MLAB 2434 – CLINICAL MICROBIOLOGY KERI BROPHY-MARTINEZ
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Transcript of MLAB 2434 – CLINICAL MICROBIOLOGY KERI BROPHY-MARTINEZ
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MLAB 2434 – CLINICAL MICROBIOLOGY KERI BROPHY-MARTINEZ
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Bacterial Cell Structure, Physiology, Metabolism, & Genetics
Taxonomy Defined as the orderly classification & grouping
of organisms into categories Based on genotype and phenotype Kingdom, Division, Class, Order, Family, Tribe,
Genus and Species ( these are the formal levels of classification)
• Family = “Clan”; has “–aceae” ending• Genus = “Human last name”• Species = “Human first name”
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Bacterial Cell Structure, Physiology, Metabolism, & Genetics (cont’d) Nomenclature
Family names: Capitalized with “aceae” endings Genus names: Capitalized
• When in print, genus and species are italicized. (Staphylococcus aureus)
• When written, genus and species are underlined. (Staphylococcus aureus)
Species names• Staphylococcus sp. is used when referring to the genus
as a whole when the species is not identified.• Can be referred to as singular or plural
• “sp.” – singular (Staphylococcus sp.)• “spp.” – plural (Staphylococcus spp.)
Abbreviations• First letter of the genus, followed by a period and the
species epithet• Ex: Staphylococcus aureus changes to S. aureus
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Bacterial Cell Structure, Physiology, Metabolism, & Genetics (cont’d)
Bacteria Identification – test each bacterial culture for a variety of metabolic characteristics and compare the results with known results.
All organisms are either “prokaryotes”, “eukaryotes”, or “archaeobacteria”
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Bacterial Cell Structure, Physiology, Metabolism, & Genetics (cont’d)
PROKARYOTES - bacteria Do not have a
membrane-bound nucleus
DNA is a single circular chromosome
Have both cell (plasma) membrane AND cell wall.
F= flagellumC=capsuleP= piliN= nuclear infoR=ribosomeCM= cytoplasmic membraneCW= cytoplasmic wall
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Bacterial Cell Structure, Physiology, Metabolism, & Genetics (cont’d)
EUKARYOTES - fungi, algae, protozoa, animal cells, and plant cells Cells have nuclei
that contains DNA and are complex
Most cells do NOT have a cell wall V=Vesicle
M=MitochondriaG= GolgiNM= nuclear membraneN= nucleusNC= nucleolusRER= rough endoplasmic reticulumPM= plasma membrane
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Bacterial Cell Structure, Physiology, Metabolism, & Genetics (cont’d)
Archaeobacteria Resembles
eukaryotes Found in
microorganisms that grow under extreme environmental conditions
Cell wall lacks peptidoglycan
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Bacterial Cell Structure, Physiology, Metabolism, & Genetics (cont’d)
Bacterial Cell Wall
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Bacterial Cell Structure, Physiology, Metabolism, & Genetics (cont’d)
Gram Positive (GP) Cell Wall Very thick protective peptidoglycan layer Many GP antibiotics act by preventing
synthesis of peptidoglycan Consists of cross-linked chains of glycan Also contain teichoic acid and lipoteichoic acid
these unique structures makes these bacteria GP
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Bacterial Cell Structure, Physiology, Metabolism, & Genetics (cont’d)
Gram Negative (GN) Cell WallTwo layers; outer is much thinner
than GP cell wallsOuter wall contains several
molecules, including Lipid A which is responsible for producing fever and shock in infections with GN bacteria
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The Cell Wall
Gram Positive Gram Negative
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Bacterial Cell Structure, Physiology, Metabolism, & Genetics (cont’d)
GP cocci in clusters →
GN bacilli (rods) →
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Bacterial Cell Structure, Physiology, Metabolism, & Genetics (cont’d)
Acid Fast Cell Wall – mainly Mycobacteria and NocardiaHave a GP cell wall structure but
also a waxy layer of glycolipids and fatty acids (mycolic acid)
Waxy layer makes them difficult to gram stain
Cannot be decolorized by acid-alcohol, hence the name “acid fast”
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Bacterial Cell Structure, Physiology, Metabolism, & Genetics (cont’d)
Absence of Cell Wall – mainly Mycoplasma and Ureaplasma Lack of cell wall results in a variety of
shapes microscopically Contain sterols in cell membrane
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Bacterial Cell Structure, Physiology, Metabolism, & Genetics (cont’d)
Some bacteria produce a capsule Protect the
bacteria from phagocytosis
Capsule usually does not stain, but can appear as a clear area (halo-like)
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Bacterial Cell Structure, Physiology, Metabolism, & Genetics (cont’d)
Some bacteria produce slime layers Made of polysaccharides Inhibit phagocytosis Aid in adherence to host tissue or
implants
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Bacterial Cell Structure, Physiology, Metabolism, & Genetics (cont’d)
Cell Appendages Flagella – exterior protein filaments
that rotate and cause bacteria to be motile• Polar
• Extend from one end• Can occur singly or in multiple tufts
• Peritrichous• Flagella found on all sides of bacteria
Pili (fimbriae) – hairlike projections that aid in attachment to surfaces
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Examples of Flagella
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Bacterial Cell Structure, Physiology, Metabolism, & Genetics (cont’d)
Microscopic Shapes Cocci (spherical) Bacilli (rod-shaped) Spirochetes (helical)
Groupings Singly Pairs Clusters Chains Palisading
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Bacterial Cell Structure, Physiology, Metabolism, & Genetics (cont’d)
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Bacterial Cell Structure, Physiology, Metabolism, & Genetics (cont’d)
Size and lengthShortLongFilamentousFusiformCurvedPleomorphic
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Bacterial Cell Structure, Physiology, Metabolism, & Genetics (cont’d)
Common Bacterial Stains Gram Stain:
• cell wall structure determines the staining characteristics
• Procedure to be covered in lab
Acid-fast • stains bacteria with high
lipid and wax content in their cell walls
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Bacterial Cell Structure, Physiology, Metabolism, & Genetics (cont’d)
Acridine Orange • stains nucleic
acid of both G+ and G- bacteria, either living or dead
used to locate bacteria in blood cultures and other specimens where background material obscures gram stains
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Bacterial Cell Structure, Physiology, Metabolism, & Genetics (cont’d)
Methylene Blue stain for
Corynebacterium diphtheriae to show metachromatic granules and as counter-stain in acid-fast stain procedures
Lactophenol Cotton Blue fungal stain
Calcofluor White fungal stain
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Bacterial Cell Structure, Physiology, Metabolism, & Genetics (cont’d)
India Ink – negative stain for capsules, surrounds certain yeasts
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I NEED A BREAK!!!!!
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Bacterial Cell Structure, Physiology, Metabolism, & Genetics (cont’d)
Microbial Growth and Nutrition NeedsSource of carbon for making
cellular constituentsSource of nitrogen for making
proteinsSource of energy (ATP) for cellular
functionsSmaller amounts of other
molecules
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Bacterial Cell Structure, Physiology, Metabolism, & Genetics (cont’d)
Nutritional Requirements for GrowthAutotrophs (lithotrophs)
• Able to grow simply, using only CO2, water and inorganic salts
• Obtain energy via photosynthesis or oxidation of inorganic compounds
• Occur in nature and do not normally cause disease
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Bacterial Cell Structure, Physiology, Metabolism, & Genetics (cont’d)
Heterotrophic• Require more complex substances
for growth• Require an organic source of carbon
and obtain energy by oxidizing or fermenting organic substances
• All human bacteria fall in this category
• Within this group, nutritional needs vary greatly
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Bacterial Cell Structure, Physiology, Metabolism, & Genetics (cont’d)
Types of Growth/Culture Media Minimal medium – simple; not usually
used in diagnostic clinical microbiology Nutrient medium – made of extracts
of meat or soy beans Enriched medium – nutrient medium
with extra growth factors, such as blood which encourages small numbers of organisms to flourish
Broths- used to detect small numbers of aerobes, anaerobes and microaerophiles
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Bacterial Cell Structure, Physiology, Metabolism, & Genetics (cont’d)
Nonselective medium- supports growth of most nonfastidious microbes.
Selective medium – contains additives that inhibit the growth of some bacteria while allowing others to grow
Differential medium – contains additives that allow visualization of metabolic differences in bacteria
Transport medium – holding medium to preserve those bacteria present but does not allow multiplication
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Bacterial Cell Structure, Physiology, Metabolism, & Genetics (cont’d)
Selection of primary culture media will vary from lab to lab
Selection of primary media will depend on anatomical site
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Bacterial Cell Structure, Physiology, Metabolism, & Genetics (cont’d)
Environmental Factors Influencing Growth pH – most media is between 7.0 and
7.5 Temperature – most pathogens grow at
body temperature; grown at 35° C in the lab• Psychrophiles: cold temperatures
• 10-20o C• Mesophiles moderate temperatures
• 20-40 o C• Thermophiles: high temperatures
• 50-60 o C
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Bacterial Cell Structure, Physiology, Metabolism, & Genetics (cont’d)
Gaseous composition• Obligate aerobes – require
oxygen• Obligate anaerobes – cannot
grow in the presence of oxygen
• Facultative anaerobes – can grow with or without oxygen
• Microaerophilic- grow better in low oxygen environments ( about 20%)
• Aerotolerant anaerobes- grows better in the absence of oxygen
• Capnophilic – grow better with extra CO2 (5-10%)
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Bacterial Cell Structure, Physiology, Metabolism, & Genetics (cont’d)
Bacterial Growth Reproduce by
binary fission Can be fast (as
little as 20 minutes for E. coli or slow as 24 hours for M. tuberculosis
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Bacterial Cell Structure, Physiology, Metabolism, & Genetics (cont’d)
Determination of Numbers Direct counting under microscope
• Estimates number of live and dead cells Direct plate count
• Determines the number of CFU (colony-forming units) in broth cultures and urine cultures
Density measurement• Useful to prepare inoculums for
antimicrobial susceptibility testing
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Is It Time For a Break?
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Bacterial Cell Structure, Physiology, Metabolism, & Genetics (cont’d)
Bacterial Biochemistry and Metabolism Metabolic reactions cause production
of energy in form of ATP Identification systems analyze
unknown specimens for:• Utilization of variety of substances as a
source of carbon• Production of specific end products from
various substrates• Production of acid or alkaline pH in the test
medium
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Bacterial Cell Structure, Physiology, Metabolism, & Genetics (cont’d)
Fermentation and Respiration (Oxidation)Fermentation
• Anaerobic process in obligate and facultative anaerobes
• The electron acceptor is an organic compound
• Does NOT require oxygen
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Bacterial Cell Structure, Physiology, Metabolism, & Genetics (cont’d)
Respiration (Oxidation)• More efficient energy-generating
process• Molecular oxygen is the final
electron acceptor• Aerobic process in obligate aerobes
and facultative anaerobes
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Bacterial Cell Structure, Physiology, Metabolism, & Genetics (cont’d)
Metabolic Pathways Embden-Meyerhoff-Parnas
• Primary cycle for bacteria• Convert glucose to pyruvic acid, a key
intermediate• Generates energy in the form of ATP
Pentose Phosphate pathwayEntner-Doudoroff pathway
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Bacterial Cell Structure, Physiology, Metabolism, & Genetics (cont’d)
From pyruvic acid: Alcoholic fermentation
(ethanol) Homolactic fermentation
(lactic acid) Heterolactic fermentation
(lactic acid, CO2, alcohols, formic and acetic acids
Propionic acid (propionic acid)
Mixed acid fermentation (lactic, acetic, succinic, and formic)
Butanediol fermentation(acetoin and 2,3 butanediol)
Butyric acid fermentation (butyric)
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Bacterial Cell Structure, Physiology, Metabolism, & Genetics (cont’d)
Main oxidative pathway is the Krebs Cycle, resulting in acid and CO2
Carbohydrate Utilization & Lactose Fermentation “Sugars” = carbohydrates Lactose fermentation – key component
in identification schemes Lactose is converted to glucose, so ALL
lactose fermenters also ferment glucose
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Bacterial Cell Structure, Physiology, Metabolism, & Genetics (cont’d)
Genetic Elements and AlterationsPlasmid
• Extra piece of DNA• Code for antibiotic resistance and
other virulence factors are often found on plasmids
• Sometimes passed from one bacterial species to another. This is how resistance is acquired.
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Bacterial Cell Structure, Physiology, Metabolism, & Genetics (cont’d)
Plasmid replication Three
methods• Transformatio
n• Transduction• Conjugation
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Bacterial Cell Structure, Physiology, Metabolism, & Genetics (cont’d)
Mutations“They don’t always read the
book”Changes that occur in the DNA
code Results in changes in the coded
protein or in the prevention of its synthesis
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References
Engelkirk, P., & Duben-Engelkirk, J. (2008). Laboratory Diagnosis of Infectious Diseases: Essentials of Diagnostic Microbiology . Baltimore, MD: Lippincott Williams and Wilkins.
http://andyannie.pbworks.com/w/page/5454436/Reproduction http://animals.howstuffworks.com/fish/eels-slippery1.htm http://fanaticstars.wordpress.com/2009/04/01/on-break/ http://www.istockphoto.com/stock-illustration-82213-coffee-break.php http://pathmicro.med.sc.edu/infectious%20disease/infectious%20diseas
e%20introduction.htm http://realneo.us/content/yellowstone-national-park-hit-swarm-earthqua
kes Mahon, C. R., Lehman, D. C., & Manuselis, G. (2011). Textbook of
Diagnostic Microbiology (4th ed.). Maryland Heights, MO: Saunders.