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Methods of culturing. The Five I's
Microbiologists use five basic techniques to manipulate, grow, examine, and characterize microorganisms in the laboratory:
Inoculation & isolation
1. Inoculation: producing a culture
a. Introduce a tiny sample (the inoculums) into a container of nutrient (6) _medium/media ( give nutrients it needs)
2. Isolation: separating one species from another
a. Separating a single bacterial cell from other cells and providing it space on a nutrient surface will allow that cell to grow in to a mound of cells (a  colony___). ex. streak plate
b. If formed from a single cell, the colony contains cells from just that species.
Streak plate method
1. Streak plate method (what we did in lab) - small droplet of culture or sample spread over surface of the medium with an inoculating loop
a. Uses a pattern that thins out the sample and separates the cells
microscopic view lets you determine bacterial morphology
Draw a 3 streak plate (4 steps) see slide #7
1. Indicate where you obtain culture
2. Indicate where you flame the loop (4 places)
3. Indicate where you overlap streaks
4. Indicate where you should isolate colonies
Loop dilation method
1. Loop dilation, or pour plate, method- sample inoculated (8)_serially (in a series)_ in to a series of liquid agar tubes to dilute the number of cells in each successive tubes
a. Tubes are then poured in to sterile Petri dishes and allowed to solidify
spread plate method
1. Spread plate method- small volume of liquid (sample over entire plate), diluted sample pipette on to surface of the medium and spread around evenly by a (9)_sterile__ spreading tool ("hockey stick")
Media: Providing nutrients in the Lab (5)
1. At least 500 different types
2. Contained in test tubes, flasks, or Petri dishes
3. Inoculated by loops, needles, pipettes, and swabs
4. Sterile (10)_technique_ necessary w/media
5. Classification of media
a. Physical state
b. Chemical composition
c. Functional type
Media=simply a nutrient source
classification of media by physical state (3)
1. Liquid media: water-based solutions, do not (11)__solidify______ at temperatures above freezing, flow freely when container is tilted
a. Broths, milks, or infusions (brain, heart added to it)
b. Growth seen as cloudiness or particulates (can have color)
2. Semisolid media: (12)__clot__-like consistency at room temperature
a. Used to determine motility/movement and to localize reactions at a specific site
3. Solid media: a firm surface on which cells can form discrete colonies
a. Liquefiable and nonliquefiable
b. Useful for isolating and culturing bacteria and fungi
classification of media by chemical content (2)
1. Synthetic media- compositions are (13) _precisely_______ chemically defined (ex. like a recipe, you can make it)
2. Complex (nonsynthetic) media- if even just one component is not chemically definable
classification of media by function (8 in total)
1. General purpose media- to grow as (14) _general__ a spectrum of microbes as possible (ex. a lot of glucose & AA)
a. Usually nonsynthetic
b. Contain a mixture of nutrients to support a variety of microbes
c. Examples: nutrient agar and broth, brain-heart infusion, trypticase soy agar (TSA). (good base & then add in blood and you get enriched media. TSA + blood = enriched media)
1. Enriched media- contain complex organic substances (for example blood, serum, growth factors) to support the growth of fastidious bacteria. Examples: blood agar, Thayer-Martin medium (chocolate agar)
2. Define Fastidious:
bacteria that require growth factors & complex nutrients
special nutritional or environmental conditions for growth
examples of enriched media (2)
1. Blood - has differential properties as well
2. Chocolate - gets its brown color from heated (16) __blood__________
selective & differential media
1. Selective media- contains one or more agents that inhibit the growth of certain microbes but not others. Promotes growth in others. Example: Mannitol salt agar (MSA), MacConkey agar, Hektoen enteric (HE) agar.
2. Differential media- allow multiple types of microorganisms to grow but display VISIBLE DIFFERENCES among those microorganisms. MacConkey agar can be used as a differential medium as well. Typically in color of agar or organism.
Mannitol salt; selective & differential for?
Salt is the selective agent
Selective for:(17) for halophyll (salt lovers). against non-halophylls (salt haters)
Differential for :(18) uses dye to change agar color to yellow. acidic by products
MacConkey agar; selective & differential for?
Selective for :(19) For gram-negative bacteria. Against gram positive bacteria
Differential for :(20) latose fermentation - take up the dyes in the agar.
miscellaneous media (4)
1. Reducing media- absorbs oxygen or slows its penetration in the medium; used for growing anaerobes or for determining oxygen requirements
2. Carbohydrate fermentation media- contain sugars (different types) that can be fermented and a pH indicator; useful for identification of microorganisms.
*Often has an inverted durham tube for capturing gases produced by bacteria
3. Transport media- used to maintain and preserve specimens that need to be held for a period of time (freeze drying)
4. Enumeration media- used to count the numbers of organisms in a sample.
incubation definition & 6 attributes
1. Incubation: an inoculated sample is placed in an incubator to encourage growth.
a. Usually in laboratories, between 20° and 40°C. (ex. increase or decrease oxygen levels)
b. Can control atmospheric gases as well.
c. Can visually recognize growth as cloudiness in liquid media and colonies on solid media.
d. Pure culture- growth of only a single known species
e. Mixed culture- holds two or more identified species
f. (21) _contaminated___ culture- includes unwanted microorganisms of uncertain identity, or contaminants.
inspection & identification (4)
1. Based on appearance
2. Metabolism (biochemical tests)
****What nutrient sources have we mentioned thus far? (22) lactose, sucrose, mannitol, glucose
3. sometimes genetic analysis
4. Sometimes immunologic testing
the microscope: 2 key characteristics
1. Two key characteristics of microscopes: magnification and resolving power
a. Results when visible light waves pass through a curved lens
b. The light experiences refraction
c. An image is formed by the refracted light when an object is placed a certain distance from the lens and is illuminated with light
d. The image is enlarged to a particular degree- the power of magnification
1. Resolution- the ability to distinguish two adjacent objects or points from one another
a. Also known as resolving power
b. Shorter wavelengths provide a better resolution
c. Numerical aperture- describes the relative efficiency of a lens in bending light rays
d. (23)___oil immersion____ lenses increase the numerical aperture. helps light come into that objective
longer wave lengths may miss. shorter wave length will cath more.
magnification & resolution (3)
1. Good resolution means being able to observe an object clearly
2. Increased magnification decreases the resolution
3. Adjusting the amount of light entering the condenser using an adjustable ___iris diaphragm___ or using special dyes help increase resolution at higher magnifications
variations on the optical microscope (2)
1. Visible light microscopes- optical microscopes that use visible light. Described by their field.
a. Four types: bright-field, dark-field, phase-contrast, and interference
b. We will only cover a couple in detail
2. Other light microscopes include fluorescence microscopes and confocal microscopes
Bright-field microscopy (4)
1. Most widely used. (used in lab, limited by longer wave lengths)
2. Forms its image when light is transmitted (24) through____ the specimen
3. The specimen produces an image that is darker than the surrounding illuminated field
4. Can be used with live, unstained and preserved, stain specimens (rotifers, stentors, etc..)
Dark-field microscopy (5)
1. A bright-field microscope can be adapted to a dark-field microscope by adding a stop to the condenser (modifies light field)
2. The stop blocks all light from entering the objective lens except for peripheral light
3. The specimen produces an image that is brightly illuminated against a dark field
4. Effective for visualizing living cells that would be distorted by drying or heat or that can't be stained with usual methods (heat fixation distorts bacterial shape. used neg. stain to see bacterial morphology)
5. Does not allow for visualization of fine internal details of cells
phase-contrast microscopy (3)
1. Transforms subtle changes in light waves passing through a specimen into differences in light intensity (differentiate internal components)
2. Allows differentiation of internal components of (25)_live_______, unstained cells
3. Useful for viewing intracellular structures such as bacterial spores, granules, and organelles
Good for inside components
fluorescence microscopy (4)
1. Includes a UV radiation source and a filter that protects the viewer's eyes
2. Used with dyes that show fluorescence under UV rays
3. Forms a colored image against a black field
4. Used in diagnosing infections caused by specific bacteria, protozoans, and viruses using fluorescent (29)_____dyes__________
electron microscopy (6)
1. Originally developed for studying nonbiological materials
2. Biologists began using it in the early 1930s
3. Forms an image with a beam of electrons
a. Electrons travel in wavelike patterns 1,000 times shorter than visible light waves
b. This increases the resolving power tremendously
4. Magnification can be extremely high (between 5,000X and 1,000,000X for biological specimens)
5. Allows scientists to view the finest structure of cells
6. Two forms: transmission electron microscope (TEM) and scanning electron microscope (SEM)
can't be done for living organisms
TEM - transmission electron microscope (6)
1. Often used to view structures of cells and viruses, resolution of 0.5nm (apart from each other)
2. ELECTRONS are transmitted THROUGH the specimen
3. The specimen must be very THIN (20-100 nm thick) and stained to increase image contrast
4. Dark areas of a TEM image represent thicker or denser parts
5. Good for viewing internal components of cells
SEM - scanning electron microscope (5)
1. Resolution of 10nm (apart)
2. Most dramatic and realistic 3D IMAGES
3. Bombards the surface of METAL COATED specimens with (32) _electrons_______
4. DEFLECTED ELECTRONS (off metal coating) are picked up by a detector and displayed on a television screen
5. Modified scanning probe allows for more magnification
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