Used for the initial detection of microbes Definitive identification of microbes

lectures 1-2 Flashcards by Ryan Bell

Sterilization

Total destruction of all microbes (bacteria, fungi,

viruses, spores, etc) present on inanimate surfaces

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Disinfectant

Destruction of microbes similar to sterilization, but at
different levels (high, intermediate, low)
− High level disinfectants approach sterilization
− Intermediate level disinfectants are used to clean
surfaces or instruments where spores or resilient
organisms are unlikely
− Low level disinfectants are used to treat non-critical
instruments (blood pressure cuffs, stethoscopes, etc.)

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Antiseptics

Reduce the number of microbes on skin surfaces
• Examples
− Alcohol
− Chlorhexidine
− Hydrogen peroxide

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Bactericidal

Substance that eradicates bacteria

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Bacteriostatic

Substance that inhibits the growth of bacteria

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Mechanism of action of sterilants,

disinfectants and antiseptics-Moist heat

Moist heat
• Modest efficacy because only a relatively low
temperature can be achieved (100° C)
• Bacterial spores may persis

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Mechanism of action of sterilants,

disinfectants and antiseptics-Autoclave

Autoclave
Uses steam under pressure to increase temperature
Causes denaturation of microbial proteins
Can achieve higher temperatures (can be > 130° C)

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Mechanism of action of sterilants,

disinfectants and antiseptics-Ethylene oxide

Ethylene oxide
• Colorless gas used to sterilize heat sensitive objects
• Slower disinfecting process and is influenced by
− Gas concentration
− Temperature
− Exposure time
− Humidity
• Requires dissipation of the gas before the item can be
used on human tissues

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Mechanism of action of sterilants, disinfectants

and antiseptics- Aldehydes

Aldehydes
• Causes alkylation of cells
• Formaldehyde and glutaraldehyde are common examples
• Formaldehyde dissolved in water creates formalin

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Mechanism of action of sterilants, disinfectants

and antiseptics- Oxidizing Agents

Oxidizing agents
• Examples include ozone, peracetic acid and hydrogen
peroxide (decomposes to form free hydroxyl radical)
• Hydrogen peroxide is used at different concentrations
− 3-6% for common use
− 10-25% kills all organisms and spores

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Mechanism of action of sterilants,

disinfectants and antiseptics- Haloggens

Halogens
• Iodine is highly reactive and precipitates proteins and
oxidates essential enzymes
• Chlorine solutions are rapidly bactericidal

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Mechanism of action of sterilants,

disinfectants and antiseptics- Quaternary Ammonium Compounds

Quaternary ammonium compounds
• Contains 4 organic groups linked to a nitrogen
• Examples
− Benzalkonium chloride
− Cetylpyridinium chloride

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Mechanism of action of sterilants,

disinfectants and antiseptics- Alcohols

Alcohols
• Germicidal activity increases with increasing carbon
chain length (5-8 carbons)
• Rapidly bactericidal for many bacteria
• Poor activity against bacterial spores, some fungi or
viruses
• Common alcohols
− Ethanol
− Isopropanol (Isopropyl alcohol)

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Microscopy

Used for the initial detection of microbes

* Definitive identification of microbes

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Brightfield microscopy

• Light source is used to illuminate the specimen
• Objective and ocular lenses are used to magnify
the image
• Multiple objective lenses can increase magnification
from about 10x to 100x
• Ocular lenses may increase magnification by another
10-15 fold
• Resolving power is approximately 0.2 um, sufficient
for most bacteria
• Limitation of brightfield micr

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Darkfield microscopy

• Uses objective and ocular lenses like brightfield
microscopy
• Uses a special condensor that prevents
transmitted light from directly illuminating the
specimen
• Scattered light reaches the specimen and
illuminates it against a black background
• Improves resolution to 0.02 um to identify small
bacteria (e.g., T. pallidum)
• Because light passes around the specimen, it can
be difficult to study internal structures

Phase-contrast microscopy

• Allows evaluation of internal structures of
bacteria
• Uses parallel beams of light passing through
different densities of structures
• This causes phase differences between the beams
of light
• A 3-dimensional image can be created and
allows greater details to be seen

Fluorescent microscopy

• Compounds called fluorochromes can absorb
short wavelength UV light and emit the energy
back at a visible wavelength
• Some bacteria have autoflorescence, while others
can be stained with fluorescent dyes and
evaluated with a fluorescent microscope
• High pressure mercury, halogen or xenon lamps
are used that emit a shorter wavelength of light,
and filters block IR light and heat
• Specimens appear as brightly illuminated against
a dark background

Electron microscopy

• Magnetic coils are used to detect a beam of
electrons from a tungsten filament, through the
specimen and onto a screen
• The very short wavelength of light allows for
increased magnitude and resolution
• Allows detection of very small specimens like
viruses
• Transmission electron microscopy allows light to pass
directly through the specimen
• Scanning electron microscopes bounce electrons off the
specimen allowing for 3D images to be produced

Gram staining

• A quick and common method to characterize
bacteria based on the nature of the cell wall
• Gram positive organisms have a thick cell wall
composed of multiple peptidoglycan layers
• Gram-negative organisms have a thinner cell wall with
much less peptidoglycan present and also an outer cell
membrane which reduces permeability of the cell
• Based on this difference, a quick gram stain can
determine whether an organism is gram+ or
gram- which can greatly assist in identification
and selection of correct antibiotic for use

Acid fast staining

• Acid fast organisms have wax-like cell walls that
contain fatty acids, waxes and complex lipids
• Such organisms require a special staining
technique
• Special stains are used that are lipid soluble and
decolorizing agents strip the stain from non-acid fast
organisms
• The most common acid-fast organisms are from
the Mycobacteria group

In vitro culture

• Allows samples taken from patients to be placed
in liquid or gel media and incubated
• Bacterial organisms then proliferate in the media
• There are many types of culture medium used
• NonSelective
− Blood agar
− Chocolate agar
− Mueller-Hinton agar
• Selective
• Differential
• Specialized

Molecular epidemiology

• DNA / RNA or proteins can be used to identify
certain microbes
• Electrophoresis
− Different DNA or RNA fragments have different sizes
and therfore different electrophoretic mobility in a gel
• Nucleic acid detection, amplification and sequencing
− DNA probes can detect, locate and quantitate nucleic
acid sequences
• Polymerase chain reaction (PCR)
− Amplifies single copies of viral DNA millions of times
− Real time PCR can quantitate the amount of DNA or
RNA

Serology

• Uses antibodies detect, identify and quantify
antigens from virus, bacteria, fungi or parasites
• Immunoassays
• Immunofluorescence
• Enzyme-linked immunosorbent assay (ELISA)
• Western blot analysis (variation of ELISA)

Bacterial Morphology | • Prokaryotic organisms

• Typically unicellular • Lack true membrane bound nucleus • Lack membrane bound organelles • DNA is not contained in chromosomes ( contained in circular plasmids) • Typically smaller cells compared to eukaryotes

Bacterial Morphology | • Eukaryote organisms

* Typically multicellular * Have true membrane bound nucleus * Possess membrane bound organelles * DNA is contained in chromosomes

Bacteria are commonly grouped into 2 main | groups: whjat are they and what is the difference?

• Gram positive − Cell walls are composed of a relatively THICK peptidoglycan layer • Gram negative − Cell walls are composed of a relatively THIN peptidoglycan layer − Also contains overlying outer membrane

Bacteria are also commonly classified depending on whether they grow in the presence or absence of oxygen, what are the classifications and what do they mean?

• Obligate aerobes = Bacteria that require molecular oxygen for metabolism and growth • Obligate anaerobes = Bacteria that cannot grow in the presence of oxygen • Facultative anaerobes = Bacteria that can grow either in the presence or absence of oxygen

Bacterial shapes are also used in classification, what are the names of the shapes and what are the shapes

* Cocci = spherical * Bacilli = rod-shaped * Spiral

Cellular arrangement based on planes of division | also help distinguish different bacteria. what are the names and what do they mean?

• Coccus bacteria = round shaped • Cocci division in a single plane are defined as Diplococci = cocci joined in pairs or Streptococci = cocci joined in linear chains • Cocci division in two planes = tetrad • Cocci division in 3 planes = sarcina • Cocci linked in random planes = Staphylococci • Rod-shaped bacteria • Bacillus = single bacilli • Streptobacillus = bacilli arranged in chains • Coccobacillus = oval bacilli similar to a coccus

 Cytoplasmic structures | • Cell wall

• Gram positive organisms − Thick peptidoglycan cell wall layer along with proteins, teichoic acid and complex polysaccharides − Functions like an exoskeleton − Porous and allows metabolites to diffuse across − Important for the structure, replication and protection of the bacteria in hostile environments − Cell wall surrounds the cytoplasmic membrane • Gram negative organisms − More complex cell walls − Has two layers external to the cytoplasmic membrane − Thin layer of peptidoglycan − No Teichoic or lipoteichoic acids present − External to the cell wall is an outer membrane unique to Gram negative bacteria − Contains lipopolysaccharide (LPS) also known as endotoxin, which can cause septic shock

capsules

 External structures • Some bacteria contain polysaccharide or protein layers known as capsules • If the polysaccharide layer is loose and nonuniform, it is known as a slime layer • Capsules and slime layers are important for survival of the bacteria − Capsules can act as a barrier − Capsules are poorly antigenic − Capsules may act as virulence factors − Capsules can promote adherence to other bacteria or to the host

Flagella

• Helically coiled proteins (flagellins) are anchored to the bacterial membranse • Allow for mobility of the bacteria

• Fimbriae (pili)

• Hairlike structures on the outside of the bacteria • Smaller in diameter compared to flagella and usually are not coiled • Promote adherence to other bacteria or to the host

Bacterial spores

• Some Gram positive bacteria may produce spores • Bacillus • Clostridium • Gram negative bacteria do NOT produce spores • Under certain harsh environmental conditions, the bacteria can convert to a vegetative or dormant state known as a spore • The spore allows the bacteria to exist in suspended animation • Spores contain: • Copy of the chromosome • Minimum amount of essential proteins and ribosomes • Inner membrane • Thin peptidoglycan layer • Protein coat • The spore protects the bacteria from • Heat • Radiation • Enzymes / chemical agents • Spores may exist for centuries • Spores are difficult to eradicate with standard disinfectants • http://www.youtube.com/watch? v=NAcowliknPs • When favorable conditions again exist, the spore germinates to form a vegetative cell