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Side ASide B
The Five I’s:
•Inoculation –Addition of microorganism to supporting medium •Incubation –Proper growth temperature and time •Isolation –Creating pure culture from a...
How do you culture a microorganism?
•To identify the agent that causes a specific disease requires that microorganisms be isolated and cultivated or cultured
Step 1 to cultivate microorganism
–Inoculum (sample to be cultured)•Environmental specimens (water, air, soil)•Clinical specimens (obtained from patient)•Stored specimens (previously cultured)
Step 2 to cultivate microorganism
–Medium(collection of nutrients allowing growth of inoculum)•Liquid broth •Solid media
Step 3 to cultivate microorganism
–Incubation (placing inoculated media at proper temperature for specific period of time)•Observable growth appears in or on medium after incubation is known as a culture
•A clinical specimen is a sample of human material to be examined or tested for the presence of a pathogen
How do you properly handle a clinical specimen? (3 Steps)
•Must take care not to contaminate the sample with organisms from the environment or other region’s of the patients body •Must be labeled properly •Must be transported...
•Obtaining Pure Cultures:
–Technique for isolating the suspected pathogen from normal microbiota in a culture
Pure culture :
–refers to a cultures composed of cells arising from a single progenitor (parent or original cell) •Progenitor is termed a CFU (colony forming unit)
refers to a set of instructions used to prevent further contamination by microbes
Two common pure culture isolation techniques:
•Streak plates •Pour plates
–Used to support the growth of microorganism or as a means of identification
–3 Physical States of Media
•Liquid •Semi solid •Solid
6 types of general culture media:
•Defined media •Complex media •Selective media •Differential media •Anaerobic media •Transport media
Physical States of Media •Liquid:
–Water-based solution –Growth appears as cloudiness or turbidity
Physical States of Media Semisolid:
–Contains agar that thickens, but does not form a solid Used to determine motility
Physical States of Media Solid:
–Agar: complex polysaccharide derived from algae –Will allow culture media to form solid surfaces
–Exact chemical composition is known –Used for organisms with strict need for specific growth factors –Not used for most clinical cultures
–Exact composition is not quite known –Yeast, meat, soy or protein extracts provide the nutrients –Trypticase soy agar (TSA), nutrient broth or nutrient agar are commonly...
•Contains substances that favor the growth of particular organisms or inhibit the growth of unwanted ones
•Presence of visible changes in the media or differences in the appearance of colonies help differentiate amongst different kinds of bacteria
Differential Media Blood agar-
differentiates between the ability of bacteria to digest, partially digest or inability to digest red blood cells
Differential Media Carbohydrate tubes:
differentiates between the ability to ferment types of sugars and the resulting products (acid or gas or both)
Differential Media MacConkey agar-
–selective for Gram – bacteria, differentiates between non-lactose and lactose fermenting Gram - bacteria
•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...
•Special media to allow for transfer of clinical specimens –Prevent contamination of other people –Prevent contamination of sample –Maintain proper growth conditions...
Special Culture Techniques Animal and cell culture:
Not all organisms will grow in media, need live cells or organism
Special Culture Techniques •Enrichment culture:
–Selective media or technique to increase the growth of bacteria found in very small numbers
•Preserving Cultures (3 steps):
–Refrigeration •Stores for short periods of time –Deep-freezing (-50°C to -95°C) •Stores for years –Lyophilization (removal of water from a frozen...
Refers to the use of light or electrons to magnify organisms not visible with the unaided eye.
General Principles of Microscopy (4):
–Wavelength of radiation –Magnification –Resolution –Contrast
General Principles of Microscopy (4) Wavelength of Radiation:
•Using radiation of smaller wavelengths results in enhanced microscopy
General Principles of Microscopy (4) Magnification:
•Increase in the size of an object (number with X, read times) •Determined by multiplying magnification power of ocular and objective lenses •Results from the passing...
General Principles of Microscopy (4) 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...
General Principles of Microscopy (4) Contrast:
•Refers to the differences in the intensity between two objects or between an object and the background –Important in determining resolution –Staining increases contrast
Types of Microscopes (3) Light Microscopes:
–Bright-field •Simple •Compound –Dark-field –Phase •Phase contrast •Differential interference contrast –Fluorescent Confocal
Types of Microscopes (3) Electron Microscopes:
–Transmission (TEM) –Scanning (SEM)
Types of Microscopes (3) Probe Microscopes:
–Scanning Tunneling (STM) –Atomic Force (AFM)
Light Microscopy –Bright-field microscopes•Simple:
–Contain a single magnifying lens –Similar to magnifying glass –Capable of 300X magnification –Leeuwenhoek used simple microscope to observe microorganisms
Light Microscopy –Bright-field microscopes •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...
Light Microscopy –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 Microscopy 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...
Phase microscopes (2 types) Phase-contrast microscope:
Phase microscopes (2 types) Differential interference contrast microscope (Normarski):
Involves prisms to split light into different colors, increasing contrast, 3Dappearance
Light Microscopy 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...
Light Microscopy –Fluorescent microscopy •Confocal microscopes:
Use florescent dyes or antibobies- Uses UV lazer-Creates 3D image
–Electron microscopes have greater resolving power and magnification (10,000X to 100,000X) –Detailed views of bacteria, viruses, internal cellular structures, molecules,...
Two Types of Electron Microscopes Transmission (TEM):
–Specimen must be very thin –Electrons are sent through specimen, similar to that of light microscopes –Electron energy is captured by a fluorescent screen, changing...
Two Types of Electron Microscopes Scanning (SEM):
–Specimen is coated with a metal –Electrons are sent across the surface of the specimen –Primary electrons excite metal to release secondary electrons –Secondary electrons...
–Magnifies more than 100,000,000 times –Detects individual molecules and atoms –Uses a probe that has been sharpened to end in a single atom
2 Types Probe 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...
2 Types Probe MicroscopesAtomic 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, what does it do?
•Increases contrast and resolution by coloring specimens with stains/dyes
Types of stains (4):
–Simple stains –Differential stains •Gram stain •Acid-fast stain •Endospore stain –Special stains •Negative (capsule) stain •Flagellar...
–Cells are suspended in liquid, one or two drops are added to slide, slide is overlaid with coverslip
–Best for observing motility –Suspended cells are added to coverslip, coverslip is inverted onto a depression slide, seal with Vaseline to prevent drying
How to prepare a specimen for staining:2 Steps
•Create a smear by spreading a thin sample of specimen on a slide and air drying •Fixation process adheres sample to slide, kills...
•Used to determine size, shape, and arrangement of cells •Composed of a single basic dye –Combine with and stain negatively charged structures –Crystal violet,...
•Used to distinguish between different types of cells or structures •Use more than one dye
Types of Differential Stains (3):
–Gram stain –Acid-fast - -Endospore
Gram Stain is used for:
•Used to differentiate between two large groups of microorganisms (Gram – and Gram +) •Typically, the first stain used to identify a bacterial pathogen
Clinical Relevance of the Gram stain: Presence of Gram negative organisms may indicate:
–Serious illnesses •Endotoxins are part of the outermost LPS layer in Gram – bacteria (Shigella, Salmonella) –Resistant to antibiotics •Outermost LPS layer ...
Gram positive bacteria:
–Final color: purple –Thick layer of peptidoglycan in cell wall –Retains dye-mordant complex ...
Gram negative bacteria:
–Final color: red/ pink –Much thinner layer of peptidoglycan in cell wall –Does not retain dye-mordant complex, needs counterstain (red)
-have hydrophobic layer •Waxy layer of acid-fast bacterial cells will retain primary dye and stain red/ pink •Non-acid-fast bacteria will not retain primary...
dehydrated structure formed inside a bacterial cell in response to harsh environmental conditions
Characteristics of Endospore (5):
–Usually found in Gram + cells –Dormant, non-reproductive –Tough cell wall is impermeable to most chemicals (stains) – Difficult to destroy (need...
Endospore Stain, what color does it stain?
•Malachite green is retained in spore •Vegetative (growing) cells will counterstain red/ pink with safranin
Special Stains Negative (capsule) stain:
•Negative stains are repulsed by negative charges on cells •Stain the background and not the cell •Cells are counterstained with another dye •Useful to determine...
Special Stains Flagellar stain:
•Used to determine the presence, number, location and arrangement of flagella •Flagella are normally invisible with light microscopy •fatens up flagella
•Staining for Electron Microscopy
-stain in the form of a metal •Prevent electrons from passing through (TEM) •Excited by primary electrons and release secondary electrons to create image (SEM) –Stains may...