Flashcards in Chapter 4 Deck (92):
A unit of measurement is?
Smaller than the object being measured
Scientists use what type of measurements?
The metric system is a?
Unit of length in the metric system is?
One tenth of a meter is?
One one-hundreth of a meter is a?
One tenth of a centimeter is?
In the metric system, we continue to divide by multiples of?
One-thousandth of a millimeter is?
A micrometer which is small enough to be useful in measuring cells
Unit used to measure the smallest cellular organelles and viruses
nanometer (nm) which is one-billionth of a meter
Microscopy refers to?
The use of light or electrons to magnify objects
Science of microscopy began when
Leeuwenhoek used primitive microscopes to observe & report the existence of microorganisms
General principle involved in both light and electron microscopy include
The wavelength of radiation, the magnification of an image, the resolving power of the instrument, and contrast in the specimen
Beams of radiation may be referred to as either
Rays or waves
rays and waves (various forms of radiation) differ in?
Wavelength, the distance between two corresponding parts of a wave
The human eye discriminates among different wave lengths of visible light & sends patterns of?
Nerve impulses to the brain, which interprets the impulses as different colors. For example we see wavelengths of 400nm as violet, & 650 nm as red. White light (composed of many colors (wavelengths), has an average wavelength of 550 nm
Electrons are negatively charged particles that orbit the nuclei of atoms. Besides being particulate, moving electrons act as?
Waves, with wavelengths dependent on the voltage of an electron beam. For example, the wavelength of electrons at 1,000,000 V is 0.001 nm. Radiation of smaller wavelengths results in enhanced microscopy
Magnification is the?
Apparent increase in size of an object. It is indicated by the number and "X", which reads "times." For example, 16,000X is 16,000 times
Magnification results when a?
Beam of radiation refracts (bends) as it passes through a lens. Curved glass lens refract light, and magnetic fields refract electron beams
A lens refracts light because?
The lens is optically dense compared to the surrounding medium (such as air); that is, light travels more slowly through the lens than through air
Because of its curvature, a lens refracts light rays that pass through its periphery more than light rays that pass through its?
Center, so that the lens focuses light rays on a focal point
The degree to which an image in enlarged depends on?
The thickness of the lens, its curvature, & the speed of light through its substance
The properties that determine the clarity of an image of a microscope, are?
Resolution and contrast
Also called the resolving power, is the ability to distinguish objects that are close together
Modern microscopes have ________ resolution than Leeuwenhoek's; they can distinguish objects as close together as 0.2 micrometers
Ability of the lens to gather light
Contrast refers to
Differences in intensity between two objects or between an object and its background. Important in determining resolution
What does phase mean?
All the waves crests and troughs are aligned
Bright-field microscopes (most common microscopes)
The background (or field) is illuminated
The specimen is made to appear light against a dark background
Phase microscopes use the
Alignment or misalignment of light waves to achieve the desired contrast between a living specimen & its background
Fluorescent microscopes use
Invisible ultraviolet light to cause specimens to radiate visible light
Microscopes that use lasers to illuminate fluorescent chemicals in a thin plane of a specimen are called
Contrast and resolution
What type of microscopy requires that specimens be treated with stains or coatings to enhance contrast?
Thin film of organisms on a slide is called a?
If the organisms are growing on a solid surface, such as an agar plate, then they are?
Mixed into a small drop of water on the slide
If the organisms are growing in a liquid, a small drop
Is spread across the surface of the slide
In heat fixation
Developed by Robert Kosh more than 100 years ago, the slide is gently heated by passing the slide, smear up, through the flame of a bunsen burner
Chemical fixation involves
Applying a chemical such as methyl alcohol to the smear for one minute
Desiccation (drying) and fixation does what to the microorganism?
Kill the microorganism, attaches them firmly to the slide, & generally preserve their shape & size
Specimens for scanning electron microscopy are?
Coated not stained
Dyes used for microbiological stains for light microscopy are usually?
Salt (usual dye) is composed of?
A positively charged cation and a negatively charged anion. At least one of the two ions in the molecular makeup of dyes is colored; this colored portion of a dye is known as the chromophore
Chromophores bind to chemicals via?
Covalent, ionic, or hydrogen bonds. For example methylene blue chloride is composed of a cationic chromophore, methylene blue, and a chloride anion. Because methylene blue is positively charged, it ionically bond to negatively charged molecules in cells, including DNA & many proteins
Anionic dyes, for example eosin, bind to?
Positively charged molecules such as amino acids
Anionic chromophores are also called?
Acidic dyes because they stain alkaline structures & work best in acidic (low pH) environments.
Basic dyes are
Positively charged, cationic chromophores. They combine with & stain acidic structures; further, they work best under basic (higher pH) conditions.
In microbiology which dyes are more commonly used more, basic or acid?
Basic dyes because most cells are negatively charged
Acidic dyes are used in?
Selectively stains membranes because it is lipid soluble and accumulates in phospholipid bilayers
Simple stains are?
Composed of a single basic dye, such as crystal violet, safranin, or methylene blue. They are "simple" because they involve no more than soaking the smear in the dye for 30-60 seconds and then rinsing off the slide with water. After carefully blotting the slide you can observe the smear under the microscope
Simple stains are used to?
Determine size, shape, and arrangement of cells
Differential stains (most common) use?
More than one dye so that different cells, chemicals, or structures can be distinguished when microscopically examined
Common differential stains are?
Gram stain, the acid-fast stain, the endospore stain, Gomori methenamine silver stain, and hematoxylin and eosin stain
Who developed the Gram stain?
In 1884, the Danish scientist Hans Christian Gram
The Gram stain differentiates between?
Two large groups of microorganisms: purple-staining Gram-positive cells and pink-staining Gram-negative cells. These cells differ significantly in the chemical and physical structures of their cell walls
Typically, a ______ stain is the first step a medical laboratory technologist performs to identify bacterial pathogens
4 Steps of Gram staining procedure
1) Flood smear with basic crystal violet for 1 min. then rinse w/water. This dye colors all cells
2) Flood smear with iodine solution for 1 min. then rinse w/water. After this step all cells remain purple
3) Rinse smear w/solution of ethanol & acetone (10-30 sec) then rinse w/water. This breaks down thin cell wall of Gram-negative cells allowing stain and mordant to be washed away. These cells are now colorless. Gram-positive cells w/their thick cell walls remain purple
4) Flood smear w/safranin for 1 min. Rinse. This red counterstain provides contrasting color to the primary stain. After this step Gram-negative cells are pink
After final step the slide is blotted dry
The primary stain of Gram stain
Iodine is a?
Mordant, a substance that binds to a dye & makes it less soluble
Acts as a decolorizing agen
Solution of ethanol and acetone
The Gram procedure works best with?
Young cells. Older Gram-positive cells bleach more easily than younger cells & can therefore stain pink, which makes them appear to be Gram-negative cells. Therefore, smears for Gram staining should come from freshly grown bacteria
The acid-fast stain is
Another differential stain because it stains cells of the genera Mycobacterium and Nocardia which cause many human diseases, including TB, leprosy, & other lung & skin infections. Cells of these bacteria have large amounts of waxy lipid in their cell walls, so they do not readily stain with the Gram stain
Acid-fast stain developed by?
Franz Ziehl and Friedrich Neelsen in 1883
Procedure of acid-fast stain
1) Cover smear with piece of tissue paper to retain dye
2) Flood slide w/red primary stain, carbolfuchsin, for several min. while warming over steaming water. This is used to drive the stain through the waxy wall and into the cell
3) Remove paper, cool slide, decolorize smear by rinsing w/solution of hydrochloric acid & alcohol. The bleaching action of acid-alcohol removes color from both non-acid fast cells and the background. Acid-fast cells retain their color because the acid cannot penetrate the waxy wall
4) Counterstain w/ methylene blue, which stains only bleached, non acid-fast cells
The Ziehl-Neelsen acid-fast staining procedure results in?
Pink acid-fast cells, which can be differentiated from blue non-acid fast cells, including human cells and tissue
The presence of acid-fast bacilli (AFBs) in sputum is indicative of?
Some bacteria-notably those of the genera Bacillus and Clostridium which contain species that cause such disease as anthrax, gangrene, and tetanus- produce?
Dormant, highly resistant cells form inside the cytoplasm of the bacteria & can survive environmental extremes such as desiccation, heat, & harmful chemicals
Endospores cannot be stained by normal staining procedures because?
Their walls are practically impermeable to all chemicals. The Schaeffer-Fulton endospore stain uses heat to drive the primary stain, malachite green, into the endospore. After cooling the slide is decolorized w/water & counterstained with safranin. This staining procedure results in green-stained endospores & red colored vegetative cells
Laboratory technicians use two popular stains to stain histological specimens (tissue samples)
Gomori methenamine silver (GMS) stain is commonly used to screen for the presence of fungi & the locations of carbohydrates in tissues. Hematoxylin & eosin (HE) stain, which involves applying the basic dye hematoxylin & the acidic dye eosin, is used to delineate many features of histological specimens, such as the presence of cancer cells
Special stains are?
Simple stains designed to reveal special microbial structures
There are three types of special stains
Negative stains, flagellar stains, and fluorescent stains
Describe the negative (capsule) stain
Most dyes used to stain bacterial cells, such as crystal violet, methylene blue, malachite green, & safranin, are basic dyes. These dyes stain cells by attaching to negatively charged molecules within them
What is repulsed by the negative charges on the surface of cells & therefore do not stain them?
Acidic dyes. Such stains are called negative stains because they stain the background & leave cells colorless. Eosin & nigrosin are examples of acidic dyes used for negative staining. A counterstain may be added to color the cells
Negative (capsule) stains are used primarily to?
Reveal the presence of negatively charged bacterial capsules. Therefore, they are also called capsule stains. Encapsulated cells appear to have a holo surrounding them
Pararosaniline and carbolfuchsin, & mordants, such as tannic acid & potassium alum are applied in a series of steps and are examples of?
Pararosaniline and carbolfuchsin, & mordants, such as tannic acid & potassium alum are applied in a series of steps and are examples of flagellar stains, these molecules bind to flagella and?
Increase their diameter, & change their color, all of which increase contrast & make them visible
Laboratory technicians increase contrast & resolution for transmission electron microscopy by?
Using stains, however these stains are not colored dyes but are chemicals containing atoms of heavy metals such as lead, osmium, tungsten, & uranium, which absorb electrons
Electron-dense stains may
Bind to molecules w/in specimens, or they may stain the background. The latter type of negative staining is used to provide contrast for small specimens (viruses & molecules)
Stains for electron microscopy can be general, in that they?
Stain most objects to some degree, or they may be highly specific. For example, osmium tetra oxide (OsO4) has an affinity for lipids & is thus used to enhance the contrast of membranes
Electron-dense stains can also be linked to?
Antibodies to provide an even greater degree of staining specificity because antibodies bind only to their specific target molecules
Reveals size, morphology, & arrangement of cell
Uniform purple stain
Crystal violet (simple stain)
Uniform blue stain
Methylene blue (simple stain)
Differentiates Gram-postivie and Gram-negative bacteria, which is typically the first step in their identification
Differential stains. Use two or more dyes to differentiate between cells or structures
1) Gram stain
2) Ziehl-Neelsen acid-fast stain
3) Schaeffer-Fulton endospore stain
Distinguishes the genera Mycobacterium and Nocardia from other bacteria
Ziehl-Neelsen acid-fast stain
Highlights the presence of endospores produced by species in the genera Bacillus and Clostridium
Schaeffer-Fulton endospore stain
Reveals bacterial capsules
Negative stain for capsules