Microscopy and differential staining Essay
1.What are the advantages and disadvantages of the different types of light and electron microscopes discussed in Chapter 3 that are used to study microorganisms? Focus your response in terms of the following parameters: ◦Range of magnification
◦Possible states of sample (e.g. whole organism, part of, living, non-living, etc.) 2.What type of microscope would you use to investigate each of the following scenarios and why (your answer to question 1 will help): ◦Determine the relative sizes of two eukaryotic cells.
◦Determine how many bacteria are dying after a chemical treatment. ◦Determine where in a eukaryotic cell a particular protein is localized. ◦Identify a virus from a contaminated sample.
◦Visualize the sensory hairs on the surface of an ant’s antennae. 3.What are advantages and disadvantages of the differential staining techniques discussed in Chapter 3 that are used to study microorganisms? Focus your response in terms of the following parameters: ◦Ease and cost of sample preparation
◦Why you would use it (i.e. what information is imparted)
◦Specificity of identification
◦Chance of false positives or negatives
1-Light Microscopes: Use visible light to observe specimens.
-Compound Light microscope:Some compound light microscopes can acheive a total magnification od 2000X with an oil immersion lens.
-the white light used in compound light microscope has a relatively long wavelength and cannot resolve structures smaller than about 0.2μm
-Specimens must be made to contrast sharply with their medium(substance in which they are suspended).The refractive index of specimens must be changed
by staining them.
-To achieve high magnification with good resolution , the objective lens must be small.
-Darkfield microscopy: used to examine live microorganisms that either invisible in the ordinary light mircrscope, cannot be stained,or are so distorted by staining that their characteristics cannot be identified.
-frequently used to examine unstained microorganisms suspended in liquid.
-One use is to examine very thin spirochetes such as Treponema Pallidum, the causative agent of syphillis
-Phase Contrast microscopy:Permits detailed examintaion of internal structures of living microorganisms.
-not necesary to fix or stain specimen.
-internal structures of a cell more sharply defined
-Electron Microscopes: Beam of electrons used instead of light.
-Objects smaller than 2.0μm such as viruses must be examined with this type of microscope-resolving power much greater because wavelength of electrons is 100,000 times smaller than the wavelengths of visible light.
-images are always black and white, but they can be colored to accentuate certain details
-use electromagnetic lens instead of glas lens used by light microscopes
– two types of electron microscopes:
Transmission electron microscope : -Can resolve objects as close as 1PM and
objects are generally magnified 10,000 to 100,000X.
-only very thin section of specimen can be studied effectiviely. Thus specimen has no three-demensional aspect.
-specimens must be fixed , dehydrated andviewed under high vacuum to prevent electron shattering and these treatments not only kill the specimen,but also cause shrinkage and distortion.
Scanning Electron Microscopy :-overcomes the problem of sectioning associated with transmission electronmicroscope
-striking three dimensional views.
-especially useful in studying the surface structures of intact cells and viruses
-resolves objects as close as 10nm and objects are genrally magnified 1,000-10,000X
2-What type of microscope would you use and why??
•Determine the relative sizes of two eukaryotic cells.Scanning electron because it is used to study surface structures •Determine how many bacteria are dying after a chemical treatment:light microscope because living cells cannot be studied in electron microscope •Determine where in a eukaryotic cell a particular protein is localized.Transmission electron microscope because it allows you to study and see different layers of the cell •Identify a virus from a contaminated sample: electron microscope because resolving power is much greater than that of light microscope •Visualize the sensory hairs on the surface of an ant’s antennae:scanning electron because it is used to study cell surfaces and has very powerful magnification 3-Gram stain:
-One of the most useful staining procedures because it clasifies bacteria
into two large groups:
gram-positive :bacteria that reatin color after alchohol has attempted to decolorize
gram-negative:bacteria that lose the dark violet or purple color
-one of the most important staining techniques in microbiology
-not universally applicable, because some bacterial cells stain poorly or not at all.
-mostconsistent when used in young growing bacteria
-Acid-fast Stain : Binds stongly only to bacteria that have a waxy material in their cell walls.
-used to identify all bacteria in the genus Mycobacterium including the causative agents of leprosy and TB
-Also used to identify pathogenic strains of the genus Nocardia