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Tools and Methods
A. Compound microscope
1. 2 lens system – objective lens and ocular lens (eyepiece)
2. Real image – created by objective lens
3. Virtual image – image produced when ocular lens magnifies the real image; the virtual image is the one we see.
B. Magnification – to make image appear larger. This is accomplished when light waves pass from one medium to another (such as air to glass lens) the light waves bend, i.e. refraction. When an object is placed within distance of the lens an image of the object is created that is larger than the actual object, i.e. it is magnified.
C. Total Magnification = Power of Objective lens x Power of Ocular lens
Total Magnification = Objective lens (4x) x Ocular lens (10x) = 400x
D. Power of Objective lenses
1. 4x scanning
2. 10x low power
3. 40x high power
4. 100x oil immersion = need to use oil to decrease refraction light
E. Power of Ocular lens is usually 10x
F. Resolving Power (Resolution) – ability to show detail; to be able to see two points as separate.
1. Ex) if the resolving power was 0.2 um (light microscope w/ oil immersion), then 2 dots can be seen as distinct as long as they were at least 0.2 um apart from each other – or else they would be seen as one dot.
2. Resolving Power = wavelength of light in nm / 2 x numerical aperture of objective
3. Visible wavelengths of light range from 400nm (violet-blue) to 750nm (red). The shorter the wavelength (violet-blue) the easier it can pass between the objects and therefore the better the resolution.
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4. Numerical Aperture – refers to the structure of the lens; determines the quantity and quality of light that enter the lens. The higher the NA the better the resolving power.
5. Overall, the shorter the wavelength of light and the higher the numerical aperture the better the resolution.
6. Magnification and Resolution are NOT the same. You can magnify something but without good resolving power the image would be blurry.
G. Types of Microscopes
1. Light microscopes
a. Bright-Field, Dark-Field, Phase-Contrast, Fluorescent
b. All have maximum magnification of 2000x and resolving power of 0.2um
c. Different type used depending on what you want to see in specimen
2. Electron Microscopes
a. Uses a beam of electrons (100,000 times shorter than visible light) to form
image. Short wavelength allows for high degree of resolution (0.5nm or 0.0005um), therefore can use high magnification, 5,000x to 1,000,000x.
b. Transmission Electron Microscope (TEM) – details of internal cell structures
c. Scanning Electron Microscope (SEM) – great surface detail
II. Preparing Specimens for Microscope
A. Depends on if specimen is living or preserved and what you want to see
B. Living Preparations
1. Wet Mounts – cells are in fluid and placed on slide with slip cover
2. Hanging Drop Slide – made with depression slide and cover slip from which a
drop of sample is suspended.
C. Fixed, Stained Smears
1. Liquid suspension of cells is smeared on slide to air dry.
2. Then Heat Fixation process kills specimen and fixes it (secures it) to the slide
without much damage or distortion to specimen.
3. Staining the smear will then allow details of cells to be seen.
III. Staining — **OPPOSITES ATRRACT**
A. 2 classes of Dyes are used for staining.
1. Basic (cationic or positively charged) dyes
a. Attracted to negative parts of the cell such as nucleic acids and proteins
b. Methylene blue, crystal violet, safranin (red), fuchsin
2. Acidic (anionic or negatively charged) dyes
a. Attracted to positively charged parts of cells
b. Eosin (red), nigrosin (blue-black), India ink (black)
c. Bacteria have negative charge on outside of cell and therefore tend to
repel acidic dyes. *Useful for Negative Staining
B. Negative vs. Positive Staining
1. Positive stain – dye binds to cell parts directly, gives them color
a. Crystal violet, Methylene blue, Safranin, Malachite green
2. Negative stain – dye does not bind to cell, but forms boundary around it.
a. Nigrosin, India ink
b. Smear is not heat fixed
c. Good to see Capsules / Spores
III. 3 classes of Positive Staining
A. Simple, Differential and Structural
B. Simple Staining
1. 1 dye colors cells same color, but reveals size, shape, arrangement
2. malachite green, crystal violet, basic fuchsin, and safranin
C. Differential Staining
1. 2 dyes
a. Primary dye
2. Used to differentiate (tell the difference between) cell types and parts
a. Gram stain, acid-fast stain, endospore stain
4. Gram Stain
a. gram-positive bacteria = purple
b. gram-negative bacteria = red
c. due to structural differences in cell wall of bacteria
5. Acid-Fast Stain
a. acid-fast bacteria = pink
b. non-acid-fast bacteria = blue
c. due to differences in outer cell wall
6. Endospore (spore) Stain
a. used to stain resistant survival cells of bacteria called spores or endospores
b. Spores are formed under adverse conditions
c. Spores are not reproductive, but are alive and very resistant
D. Structural Stains
1. Used to emphasize cell parts such as flagella and capsules
IV. Gram Staining
A. Step 1 – use Crystal Violet (purple primary dye) to stain all cells in smear
B. Step 2 – use Gram’s Iodine as a mordant; causes dye to get trapped in cell wall
1. Gram positive cell walls are thicker and more dye gets trapped than in the cell walls of
Gram negative bacteria cell walls.
C. Step 3 – use alcohol to dissolve lipids in outer membrane; removes dye in gram negative cells,
but not gram negative cells. Gram positive cells stay stained purple but gram negative cell become colorless.
D. Step 4 – Use Safranin (red counterstain) to color gram negative cells
E. Results: Gram positive cells stain purple and Gram negative cells stain red
V. “The Six I’s” of Microbial Techniques
A. Inoculation – put sample of microbes in medium. 1st stage in culturing.
1. Medium – nutrient gel/liquid used to grow organisms
2. Culture – observable growth of microbes that appear on medium
a. sputum, urine, feces, and infected tissue are often cultured
B. Incubation – promote growth of inoculated medium by placing in optimal growth conditions.
1. Incubator – controls temperature
C. Isolations – separating individual microbes to achieve isolated colonies that can be distinguished macroscopically.
D. Inspection – Look at cultures macroscopically for growth characteristics and microscopically for details such as cell type and shape.
E. Information Gathering – Testing cultures to analyze biochemical and enzyme characteristic, drug sensitivity, DNA analysis, etc.
F. Identification – Determine type of microbes present.
1. use an identification key, “keying out”
A. Sterile – complete absence of viable microbes
B. Aseptic – prevention of infection. *nurse uses aseptic technique to insert a catheter
C. Pure culture – growth of a single species of microbe
D. Subculture – technique to make a pure culture. Transfer a sample of a well isolated colony to another medium and incubate to create a pure culture.
E. Mixed culture – container of 2 or more easily differentiated microbes.
F. Contaminated culture – culture with unwanted microbes (contaminants)
Doctor says, “I’m going to culture that infection on your hand.” Doctor then uses a sterile swab to capture microbes from the infection and places it into a sterile container. The container then goes to the lab. At the lab they take the swab covered with microbes and inoculate a medium with the swab, i.e. they transfer the microbes from the swab to a nutrient gel/liquid to promote growth. The growth will then be analyzed to determine specific organisms that are present in the wound. This will provide which antibiotic is best to use, i.e. are there drug resistant bacteria present?
V. Isolation techniques – an individual bacterial cell is separated from other cells and placed onto a nutrient surface (agar in a Petri dish) where it divides into a discrete mound of cells, i.e. colony. One species is now isolated.
A. Streak plate method – droplet of sample is spread with an inoculating loop over surface of agar plate in a specific pattern that separates cells over plate.
B. Loop dilution (serial dilution) or pour plate technique – use inoculating loop to inoculate series of liquid agar tubes to dilute the number of cells in tube series.
1. Inoculated tubes are then poured into sterile Petri dishes and allowed to harden.
2. The diluted plates will allow for separate colonies to grow.
3. Colonies will grow deep in medium as well as on surface.
C. Spread plate technique – a small liquid sample is pipette onto suregace of medium and
spread evenly with sterile spreading stick (hockey stick). Goal again is to form isolated colonies.
A. Over 500 variations; depends on requirement of specific microbes
B. Media contained in test tubes, flasks, and Petri dishes
C. Tools for inoculating media – loops, needles, pipettes, swabs
D. Physical States of Media
1. Liquid media – water based, not solid at room temp
a. Broths, milks, infusions
b. Growth occurs through media; cloudy appearance
c. Nutrient broth – contains beef broth & peptone (digested proteins)
2. Semisolid media
a. Clotlike, but not firm; contain some agar or gelatin
b. Used to test Motility of bacteria
3. Solid media
a. Firm surface to isolate and culture bacteria and fungi
b. Agar – polysaccharide, solid at room temp, liquid at 100 C
1) Nutrient agar – media w/ 1-5% agar, beef broth & peptone
E. Chemical Content of Media
1. Synthetic media
a. Exact chemical composition is known
2. Nonsynthetic / Complex
a. Exact chemical composition in NOT known
b. Blood agar, serum, milk, peptone, nutrient broth
3. General-purpose Media
a. Used to grow broad spectrum of microbes
b. Nutrient agar, broth
4. Enriched media
a. Contains special nutrients/growth factors for Fastidious Bacteria
b. Fastifious Bacteria require special nutrients/factors to grow
F. Selective Media
1. Media that inhibits growth of certain microbes, but selects (or favors) another
2. Ex) Mannitol salt agar (MSA) – high NaCl content inhibits most human pathogens, but
allows Staphylococcus to grow.
a. Therefore selects for the growth of Staphylococcus
G. Differential Media
1. Allow several types of microbes to grow, but bring out visible differences between them.
2. Differences include
a. Colony size and color
b. Color change of media
c. Formation of gas bubbles or precipitates
3. Differences occur because of the way different microbes metabolize different chemicals in the media.