Microscopy - reverse and invert
Bright- field (Compound Microscope) - use light form image through sample. Specimen makes image darker
than background. Used w/ live, unstained, preserved, stain specimen. (Vis. Light)
The image is produced by light passing through the sample, with little contrast in unstained samples.
Phase Contrast - for live specimens. Contracted against gray background. Great for internal cell details. (Vis.
Light) [2000X]
Fluorescence - UV radiation source w/ dyes used. Colored image against black background. Diagnosed
infections from bacteria, protozoa, viruses using fluorescent antibodies. (UV rays) [2000X]
Transmission Electron Microscope (TEM) [100,000X] - structure of cell/viruses. Electrons through thin stained
specimen (20-100 nm) Dark areas=thick/dense parts. No color.
Scanning Electron Microscope (SEM) [650,000X] - detailed 3d of object. Electron cover surface, deflected
ones picked up by detector →image to computer. Outside only. No color. Characterized by lower magnifying
power, but can provide 3-dimensional viewing of objects. Captures the image of the object in black and white
after being stained with Au and Pd
Optical Microscope: Optical microscopes use visible light (or UV light in the case of fluorescence microscopy)
to sharply magnify the samples. The light rays refract with optical lenses. The first microscopes that were
invented were found to belong in this category. Optical microscopes can be further subdivided into several
categories:
- Compound Microscope: The compound microscope is built of two systems of lenses for greater
magnification. The utmost useful magnification of a compound microscope is about 1000x.
- Stereo Microscope (dissecting microscope): Optical microscope which magnifies up to about maximum
100x and provides a 3-dimensional view of the specimen.
Confocal Laser scanning microscope: Unlike compound and stereo microscopes, Confocal Laser scanning
microscopes are reserved for research organizations. Such microscopes are able to scan a sample in depth,
and a computer can then assemble the data to create a 3D image.
Electron Microscope: Most advanced microscopes used in modern science. Accelerated electrons strike any
objects in path, to magnify them up to 2 million times due to the small wavelength of high energy electrons.
Specifically for studying cells and small particles of matter, as well as large objects. The high energy electrons
are quite tough on the sample being observed. The electron microscope has a higher resolving power than a
light microscope. To reveal the structure of objects, it may take a long time to completely dehydrate and
prepare the specimen; a sleek layer of a metal can be used to coat some of the biological specimens for easy
observation..
- Reflection Electron Microscope: Designed on the principle of electron beams, but they are different from
transmission and scanning electron microscopes bc it is built to detect electrons that have been scattered
elastically.
X-ray Microscope: Uses a beam of x-rays to create a high-resolution 3D image. Due to the small wavelength,
the image resolution is higher as compared to optical microscopes. Magnification is between optical and
electron ones. Allows observing the structure of the living cells. Puts together thousands of pictures to make
a 3D image.
Scanning Helium Ion Microscope (SHIM or HeIM): Uses a beam of Helium ions to generate an image. Sample
is left mostly intact (due to the low energy requirements) and gives a high resolution. The first commercial
systems were released in 2007.
Scanning acoustic microscope (SAM): Focused sound waves generate an image. Has a wide range of
applications in materials science to detect small cracks or tensions in materials. Can also be used in biology
to study the physical properties of the biological structure and help uncover tensions, stress,elasticity inside.
Neutron Microscope: Still under an experimental stage. Generate a high-resolution image and may offer better
contrast. Would use neutrons instead of beams of light or electrons to generate images.
Scanning Probe Microscopes: Helps visualize individual atoms. The image of the atom is
computer-generated. A small tip measures the surface structure of the sample. High image magnification to
observe three-dimensional specimens. The amount of current that flows is proportional to the height of the
structure. A computer then assembles the position data of the tip.
Organisms
- Signs of life for all cells, including basic molecules.
[virus]-Often nucleic acid in protein, use components of other living cells to reproduce. [prions -proteinaceous
infectious particles]-infectious agents in mostly protein, induce polypeptides to host to take on form.
Cellular (cell) -bacteria and archaea → prokaryotes [single, nuclear material, no organelles bound]. Algae,
fungi, protozoa → eukaryotic. Algae are autotrophic, fungi heterotrophic.
Cilia - small hair, to move material over surface. Can be short or long.
Flagellum - tail, move in direction
RNA - transcription DNA → proteins
Pilus - hair outside to help bacterial cell stick to surfaces/other cells AND transmit genetic material
Fimbria - hair outside. Cell to cell or cell to host attachment. Partake in pathogenesis when attached.
Theory - E. from P. cells
Mitochondria and Chloroplast can reproduce independently of rest of cell.
Bacteria - unicellular, live in all different types of environment. Often 3 shapes. Some are immobile while
others can move. Some form spores. Individual or together in shapes. Photoautotrophic for own food and
give off oxygen. Cyanobacteria [-, in water, coloring] use oxygen and photosynthesis. Chemoautotrophic food
from chemical reactions.
Nucleolus (contains DNA) surrounded by cytoplasm containing ribosomes floating. Then by cell membrane,
mesosome, cell wall, then capsule. Have flagella and pili. In humans to degrade food, make nutrients, and
neutralize toxins.
Archaea: Very similar structure to bacteria, but without a mesosome. Their main functions are nutrient
cycling, stress response, and phytohormone biosynthesis. Their cell wall is made of pseudopeptidoglycan.
Algae Cells: SImilar structure to plant cells, but its DNA exists in less complex strands found more commonly
in prokaryotes, contains a pyrenoid (photosynthesis enhancer), an eyespot (region where light is taken in), and
two flagellum. Microalgae are being studied as an alternative to nonrenewable fuels because of their ability to
grow in artificial light, can be grown on the ocean, and high oil content.
Fungi - Similar to animal cells, but with a peroxisome, cell wall, cytoskeleton, and buds. Cells communicate
with external environments with diff. molecules, using proteins and breaking down dead plants and animals
to redistribute their nutrients.
Viroids - single strand, covalent closed circular/linear. RNA molecules with expensive regions of
intermolecular complementarity. Exist in native state as highly base-paired rods. Smallest agents of infectious
diseases.
Virus - virion containing unwound DNA or RNA (never both), protected by a geometrical capsid, then an
envelope (from host cell), with a tail and tail fibers (legs!). Attach themselves to host cells and inject their DNA,
converting the cell and multiplying their DNA.
Baltimore Classification:
- Class l viruses are double stranded DNA viruses
- Class ll viruses are single stranded DNA
- Class lll viruses are double stranded RNA viruses
- Class lV viruses are positive sense (similar to mRNA) single stranded RNA viruses
- Class V viruses are negative sense (complementary to mRNA) single stranded RNA viruses
- Class Vl viruses are RNA retroviruses
- Class Vll viruses are DNA retroviruses
Virus Replication: Lytic or Lysogenic
Lytic: virus injects its genome into the host cell (not able to differentiate the virus from its own DNA). Cells
begin to make the mRNA from the viral DNA. Messes up the central dogma and kills the cell’s DNA. Cell shuts
down, but virus still uses it to replicate until enough viruses make the cell lyse and gin to infect other cells.
Lysogenic: Viral DNA is known as prophage. Remains dormant in the cell for generations before becoming
active, leaving the cells DNA and directing the synthesis of new viral proteins. The viral genome integrates into
the host genome and replicates along with the host.
Bacteriophages do both.
Bacteriostatic - agent prevents the growth of bacteria, keeping them in the stationary phase of growth
Bactericidal - kills the bacteria
GET CULTURED!! (Bacteria Culturing) - the process of growing or propagation in the lab. Includes liquid and
agar.
Differential Media - uses special substances in the media to different intended bacteria
Selective Media - contains specific blends of compounds or antibiotics to prevent growth of other bacteria
Broth Culture - nutrient rich liquid inoculated onto bacterial smear, then left to incubate at optimal temp. for 24
hrs or more for reproduction. Broth is mostly water usually containing beef extract that contains broken down
proteins. Many bacteria can grow with this, even with varying oxygen requirements.
Culture based methods can be disadvantageous due to its cost, sensitivity, and the safety concerns with
pathogens. Is also a tedious and lengthy culturing process, making it prone to contamination, reliance on
phone types, and inadequate since about only 2% of the microbe population can be isolated and cultured.
Agar Culture: Made using a broth and adding the polysaccharide agar. This is spread on a dish or test tube
and allows bacteria to grow atop it. Beneficial in revealing their colony morphology, aka their visual
characteristics when growing from a parent cell.
Fed-Batch Culture: Nutrients are added periodically, removing nutrient supply as a limiting factor. However,
this can result in a lot of waste products and it is a very easy point of contamination.
Measurements of Bacterial Growth
Optical Density: use spectrophotometer to measure turbidity (cloudiness) of culture.
Plate Count: dilute and plate bacterial cultures, count the # of colonies that form and determine the Colony
Forming units per mL (CFU/mL)
Quantifying DNA or Protein: extract DNA and protein from bacterial culture and quantify using laboratory
assays.
Serial dilution and plating: dilute culture 10-fold (ex: 1mL of culture into 9mL of fresh medium); transfer same
volume of first dilution to a second tube with the same amount of fresh media, generating a 100-fold dilution,
continue until 10º dilution has been made; spread volumes of each dilution on plates; count colonies that
form; determine Colony Forming Units per mL of medium (CFU/mL).
(Binary Fission - one cell splits into two cells)
- growth equation ? Or use Monod Equation
𝑃=𝑃0·2(𝑡/𝑔)
Batch vs. Chemostat Growth - batch culture (aka closed culture) involves bacteria inside a closed container
using a fixed volume of medium and not adding new chemicals, etc, throughout the growth process.
Chemostat (aka open or continuous), nutrients continuously added or removed from the medium to achieve
constant environmental conditions
Defined vs. Complex Media - defined media made the same way following the recipe each time while complex
media contains ingredients that have varying chemical compositions each time.
4 Stages of Microbial Growth:
1. Lag Phase - cells mature for doubling, synthesis of RNA, enzymes, etc
2. Exponential Growth (Log) Phase - microbial population undergoes constant doubling. More better
conditions = longer slope, faster growth = steeper slope. Generation time can be calculated here.
3. Stationary Phase - top flat portion where there is no significant increase in the number of cells, stabilization
of population because rates of cell death and division are nearly equal = no effect change in population size.
4. Decline or Death Phase - the non constant down slope where the rates of death are greater than the rate of
cell division. Since the bacteria don't have an infinite source of nutrients (chemostat growth) living cells take
the nutrients and increase the amount of bacterial waste resulting in an unfavorable environment.
*5. Long-term stationary Phase - This stage occurs for some bacteria (ex. E-coli). This phase has little cell
division and has high rates of both division and death. (Some bacteria, after decline, can still persist in closed
systems for some period of time). This stage looks like rounded bumpy mountain tops or straight lines well
above the Lag Phase.
Gram Staining - Classifying bacteria by their cell wall structure. The procedure involves first heat-fixing
bacteria (so they can hold their primary stain and keep them in place) before sequentially treating them with
four different reagents.
Gram +/- Staining procedure to check for bacteria at site of potential infection such as the throat, lungs, or
genitals.
1) A cationic (i.e., positively charged) primary stain (often crystal violet, which stains cells purple, or methylene
blue) that is taken up by both Gram-positive and Gram-negative bacteria. Cells are usually incubated with the
dye for at least 1 minute. In an aqueous solution, crystal violet dissociates into CV+ and Cl-ions. These ions
penetrate through the cell wall and CV+ associates with negatively charged functional groups in bacterial cell
walls.
2) A mordant (usually Gram's iodine solution) containing anions that are complex with the positively charged
primary stain inside of Gram-positive cell walls, preventing easy removal of the primary stain when cells are
washed with a decolorizer. The mordant essentially acts as a trapping agent for the primary dye, and cells are
usually incubated for at least one minute before rinsing off any excess mordant.
3) A decolorizer (often ethanol or acetone) to wash the primary stain off the surface of Gram-negative
bacteria, which cannot remove any primary stain molecules that were fixed inside Gram-positive cell walls by
the mordant Alcohol dissolves the outer membrane of Gram-negative bacteria, effectively removing any
primary stain on Gram-negative cells, while the primary stain remains trapped in the thick cell walls of
Gram-positive cells.
4) A counterstain (often safranin, basic fuchsin, or carbol fuchsin, all of which stain cells red/pink). The
counterstain stains both Gram-positive and Gram-negative cells, but is not visible on Gram-positive cells due
to the darker color of the primary stain. This procedure results in Gram-positive bacteria being stained purple,
while Gram-negative bacteria stain red/pink color.
- If the Gram Stain appears blue or purple, it is likely gram positive, meaning it has a thick cell wall of
peptidoglycan. If it’s red or pink, it is gram negative with a thin peptidoglycan wall but a higher fatty acid
content.
Bacterial Processes/Division/Fission
Central Dogma - DNA →RNA→Protein: Replication, Transcription, Translation
Replication - Helicase breaks apart bonds in the parent DNA. Polymerase reads it and builds new strands
resulting in two daughter double helices. The process is semiconservative as only half of the parent's DNA is
in each daughter's DNA.The origin of replication is where DNA replication begins. The Origin Recognition
Complex (ORC) binds to mark the starting point. DNA helicase unwinds the DNA strands, creating
single-stranded templates. Single-Strand Binding Proteins (SSBs) prevent the strands from reannealing. DNA
primase synthesizes RNA primers, allowing DNA polymerase to start adding nucleotides, ensuring accurate
and efficient replication.
Transcription - DNA to RNA.
1. Initiation - Polymerase binds to the promoter region of the gene (TATA) to have the unwound DNA ready
(DnaA here as a protein that starts this process)
2. Elongation - Polymerase makes the mRNA that is complementary to the DNA.
3. Termination - Polymerase creates an end cap that completes the mRNA making it detach from the DNA
Transcription
Genetic code in mRNA is read to be made into a protein, from nucleotide to amino acids, through a ribosome
with the help of tRNA
Bacterial Reproduction
In bacteria, both transcription and translation can happen simultaneously. This is known as
transcription-translation coupling.
Bacterial Gene Regulation
Each operon contains regulatory DNA sequences, which act as binding sites for regulatory proteins that
