Methods of culturing. The Five I's
Microbiologists use five basic techniques to manipulate, grow, examine, and characterize microorganisms in the laboratory:
(1) inoculation
(2) incubation
(3) isolation
(4) inspection
(5) identification
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 cel
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 m
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. F
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
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-hear
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:
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 type
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; usef
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 clo
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
Or
4. Sometimes immunologic testing
the microscope: 2 key characteristics
1. Two key characteristics of microscopes: magnification and resolving power
2. Magnification
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
resolution
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 lig
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 resol
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 fluor
4 types of visible light microscopes
bright-field
dark-field
phase-contrast
interference
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,
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 tha
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 intr
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,
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
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 o
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 scree
Methods of culturing. The Five I's
Microbiologists use five basic techniques to manipulate, grow, examine, and characterize microorganisms in the laboratory:
(1) inoculation
(2) incubation
(3) isolation
(4) inspection
(5) identification
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 cel
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 m
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. F
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
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-hear
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:
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 type
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; usef
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 clo
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
Or
4. Sometimes immunologic testing
the microscope: 2 key characteristics
1. Two key characteristics of microscopes: magnification and resolving power
2. Magnification
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
resolution
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 lig
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 resol
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 fluor
4 types of visible light microscopes
bright-field
dark-field
phase-contrast
interference
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,
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 tha
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 intr
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,
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
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 o
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 scree