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41 Cards in this Set
- Front
- Back
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Micrometer (mm) = 10‐6m
Nanometer (nm) = 10‐9m |
Units of Measurement
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visible light passes through the specimen
most common microscope is the bright‐field microscope |
Compound light microscope
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objective mag x ocular mag
4X 10X 40X 100X (oil) |
Total Mag
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The usefulness of a microscope depends on its resolving power (min distance between 2 objects
that can be “resolved”) the ability to distinguish between two adjacent objects |
Resolution
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Lack of contrast is a problem when viewing unstained bacteria. Stains can add contrast but kill.
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Contrast
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Light passing from one medium into another may bend
oil refraction = glass so light does not bend air refraction < glass |
Refraction
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When the background is tainted dark and iluminates the bacteria a bright color
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Darkfield Microscopy
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The phase contrast microscope has optical devices that amplify differences in refraction
Phase device is located between light source and slide |
Phase‐Contrast Microscopy
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Fluorochromes = group of fluorescent dyes
(a) live fluoresce green, dead are red (b) auramine on Mycobacterium modified acid‐fast (c) antibodies tagged on Streptococcus pyogenes look closely you can see cells not bound by Ig |
Fluorescent dyes and tags
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line of vision= binocular lens
objective lenses stage condenser lenses imulinator |
Brightfield Ilumination
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An electron microscope that provides high magnifications (10,000‐100,000X) of thin sections of a
specimen hint: internal structures |
Transmission Electron Microscopy
TEM |
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An electron microscope that provides three‐dimensional views of the specimen magnified 1,000‐
10,000X hint:3D image of surface structures |
Scanning Electron Microscopy
SEM |
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have + chg drawn to N.A.’s (nucleic acid), and parts of proteins
stain adheres to cell Methylene blue Crystal violet Safranin Malachite green |
Basic dyes
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have – chg
often stain background |
Acid dyes
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uses just one stain or dye
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Simple staining
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Used to distinguish one group of bacteria from another
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Differential stains:
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stain purple
have cell membrane;thick wall; many layers of peptidoglycan |
Gram Stain positive
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*Primary stain: crystal violet
*Mordant = Iodine increases affinity of dye for cell components --- still purple *Decolorizer = alcohol --- still purple *Safranin = counterstain stays purple |
Gram + positive stain
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*Primary stain: crystal violet
*Mordant = Iodine increases affinity of dye for cell components---still purple *Decolorizer = alcohol Gram - will loose violet color---colorless *Safranin = counterstain Gram - turns pink |
Gram - negative stain
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stain pink
have cell membrane;have thin cell wall (1 or 2 layers of peptidoglycan) and outer membrane |
Gram - negative
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used to stain organisms, such as Mycobacterium (causes tuberculosis and lepracy), which do not take up stains readily
acid‐fast organisms stain pink and all other organisms stain blue |
Differential: Acid Fast
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an example of a negative stain it colors the background, allowing the capsule to stand out as a
halo around an organism |
Capsule Stain
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uses heat to stain endospores
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Spore stain
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The flagella stain employs a mordant that enables the stain to adhere to and coat the otherwise thin flagella
Location and number of flagella can help identify bacteria |
Flagella Stain
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How to prepare a stain
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1. One loopful liquids plus microbes.
2. Spread and air dry 3. Heat fix 4. Stain, rinse, dry 5. carefully view w/ scope |
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Oil immersion ________________ (increases/decrease) the numerical aperture, which __________(increases/decrease) resolution because ___________(more/less) light rays are involved.
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increases
increases more |
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provide a DARK background for small or colorless specimens
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Darkfield microscopes
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Uses a series of lenses for magnification
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Compound microscopes
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provides an image produced by the TRANSMISSION of electrons through a thinly sliced, dehydrated specimen
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TEM - transmission electron microscope
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provides a three dimensional image by scattering electrons from the metal-coated surface of specimen
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SEM - scanning electron microscope
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also called resolving power, is the ability to DISTINGUISH between objects that are CLOSE TOGETHER
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Resolution
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determined by multiplying the magnification of the objective lens by the magnification of the ocular lens
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total magnification
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magnification is achieved as light rays pass through a specimen and into an objective lens (4 objectives - lab)
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light microscopy
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provides detailed view of the smallest bacteria, viruses, internal cellular structures and even molecules and large atoms.
Magnify objects 10,000X to 100,000X |
Electron microscopy
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bright specimen against dark background
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Dark field microscope
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monotone, 2 dimensional, highly magnifies images, may be color enhanced
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TEM microscope
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monotone, three dimensional, surface images , may be color enhanced
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SEM microscope
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Gram Staining Step 1:
Slide is flooded with crystal violet for ONE minute then rinsed with water. What is the result? |
All cells are stained PURPLE
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Gram Staining Step 2:
Slide is flooded with IODINE for ONE minute , then rinsed with water. What is the result? |
Iodine acts as a MORDANT; all cells REMAIN PURPLE
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Gram Staining Step 3:
Slide is flooded with solution of ethanol and acetone for TEN to THIRTY seconds , then rinsed with water. What is the result? |
Smear is decolorized ; GRAM (+) cells remain PURPLE but GRAM (-) cells are now colorless.
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Gram Staining Step 4 :
Slide is flooded with safarin for ONE minute, then rinsed with water and blotted dry. What is the result? |
Gram (+) cells remain PURPLE, Gram (-) cells are pink
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