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    Micro bio dentistry.ppt Micro bio dentistry.ppt Presentation Transcript

    • Introduction and Historical Development in Microbiology Organism - object / structures invisible to the naked eye are called microorganism = human eye cannot see object smaller than 30u (1/1000 of an inch in dia.) Microbiology – science that deals with the study of microorganisms and their activities Medical Microbiology – deals with the study of disease producing organism affecting human = also concerned with prevention and control of disease.
      • Scope of study (order of decreasing size)
              • a) Algae (Phycology)
          • b) Fungi (Mycology)
          • c) Protozoa (Protozoology / Parasitology)
          • d) Bacteria (Bacteriology)
          • e) Virus (Virology)
          • Features of Microorganism :
          • * All microorganisms are not alike
          • * Some of them are very small while others are
          • relatively larger in size
          • * Some of them possess plant-like characteristic,
          • while others have animal-like and few of them
          • have neither plant-like nor animal-like characters
    • BRIEF HISTORY
      • before microorganism were discovered, ancient people regarded diseases as a form
      • of punishment sent by GOD for the sins of men
      • disease were not new to man
      • Biblical disease - Leprosy
      • - Tuberculosis
      • - Syphilis
      • - Plague
      • treatment and prevention of these diseases were sought by sacrifices to appease the anger of God
      • Varro = during the 2 nd century B.C.– postulated the
      • concept of contagion
      • diseases are transmitted
          • by invisible creature
      • Roger Bacon (13 th century) = postulated that invisible
      • living things called “germs ” produce disease
      • Fracastorius (1546) = postulated that disease is caused
      • by invisible living things and can be transmitted
      • by direct contact (person to person contact)
      • Spontaneous Generation Theory
      • Aristotle – proposed the
      • “ Spontaneous Generation Theory”
      • also called Abiogenesis
      • state that living things could develop spontaneously from non-living materials
      • supported by appearance of living creatures in decaying meat, stagnating ponds, fermenting grain, and infected wounds
    • Experiments to disprove Spontaneous Generation Theory
      • Francesco Redi 1668
        • did an experiment with flies and wide-mouth jars containing meat
        • he demonstrated the appearance of maggots in decomposing meat as a result on the deposition of eggs by flie
      • Rudolph Virchow 1858
        • 1 st person to propose the Theory of Biogenesis
          • ‘ Cells can only arise from preexisting cells’
      • John Tyndall
        • proved that dust carried germs
        • also demonstrated the great resistance of spore to heat
        • introduced (Tyndallization/Fractional Sterilization) a method by which spore can be destroyed by heating using a free-flowing steam for 30-60 minutes at 100 ° C for 3 consecutive days.
      • Anton Van Leeuwenhoek
        • 1 st to describe bacteria in 1677 with the use of a simple microscope utilizing crude lenses
        • considered as the Father of Bacteriology for being the first person described the 3 morphologic forms of bacteria: rod, spherical, spiral
      • Robert Hooke 1678
        • developed the compound microscope and was able to confirm Leeuwenhoek’s discoveries
        • first person to coin the word “cell” to describe the tiniest components of the living system
      • Agostino Bassi 1800
        • proved that a fungus cause a disease in “silkworm” called Muscardine
      • Rayer and Davaine 1850
        • were able to observed the presence of microorganism in blood of animals dying from a disease called Anthrax.
      • Edward Jenner - developed the concept of vaccination
      • Paul Erlich - developed the used of staining to demonstrate bacterial cell morphology for better visualization
      • Louis Pasteur
        • published the results of an experiment he did to disprove spontaneous generation in microscopic organisms
        • introduced Pasteurization a method of killing organisms found in dairy products
      • Joseph Lister 1860s
        • demonstrated the importance of using antiseptic to control spread of disease-producing organism
        • uses a chemical disinfectant (aqueous phenol) to prevent surgical wound infections.
        • also developed the first pure culture technique using liquid medium which was the key to identification of bacteria
      • Robert Koch 1876
        • establish proof of microbial etiology of 3 important diseases:
            • Cholera ( Vibrio cholerae)
            • Tuberculosis ( Mycobacterium tuberculosis)
            • Anthrax ( Bacillus anthracis)
        • formulated the Koch’s postulates which provides proof that a specific bacterium caused a particular disease.
        • also developed a solid culture medium for isolation of bacteria in pure culture.
        • Koch’s postulates :
      • 1. The organism must always be found in diseased
      • animals and not in healthy one
      • 2. Organisms isolated from the diseased animals
      • must be grown in pure culture away from the
      • diseased animals
      • 3. Organisms grown in pure culture must initiate and
      • reproduce the disease when re-inoculated into
      • susceptible animal
      • 4. Organisms must be re-isolated from experimentally infected animals
      • Exceptions to the Kock’s Postulate:
        • #1) Carrier - some individual harbors the organism but do not
        • manifest the disease and can transmit the disease to others
        • #2) Some or certain organisms/bacteria cannot be cultured in
        • vitro (cannot grow in artificial culture media)
      • Ex. Mycobacterium leprae / Treponema pallidum
        • #3) Certain animals not susceptible to certain microorganism some animals are by nature immune
          • - cannot replicate even with the same specie
          • some bacterial specie are host specific
            • Ex. Vibrio cholerae cannot cause chicken cholera or vice versa
    • Classification and Identification of Bacteria Protist = group of single–celled microorganism which do not form highly differentiated tissue and organ system. In the early history living organism were classified into 2 kingdoms: 1. Plant 2. Animal = after the discovery of microorganism, it was found that many microorganism possess both plant and animal characteristic or neither plant or animal characteristic. So a new kingdom was proposed for microorganism (Kingdom Protista) = all bacteria, fungi, algae and protozoa are group in kingdom protista = viruses are not included because they do not have cellular organization.
      • = Kingdom Protista is divided into 2 groups based on cellular organization:
      • 1. Procaryotes – lower protista
      • 2. Eucaryotes – higher protista
      • Eucaryotes – Higher protista
          • Characteristics
            • 1) Size greater than 5um
            • 2) Possess a well developed true nucleus enclosed in a nuclear membrane
            • 3) Contain sets of chromosomes
            • 4) Cytoplasm contains mitochondria endoplasmic reticulum and vacuoles
            • 5) Cell membrane continous with well-defined
            • endoplasmic reticulum and nuclear membrane
            • 6) Ribosome (sedimentation coefficient) 80s
            • 7) Motility organelle multistranded
            • Eg: Algae (Red – Brown), fungi, protozoa, slime mold
      • B) Procaryotes – Lower protista
          • Characteristic:
            • 1) Size less than 4um
            • 2) Nucleus primitive and is homogenous with the
            • cytoplasm of cell and not enclosed in a nuclear
            • membrane (naked)
            • 2) Posses single chromosome (haploid)
            • 3) Do not have mitochondria and endoplasmic reticulum
            • 4) Cell wall made up of complex rigid layer
            • 5) Ribosome (sedimentation coefficient) 70s
            • 6) Motility organelle unstranded
            • Eg: Bacteria, Blue – Green algae
    • Laboratory Procedure Employed in the Identification of Bacteria 1. Isolation of bacteria in pure (axenic) culture. = depends on source of clinical specimen = blood, spinal fluid and closed abscesses yield pure bacterial culture of microorganism = sputum, skin and body fluids, stool usually contains mixed organism.
      • 2. Bacterial colony morphology (macroscopic)
            • = colony is composed of descendant of a single cell,
            • a clone or lump of cell
            • = usually the result of rapid multiplication of cell
            • when inoculated in a medium containing 2%
            • agar incubated for 18 – 24 hours in favorable
            • atmosphere.
            • = charac. microbial growth pattern on artificial media as observe when inspected with the
            • unaided eye
            • = determine size, shape, texture, presence/absence
            • of pigmentation, odor of the colonies
      • 4 types of bacterial colony :
          • 1) Smooth (S-colonies) - gives the appearance of
          • homogeneity and uniform texture without
          • appearing as liquid or mucoid colonies.
      • = characteristic of freshly isolated wild – type
      • organism.
      • Ex. Shigella, Salmonella, Proteus and E. coli
          • 2) Rough (R-colonies) granulated and rough in appearance.
            • = produced by mutant strain of organism
          • 3) Mucoid (M-colonies) - exhibits a water-like glistening
          • confluent appearance.
          • = seen among organism that forms well-defined capsule or slime layer.
      • 4) L colonies – seen among cell-walless bacteria.
          • Ex. Mycoplasma
          • 3. Microscopic morphology and staining reaction.
            • = determines shape, arrangement and staining reaction
            • = light microscopic examination of gram-stained preparation with the oil immersion lens and without
            • coverslip is routinely used to determine bacterial morphology.
      • = staining artificially coloring the organism with dyes
      • = advantages:
      • 1) organism are made more clearly visible
      • 2) differences between all can be demonstrated
            • 3 types of staining procedure :
            • 1. Simple – use of 1 dye only.
            • 2. Differential – used of 2 or more dyes.
              • A)gram – differentiates gram positive from gram
              • negative bacteria.
              • B)acidfast – differentiates acidfast from non-acidfast organism.
            • 3. Special – used to detect special bacterial structure (capsule, flagella, spores, and intracellular)
            • inclusion granule
      • A) Negative
      • B) Positive
      • 4. Biochemical
      • = detect different metabolic products and sugar
      • fermentation reaction of various species
      • of microorganisms.
          • = 60% of common pathogens are identified by metabolic test.
          • = commercial kits now available especially for identification of enterobacteria.
          • 5. Serological – detect presence of antibodies in patient
          • serum against specific antigens.
            • Ex. Widal test, TPI, VDRL
          • 6. Animal pathogenicity
          • 7. Antibiotic susceptibility test – important use for the
          • purpose of treatment.
          • 8. Epidemiologic test – useful in tracing source of
          • outbreak of certain bacterial diseases.
    • Specimen smear cultivation (pure culture) microscopic colonial morph. exam. biochemical serologic animal path. antimicrobial susceptibility
    • Bacterial Morphology Bacterial cell is a complete unit of any living organism. All its functions are genetically controlled and performed by that particular cell structure whether it be physiologic or biochemical.
    •  
      • Bacterial morphology includes:
      • Form, Arrangement, Size and Staining reaction
      • I. Form
      • = determine by heredity
      • = genetically most bacteria are monomorphic
      • (maintain a single shaped)
      • = however some environmental conditions can alter shape
      • = some are genetically pleomorphic
      • (can have many form)
      • = determined by the configuration of the cell wall
      • = detected by brightfield microscopy of stained smear
      • A. Coccus (spherical-shaped)
      • = round-like a ball, perfect sphere, globe
      • = can be oval, elongated, or flattened on one side
      • Variations :
          • 1. Ovoid shape- both sides rounded ends are pointed.
              • Ex. Streptococcus
              • 2. Lancet-shape - one end is pointed, other end is flat.
              • Ex. Pneumococcus
              • 3. Coffee-bean shape- flat on one side, opposite side convex or appear as letter “D” form.
              • Ex. Neisseria
          • B) Bacillus (rod-shaped)
          • = cell appears longer than wide or cylindrical form
          • = both sides parallel and ends are convex
          • = varies in actual form depending on the species
          • Variations:
              • 1. Clubbed/drumstick shaped–swollen on one end.
              • Ex. C. diphtheriae / C. tetani
              • 2. Corset-shaped – both sides swollen, end flat or
              • concave. Ex. Bacillus anthracis
              • 3.Fusiform - both sides parallel ends pointed
              • (thick at the center and tapered)
              • towards the end
          • C) Spiral
          • = bacteria w/ more than one somatic curved
          • = may be regarded as bacillary form twisted to
          • form a helix.
          • 2 types:
          • 1. Flexible – spirals that can contract and relax
          • whose long axis bends when in motion.
      • Ex. Spirochetes
      • Genus Treponema - tightly coiled spiral
      • with corkscrew appearance
      • Ex. T. pallidum
      • Genus Borrelia - much less tightly coiled having
      • the appearance of an extremely long
      • undulating pole. Ex. B. recurrentis
      • Genes Leptospira - tightly coiled spiral with
      • sharp-hooked like bends.
      • Ex. L. interrogans
      • 2. Rigid – spirals that cannot contract and relax and
      • move by rotation in corkscrew-like
      • (long axis remains) rigid when in motion
            • Ex. Spirillum minor
      • D) Intermediate forms
      • A. Coccobacillary–when a rod is short thick, wide/plump.
              • = these form is intermediate between a
              • spherical and rod.
              • Ex. Haemophilus, Brucella
              • B. Vibrio - a gently curved bacteria (comma-shaped)
              • Ex. V. cholerae
    • II. Arrangement = is the result of the number of planes in which the cell divides and how they remain attached afterwards.
      • A) Sphericals
      • 1. Singly – occurs as a single spherical cell
      • 2. Chain – common among ovoid-form resulting
      • in one plane division with daughter cells
      • remained attached to one another
      • (Streptococci)
      • Ex. Streptococcus pyogenes
          • 3. Pair –common with lancet-shaped and coffee-
          • bean shaped form resulting from one
          • plane division with daughter remain in
          • pair after dividing (Diplococci)
            • Ex. Streptococcus pneumoniae
            • Neisseria gonorrheae
            • 4. Cluster – common with sphericals resulting from
            • many plane division with daughter cell in
            • grape-like agglomeration bunch.
            • (Staphylococci) Ex. Staphylococcus aureus
            • 5. Tetrads – (Packets of 4) – result from 2 plane
            • division with daughter cell separating from
            • one another to form group of 4 cells.
            • Ex. Graffya tetragena
            • 6. Sarcinae – (Packets of 8) – results in 3 planes
            • division with cells remain attached in cube-
            • like group of 8.
            • Ex. Sarcina lutea
          • B) Rods
          • = divides only across their short axis
            • = fewer arrangement of bacilli than cocci
            • = most bacilli appears as single rods
            • 1. Chain – result in one plane division with daughter
            • cell remain attached to one another.
          • Ex. Bacillus anthracis
          • 2. Palisade – arrangement like fence due to slipping
          • movement of daughter cells.
          • Ex. Diptheroids
          • 3. Chinese-letter arrangement – common with clubbed-
          • shaped rods resulting from a snapping post
          • division movement of the daughter cells.
          • Ex. Corynebacterium diptheriae
          • 4. Packets of cigarette – arrangement like bundles.
      • Ex. Mycobacterium leprae
      • 5. Serpentine – chain arrangement commonly seen
      • among virulent strain of Myco. tuberculosis
      • C) Spirals
      • = no characteristic cell arrangement, most occur
      • singly
      • = different species vary in size, length, rigidity and
      • amplitude of their coils
      • III. Bacterial size
            • 1. Micrometer
            • 2. Ultrafiltration
            • 3. Ultracentrifugation
            • Cocci – 0.5 – 3.0um dia.
            • Bacilli – 0.2 – 2.0um dia. X 0.5 – 20um in length
            • Vibrio and Spirilla – 0.2-2.0um dia. X 0.5-100um L
            • Spirochetes – 0.1 – 3.0um dia. X 0.5 – 250um L
    • IV. Staining Reaction = putting a color to facilitate identification = unstained bacteria are colorless and transparent = can be determined through the used of basic aniline dyes
      • Types:
      • 1. Simple = employs one dye only
              • = sufficient to det. shaped and arrangement
              • 2. Differential = employs the use of more than one dye
              • = enables microbiologist to differentiate one
              • group from the other by the color as well
              • as shape
              • = it is based on relative affinity of diff. bacterial
              • cell for the stained used
      • 2.a) Gram = diff. gram (+) from gram (-) bacteria
      • = gram (+) those that retain the primary
      • stain and deep violet or purple color
      • = gram (-) those decolorize with ethyl
      • alcohol and stained by the counter
      • stain appearing pink or red in color
      • 2.b) Acidfast = differentiate acidfast from
      • non-acidfast bacteria
      • 3. Special
      • = use to color and determined bacterial structures like capsule, flagella,
      • inclusion granule, endospore and etc.
              • 3.a) Negative staining
              • 3.b) Positive staining
    • BACTERIAL CELL ULTRASTRUCTURES
    •  
    • General Division: I. External - Capsule - Fimbriae - Flagellum II. Internal - Cell Wall - Cytoplasmic Membrane - Cytoplasm - Mesosome - Nuclear Body - Ribosome - Plasmid - Inclusion Granule - Endospore
          • BACTERIAL ULTRASTRUCTURE
          • Bacterial Surface Coating:
          • = are extracellular polysaccharide polymers which
          • surrounds the bacterial cell
      • Capsule
      • = well-formed thick viscous jelly-like structure firmly
      • attached to the cell wall surrounding the cell
      • = not readily removed.
      • = easily visualized by negative staining using India
      • ink method
          • Slime layer
          • = loose and irregularly arranged meshwork of
          • fibrils totally detached from the cell but
          • still surrounds the cell
          • = when present are more easily washed off
      • Chemical Comp.:
      • = water – main component 98-99%
      • = 1-2% of bacterial capsule are chemically
      • polysaccharide, Except capsule of:
      • B. anthracis-polypeptide (D-glutamic acid)
      • S. pyogenes-Hyaluronic acid
      • Function:
      • = protection from phagocytosis
      • = correlates with virulence
      • = act as antigen (used in the identification)
      • and typing of capsulated bacteria
      • = allows bacteria to adhere/attach to various
      • surfaces in its natural environment in
      • order to survive
      • Detection:
              • 1. Negative staining (India Ink)
              • 2. Special staining (Capsular method)
              • 3. Serological (Quellung reaction)
      • B. Appendages Flagellum & Axial filaments
      • Fimbriae
      • FLAGELLUM:
      • = long, thick, helical protein filament of uniform length
      • and diameter
      • = commonly seen among free-swimming bacteria
      • = originates in cytoplasmic membrane
      • = composed of 3 parts:
          • 1. Basal body – anchors the flagellum to the
          • cell wall and plasma membrane
          • 2. Hook – attached to the basal body and
          • connects to the filament
          • 3. Filament – external to the cell and contains
          • the flagellar protein
      • Chemical Comp.: protein monomer - Flagellin
      • Function - Motility organelle of many pathogenic bacteria
      • Detection:
              • 1. Darkfield / Phase contrast microscopy
              • (Wet mount / Unstained smear)
              • 2. Brightfield/Electron Microscopy – using stained
              • specimen
              • 3. Serological – use of specific antisera against
              • flagellar (H) antigen
              • 4. Swarming phenomenon
              • 5. Motility medium
              • 6. Hanging drop preparation
    • Types and Arrangement of flagella in relation to the distribution and number: I. Monotrichous – single polar flagellum at one end II. Multitrichous – more than one flagella
      • Types:
          • A) Lophotrichous – tuft of flagella at one polar end
          • B) Amphitrichous – single flagellum or tulf of
          • flagella at both polar end
          • C) Peritrichous – flagella distributed all over the
          • body of the bacteria
          • Axial Filaments
          • = flagella-like sheathed filaments located in
          • the periplasmic space (between the inner)
          • and outer membrane of the cell
          • = move by traveling helical wave on opposite
          • direction
      • = motility organelle of spirochete
      • Fimbriae / Pili / Microfibrils
      • = short, straight, thin hair-like filaments usually
      • distributed around the body of bacteria.
      • = originates in the cytoplasmic membrane
      • = found virtually among all gram (-) bacteria but not in
      • gram (+) bacteria
      • 2 types according to function:
      • 1. Common/ordinary – for attachment or adherence
      • to mucosal surfaces of host cell during
      • colonization and infection.
      • 2. Sex pili – responsible for attachment of donor and
      • recipient cell during bacterial conjugation
      • whereby genetic material (DNA) from one cell
      • is transferred to another (reproduction).
      • Chem. Comp. - Protein (Pilin)
          • Detection: Electron Microscopy
      • Cell wall (Peptidoglycan, Murien, Mucopeptide / Glycopeptide)
      • = complex, rigid, multilayered structure that protects the
      • protoplast and the underlying fragile plasma
      • membrane
      • = found in all pathogenic free-living bacteria except
      • Mycoplasma (cell-walless bacteria)
      • Chemical Comp.:
          • 1. Protein (Mesodiaminopimelic acid,Isomers of D-glutamic)
          • acid and D-alanine
          • 2. Polysaccharide
          • (N-acetylmuramic acid and N-acetylglucosamine)
          • = responsible for rigidity of cell wall
          • 3. Lipids
          • Function :
          • 1. Responsible for the characteristic shape of bacterium
          • 2. Provides strong structural support necessary to keep
          • bacterial cell from rupturing due to changes in
          • the environmental osmotic pressure
          • 3. Contain somatic O antigen that can serologically
          • identify particular bacteria
          • 4. Site of action of some antibiotics
          • 5. Determines differences in gram staining reaction
      • Detection :
          • 1. Microscopy of smear prepared from special
          • staining method.
          • 2. Electron microscopy.
          • 3. By chemical methods using lysozyme.
    • COMPARISON OF GRAM POSITIVE AND GRAM NEGATIVE CELL WALL Gram Positive Gram Negative Peptidoglycan
      • Very thick homogenous single layer
      • extensively crossed-linked.
      • Constitute 50-90% of the cell wall
      • material.
      • (NAM, NAG, D-alanine)
      • Diaminophemilic acid
      • More complex layer composed
      • of thin peptidoglycan with few
      • crossed-linked and an outer
      • membrane layer.
      • Constitute 5-10% of the cell
      • wall material.
      Auxiliary compounds
      • Teichoic acid (Polymer of ribitol and)
      • glycerol phosphate
      • Lipoteichoic acid
      • Surface protein
      • No teichoic acid
      • Outer membrane contains:
      • Phospholipids-located in the
      • inner membrane
      • Lipoprotien – connects OM to
      • the peptidoglycan
      • Lipopolysaccharide
      • (LPS/Endotoxin layer)
      • – located in the outer
      • layer of the OM
      • - contains the lipid A
      Penicillin sensitivity
      • Sensitive
      • Resistant
      Response to lysozyme
      • Digest/ Degrade
      • Resistant
    •  
    • Gram Positive Gram Negative
      • Damage to the cell wall may result to:
          • = when a gram positive bacteria is exposed to
          • lysosyme will degrade the peptidoglycan
          • layer resulting to complete removal of the cell
          • wall producing a osmotically fragile wall-less
          • spherical body called Protoplast
          • = since all cell wall components is removed,
          • therefore incapable of regeneration
      • Spheroplast
          • = when a gram negative bacteria is exposed to
          • lysozyme it looses the peptidoglycan layer
          • but retain the outer membrane leaving a
          • less fragile spherical body capable
          • of regenerating the cell wall.
    •  
    • Protoplast: comprises the naked cytoplasmic membrane and its content PERIPLASM = space between the plasma inner membrane and the outer membrane layer = readily observe among gram negative bacteria = consist of a gell-like substances which help secure nutrients from the environment CYTOPLASMIC/PLASMA/CELL MEMBRANE: = thin elastic bilayered semi-permeable membrane lying underneath the cell wall enclosing the cytoplasm of the cell. Chemical Composition - Lipoprotein - (composed of phospholipids and protein molecules)
      • Functions:
          • 1. Serve as selective permeability barrier
          • (transport of nutrient material in & out of cell)
          • 2. Site of electron transport and oxidative phosphorylation of substances involve in the
          • generation of chemical energy (ATP)
          • 3. Helps in the excretion of metabolic waste product
          • 4. Site for excretion of enzyme involved in OM
          • synthesis, CW synthesis and in the secretion
          • of extracytoplasmic & extracellular substances:
            • A. Permease for active uptake of nutrient
            • B. Respiratory
            • C. Polymerase that manufacture substances
            • of the cell wall
            • D. Hydrolytic
      • Detection - Electron microscopy
      • MESOSOME:
      • = usually seen as an invagination of the membrane
      • associated cytoplasmic sac seen in Gram (+) cell
      • = contains lamellar, tubular and vesicular structure
      • = often associated with division septa
      • Chemical Composition - Lipoprotein
      • Functions:
          • 1. Site for the synthesis of cytochrome oxidase
          • and reductase enzyme
          • 2. Provide support for enzymes present in the cell
          • membrane
          • 3. Responsible for compartmenting DNA during
          • cell division and sporulation
      • Detection - Electron microscopy
      Cytoplasm = refers to everything that is enclosed by the cytoplasmic membrane = site for most bacterial metabolism = 80% is composed of water
      • NUCLEAR BODY: (Nucleus / Nucleoid)
      • = genetic material of bacteria
      • = contains a single circular molecule of double
      • stranded DNA network which runs parallel to the
      • long axis of the cell
      • = found homogenous within the cytoplasm not
      • enclosed in a nuclear membrane.
      • = constitute 2-3% of the cell weight.
      • = no definite form and seen at all stages of growth
      • cycle of the bacteria.
      • Chemical Composition – DNA
      • Function:
            • 1. Controls the growth and metabolic activity of
            • the bacteria
            • 2. Responsible for hereditary characteristics of the cell.
      • Detection - Electron microscopy, Feulgin staining (+)
      • PLASMID/EPISOME:
      • = extrachromosomal genetic material capable of
      • autonomous replication usually located near
      • the chromatin body.
      • Chemical Composition – DNA
      • Function:
          • 1. Transfer of genetic material from one cell to another
          • by conjugation (Transmissible Plasmid)
      • 2. Carry genes for activities like:
              • a) Antibiotic resistance
              • b) Toxin production
              • c) Synthesis of enzyme
              • d) Tolerance to toxic metals
      • Detection – Feulgin staining (+), Electron microscopy
    • RIBOSOME: = histone-like particles composed of ribosomal RNA (rRNA) and protein molecules found in the cytoplasm of the cell. = site of action for many antibiotics that inhibit protein synthesis. = have a sedimentation coefficient of 70S and are composed of 50 S and 30 S subunits containing 16 S, 23 S and 5 S RNA respectively = procaryotic ribosome - 70s = eucaryotic ribosome - 80s Function – Site for protein synthesis Detection – Feulgin staining (-), Electron microscopy
      • INCLUSION / CYTOPLASMIC GRANULE:
      • = large granules found anywhere in the cytoplasm
      • = are accumulation of organic and inorganic substances
      • which serves as source of energy and nutrient
      • supply to the organism
      • Kinds:
      • 1. Babes Ernst / Volutin / Metachromatic granules
          • Chemical Composition – polymerized
          • metaphosphate which can be used in the
          • synthesis of ATP
          • Function – reserve energy supply
      • Demonstration – simple staining / special
      • staining (Albert’s stain)
            • 2. Lipid granules
              • Chemical Composition – polymer of beta-
              • hydroxybutyric acid
              • Function – reserve food supply.
            • 3. Sulfur granules – derived energy by oxidizing
            • sulfur and sulfur-containing
            • compounds.
              • Function – reserve energy supply for sulfur-
              • containing bacteria.
            • 4. Glycogen granule – polymer of glucose.
              • Function – reserve food supply.
    • ENDOSPORES: = highly refractile body formed within vegetative bacterial cells in response to adverse environmental condition and due to deficient nutrient supply = known as endospore, because it is formed inside the bacteria cell and is genetically controlled = are metabolically inactive bacterial cell that are highly resistant to desiccation, heat and various chemical agents = found in the cytoplasm of rod-shaped sporeforming bacteria of the genus Bacillus & Clostridium
      • = Composed of five parts:
      • 1. Core – located at the center which is the spore
              • cytoplasm/protoplast.
              • - contains the nucleus and enzyme dipicolinic acid
            • 2. Spore wall – enclosing the core.
            • 3. Cortex – laminated structure surrounding the spore
            • wall
            • 4. Spore coat – multilayered membrane enclosing the
            • cortex
            • 5. Exosporium – the outermost covering of the spores
            • which give the spore a rigid appearance.
      • = contains Calcium Dipicolinate which aids in heat
      • resistance within the core by making the protein
      • and nucleic acid more resistant to denaturation
      • = germinate under favorable nutritional condition after
      • an activation process that involves damage to the
      • spore coat
      • = helpful in identifying some specie of bacteria especially
      • those sporeforming bacilli (Clostridium and Bacillus)
      • Classification accdg. to:
      • A. Location - Central, Subterminal, Terminal
      • B. Shape - a) Ovoid
      • b) Circular
      • C. Swollen/not swollen
      • (swollen when the diameter is more than the of the)
      • bacterial cell, not swollen diameter the same as bacterial cell
      • Detection:
          • 1. Microscopy of stained smear
            • A) Negative staining
            • B) Positive staining
            • 2. Phase contrast microscopy
          • PHYSIOLOGY OF BACTERIAL CELL
          • Nutrition
          • = process by which chemical substances (nutrients)
          • either organic or inorganic are use in cellular
          • activities of microorganism for metabolism
          • and growth
          • Growth
      • = orderly increase of all chemical constituents of the
      • cell including size and number
      • = process entails replication of all cellular structures,
      • organelles and protoplasmic components of
      • the cell
      • Generation time
      • = measure of the growth rate of microbial species.
      • = varies in length of time accdg. to environmental
      • conditions.
          • Bacterial growth requirement:
          • 1) Water – most important requirement
          • = vehicle for the entry of all nutrients into the
          • cell and elimination of their waste product
          • = form an integral part of the cell protoplasm
          • 2) Nutrient requirement :
      • = source of Carbon and Nitrogen
      • a) Carbon = major building block for constructing
      • cell material
      • Types of bacteria acdg. to carbon requirement
            • 1. Autotroph (Lithotroph)
      • = req. only H 2 O, CO 2 & inorganic substances
      • = utilize CO 2 as source of carbon
      • = Photoautotroph- light as energy source = Chemoautotroph - chemical reaction as
      • energy source
      • 2. Organotroph
      • = requires organic substances for growth
      • = unable to utilize CO 2 as energy source
      • Photoorganotroph - light as energy source
      • Chemoorganotroph - chemical reaction as
      • energy source
      • 3. Heterotroph
      • = requires both organic and inorganic
      • substances for growth
    • b) Nitrogen Requirement = main reservoir of nitrogen is nitrogen gas (N 2 ) which make up 79% of earth atmosphere = must be degraded into their basic building block (Protein  Amino acid; Nucleic acid  Nucleotides) 3) Inorganic Ions = small amount needed ex. Sulfur, Phosphorous, Magnesium, Calcium, Manganese, Zinc, Cobalt, Copper
    • 4) Growth Substances = organic nutrient essential to an organism metabolism that cannot be synthesized and must be provided in the culture medium Ex. yeast extract, whole blood, serum, B-complex vitamins, amino acids, purins and pyrimidines Prototrophic = bacteria that do not require exogenous source of growth factor because they are capable of synthesizing their own Auxotrophic = bacteria that requires additional growth factor in the culture medium for growth to occur
      • II. Physical Requirement
          • A)Temperature
              • 1. Phychrophile/Cryophile
              • = 5-30 0 C opt. 10-20 0 C
              • = grow at refrigeration temp. of 4 0 C - 8 0 C
              • = responsible spoilage food refrigeration
              • = cold –loving bacteria
              • = found normally in cold water
      • 2. Mesophiles
      • = 10 – 45 0 C opt. 20-40 0 C
      • = saprophytic – 26-35 0 C
      • = parasitic – 37-45 0 C
      • = bacteria pathogenic for human
    • 3. Thermophiles = 25-80 0 C opt. 50-60 0 C = hotspring, tropical soil, hot water heater 4. Thermoduric = 80-100 0 C = resist high temperature but cannot grow and multiply
          • B) Oxygen
            • = required by particular bacterium to satisfy its energy
            • needs 5 groups of bacteria on the basis of their O 2
            • requirement
              • 1) Obligate anaerobes
              • = grows only under condition of high
              • reducing intensity and for which
              • oxygen is toxic
              • (complete absence of oxygen)
              • = requires oxygen - free environment to
              • survive
              • 2) Aerotolerant anaerobes
              • = organism that are not killed by exposure
              • to oxygen
          • 3) Facultative anaerobes
          • = capable of growth under both aerobic and
          • anaerobic condition
          • 4) Obligate aerobes
          • = requires oxygen for growth
          • 5) Microaerophilic
          • = organism that grows best at low oxygen tension
          • 6) Capnophiles
          • = requires 5-10% carbon dioxide and oxygen on
          • primary isolation
          • C) Hydrogen Ion Concentration (PH)
            • = optimum ph for pathogenic bacteria 7.2 – 7.6
              • 1. Acidophilic – below ph 6.5 – 7.6
              • 2. Basophilic – (alkalophilic) – 8.4 – 9.0
              • 3. Normophilic (neutrophilic) – ph 7.5 – 8.0
          • D) Osmotic Pressure
            • = direct pressure – resistant
            • = osmotic pressure – sensitive
            • = plasmolysis – shrinkage
            • = osmophile – bacteria that can grow on high
            • osmotic pressure
            • = Ex. Halophile – seawater bacteria grow best at
            • high concentration of salt
      • Measurement of Bacterial Growth
          • 1. Cell Concentration (cell number) number of cell per unit
          • volume of culture. Can be counted directly by a
          • microscope counting chamber.
          • DETERMINATION:
            • 1) Total direct plate count
              • A) Bacterial Counting Chamber
              • (Petroff-Hauser Counter)
              • B) Coulter Counter (Electron Particle Counter)
              • = measures both distribution of size
              • and number of bacteria in
              • bacterial suspension
            • 2) Indirect Viable Count
              • = plate sample of culture and make dilutions
              • of microbial population  inoculate
              • suitable solid medium  incubate 
              • formation of viable colony
      • USES:
          • A) Solving problems on bacterial cell division
          • B) Genetic
          • C) Infection
          • D) Microbial Inactivation
      • 2) Cell Density (Cell Mass)
      • = total protoplasm/dry weight of the cell per unit
      • volume of culture
      • = include living and dead cell
          • DETERMINATION:
          • 1. Absorbance Spectrophotometer
              • = measuring optical density of broth culture of
              • microorganism
          • 2. Turbidimetric Technique
              • = useful in determining mass of cells during bacterial growth
          • 3. Nitrogen Determination
          • 4. Centrifugation
      • USES:
          • 1. Study of bacterial nutrition
          • 2. Biochemistry
      • Bacterial Growth Curve
      • (5 PHASES)
          • 1. Lag phase (Phase of Rejuvenescence/Phase of)
          • Physiologic Growth
            • = after inoculation  bacteria requires a period of
            • adjusting and adapting to new environment
            •  marked increase macromolecular
            • component of the cell  increase in cell size
            • but no detectable increase in cell number
      • = no cell division occur
          • 2. Exponential phase (Logarithmic phase)
            • = cell in state of balanced growth
            • = characterized sharp rise in growth curve indicating
            • rapid growth and multiplication
      • = bacterial cell double its growth per unit of time
          • 3. Stationary phase
          • (phase of equilibrium/”plateau”)
            • = manifestation of unbalanced growth  growth rate zero
            • = viable count remains constant for a short period eventually gives way to decreasing population
      • = number of living bacteria equals number of
      • bacteria dying
          • 4. Phase of Decline (Death phase)
            • = growth rate decreases  complete cessation of multiplication
            • = result in the decline in total viable count
            • = most bacteria dies due to :
              • 1. lack of nutrient material in the medium
              • 2. accumulation of waste product excreted by
              • the bacteria
              • 3. change in ph of the environment
    •  
      • Consequence of growth & multiplication is reproduction
      • 3 methods of bacterial reproduction
          • 1. Binary fission
          • 2. Branching
          • 3. Sporulation
      • 2 methods of bacterial multiplication
          • 1. Binary fission
          • 2. Branching
    • STERILIZATION AND DISINFECTION
    • STERILIZATION - the process of destroying all forms of microbial life in terms of their ability to reproduce and multiply including spores PHYSICAL AGENTS FOR STERILIZATION A. HEAT: = most reliable method of sterilization = method of choice for material which are not damage by heat = denature proteins and damage membrane 2 types of heat: 1. DRY HEAT: = preferred method for sterilization of glasswares, such as glass syringes, test tube, petri plates and material such as oil, jellies and powders = less effective than moist heat as it requires longer time of heating and higher temperature = kills the organism by oxidizing their chemical constituent
    • a) Hot air oven: = most widely used method of sterilization by dry heat = used for sterilization of glasswares such as flask, pipettes, test tube, swab = principle of killin g: heating at 160 O C or 180 O C for 30 minutes b) Direct Flamming/Heating = direct heating of instrument or object in a flame till it becomes red hot = easy way of sterilization but has limited application = useful method of sterilization for non-inflamable material such as bacteriological loop or needle, forceps, scissors c) Incineration = direct burning of contaminated materials = efficient method for sterilization and disposal of soiled dressing, bedding of patient and animal carcasses
      • 2. Moist Heat = kills bacteria faster than dry heat
          • Boiling
          • = 30 minutes boiling at 100 0 C is adequate to kill
      • vegetative cell of bacteria, fungi, protozoa
      • = sporeformer requires 5½ hours boiling to kill the
      • organism
          • 2. Free – Flowing stream
      • = live stream  Arnold sterilizer
      • = same sterilizing action with boiling
      • 3 . Steam Under Pressure
      • = heating material at 15 lbs/square inch pressure for 15 – 20 minutes at 121 0 C
      • = most efficient and reliable method of sterilization = commonly used for sterilizing of:
            • a. Hospitals surgical & medical instrument
            • b. Bacteriological media not destroyed by heat
            • c. Hospital supplies
      • 4. Tyndallization (Fractional/Intermittent sterilization)
      • = for sterilization of liquid & semi-solid material easily
      • destroyed by heat .
      • Ex. culture medium contg. egg, serum,
      • carbohydrate
      • = heating material at 80-100 0 C 30 minutes 3 conc. days
      • = kills both sporeformer & non-sporeformer bacteria
          • 5. Pasteurization
      • = consist of heating material at 62 0 C for 30 minutes
      • followed by rapid cooling
      • = use to kill only disease – producing organism found in
      • milk, beverages, milk products and in preparation
      • bacterial vaccine
      • Mechanism of moist heat: Denetuaration and coagulation of
      • protein inside bacterial cell
          • B) Radiation
          • = produce damage to DNA resulting to killing of the organism
      • = 2 types :
            • A. Non-ionizing radiation
            • Ex. Ultraviolet rays – have low penetrating power
            • = used to destroy bacteria, fungi, viruses that
            • are sensitive to U-V treatment
      • = primarily used to control airborne infections in
      • nurseries, hospital wards & OR
            • B. Ionizing radiation
            • Ex: X-ray, alpha, beta and gamma rays
      • = have greater penetrating power
      • = use extensively for sterilization of cutgut ,
      • plastic syringe, catheter, prosthesis,
      • swab and dressing pack
      • C. ) Filtration = process of separating microorganism from
      • contaminated solution
      • = employs the use of filters for removal of microorganism
      • that are sensitive to heat
      • = for sterilization of heat – labile C.M containing serum,
      • plasma, urea
          • D) Ultrasomic vibration
      • = are sound waves with high frequency
      • causes desruption & disintegration of the cell
      • = uses 1. research laboratories
      • 2. treating sewage H 2 O
          • E) Lyophilization ( Freeze-drying )
      • = not reliable
      • = causes protein denaturation & damage of the
      • membrane w/ leakage of intracellular
      • organic compounds
      • F) Laser’s
      • = sterilization of medical instrument and operating
      • room
    • Chemical agents : Disinfection = process destroying / eliminating potentially hazardous & pathogenic organism = aims to destroy the disease-producing organisms Disinfectant = agents that kills growing (vegetative) form of organism but not necessarily sporeformer = applied to non-living/inanimate object Bactericidal / Germicidal = implies the killing action of chemical substances Bacteriostatic = substances that inhibit / prevent growth & multiplication of bacteria Antiseptics = subs. that either kill / prevent growth = applied to living tissue (animate object) found in the surface of skin and mucous membrane
      • Mech. of action:
          • 1. damage cell membrane function
          • 2. denatures protein
          • 3. induce extensive nucleic acid damage
      • Agents that damage cell membrane function
      • I – Surface active agents : = destroys structural integrity of cell membrane
      • (protein & lipids)
      • = interferes w/ normal membrane function
      • = widely used as detergent, wetting agent and emulsifier
      • 3 types:
      • cationic = most useful
      • = includes Quarternary ammonium cpd.,
      • Benzalkonium chloride ( Zephiran )
      • anionic = soap, surfactant, sanitizer
      • = help remove microbe by rubbing
      • nonionic = not effective, non-toxic
      • II – Phenolic compound
      • = disrupt cell membrane function  leaking cell content
          • A) Carbolic acid (phenol)
      • = excellent for disinfecting feces, blood, pus,
      • sputum and other proteinous material
      • = primarily use for testing new bactericidal agent
          • B) Cresol / Creolin
      • Lysol - alkyl derivative phenol
      • = disinfect inanimate object esp.
      • contaminated with TB bacilli
      • Creolin - septic tank disinfectant
          • C) Diphenyl compound (Hexachlorophene)
      • = effective against gram + bacteria (staph & strep)
    • III – Alcohol = disorganize lipids  cell membrane = denature protein = widely used antiseptics = has ability to remove lipids from skin surfaces Ethanol / Ethyl alcohol = skin disinfectant = disinfection clinical thermometer = effective against gram (+) , gram (-) , AF bacteria Isopropyl alcohol = most effective, less volatile = bactericidal property greater than ethanol = used as skin antiseptic = has a better fat solvent property and more bactericidal = effective at a concentration of 50-70%
      • Agents that denatures protein
          • 1. Acid & alkalies
          • 2. Alcohol
          • 3. Acetone
      • Agents that modify functional group of protein & nucleic acid
      • 1. Salts of heavy metals – mercury, silver , arsenic
      • Silver cpd.  widely used as antiseptic
      • AgNO 3 (silver nitrate) – routinely used as
      • prophylaxis against opthalmia neonatorum
          • 2. Oxidizing agents (Halogen Compound)
      • iodine – wound/skin disinfectant
      • - active against tubercle bacilli
      • chlorine – H 2 O disinfectant
      • Hydrogen peroxide – use for cleaning wound
          • 3. Dyes
            • Triphenyl methane dyes (aniline acid)
            • Acidine dyes (Flavines) – yellow color
          • 4. Alkalyting agent (Formaldehyde, Glutaraldehyde,)
            • Ethylene oxide
            • = exert lethal effect on protein substances
            • Formaldehyde – in aqueous solution is bactericidal,
            • sporocidal and also effective against
            • viruses
            • = use for inactivating vaccine and
            • preservation of fabrics
            • formalin = in aqueous solution of 37%
            • formaldehyde is used for preserving
            • fresh tissue specimen
            • = as gas, used for fumigating operating
            • room, hospital ward, laboratories
          • Glutaraldehyde
          • = used as cold sterilant for sterilizing medical
          • and surgical instruments
          • Ethylene Oxide
          • = used for sterilization of polyethylene tubing,
          • heart lung machine, electronic hospital
          • devices and sensitive medical instrument
      • Chemotherapeutic Agent
      • = one of the most valuable method of treating infection
      • Antibiotics : are naturally occurring metabolic products of
      • primarily soil bacteria & fungi
      • Desirable Properties :
          • 1. Selective Toxicity
          • 2. Bactericidal
          • 3. Should not be allergenic and with no adverse reaction
          • on large doses or prolonged use
          • 4. Active in the presence of plasma, body fluids &exudates
          • 5. Should have broad-spectrum activity
          • 6. Water soluble & stable
          • 7. Susceptible organism should not become resistant
          • 8. Cheap
              • Antibiotic producers
      • 1. Penicillium
      • 2. Bacillus
      • 3. Streptomyces
      • 4. Cephalosporium
      • Mech. of action :
      • 1. Interfere with cell wall synthesis
      • 2. Interfere with cell membrane function
      • 3. Interfere with Protein synthesis
      • 4. Interfere with nucleic acid metabolism
      • Cell Wall Inhibitors :
          • 1. Beta – lactam antibiotics
            • A. Penicillin
            • B. Cephalosphorins
          • 2. Fosfomycin
          • 3. Cycloserine
          • 4. Vancomycin
          • 5. Bacitracin
      • Penicillins
          • 1. Natural PCN – Benzyl PCN G
      • - Phenoxymethyl PCN V
          • 2. Semi – synthetic PCN
            • A) Penicillinase – resistant PCN’s
      • Methicillin
      • Nefcillin
      • Isoxazolyl PCN – Cloxacillin
      • Drug of choice - Dicloxacillin
      • For PCN resistant - Oxacillin
      • S. aureus & S. epidermides
            • B) Extended spectrum PCN’s
      • Ampicillin
      • Amoxicillin
            • C) Antipseudomonas
      • Carbenicillin
      • Ticarcillin
      • Piperacillin
    • Cephalosporins = fermentation product fungus  Cephalosporium First Generation – bactericidal against most gram (+) and many gram (-) bacteria , Except : Enterobacter Proteus ( Indole + ) Pseudomonas Cephalothin Cephapirin Cefazolin Cephradine Cephalexin Cefadroxil
    • Second Generation – Gram (-) bacilli – Enterobacter - Proteus Cefamandole - Hemophilus Cefoxitin – N. gonorrhea Cefaclor Serratia Cefuroxime Bacteroides Third Generation – less active against gram (+) but more active against gram (-) bacteria Cefoperazone Ceftazidine more active against Cefpiramide Pseudomonas Cefpirome
      • II : Cell Membrane Inhibitors
          • 1. Polymixins – reserved for serious pseudomonas infection
          • 2. Polyenes
      • Amphotericin B – for deep seated mycoses
      • Nystatin – seperficial & cutaneous fungus infection
          • 3. Azoles – Imidazole antifungal agent – Ketaconazole
      • - Miconazole
      • - Clotrimozole
          • 4. Triazoles – Itraconazole
      • - Fluoconazole
      • III : Inhibitors of DNA function
      • 1. Mitomycin
      • 2. Nalidixic acid & Quinolones
      • 3. Norfloxacine & Ciprofloxacin
      • 4. Metronidazole – for anaerobic infection
      • 5. Novobiocin – gram (-)
      • 6. Griseofulvin
              • IV : Inhibitors Protein Synthesis
      • A. Actinomycin – active against gram (+) & gram ( -)
      • B. Rifampicin – major drug for TB & Leprosy
      • C. Streptomycin
      • Gentamycin Aminoglycosides
      • Tobramycin
      • Amikacin
      • D. Chloramphenicol
      • E. Nitrofurans
      • F. Erythromycin
      • G. Clindamycin / Lincomycin
              • V : Metabolite Analogue
      • 1. Sulfonamide
      • = gram (+) gram, (-) Nocardia, Chlamydia, Pneumococcus
      • 2. Sulfone
      • = Myco. leprae
      • 3. Para-aminosalicyclic acid
      • (PAS) – tuberculosis