly gram positive -> cell wall completely removed

why are gram positive more susceptiel to lsozyme and penicillin than gram negative bacteria

penicilli

-binds irreversibly to penicillin binding proteins (PBPs) Transpeptidases, enzymes needed for peptidoglycan synthesis

Penicillin forms an inactive complex with the the transpeptidase inhibiting formation of peptide cross linkages to prevent complete cell wall formation. Results in defective cell walls with no protection to osmotic shock which leads to cell death -This only works on growing cells Penicillin will have no effect on mycoplasma species because they lack a cell wall


periplasmic space

space between inner and outer membranes of gram negative bacteria but is also present in some gram positive gel like area of loose network of peptidoglycans contains nutrient transport proteins, preoteases, detoxifying enzymes, membrane derived oligosaccharides and osmoprotectants

axial filaments

-flagella like filaments (similar chemically and structurally) Composed of a long thin microfibril inserted into a hook entire structure is in the periplasmic space and is referred to as the endoflagellum they have a corkscrew-like motion

cytoplasmic membrane

aka plasma membrane/inner membrane, or cell membrane

a phospholipid bilayer 4-5nm thick, phospholipid 30-40% & protein 60-70% proteins intergral and peripheral

      • no sterols except mycoplasma species

semipermeable barrier

Functions

  1. active transport
  2. secretion of extracellular enzymes and toxins
  3. oxidative phosphorylation ...bacteria do not have mitochondria (cytochromes and dehydrogenase enzymes are in the cyltoplasmic membrane
  4. biosynthesis and export cell wall components (phospholipid biosynthesis
  5. anchoring dna (during cell divison ) (mesasome)
  6. chemotactic response - receptors located wthin the cytoplasmic membrane

internal contents

  1. protoplasm/cytosol

granular appearance due to free ribosomes site of all biochemical activity of a bacterial cell composed of 70-80% water which acts as a solvent

  1. Ribosomes

RNA/protein bodies sites of protein synthesis 70S ribosomes made up of 50S and and 30S subunits eukaryotic -> 80S from 60S + 40S another site for antibiotics

  1. Mesosomes

Extensive invaginations of cytoplasmic membranes mainly seen in gram positive bacteria -unknown function, may anchor dna during cell division

  1. chromatin area

prockaryotes do not possess a distinct membrane enclosed nucleus and no mitotic apparatus DNA is aggregated in one area known as a nucleoid

  1. bacterial chromosome

-single haploid circular dna known as chromatin body -some exceptions: streptomyces and borrelia species (linear) rhodobacter sphaeroides (2 seperate chromosomes) size on average 3x10^9 daltons smaller than eukaryotes all genes are linked no histone proteins (instead have Mg+2 polyamines which acts like histones)

  1. Plasmids

circular extrachromosomal DNA smaller and seperate from chromosome capable of self replicating carry supplemental genetic information (antibiotic resistance (r-plasmid) Production of toxins Mating capabilities (F-plasmid -> F-pilus0 Tolerance to toxic metals

  1. Inclusion bodies - not always found in a bacterial cell

storage granules : energy and nutrient stores seen under light microscopy not permanent structures ie poly beta hydroybutyrate (PHN polymetaphosphate glycogen starch

  1. vegetative bacterial cells -> Bacterial cells capable of growth

Endospores

-not a structure of the bacterial cells specialized structure that enable survival produced by bacillus and clostridium species Resistant to UV, irradiation, chemical disinfection and drying require a speicalized endospore stain (Light microscopy)

structure

coat- - keratin like protein provides an impermeable layer (resistance to antibacterials) cortex - type of peptidoglycans (fewer crosslinks) spore wall peptidoglycan layer (taken from cell wall of vegetative cell) core - contains complete nucleus protein synthesizing apparatus energy-generating system (glycolysis) and calcium dipicolinic acid

differences between veg cells and endospores property vegatative cell endospore surface coat typically gram+ thick spore coat + peptidogylcan spore wall microscopic appearance non-refractile refractile Calcium dipicolic acid absent present in core Enzymatic activity present absent Heat resistance low high radiation resistance low high resistance to chemicals (acids) low high sensitivity to lysozyme sensitive resistant sensitivity to stains/dyes sensitive resistant

sporulation / sporogenesis

true differentiation ...1 veg cell -> 1 spore this is not growth takes 6-8 hours to complete sporulation

  -dna condences
- transverser wall  ??????????
- spore forms inside vegatative cell
vegatative cell grows around the spore forms multi layered container

cell lysis


germination

outgrowth from spore 1 spore -> vegatative cell only occurs when environmental conditions are suitable for bacterial cell growth takes 2 hours to complete

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