Lecture #5: Tools & Techniques Flashcards
Whole Functional Groups Relate to Gram Staining Reactions
Gram + Cocci – Bacteria of the skin, throat.
Gram + Rods – Soil Bacteria.
Gram – Rods = Enterics.
Why Do We Care What Color They Stain or How Thick the Cell Wall Is?
It relates to specific groups and can be diagnostic.
If you find a GP inside the body (GP bacteria typically inhabit the skin), may indicate an infection. Example is a Staph infection.
If you find a GN bacterium that cannot ferment lactose in the gut of a person complaining of food poisoning, it is probably a pathogen. GN bacteria occur in your gut and can utilize lactose (in the dairy products you consume). Contaminants from soil or water don’t use lactose.
GP rods occur in the soil and many are pathogenic to humans. So a GP rod in a stool sample suggests food poisoning.
Streaking for Isolation
Streaking for Isolation means taking cells from one colony, delivering the cells to a new food source (your plate), diluting it down to a single cell by means of streaks.
Stains & Stain Protocols
Provide increased contrast.
Simple – single stain. Quick, easy and can show shape and size, orientation of cells effectively for little effort.
Differential – Sorts organisms based on some characteristic. Two stains used; one has an affinity for a particular structure or condition, while the other is a counterstain.
- Gram Stain
- Endospore Stain
- Acid-Fast Stain
Simple, Differential Stains
These 2 terms are not logical opposites.
Simple means one stain but differential does not really mean more than one.
Differential means that it differentiates (sorts) based on a key characteristic. The staining protocols happen to include a second stain, but differentiation would be present without it.
Simple Staining
Positive Stain Protocol provides direct contrast. Sticks to the structure of interest.
Negative Stain Protocol enhances a structure by NOT staining it. Stain is repelled and stains around it, allowing you to detect the structure or condition.
Differential Staining Protocols
Differentiate between 2 different conditions. Gram stain for Gram + and Gram -. Presence or absence of a thick cell wall. If present, retains the CV stain.
Acid Fast Staining differentiates between bacteria with a waxy lipid in walls. Hard for stains to get in, but once in, hard to remove. If retain the stain, have mycolic acid (waxy lipid).
Endospore Stain protocol drives Malachite Green into the thick-walled endospores. Due to the thick covering with a special substance, won’t wash out.
Acid-Fast Staining
Forms that have Mycolic Acid.
Employs special stains, harsh treatment.
Basic Fuchsin stain is applied and enters cells by weakening (phenol) and heat. If Mycolic Acid is in the walls, stain is retained. Differentiates Mycobacterium form other bacteria. (TB and leprosy; Mycobacterium tuberculosis, M. leprae).
Stain/Decolorizer/Counterstain
The pinkish-purplish cells are acid fast (B).
The other cells (bacteria) are the color of the counterstain (Methylene Blue).
Endospore Stain
Presence of a thick cell wall, this time with Dipicolinic Acid. Endospores have thick walls and resist most stains. Again, use heat to drive the stain in. No decolorizer, just a rinse. If thick wall (endospore) the stain (Malachite Green) is retained. Cells without the thick cell wall of an endospore, will lose the green color.
Endospores are Green, Cells are the Counterstain Color.
Dark Field Microscope
Produces detailed images of living, unstained cells and organisms by simply changing the way in which they are illuminated. A hollow cone of light is focused on the specimen in such a way that unreflected and unrefracted rays do not enter the objective. Only light that has been reflected or refracted by the specimen forms an image.
The field surrounding a specimen appears black, while the object is brightly illuminated. This microscope can reveal considerable internal structure in larger eukaryotic microorganisms.
Phase-Contrast Microscope
Converts slight differences in refractive index and cell density into easily detected variations in light intensity. The condenser of a phase-contrast microscope has an annular stop, an opaque disk with a thin transparent ring that produces a hollow cone of light. As this cone passes through a cell, some light rays are bent due to variations in density and refractive index within the specimen, and are retarded by about 1/4 wavelength. The deviated light is focused to form an image of the object. Undeviated light rays strike a phase ring in the phase plate, an optical disk located in the objective, while the deviated rays miss the ring and pass through the rest of the plate. If the phase ring is constructed in such a way that the undeviated light passing through it is advanced by 1/4 wavelength, the deviated and undeviated waves will be about 1/2 wavelength out of phase and will cancel each other when they come together to form an image. The background, formed by undeviated light, is bright, while the unstained object appears dark and well defined. Th This type of microscopy is called dark-phase-contrast microscopy. Color filters improve the image.
Phase-contrast microscopy useful for studying microbial motility, determining shape of living cells, and detecting bacterial structures such as endospores and inclusion bodies. These are clearly visible because they have refractive indices markedly different from that of water. Also widely used to study eukaryotic cells.
Differential Interference Contrast (DIC) Microscope
Similar to the phase-contrast microscope in that it creates an image by detecting differences in refractive indices and thickness. Two beams of plane-polarized light at right angles to each other are generated by prisms. In one design, the object beam passes through the specimen, while the reference beam passes through a clear area of the slide. After passing through the specimen, the two beams are combined and interfere with each other to form an image. A live, unstained specimen appears brightly colored and 3D.
Fluorescent Light
Objects can also be seen because it emits light: this is the basis of fluorescence microscopy. When some molecules absorb radiant energy, they become excited and release much of their trapped energy as light. Any light emitted by an excited molecule has a longer wavelength (i.e., has lower energy) than the radiation originally absorbed.
Fluorescent light is emitted very quickly by the excited molecule as it gives up its trapped energy and returns to a more stable state.
Fluorescence Microscope
Exposes a specimen to ultraviolet, violet, or blue light and forms an image of the object with the resulting fluorescent light. The most commonly used fluorescence microscopy is epifluorescence microscopy, also called incident light or reflected light fluorescence microscopy. Epifluorescence microscopes employ an objective lens that also acts as a condenser. A mercury vapor arc lamp or other source produces an intense beam of light that passes through an exciter filter. The exciter filter transmits only the desired wavelength of excitation light. The excitation light is directed down the microscope by the dichromatic mirror. This mirror reflects light of shorter wavelengths (i.e., the excitation light) but allows light of longer wavelengths to pass through. The excitation light continues down, passing through the objective lens to the specimen, which is usually stained with molecules called fluorochromes. The fluorochrome absorbs light energy from the excitation light and fluoresces brightly. The emitted fluorescent light travels up through the objective lens into the microscope. Because the emitted fluorescent light has a longer wavelength, it passes through the dichromatic mirror to a barrier filter, which blocks out any residual excitation light. Finally, the emitted light passes through the barrier filter to the eyepieces.
The fluorescence microscope has become an essential tool in microbiology. Bacterial pathogens can be identified after staining with flurochromes or specifically tagging them with fluorescently labeled antibodies using immunofluorescence procedures. In ecological studies, the fluorescence microscope is used to observe microorganisms stained with fluorochrome labeled probes or fluorochromes that bind specific cell constituents. In addition, microbial ecologists use epifluorescence microscopy to visualize photosynthetic microbes, as their pigments naturally fluoresce when excited by light of specific wavelengths. It is possible to distinguish live bacteria from dead ones by the color the fluoresce after treatment with a specific mixture of stains. Thus, the microbes can be viewed and directly counted in a relatively undisturbed ecological niche.
Localization of Proteins with Fluorescence Microscopy
Another important use of fluorescence microscopy is the localization of specific proteins within cells. One approach is to use genetic engineering techniques that fuse the gene for the protein of interest to a gene isolated from jellyfish belonging to the genus Aequorea. The jellyfish gene encodes a protein that naturally fluoresces green when exposed to light of a particular wavelength and is called green fluorescent protein (GFP). Since its isolation, the gene has been altered to create proteins that fluoresce different colors.
GFP and its variants have been used extensively in studies on bacterial cell division and related phenomena. In fact, the 2009 Nobel Prize in chemistry was awarded to Osamu Shimomura of Japan and Americans Martin Chalfie and Roger Tsien for their development of this tool.
