The 5 I’s of culturing microbes 1. Inoculation – introduction of a sample into a container of...
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Transcript of The 5 I’s of culturing microbes 1. Inoculation – introduction of a sample into a container of...
The 5 I’s of culturing microbes
1. Inoculation – introduction of a sample into a container of media
2. Incubation – under conditions that allow growth
3. Isolation –separating one species from another
4. Inspection5. Identification
Fig. 3.1a
Fig. 3.1b
Fig. 3.1c
Isolation
If an individual bacterial cell is separated from other cells & has space on a nutrient surface, it will grow into a mound of cells- a colony
A colony consists of one species
Isolation technique
Fig. 3.3
Fig. 3.1d
Media – providing nutrients in the laboratory
Most commonly used:– nutrient broth – liquid medium containing beef extract &
peptone– nutrient agar – solid media containing beef extract,
peptone & agar agar is a complex polysaccharide isolated from red
algae– solid at room temp, liquefies at boiling (100oC), does not
resolidify until it cools to 42oC– provides framework to hold moisture & nutrients– not digestible for most microbes
Types of media
synthetic – contains pure organic & inorganic compounds in an exact chemical formula
complex or nonsynthetic – contains at least one ingredient that is not chemically definable
general purpose media- grows a broad range of microbes, usually nonsynthetic
enriched media- contains complex organic substances such as blood, serum, hemoglobin or special growth factors required by fastidious microbes
Enriched media
Blood agar Chocolate agar
selective media- contains one or more agents that inhibit growth of some microbes and encourage growth of the desired microbes
differential media – allows growth of several types of microbes and displays visible differences among desired and undesired microbes
Selective media
Mannitol salt agar
MacConkey agar
Differential media
TSIA
CHROMagar Orientation
Miscellaneous media
reducing medium – contains a substance that absorbs oxygen or slows penetration of oxygen into medium; used for growing anaerobic bacteria
carbohydrate fermentation medium- contains sugars that can be fermented, converted to acids, and a pH indicator to show the reaction; basis for identifying bacteria and fungi
transport media
Carbohydrate fermentation media
Fig. 3.12
Cultures
Pure culture- only grows one type of microbe Mixed culture- contains 2 or more microbes Contaninated culture- was once pure, but
unwanted microbes became introduced– Axenic- free of other living things except the one
being studied
Maintenance and disposal of cultures
Catalogues of microbes are maintained at the American Type Culture Collection
Microbes are disposed of by various forms of sterilization
Why use animals?
You can test the effects of drugs, etc without risking the lives of humans
To evaluate disease, or study the process and cause of disease
Source of antibodies, antitoxins May be used to determine the pathogenicity or
toxicity of certain bacteria To grow microbes which cannot be grown on artificial
media
compound light microscope
magnification – ability to enlarge objects resolving power – ability to show detail refraction- bending ability of light
Pathway of light
Total MagnificationOcular power Objective power Total
magnification
10x 4x
Scanning
40x
10x 10x
Low
100x
10x 40x
High and dry
400x
10x 100x
Oil immersion
1000x
resolution
The human eye can only resolve objects about 0.2mm apart
Oil immersion allows resolution to about 0.2um
RP= wavelength of light (nm)/2 x NA NA is the cone of light entering the lens
Effect of wavelength on resolution
Oil immersion lens
Other constraints for a clear image
Other things needed for a clear image are– Quality of the lens– Quality of the light source– Lack of contrast in the specimen
Effect of magnification
Types of light microscopes
Bright-field – most widely used, specimen is darker than surrounding field
Dark-field – brightly illuminated specimens surrounded by dark field
Phase-contrast – transforms subtle changes in light waves passing through the specimen into differences in light intensity, best for observing intracellular structures
3 views of a cell
Brightfield 400x
Darkfield 400x
Phase contrast 400x
Types of light microscopes
Differential interference contrast- uses light waves and 2 prisms which produce contrasting colors; well defined, colored, 3D images
Fig. 3.21
Phase contrast 600x
DIC 160x
Fluorescence Microscope
Modified compound microscope with an ultraviolet radiation source and a filter that protects the viewer’s eye
Uses dyes that emit visible light when bombarded with shorter uv rays.
Useful in diagnosing infections
Cheek cells and streptococci
Light Microscopes
Confocal- uses a laser to scan sections of the specimen
Electron microscopy
Forms an image with a beam of electrons that can be made to travel in wavelike patterns when accelerated to high speeds.
Electron waves are 100,000X shorter than the waves of visible light.
Electrons have tremendous power to resolve minute structures because resolving power is a function of wavelength.
Magnification between 5,000X and 1,000,000X
2 types of electron microscopes
Transmission electron microscopes (TEM) – transmits electrons through the specimen; darker areas represent thicker, denser parts and lighter areas indicate more transparent, less dense parts
Scanning electron microscopes (SEM)– provides detailed three-dimensional view. SEM bombards surface of a whole, metal-coated specimen with electrons while scanning back and forth over it.
Scanning Electron Micrograph
paramecium
Table 3.6
Table 3.5
Specimen Preparation
The way a slide is prepared depends upon– The condition of the specimen– The needs of the examiner– The type of microscope available
Specimen preparation
wet mounts & hanging drop mounts – allow examination of characteristics of live cells: motility, shape, & arrangement– Wet mount- a drop of culture is placed on a slide
with a cover glass Quick and easy to prepare Cover glass may damage larger cells Slide is susceptible to drying and can contaiminate the
handler’s fingers
Specimen preparation
Hanging drop- uses a special slide with a depression; sample is placed in the depression and vaseline is placed around it; coverslip overlies the sample
Specimen preparation
fixed mounts are made by drying & heating a film of specimen. This smear is stained using dyes to permit visualization of cells or cell parts.– Kills the microbe– Allows adherence to the slide– Preserves cellular components in a natural state
with minimal distortion
Staining
cationic dyes - basic, with positive charges on the chromophore
anionic dyes - acidic, with negative charges on the chromophore
surfaces of microbes are negatively charged and attract basic dyes – positive staining.
negative staining – microbe repels dye & it stains the background
– Simple– Reduced shrinkage or distortion of cells– Can observe capsules
Staining
simple stains –one dye is used differential stains – use a primary stain and
a counterstain to distinguish cell types or parts. examples: Gram stain, acid-fast stain and endospore stain
special stains: capsule and flagellar stains
Table 3.7