Study sets, textbooks, questions
Upgrade to remove ads
Principles of Microscopy
Terms in this set (56)
The use of light or electrons to magnify small objects
What are the two types of microscopy that are used to view microorganisms?
1) Light Microscopy
2) Electron Microscopy
The process of enlarging an object in appearance only
What is the equation for total magnification?
total magnification = objective lens x ocular lens
When do we use immersion oil?
When the objective lens is on 100x
The ability of the lenses to distinguish two points as distinct and separate
What does the resolving power depend on?
1) The wavelength of the electromagnetic radiation
2) The numerical aperture (NA) of the lens (its ability to gather light)
What is the maximum resolution?
0.2 microns or 200 nanometers
increases resolution on the 100x objective lens by filling the air gaps between the slide and objective lens (prevents bending of light)
What are 3 main types of light microscopy?
- a microscope that uses visible light for illumination
- the specimens are viewed against a white background
- visualize pigmented or stained (killed) specimens
- a compound light microscope that allows examination of structures inside cells through the use of a special condenser
- use to see living cells, how they move, and their internal structures
- no staining required
- a microscope that uses a UV light source to illuminate specimens that will fluoresce (emit visible light)
- some organisms will fluoresce naturally
- other cells need to be stained with fluorescent dyes (fluorochromes)
fluorescent dyes used to stain cells
Fluorescence-Antibody (FA) Technique
- aka: immunofluorescence
- a diagnostic tool using antibodies labeled with fluorochromes and viewed through a fluorescence microscope
- a microscope that uses electrons instead of light to produce an image
- moving electrons act as waves
- uses electromagnetic lenses to focus electron beam
How does a transmission electron microscope work?
- specimens are thinly sectioned, stained with metallic stains, and put onto a grid
- electrons pass through the specimen and are scattered
- electromagnetic lens focuses the electrons onto a screen/plate
Transmission Electron Microscopy (TEM)
- an electron microscope that provides high magnifications (10,000-100,000x) of thin sections of a specimen
- 2D view
- good for viewing internal structures of cells & viruses
- cannot visualize live microbes b/c they need to be stained
Scanning Electron Microscopy (SEM)
- an electron microscope that provides 3D views of the specimen magnified 1000-500,000x
- specimen is coated with metal, the primary beam of electrons scans the surface, and the secondary electrons emitted from the specimen are collected by the screen
- cannot visualize live microbes b/c they need to be stained
- are salts made of positive (+) and negative (-) ions, one of which is more colored (chromophore)
- chromophore is the (+) ion, used to directly stain bacterial cells
- the chromophore is attracted to the slightly (-) charged bacterial cell
- e.g., crystal violet, methylene blue, malachite green
- chromophore is the (-) ion, used to stain background around cells
- e.g., in negative staining
- the colored ion in a stain
What are the 6 steps of preparing specimens for light microscopy?
1. Prepare smear on slide
2. Air dry the smear
3. Heat fix to kill the microbes and attach the cells to the slide
6. Blot dry
then we can add the immersion oil
What are the 3 staining techniques?
1. Simple Stains
2. Differential Stains
3. Special Stains
- a method of staining microorganisms with a single basic dye
- e.g., crystal violet (purple), methylene blue (blue), safranin (pink)
- can also be used with a mordant
What is a mordant used for?
- to increase affinity of stain for cell (allows it to bind tighter to cells)
- or to coat a structure to make it easier to see
- a stain that distinguishes objects on the basis of reactions of the staining procedure
- uses two different colored dyes (+ mordant) that react differently with different types of bacteria
- e.g., gram stain and acid-fast stain
- negative staining
- endospore staining
- flagella staining
- a differential stain that classifies bacteria into two groups, gram-positive and gram-negative
- the basis of bacterial classification and identification
What are the 2 main differences between gram-positive and gram-negative bacteria?
1) different cell wall structures
2) different susceptibility to antibiotics
What are the 4 steps of the gram stain procedure?
1) crystal violet (primary stain): 1 min, then rinse with water
2) gram's iodine (mordant): 1 min, then rinse with water
3) 95% ethanol (decolourizing agent):
- 1 x 10 sec, then rinse
- 1 x 10 sec, then rinse
4) safranin O (counterstain): 30 sec, rinse, then blot
- first stain used that dyes all of the cells
- used to increase the affinity of stain for the cells (binds more tightly)
- a solution used in the process of removing a stain
- will strip the primary stain off of certain cells
- a second stain applied to a smear
- provides contrast to the primary stain
- colors the cells that were colorless as a result of the decolourizing agent
How do we interpret the results of a gram-stain test?
- gram-positive cells retain crystal violet (CV-1) complex and will stain purple
- gram-negative cells are decolourized by alcohol as they lose the CV-1 complex and are counterstained pink
- a differential stain used to identify bacteria that are not decolourized by acid-alcohol
- differentiates into acid-fast and non-acid-fast
- used to identify TB (mycobacterium tuberculosis) and leprosy (mycobacterium leprae)
What makes mycobacteria different?
- they have waxy lipids in their cell wall called mycolic acids
- this makes them difficult to stain using normal methods
What are the steps for the Ziehl-Neelson Acid-Fast Stain Method?
1) Apply carbolfuchsin (primary stain), heat for 5 min, then rinse (will be red)
2) Apply acid-alcohol (decolourizing agent), 2 x 15 sec, & rinse after each decolorization
3) Apply methylene blue (counterstain) for 1 min, rinse, and blot
How do we interpret colors in an acid-fast stain?
- After the primary stain, both acid-fast and non-acid fast bacteria will be red
- After the decolourizing agent, acid-fast will be RED and non-acid fast will be COLOURLESS
- After the counterstain, acid-fast will be RED and non-acid fast will be BLUE
Negative Staining for Capsules
- use Nigrosin (acidic stain) which stains the background and leaves the cells unstained (negative staining)
- to view capsules, we stain cells with basic dye (safranin O)
- the capsules will then appear as unstained halos surrounding the cells
- polysaccharide (sugar) covering on some microbes
- contribute to virulence
- a dormant structure found in some bacteria (bacillus species & clostridium species)
- special resistant
- cannot be stained by normal methods
What do endospores enhance bacterias survival from??
What are the 2 steps in endospore staining?
1) apply malachite green (primary stain), heat for 5 min, then rinse for 30 sec (longer than usual)
2) apply safranin (counterstain) for 1 min, then rinse and blot dry
How do we interpret the results of endospore staining?
- endospores will be green
- rest of cells are pink
Why do we heat and rinse the primary stain on endospores for longer than usual?
- heat allows the stain to penetrate the endospore as they are located within the cells
- rinsing longer allows us to get the stain removed from parts of the cell that it is heated onto very well
Clostridium difficile (C. difficile)
- gram-positive rod
- forms endospores which make it resistant to disinfectants and allows it to survive in the environment
- found naturally in the gut of some people
Where is C. difficile prominent?
- nursing homes
- LTC facilities
often referred to as a HAI
What are the risk factors for C. difficile?
- age (elderly)
- underlying illness
- prolonged antibiotic use
- chemotherapy (immunosuppression)
What are the usual S&S of C. difficile?
- mild to severe watery diarrhea
What are the serious complications of C. difficile?
- pseudomembranous colitis
- bowel perforation
What are the steps for flagella staining?
1) apply a dormant to build up the diameter of flagella to make them visible microscopically
2) stain flagella with carbolfuchsin
- structures used for cellular motility
Sets found in the same folder
Topic 2: Principles of Microscopy
Functional Anatomy of Prokaryotes and Eu…
Topic 6: Microbial Genetics
Sets with similar terms
Micro Unit 3
Microbiology Chapter 3
BSC 242 Lab Exam 1
Other sets by this creator
Bacterial Growth Terms
STATS Chapter 15
STATS Chapter 14
STATS Chapter 12
Other Quizlet sets
Advanced Cyber Chapters 14
American Republic Ch. 13 - Storm Clouds Over The N…