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Exam 1 - Chapter 3

Culturing- The Five I’s Inoculation Incubation Isolation Inspection Identification
Inoculation Addition of microorganism to supporting medium
Incubation Proper growth temperature and time
Isolation Creating pure culture from a mixed population
Inspection Macroscopic and microscopic observations
Clinical Specimens and the Methods Used to Collect Them - Blood Needle aspiration from vein
Needle aspiration from subarachnoid space of spinal column
Clinical Specimens and the Methods Used to Collect Them - Stomach Intubation, which involves inserting a tube into the stomach, often via a notril
Clinical Specimens and the Methods Used to Collect Them -Urine In aseptic collection, a catheter is inserted into the bladder through the urethra; in the "clean catch" method, initial urination washes the urethra, and the specimen is midstream urine.
Clinical Specimens and the Methods Used to Collect Them - Lungs Collection of sputum either dislodged by coughing or acquired via a catheter
Clinical Specimens and the Methods Used to Collect Them - Diseased Tissue Surgical removal (biopsy)
Pure Cultures (Isolation) -Technique for isolating the suspected pathogen from normal microbiota in a culture (mixed culture) -Pure culture refers to a culture composed of cells arising from a single original cell -Original cell termed a CFU (colony forming unit)
Obtaining Pure Cultures (Isolation) Use aseptic technique and sterile media & equipment to prevent further contamination
2 common Pure Isolation Techniques Streak plates Pour plates
Types of media for culturing organisms a. Agar b. Defined vs. complex c. Selective d. Differential e. Anaerobic f. Transport g. Special techniques (viral and enrichment) h. Preserving these cultures
Physical States of Media Liquid Semi solid Solid
Six types of general culture media Defined media Complex media Selective media Differential media Anaerobic media Transport media
Liquid Water-based solution Growth appears as cloudiness or turbidity
Semisolid -Contains agar that thickens, but does not form a solid -Used to determine motility
Solid -Agar: complex polysaccharide derived from algae -Will allow culture media to form solid surfaces
Defined Media -Exact chemical composition is known -Used for organisms with strict need for specific growth factors -Not used for most clinical cultures
Complex media -Exact composition is not quite known -Yeast, meat, soy or protein extracts provide the nutrients -Trypticase soy agar, nutrient broth or nutrient agar are commonly used
Selective Media -Contains substances that favor the growth of particular organisms or inhibit the growth of unwanted ones -Add high concentration of salt to selectively allow for the growth of halophiles (Staph. aureus) -Sabouraud dextrose agar has a lower pH to select
Differential Media Presence of visible changes in the media or differences in the appearance of colonies help differentiate amongst different kinds of bacteria
Anaerobic Media -Obligate anaerobes need to be cultured in the absence of oxygen Agar Stab Reducing media that chemically combine with free oxygen and remove from media Anaerboic culture system (Gas Pak)
Transport Media -Special media to allow for transfer of clinical specimens -Prevent contamination of other people -Prevent contamination of sample -Maintain proper growth conditions for survival
Special Culture Techniques -Animal and cell culture Not all organisms will grow in media, need live cells or organism Examples: Mycobacterium leprae (leprosy), Treponema pallidum pallidum(syphillus) and viruses -Enrichment culture Selective media or technique to increase the
Preserving Cultures- Refrigeration Stores for short periods of time -Deep-freezing (-50°C to -95°C) Stores for years -Lyophilization (removal of water from frozen culture) Stores for decades
Microscopy Refers to the use of light or electrons to magnify organisms not visible with the unaided eye.
General Principles of Microscopy Wavelength of radiation Magnification Resolution Contrast
Wavelength of Radiation Using radiation of smaller wavelengths results in enhanced microscopy
Magnification -Increase in the size of an object -Determined by multiplying magnification power of ocular and objective lenses -Results from the passing of radiation (light or electrons) through a lens or series of lenses
Resolution -Resolution or resolving power is the ability to distinguish between objects that are close together -Dependent on the wavelength of radiation and numerical aperture (na)of lens -Ability of lens to gather light
Contrast -Refers to the differences in the intensity between two objects or between an object & the background -Important in determining resolution -Staining increases contrast
Light Microscopes- Simple Simple: -Contain a single magnifying lens -Similar to magnifying glass Capable of 300X magnification -Leeuwenhoek used simple microscope to observe microorganisms
Light Microscopes- Compound Compound: -Series of lenses for magnification -Light passes through specimen into objective lens -Have one or two ocular lenses -Most have condenser lens (direct light through specimen) -Capable of 2,000X magnification -Background bright, specimen
Light Microscopes- Dark-field Dark-field microscopes: -Best for observing pale objects -Only light rays scattered by specimen enter objective lens -Increases contrast and enables observation of more details -Specimen appears light against dark background
Light Microscopes - Phase Phase microscopes -Used to examine living organisms or specimens that would be damage by attaching them to slides or staining -Light rays in phase produce brighter image, while light rays out of phase produce darker image -Contrast is created because l
Two types of Phase microscope -Phase-contrast microscope Simplest -Differential interference contrast microscope (Normarski) Involves prisms to split light into different colors, increasing contrast, three-dimensional appearance
Light Microscopes - Fluorescent Fluorescent microscopes -Direct a UV light source at specimen -Specimen emits visible light when bombarded by short UV rays -Some cells are naturally fluorescent; others must be stained -Used in immunofluorescence to identify pathogens or to make v
Fluorescent microscopy - Confocal microscopes Confocal microscopes -Also uses fluorescent dyes or antibodies -Differences from conventional fluorescent microscope UV laser is used to illuminate the fluorescent chemicals in a single plane, creates a sharper image Computer constructs 3-D image from
Electron Microscopy -Electron microscopes have greater resolving power and magnification (10,000X to 100,000X) -Detailed views of bacteria, viruses, internal cellular structures, molecules, and large atoms -Lenses are magnets, not glass -Energy beam in form of electrons,
Two Types of Electron Microscopes Transmission (TEM) Scanning (SEM)
Transmission (TEM) -Specimen must be very thin -Electrons are sent through specimen -Electron energy is captured by a fluorescent screen -More dense the specimen, more electrons blocked, seen as darker on the final image -Finest detailed structures of cells can be obser
Scanning (SEM) -Specimen is coated with metal -Electrons are sent across the surface of the specimen -Primary electrons excite metal to release secondary electrons -Secondary electrons are captured by detector -Final image is three dimensional, but of surface of s
Probe Microscopy Magnifies more than 100,000,000 times Detects individual molecules and atoms Uses a probe sharpened to end in a single atom
Two types of Probe Microscope Scanning tunneling microscopes Atomic force microscopes
Scanning tunneling microscopes -Probe is passed back and forth and slightly above specimen -Measures the flow of voltage back and forth from the probe to specimen, translates to an image
Atomic force microscopes -Probe is passed along the surface of the specimen -Tip moves up and down with contour of surface, movement is translated to image
Staining Increases contrast and resolution by coloring specimens with stains/dyes
Types of stains -Simple stains -Differential stains Gram stain Acid-fast stain Endospore stain -Special stains Negative (capsule) stain Flagellar stain -Electron microscope stains
Wet mount Cells are suspended in liquid, one or two drops are added to slide, slide is overlaid with coverslip
Preparing a specimen for staining -Create a smear by spreading a thin sample of specimen on a slide and air drying. -Fixation process adheres sample to slide, kills organism, preserves shape and size
Simple Stains -Used to determine size, shape, & arrangement of cells -Composed of a single dye: Combine with and stain negatively charged structures Crystal violet, safranin, or methylene blue
Differential Dyes -Used to distinguish between different types of cells or structures -Use more than one dye
Types of Differential Stains Gram stain Acid-fast Endospore
Gram Stain -Used to differentiate between two large groups of microorganisms (Gram negative and Gram positive) -Typically, the first stain used to identify a bacterial pathogen -Staining is dependent on the thickness of the cell wall (peptidoglycan)
Gram positive bacteria Final color: purple Thick layer of peptidoglycan Retains dye-mordant complex (purple)
Gram negative bacteria Final color: red/ pink Thin layer of peptidoglycan Does not retain dye-mordant complex, needs counterstain (red)
Acid-Fast Stain -Used to stain pathogens of the Mycobacterium (TB, leprosy) and Nocardia (lung & skin infections) -Thick waxy lipid in cell walls of these bacteria significantly affects staining abilities, heat must be applied
Endospore Stain Endospore: dehydrated structure which forms inside a bacterial cell in response to harsh environmental conditions. -Dormant, non-reproductive, difficult to destroy -Tough cell wall is impermeable to most chemicals (stains), need to apply heat -Exampl
Special Stains -Stains designed to stain special structures within a microbe -Types of special stains Negative (capsule) stain Flagellar stain
Negative (Capsule) Stain -Capsular stains are repulsed by negative charges on cell -Stain the background and not the cell -Cells are counterstained with a second positive dye -Useful to determine the presence of a capsule (does not stain)
Flagellar Stain -Used to determine the presence, number, location and arrangement of flagella -Flagella are normally invisible with light microscopy -A series of stains and mordants are applied to increase the diameter, allowing the flagella to become visible
Staining for Electron Microscopy -Chemicals containing heavy metals used for electron microscopy: Prevent electrons from passing through (TEM) Excited by primary electrons and release secondary electrons to create image (SEM) -Stains may bind molecules in specimens or the background
Created by: bonitasoul