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Staining
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- 1Microscopy refers to the use of light or electrons to magnify objects.General principles
wavelength of radiation,
magnification of an image,
resolving power of the instrument
,contrast in the specimen. - 2Magnification is the apparent increase in size of an object. It is indicated by a number and “×”- “times.”Magnification i when a beam of
radiation refracts as it passes
through a lens. Curved glass lenses refract
light, and magnetic fields
refract electron beams - 3Resolution-resolving power-ability to distinguish between objects that are close together. Better resolution, the better the ability to distinguish two objects that are close to one another. The limits of resolution
(1) the wavelength of the
electromagnetic radiation and
(2) the numerical aperture of
the lens, which refers to the
ability of a lens to gather light. - 4Contrast refers to differences in intensity between two objects, or between an object and its background. Contrast is important in determining resolution. Contrast by staining .
The use of light that is in phase – that is, in which all of the waves’ crests
and troughs are aligned–can also enhance contrast. - 5Compound microscopeThe total magnification of a compound microscope is determined by
multiplying the magnification of the objective lens by the magnification of the
ocular lens. - 6Electron microscopesmagnification magnify objects 10,000× to 100,000- detailed views.Cell’s
ultrastructure seen.
2 typestransmission electron microscopes and scanning electron
microscopes. - 7StainingMicroorganisms=colorless . Stains are used to make microorganisms and their parts more
visible because they increase contrast between structures, and between a
specimen and its background. - 8Preparing a specimen for staining:Spread in liquid across the surface of slide using a
circular motion,dryin the air,passed through the flame to the glass. - 9Simple stains are composed of a single basic dye such as crystal violet, safranin, or methylene blue. “simple”- soaking the smear in the dye for 30–60 seconds and then rinse.
Quickly, increase contrast, and
allow determination of size, shape, and arrangement of cells. - 10Differential stains-use more than one dye Common differential stains are the Gram stain, the acid-fast stain, and the
endospore stain. - 11The Gram stain differentiates between two large groups of microorganisms: purple-staining Gram-positive cells and pink-staining Gram-negative cells.1st step to identify bacterial pathogens.Works best with
young cells. - 12Physical Characteristics Different physical characteristics are used to identify microorganisms.Identify on morphology (shape).
Stains used to view the size/shape and show
presence/absence of like endospores and flagella.Colony shape - 13Biochemical Tests Microorganisms can be separated on the basis of differences in their ability to utilize or produce certain chemicals.Biochemical tests
that determine an organism’s 1) ability to
ferment; 2) utilize
various substrates such as specific
amino acids;
or 3) produce waste products (H2S) gas. - 14Microbial GrowthGrowth is an increase in a population of microbes rather than to anincrease in size of indivual. The reproduction of individual microorganisms results in the growth ofa colony.
- 15Growth requirementsOrganisms use nutrients to meet their energy needs and to build organic
molecules and cellular structures. - 16Autotrophs: organisms that utilize an inorganic source of carbon as their sole source of carboneg CO2 and thus need not feed
on organic compounds from other organisms to acquire carbon. - 17Heterotrophs:organisms that catabolize reduced organic molecules (such as
proteins, carbohydrates, amino acids, and fatty acids) they acquire from other
organisms. - 18Chemotrophs:organisms that acquire energy from redox reactions involving
inorganic and organic chemicals. - 19Phototrophs:Phototrophs: organisms that use light as their energy source
- 20Obligate aerobes:oxygen is essential-because it serves as thefinal electron acceptor of electron transport chains, which produce most of the ATP in
these organisms. - 21Obligate anaerobes: oxygen is a deadly poison for these organisms, they cannot grow in its presence.4 toxic forms of oxygen: Singlet oxygen
Superoxide radical
Peroxide anion
Hydroxyl radical - 22Facultative anaerobes:Some aerobic organisms can maintain life via fermentation or
anaerobic respiration, though their metabolic efficiency is often reduced in the absence
of oxygen. e.g. E. coli. - 23Aerotolerant anaerobes do not use aerobic metabolism, but they tolerate oxygenThey have some enzymes that detoxify oxygen’s poisonous forms. Grown without the special conditions required by strict anaerobes.
e.g. lactobacilli. - 24Microaerophiles: require oxygen levels of 2% to 10%. This concentration of oxygen is found in the stomachDamaged by the 21%
oxygen in the atmosphere- limited ability to detoxify
hydrogen peroxide and superoxide radicals. e.g. Helicobacter pylori. - 25Nitrogen-essential element,contained in many organic compounds, including the amine group of amino acids and as part of nucleotide bases. Organisms acquire nitrogen from organic and inorganic nutrients,. In addition, all cells recycle nitrogen from their amino
acids and nucleotides. - 26Temperature plays an important role in microbial lifeMin growth temp: lowest temp to conduct metabolism.Max growth temp: the highest temp at which an organism
continues to metabolize.=permanently denatured+dies.Optimum growth temp: metabolicactivities produce the highest growth rate. - 27pHOrganisms sensitive to changes in acidity- interfere with hydrogen bonding within the molecules of protein
- 28Neutrophiles: Most bacteria and protozoa, including most pathogens, growbest in a narrow range around a neutral pH–that is, between pH 6.5 andpH7.5, which is also the pH range of most tissues and organs in the human body.
- 29Acidophiles:Organisms that grow best in acidic habitats; e.g. bacteria and
many fungi.obligate acidophiles- require an acidic enviro
acid-tolerant microbes - survive in acid without preferring it. - 30Alkalinophiles:grow best in alkaline environments, e.g. alkaline soils and
water up to pH 11.5. - 31WaterMicroorganisms require water-metabolically
active. - 32Osmotic PressureThe osmotic pressure=pressure
exerted on a semipermeable membrane by a solution containing solutes
(dissolved material) that cannot freely cross the membrane. - 33Obligate halophiles,Adapted to growunder high osmotic pressure = Great Salt Lake and smaller salt ponds. They may grow in up
to 30% salt and will burst if placed in freshwater. - 34Facultative halophilesdo not require high salt concentrations but they
can tolerate them. E.g. S. aureus can tolerate up to 20% salt, which allows it
to colonize the surface of the skin - 35RelationshipsAntagonistic -one organism harms or evenkills another .Synergistic- cooperate+ receives benefits that exceed those that would result if each lived by itself. Symbiotic relationships,-organisms live in such close nutritional or physical contactthat they become interdependent
- 36 Biofilms are examples of complex relationships among numerous individuals,Often different species, that attach as a group to surfaces and display metabolic
and structural traits different from those expressed by any of the microorganisms
alone. Many bacterial diseases are caused by biofilms - 37CultivationTo cultivate microorganisms, a sample
called an inoculum is introduced a medium=culture;Can be grown in liquid media=
broth - 38Clincal samplingClinical specimen is a sample of human
material that is examined or tested for
the presence of microorganisms.
Clinical specimens are often transported in
special transport media. - 39Obtaining pure samplesPure cultures-arising from a single progenitor.:
colony-forming unit (CFU).
All media, vessels, and instruments must be sterile - 40Isolation techniques: streak plates and pour plates.Isolation techniques work well only if a relatively large number of CFUs of the
organism of interest are present in the initial sample, and if the medium
supports the growth of that microbe. - 41Streak platesAn inoculum is spread across the surface of an agar plate in a sequential pattern of streaks.
The loop is sterilized between streaks. Effect: dilution of the number of cells each time. - 42Pour platesCFUs are separated from one another using a series of dilutted,mixed in Petri dishes with sterile, warm medium
containing agar–a gelling agent.
Individual CFUs form
colonies in and on the agar - 43Agar, a complex polysaccharide, is a useful compound in microbioly: Most microbes cannot digest agar
- 44Types of culture mediaA defined medium (also called a synthetic medium) is one in which the
exact chemical composition is known. Complex media contain nutrients released by the partial digestion of nutrients - 45Selective mediatypically contain substances that either favor the growth of particular
microorganisms or inhibit the growth of unwanted ones. - 46Differential mediaare formulated such that either the presence of visible changes in
the medium or differences in the appearances of colonies helps microbiologists
differentiate among different kinds of bacteria growing on the medium. - 47Reducing media contain compounds, such as sodium thioglycollate, that chemically combine with free oxygen and remove it from the mediumHeat is
used to drive absorbed oxygen from thioglycollate immediately before such a
medium is inoculated. - 48Transport media is used to carry clinical specimens of feces bodily fluids in such a way as to ensure that people are not infected and that the specimens are not contaminated.Speed is extremely important
because pathogens often do not survive outside the body - 49Refrigerationis often the best technique for storing bacterial cultures for
short periods of time. - 50Deep-freezing involves freezing the cells at temperatures from –50°C to –95°C.Deep-frozen cultures can be restored years later by thawing them and
placing a sample in an appropriate medium. - 51Lyophilization (freeze-drying) involves removing water from a frozen culture using an intense vacuum.Lyophilized cultures can last for decades and are
revived by adding lyophilized cells to liquid culture media - 52Binary fissionUnicellular microorganisms reproduce by
binary fission- cell grows to
twice its normal size and divides in half to produce
two daughter cells of equal size. - 53Population growthWith binary fission, any given cell divides to form two cells; then each of
these new cells divides in two,This type of growth is called logarithmic growth or exponential growth.The number of cells arising from a single cell reproducing
by binary fission is calculated as 2n - 54Generation timeis the time required for a population of cells to double in
number. - 55A graph that plots the numbers of organisms in a growing population over time is known as a growth curve.4stages the lag, log, stationary, and death
phases. - 56MeasuringDirect Methods= viable plate counts, membrane filtration,
microscopic counts.Indirect Methods=metabolic activity,
dry weight, turbidity, or MPN tubes - 57Turbidity As bacteria reproduce in a broth= turbid. greater the bacterial population, the more turbid a broth will be. A spectrophotometer=measure
changes in turbidity.
Spectrophotometry does not distinguish between living and
dead cells. fast and simple
Información
- Descripción
- University Infection Prevention and Control Fichas sobre Staining, creado por Eimear O'Donovan el 10/12/2014.
- Creado por
- Eimear O'Donovan
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