Microbiology Chapter 3

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The Five I’s:
•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 •Identification

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
Clinical Specimens:
•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 quickly to the lab to avoid death of microbe and minimize growth of normal microbiota
•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)
Aseptic technique:
refers to a set of instructions used to prevent further contamination by microbes
Two common pure culture isolation techniques:
•Streak plates •Pour plates
•Culture Media:
–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

–Water-based solution –Growth appears as cloudiness or turbidity

Physical States of Media

–Contains agar that thickens, but does not form a solid Used to determine motility

Physical States of Media

–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 (TSA), 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
Differential Media:
•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
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 machine)
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
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 culture) •Stores for decades
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)

•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 of radiation (light or electrons) through a lens or series of lenses •Magnification is limited by resolution

General Principles of Microscopy (4)
•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

General Principles of Microscopy (4)

•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 through specimen) –Capable of 2,000X magnification –Background bright, specimen darker
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 against dark background
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 out of phase produce darker image •Contrast is created because light waves are out of phase

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 immunofluorescence to identify pathogens or to make visible a variety of proteins
Light Microscopy –Fluorescent microscopy •Confocal microscopes:
Use florescent dyes or antibobies- Uses UV lazer-Creates 3D image
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 is in form of electrons, not visible light –Requires a vacuum, therefore only dead specimens can be observed

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 some energy to visible light –More dense the specimen, the more electrons blocked, seen as darker on the final image –Finest detailed structures of cells can be observed
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 are captured by detector and photomultiplier –Final image is three dimensional, but of surface of specimen only
Probe Microscopy:
–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 to an image
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 stain –Electron microscope stains
Wet mount:
–Cells are suspended in liquid, one or two drops are added to slide, slide is overlaid with coverslip
Hanging drop:
–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 organism, preserves shape and size –Heat fixation- briefly pass slide through flame –Chemical Fixation- dip slide in methanol or formalin
Simple Stains:
•Used to determine size, shape, and arrangement of cells •Composed of a single basic dye –Combine with and stain negatively charged structures –Crystal violet, safranin, or methylene blue
- 1 dye
-neg cells attract pos dye
Differential Dyes:
•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 protects the peptidoglycan layer from antimicrobials –Presence of a coliform •Indicator of unsanitary conditions of food or water (E. coli)
Gram positive bacteria:
–Final color: purple –Thick layer of peptidoglycan in cell wall –Retains dye-mordant complex (purple)
Gram negative bacteria:
–Final color: red/ pink –Much thinner layer of peptidoglycan in cell wall –Does not retain dye-mordant complex, needs counterstain (red)
Acid-Fast Stain:

-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 dye and will counterstain blue
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 to autoclave) –Examples: Bacillus (anthrax) and Clostridium (botulism) species
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 the presence of a capsule

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 bind molecules in specimens or the background

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