Lecture 2 Flashcards
What are the 5 techniques performed in the lab?
innoculation, incubation, isolation, inspection, identification
What is inoculation?
The placement of microorganisms into or onto culture media
What is a culture?
The growth of microorganisms with various media
What is a medium?
a nutrient used to grow microorganisms outside their natural habitat
What is an incubator?
chamber that is temperature-controlled (and sometimes control of other environmental factors), use for growth of microbes
What temperature is used for human pathogens?
temperatures fall between 20°C and 40°C for human pathogens
What happens during incubation?
During incubation, microbes grow and multiply, producing visible growth in the media; *growth indicates increase in NUMBER rather than increase in SIZE
What is a pure culture
just the one microorganism in the container
What is a mixed culture?
2 or more identified organisms in a container
What is minimal media?
media that only contains a few components
What is complex media?
contain at least one ingredient that is not chemically definable
What is selective media?
contains one or more components that inhibit the growth of a certain microbes but not others
What is differential media?
allows multiple types of microorganisms to grow but are designed to display differences among those microorganisms so that they can be distinguished from one another
What is reducing media?
contains a substance (i.e. thioglycollate) that absorbs oxygen or slows the penetration of oxygen
What is fermenation media?
contain sugars that can be fermented and a pH indicator that shows acid production as the
result of this reaction
what is transport media?
used to maintain and preserve specimens that have to be held for a period of time before clinical analysis
What is a colony?
a visible cluster of microbes on a solid medium
What is isolation?
Requires solid media with enough surface area to spread a culture for separation
Allows for identification of multiple organisms in mixed cultures
What is inspection and indentification?
Microbes can be identified through a combination of
microscopic appearance – wet mounts or staining
use of selective and differential media
characterization of cellular metabolism and products produced during growth, presence of certain enzymes
genetic and immunological characteristics
*A combination of these methods is used as usually one alone is not sufficient for positive identification
What is resolution?
Ability to distinguish detail
Higher the resolution, clearer the picture
Resolving power – closest 2 points can be to each other and still be distinguished
What is resolving power?
1) size of objective lens (larger the lens the greater the resolving power)
2) wavelength of light passing through specimen (shorter wavelengths tend to give better resolution)
3) refractive index of the material between the objective lens and specimen – oil, air…
What is magnification?
Enlargement of an image
Requires a convex lens (thicker in center than at the edge)
What is total magnification?
Number of times larger the image seen appears compared to the actual sample
What is a wet mount?
Drop of liquid containing microorganisms on a slide with a coverslip on top
Allows you to see the true size and shape, motility
What is a stain?
Increase contrast, bind to certain parts of microorganisms, allow them to be visible
Vital stains – stain living cells, can be added directly to a wet mount
Some staining requires fixation of the cells
What is fixation?
Getting the organism to stay on the slide
What are the types of dyes?
Basic dyes – positively charged (safranin, basic fuchsin, crystal violet, methylene blue)
Acidic dyes – negatively charged (Eosin, acid fuchsin, congo red)
Mordants – intensify staining, increase the cell’s affinity for a dye
What are the types of stains?
Simple
Differential
Special
What are simple stains?
Basic dyes that allow the cell to be visible, all cells are stained the same color
What are differential stains?
Distinguish between types of organisms based on how they stain
What are the 3 steps of differential staining?
Three steps:
primary staining – same as simple staining
destaining – removes stain in certain cells
counterstaining – stains parts of cells that were destained to allow them to be seen
What is Gram’s Stain?
Divides bacteria into 2 groups, Gram positive and Gram negative
What is acid fast stain?
Used to identify Mycobacterium tuberculosis, only stains mycobacteria and some actinomycetes (called acid fast bacteria)
These bacteria resist destaining because of a waxy material in their envelope
Acid fast bacteria stain red, non-acid fast stain blue
What are structural stains?
cell wall, flagella,
capsule
What is an example of a hard to stain bacteria?
spirochetes
What is negative stain?
stains everything except structure you’re looking for (capsule stain)
What are the types of microscopy?
Brightfield, Darkfield, Phase contrast, Differential,
Fluroscent, confocal, electron, transmission, and scanning
What is Brightfield Microscopy?
Brightfield is a stain and the light waves move in sync.
You have to have stain in this type and you have to set the right settings
What is darkfield microscopy?
Live organisms (wet mount) Makes the field dark and the organisms glow
What is phase contrast microscopy?
Allows visualization of live, unstained organisms (wet mount)
Phase rings in objective lens and condenser that send some light rays through specimen, some around it (where as brightfield sends rays straight through the specimen), allowing for visualization)
What is differential microscopy?
Like the phase-contrast microscope, the differential interference contrast (DIC) microscope provides a detailed view of unstained, live specimens by manipulating the light.
two prisms that add contrasting colors
What is fluorescence microscopy?
Used in research and diagnostically. Stain only binds to a certain organism so when you had the stain to the sample and something glows then that is what you are looking for. You are using dyes that glow under certain lights. Different wavelengths cause different things to glow in different colors
What is confocal microscopy?
Really nice clean images.
Z stacks take a bunch of pictures and stack them to make a 3d image
What is electron microscopy?
Used for examining objects smaller than 2uM.
Uses a beam of electrons instead of light (much shorter wavelength, so get much higher resolution)
What is transmission microscopy?
Fixed samples that are dead
What is scanning microscopy?
Beam of electrons is directed over the surface of the specimen, knocking electrons out of the surface of the specimen. These secondary electrons are transmitted to an electron collector, amplified, and used to produce a 3-D image
