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Introduction to Microbiology
What is Microbiology? Study of microscopic organisms
– 5 Groups of microbes:• bacteria, fungi (yeasts & molds), viruses,
protozoa, algae
Clinical microbiology– study of microscopic organisms in
pathogenic (disease causing) processes in humans
Purpose of Clinical Microbiology To work with health care professionals to
provide accurate and rapid diagnostic aid and to manage information about infectious diseases– prompt diagnosis leads to early treatment– reduces morbidity (course of illness) and mortality
(death) To prevent or control the spread of pathogens
– In the community and/or hospital-acquired (nosocomial)
To track organisms’ resistance – to antimicrobial/antifungal agents
Basic Bacteriology includes:
Inoculation Incubation Isolation Inspection Interpretation Identification
Microbial life characteristics
All living things have similar “life characteristics” Respiration Reproduction Growth Motion Nutrition Excretion
Respiration Process organisms use to convert energy in
chemical bonds of O2 or an organic molecule to make ATP energy
Aerobic organisms use O2 during respiration organisms are incubated in ambient air
(room air) Anaerobic organisms
use organic molecules (N2) during respiration
intolerant of oxygen
RespirationOrganism can be:
Obligate aerobe Facultative anaerobe Microaerophilic Capnophilic Aerotolerant anaerobe Obligate anaerobe
Facultative anaerobic organisms capable of adaptive behavior can grow with or without oxygen
Microaerophilic organisms grow best at O2 tension and CO2
example: Campylobacter species
Capnophilic organisms require CO2 (5-10%) for growth
example: Neisseria /Haemophilus species
Respiration
Respiration
Aerotolerant anaerobescant growth in room air or 5-10%
CO2example: Clostridium carnis
Obligate (strict) anaerobecannot tolerate oxygenexample: Clostridium
hemolyticum
Generating Anaerobic Conditions Anaerobic chamber or glove box
Attached to N2 and Mixed gas tanks Gas-Pak anaerobic jar
Na borohydride + Na bicarbonate + citric acid +(add) H2O in the presence of a palladium catalyst forms
H2 + CO2 + N2
H2 + O2 (from the environment) H2O palladium catalyst becomes inactivated by water
reactivate by heating to 160-1700C for 2-4 hours indicator system: methylene blue/resazurin 90-100 minutes to achieve anaerobic conditions
Generating Anaerobic ConditionsAnaerobic chamber or glove box
Generating Anaerobic ConditionsA-Jar ANO2 Gas-Pouch
Generating Anaerobic ConditionsThe Gas-Pak
Generating Anaerobic ConditionsThe A-jar
Anoxomat
Anoxomat
Uses 3 cycles to flush jar Achieves anaerobic conditions in 1-3 minutes,
microaerophilic in 20 seconds Uses a rechargeable catalyst Uses gas mixture of: 10% CO2, 5% H2, 85% N2, Uses less gas than an anaerobic chamber
Generating Capnophilic Conditions CO2 incubator (5-10%)
tank is attached to an incubator, a regulator adjusts gas flow
CO2 packs (5%) similar principle as Gas-Pak
Candle jar (3%) place lit candle in jar with media; as flame
burns, it consumes O2 and produces CO2
Generating Capnophilic Conditions
Candle Jars CO2 Gas-pouch
Reproduction During bacterial reproduction, DNA is
replicated, the cell grows and then divides into 2 identical daughter cells changes in nucleotide sequences of the DNA
molecule may occur changes may be spontaneous or result of the
influence of external agents(i.e antibiotics) bacteria can also exchange genetic material
between other organisms
Reproduction Bacteria reproduce by binary fission
cell doubles in size, then divides into two identical daughter cells
doubling or generation time = time required for population of bacteria to double in number• varies from organism to organism• E. coli: 20 minutes• M. tuberculosis: 16-20 hours
Phases of Bacterial Growth Lag phase
growth does not begin immediately after inoculation to media
organisms are acclimating to their new environment
They are enlarging and producing energy Log (exponential) phase
each cell in population divides in two growth occurs exponentially organisms are most susceptible to damage
at this point, especially from antibiotics
Phases of Bacterial Growth Stationary phase
cells stop metabolizing as a result of food and nutrients depletion
growth stabilizes: rate of doubling equals rate of death
Death phase food is depleted and toxic waste product
build up more orgs are dying than doubling actual numbers of viable organisms decline
Phases of Bacterial Growth
Genetic Recombination Transfer of DNA from one organism to another
Include:
• Transformation
• Conjugation • Transduction
Transformation:
Free DNA fragments from dead Bacterium enter a related species across the cell wall
DNA fragments are exchanged for a piece of the recipient’s DNA
Ex: Strep pneumoniae, non encapsulated strains acquire a capsule
Transformation
Conjugation Transfer of DNA from one donor bacterium
to a recipient via a sex pilus Plasmids coding for antibiotic resistance
may also be transferred in this process.
Transduction DNA is transferred from one cell to another
by a bacteriophage (bacterial virus)
Genetic Recombination
Conjugation
Transduction
Motion
Movement of microorganisms is accomplished by either flagella, pseudopodia or cilia
Bacteria move by means of flagella– classification can be based on location of
the flagella
Flagella Location Atrichous
no flagella
Monotrichous single flagellum at either end
Amphitrichous flagella at both ends; singly, in pairs or clumps
Peritrichous flagella surrounding the entire organism
Flagella Location
Nutrition Bacteria obtain nutrients by taking up small
molecules across the cell wall Nutrition + Respiration ATP (energy) Nutrition is obtained from the culture media
organic and inorganic materials adequate moisture to maintain osmotic pressure
• Most bacteria requires an isotonic/hypotonic environment
pH must be properly adjusted bacteria: 6.5-7.5 fungi: 5-6
Hypotonic movement
http://student.ccbcmd.edu/courses/bio141/lecguide/unit1/prostruct/hypotonic_flash.html
Metabolic Pathways
Bacteria can use 3 pathways for glucose degradation
Metabolic pathways
The result of any metabolic process is: Hydrogen ions are transferred to
compounds of higher redox potential The result is release of ATP energy.
Embder-Meyerhoff Pathway
Glycolytic or anaerobic pathway (fermentation)
Glucose is degraded without the presence of oxygen
End products are acids which are detectable by pH changes:
A. acetoin (VP pos bacteria) (mildly acidic) B. Mixed acids (MR pos bacteria) (pH<4.4) Examples: Enterics, Anaerobes
Entner-Douderoff Pathway
Aerobic pathway Oxygen is required for glycolysis End products are weak acids Examples: non-fermenting GNR
Warburg-Dickens (Hexose Monophosphate shunt)
Aerotolerant Nonoxidative bacteria that are capable
of growing in the presence of oxygen but grow better anaerobically
Examples: Aerotolerant anaerobes
Excretion
Removal of waste products For bacteria, this is usually
accomplished by either active or passive transport
Cellular Organization
Cell is the basic unit of life All living cells are divided into 2 groups:
Eukaryotesanimal, plants, fungi, protozoans, and algae
Prokaryotesbacteria
Cellular OrganizationProkaryotes vs. Eukaryotes Prokaryotes (bacteria)
genetic material is not enclosed within membrane DNA is not associated with histone proteins cell walls contain peptidoglycan no membrane-bound organelles
Eukaryotes (fungi, parasites, plants, animals) genetic material is within a membrane and
organized into chromosomes DNA is associated with histone proteins Cell walls(plant,fungi, algae)/No cell walls
(animal,protozoans) Cell walls have no peptidoglycan contain nucleus and membrane-bound organelles
Bacterial cell wall Rigid structure that gives the organism its
shape Contains peptidoglycan (a.k.a murein)
mucopolysaccharide Prevents the cell from bursting in hypotonic
conditions Maintains osmotic pressure within the cell Types of cell walls
– based on their staining ability Gram positive Gram negative Acid fast
Gram-Positive Cell Wall Composed of dense layer of peptidoglycan
and teichoic acids Gram stain: resist decolorization and retain
crystal violet dye (purple) Thick peptidoglycan (murein) layer (60-90%
of G+ cell wall) forms rigid structure Teichoic acids
link the cell wall peptidoglycan to cell membrane regulate the movement of ions into and out of the
cell Triggers immune response (i.e initiate
inflammation)
Gram-Positive Cell Wall
Gram-Negative Cell Wall Gram stain: easily decolorized and absorbs
counterstain, safranin (pink) Cell wall consists of two layers
thin inner layer of peptidoglycan (10-20% of G- cell wall)
outer membrane of proteins, phospholipids and lipopolysachharides (LPS)
Outer membrane is a barrier to toxic substances(I.e penicillin G) is a sieve, allowing water soluble molecules and
proteins into the cell provides attachment sites to allow adhesion to
host cells and triggers immune response (i.e fever, inflammation)
Gram-Negative Cell Wall
Cell Walls
Lipopolysaccharide (LPS)
Lipopolysaccharide (LPS) contains 3 regions antigenic O-specific polysaccharide
• specific to each species core polysaccharide
• common to all Gram negatives inner lipid A (endotoxin)
• causes fever and shock associated with Gram negative sepsis
Acid-Fast Cell Wall Contains small amounts of peptidoglycan and
large amounts of glycolipids (mycolic acid) Mycolic acid waxy lipid (60% of cell wall)
Make cell wall impermeable Difficult to stain, appear as “ghost cells” on Gram
stain Block entry of chemicals and cause organism to
become more resistant to phagocytosis
Acid fast stain: resist decolorization by acid-alcohol mixture, retain carbol fuchsin (red) Ex: Mycobacteria, Nocardia species
Bacterial Taxonomy Comprise of classification, nomenclature, and
identification Classification is the arrangement of organisms
into specific groups based on their common morphologic, physiologic, and genetic traits classically based on phenotypic behavior (i.e.,
biochemical reactions, etc.) newer techniques for classification are based on
genotypic characteristics (i.e. how closely the organisms are genetically related)
NomenclatureBinomial system
Binomial System Two-name systemGenus and species
Genus: name given to related organisms- ALWAYS capitalized first letter- Can be abbreviated
Species: name given to each type of organism within a genus
- NEVER capitalized Either italicized or underlined
- Ex. Proteus (genus) mirabilis (species) or P. mirabilis System is constantly changing
- Neisseria catarrhalis Moraxella catarrhalis Branhamella catarrhalis Moraxella catarrhalis
Classification: Kingdom Division Class Order Family Tribe Genus Species