Microbiology Techniques Flashcards
What is microbiology?
study of uni/multicellular organisms too small to be seen with the naked eye (with the exception of fungi and algae)
What techniques define microbiology and what are they used for?
Culture Media: used to isolate and grow organisms in pure culture
Biochemical Techniques: used to study cell components
Molecular and Genetic techniques: used to study DNA sequencing
What kind of morphology is shown on an agar plate vs microscopic image?
Colony morphology is seen on an agar plate.
Cell morphology is seen in a microscopic image.
What are five reasons why microbiology is significant?
- Oldest form of life
- Largest mass of living material on Earth
- Carry out major processes for biogeochemical cycles
- Survive in typically unfavourable environments
- Required of other organisms to survive.
How are rRNA genes sequenced?
- DNA is collected from a pure culture.
- SSU rRNA gene is amplified using PCR
- Gene is sequenced
- Sequence is aligned with sequences from other organisms. The number of differences is used to calculate evolutionary distance
What are the rules of classification and nomenclature of microorganisms?
- Name is binomial ad latinized
- Name is italicized or underlined
- Genus is capitalized but species epithet is not.
- Genus may be abbreviated the second time it is used.
- Trivial names can be used that do not follow the above rules.
Who was Robert Hooke and what did he discover? What did he observe and how did he observe it?
17th century scientist who was the first to describe microbes. He used a compound (2 lens) microscope to magnify the image up to 30x to observe cells in cork and bread mold filaments.
Who was Antoni van Leeuwenhoek and what did he discover? What did he observe and how did he observe it?
17th century scientist who was the first to discover bacteria. He built microscopes that magnified specimen by 50-300x and observed single-celled organisms, calling them animalcules.
Who was Louis Pasteur and what did he study? What did he develop?
19th century scientist who studied wine and beer production through the fermentation of yeast (yeast converts sugar to alcohol in absence of oxygen). He 9developed pasteurization (heating to kill bacteria).
How did Pasteur disprove spontaneous generation?
Pasteur Prepared infusions in swan-necked flasks and boiled the infusion to sterilize it by trapping microbes in the bend. When the infusions touch the bent end, the liquid putrefies, but if it doesn’t touch it, the liquid remains sterile. This proved that the microbes were putrefying the infusions.
Who was Robert Koch and what did he study? What did he develop?
20th century scientist who studied anthrax (disease). He developed Koch’s postulates: criteria for relating a specific microbe to a disease and found that solid media was a suitable way to obtain pure cultures.
What are Koch’s postulates?
- Pathogen must be present in all cases of disease, and absent from healthy animals
- Pathogen must be grown in a pure culture.
- Cells from pure culture of the suspected pathogen must cause disease in a healthy animal when injected.
- Suspected pathogen must be re-isolated from animal injected with and shown to be the same as the original.
What is a pure culture?
A pure culture is a population of cells growing in the absence of other species or types.
What is the solid media used to grow a pure culture?
The solid media is a mixture of broth medium (nutrients) and 1.5% agar (marine algae polysaccharide)
Why is agar used to create solid media in a pure culture?
Agar cannot be used as an energy source by many bacteria, and can be easily solidified since it melts at 97°C and solidifies at 43°C.
What are three ways to isolate a pure culture? How are they different?
- Streak plate technique: One edge of plate is inoculated with a concentrated sample of bacteria and is diluted by streaking it across the surface of the plate.
- Spread plate technique: Diluted sample is spread over the plate with a spreader (0.1 mL or less)
- Pour plate technique: Diluted sample is mixed with molten agar which solidifies, allowing colonies to form within the agar.
What is bacterial titre?
It is the concentration of bacteria in a population expressed in colony forming units (cfu)/mL. It is calculated as follows:
titre = (# colonies) / (volume * dilution)
How are plates counted?
Plates are counted between 30-300 colonies. When there is more than one countable plate, we calculate titre from each and take the average of the titres.
What are the 4 types of light microscopy?
- bright-field
- phase-contrast
- dark-field
- fluorescence
How are specimens visualized in a brightfield microscope?
Specimens are viewed through a compound microscope through the difference in contrast between specimen and surroundings.
What are the two sets of lenses used to form the image and their magnifications? What is the highest possible total magnification
objective lens: 10x-100x
ocular lens: 10-20x
max mag: ~2000x
What is the difference between magnifications and resolution?
Magnification: zoom factor of an object
Resolution: clarity of the image
What is the limit of resolution for light microscope?
0.2 micrometers
What does the 0.2micrometer limit of resolution mean?
Two points can be distinguished if they are at least 0.2 micrometers apart.
What is the effect of wavelength on resolution?
As wavelength decreases, resolution improves.
How is contrast improved in a light microscope?
Dyes (organic compounds) bind to specific cellular materials to improve the contrast of the image. Some microbes already have a natural stain.
What are 3 examples of common stains?
- Methylene Blue (blue)
- Safranin (pink)
- Crystal Violet (violet)
What is the difference between simple and differential staining?
simple staining: one dye is used to colour a specimen.
differential staining: multiples dyes are used
What is a chromophore?
A chromophore is the coloured portion of a dye.
What are two types of chromophore used in staining? What do they bind to?
Basic Dye: positively charged chromophore, binding to negatively charged molecules on cell surface (aka positive stain)
Acidic Dye: negatively charged chromophore is repelled by cell surface and stains the background (aka negative stain)
3 Steps used to prepare samples for staining
- Prepare smear: spread thin film of culture then air dry
- Heat fixing + staining: pass slide through flame, flod slide with stain, rinse, dry.
- Microscopy: place drop of oil on slide and examine with lens
What are 3 types of differential stains? What differentiates them?
- Gram stain: separates bacteria via cell wall structure
- Acid fast stain: detects mycolic acid in the cell wall of microbes in the mycobacterium genus
- Endospore stain: stains the endospore of a bacteria
What is the difference between a gram positive stain and a gram negative stain?
Gram positive: cells have a thick layer of peptidoglycan and retain the primary stain (purple)
Gram negative: cells have a thin layer of peptidoglycan and take the colour of the counter stain (pink)
What colour dyes are used in acid fast stains?
Fuschia (primary stain) - retained by mycobacterium
Blue (counter stain) - retained by anything else on the slide
What colour dyes are used in endospore stains?
Green (primary stain) - retained by endospores
Pink (counter stain) - retained by other cells in the sample
What is phase-contrast microscopy? What does the specimen look like?
Type of microscopy that creates a phase ring around the refractive index of a cell and surroundings which allows for the visualization of live samples. Specimen is dark, background is light.
What is dark-field microscopy? What does the specimen look like?
Type of microscopy that illuminates the specimen through a hollow cone of light, letting only refracted light enter the objective. Specimen is bright, background is dark.
What is fluorescence microscopy?
Type of microscopy that is used to visualize specimens that fluoresce (emit light when illuminated with a specific wavelength). Cells can fluoresce naturally, or via fluorescent dye.
What is an examples of a cell that fluoresces naturally?
Photosynthetic cyanobacteria have chlorophyll that absorb light at 430 nanometers (blue-violet) and emits 670 nanometers (red)
What is Differential interference contrast (DIC) microscopy?
A type of microscopy that uses a polarizer to create two distinct beams of polarized light to give endospores, vacuoles, and granules a 3D appearance.
What is Confocal scanning laser (CSL) microscopy? What is its resolution?
A type of microscopy that uses a computerized microscope and a laser source to focus on single layers of a specimen and generate a 3D image. Resolution is 0.1 micrometers.
What is electron microscopy? What are the two types?
A type of microscopy that uses electrons instead of photons to visualize cells and structures. Either transmission electron microscopes or scanning electron microscopes.
Why does electron microscopy have a higher resolution than light microscopy?
Wavelength of electrons is much shorter than light. Since smaller wavelengths result in a clearer image, electron microscopy has a higher resolution than light microscopy.
How does a transmission electron microscope differ from a compound light microscope?
Electron beam is focused on a condenser lens, objective lens, and ocular lens made of electromagnets rather than glass, and the electrons strike a viewing screen rather than the naked eye.
What is the resolution of a transmission electron microscope? What must be for them to be seen?
The resolution is 0.2 nm and must be very thin (20-60nm) and stained with metals (lead/uranium) to be seen.
Why must cells be metal-stained to be seen under a transmisison electron microscope?
Metals bind to cell structures to make them more electron dense which enables the visualization of structures at molecular level.
How does a transmission electron microscope differ from a scanning electron microscope?
TEM: used to view internal structures, coated with metal
SEM: used to view external structure, coated with heavy metal
