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58 Cards in this Set
- Front
- Back
Purpose of Isolation Technique
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to obtain pure culture
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Define: Pure Culture
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all cell of a colony or culture had a common origin and are all descendants from one same parent cell
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Define: Colony
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Composed of billions of cells that originate from one parent cell
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Pure Culture Allows one to study
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1. Macroscopic (Colony) Characteristics
2. Microscopic (Morphology) Characteristics 3. Physiological (Biochemical) Characteristics |
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Method of Obtaining a Pure culture and enumeration
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dilute out mixed bacterial culture to get individual, isolated colonies growing on agar plates
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Streak Plate Procedure (Isolation Technique)
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individual cells are spread out and separated which then develop into individual, isolated colonies, composed of billions of cells
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Pour Plate Procedure (Isolation and Enumeration Technique)
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Individual colonies develop and are separated (isolated) on the surface and below the agar surface (within the agar)
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Spread Plate Procedure (Isolation and Enumeration Techniques)
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individual colonies develop in the surface of agar plate
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Purpose of Enumeration Techniques of Microbes
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to find tilter in cells/ml of an unknown bacterial solution
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Cell Count Method
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a. counts number of bacteria in specific number of squares
b. use math formula to determine the titler (cels/ml) c. Advantages -quick and easy d. Disadvantages -exposed to living cells -counts only small portion of the sample -counts both living and dead cells |
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Turbidometric Methods
Turbidity |
To determine cell densities
the cloudiness, which represents bacterial growth |
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Spectrophotometer
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used to measure turbidity
1. determines the amount of light blocked by particulate material (ie) bacteria in a sample 2. measure the amount of light absorbed by microbes or the percentage of light transmittance |
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Spectrophotometer
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note: absorbance increases linearly as bacterial concentration increases
Advantages: quick and easy Disadvantages counts both living and dead cells not that accurate expensive |
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Coulter Counter
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electronic counter machines that measures and counts particles
used in hospitals to count red blood cells Advantages quick and easy Disadvantages counts both living and deal cells expensive |
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Membrane Filter Method
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Disadvantages
incubation time 24 to 48 hours hard to count colonies - small filter diameter osmotic shock (bacteria lysis) fastidious microbes will not grow |
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Pour Plate Method
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countable colonies from 30 to 300
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Pour Plate Method
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Advantages
only counts living cells standard text Disadvantages heat kills time of incubation 24 to 48 hours osmotic shock |
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Spread Plate
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Advantages
doesn't involve heat counts only living cell Disadvantages incubation time 48 hours osmotic shock |
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Dilution Factor
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all individuals dilutions need to get countable
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Platting Factor
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amount of stock solution pipetted and plated into plate
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Cultivation of Anaerobic Bacteria
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purpose: to determine the oxygen requirements of bacteria
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Obligate (Strict) Aerobes
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require 16% oxygen
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Obligate (Strict) Anaerobes
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require lack of oxygen
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Facultative Anaerobes
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(either/or) with or without oxygen (most prefer oxygen)
a. enzymes system allows them to utilize free oxygen or some alternate oxygen source such as nitrate b. most grow best in the presence of oxygen c. indifferents- show no preference of oxygen or alternate form |
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Microaerophilic
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grow in the presence of minute quantities of free oxygen
require oxygen is small quantities (6%-10% of oxygen) |
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Aerobic Bacteria
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have the enzyme catalase to breakdown acccumulation of toxic hydrogen peroxide from respiration
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Anaerobes Bacteria
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lack the enzymes catalase and are killed by the buildup of toxic H2O2
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Thioglycollate Broth Tubes
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1. glucose
2. cystine 3. sodium thioglycollate 1. lowers oxidation and reduction potential |
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Resazurin Indicator
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Aerobic = red/pink color
Anaerobic = clear |
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Aerobic Bacteria
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grows at the top and in the red area
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Microaerophilic Bacteria
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growth just below the surface in the red area
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Anaerobic Bacteria
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growth only in the clear area
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Facultative Anaerobes
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growth throughout the entire tube - red to clear area
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Brewers Anaerobic Jar
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creates only anaerobic conditions
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Platidium
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oxygen is converted to water on surface of platidium pellets
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Gas Pak
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add water, and it creates an anaerobic environment by releasing CO2 and H2 gases
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Indicator strip
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methylene blue
white color-anaerobic conditions blue color - aerobic |
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Candle Jar (microaerophilic environment)
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used to reduce the oxygen concentration to between 6% to 10% by increasing co2
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What does staining do?
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microorganisms are usually transparent so staining can also increase the size of certain features such as flagella which other wise would be too small to be observed with the compound light microscope.
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Stain
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an organic compound that contain a chromophore and an auxochrome group lined to benzene rings.
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Chromophore
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the portion of the stain which gives it color
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Auxochrome
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gives the stain the salt forming properties which is neccessary for the compound to adhere to the substrate
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electrical charge
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the electrical charge on bacteria is negative due to their protein and DNA molecules
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Basic Stains
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the balance of the electrical charge on the dye molecules is positive. it will for a salt with a metallic anion.
(ie) methylene blue (+) - chloride (-) stain negatively charged bacteria due to protein and DNA |
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Acidic Stains
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The balance of electrical charge on the dye molecules is negative (-). it will form a salt with a metallic cation
(ie) Na+ sodium + - eosinate (-) stains red blood cells |
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Neutral Stains
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a complex dye containing a dye acid with a dye based with an over all neutral charge
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Process od Heat Fixation
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1. adhering/fixing cells onto the slide
2. killing microbes (pathogens) 3. unfolding protein molecules and exposing postive and negative charges and this increasing the affinity of the specimen for the stain 4. preserve cell structure |
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Simple stain
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a single stain used to stain a heat fixed smear (cells) in one application
(ie) methylene blue |
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Negative Stain
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living organism are made visible by staining the background not the organism (ie) spirochetes
advantages 1. observe living organism 2. observe true cell morphology (no distortion of cell morphology from heat fixation or staining) |
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Differential Stain
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stains which use more than one dye and
a. differentiates or seperates microbes into major groups (ie) gram stain b. distinguish/differentiates structures (ie) spore stain. capsule stain |
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Primary Stain
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first stain applied during a differential stain procedure
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mordant
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any chemical substance
(ie) iodine or physical agent (ie) heat that allows the primary stain to adhere to substrate (ie) cell wall, spore a chemical mordant forms a complex with the primary stain and a physical mordant such as heat drives the primary stain into the substrate |
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destain
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functions to remove the primary stain during differential stain procedure
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Secondary Stain
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a background stain applied to stained material to increase contrast
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Gram positive bacteria
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organism which retain the crystal violet iodine complex after using the ethyl alcohol acetone destain and remain purple
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Gram negative bacteria
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organisms which are destained by the ethyl alcohol acetone destain that results in the loss of crystal violet iodine complex. they are then counter stained with safranin and become pink/red
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Acid-Fast Bacteria
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organisms which are not readily stained but when stained during the acid fast staining procedure retain the color of the primary stain carbolfuchsin and stain red. acid fast positive bacteria are resistanct to decolorization with acid alcohol and they contain mycolic acid
(ie) mychobacterium |
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Spore Stain
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the species of bacillus and clostridium produce endospores and exospores which resist staining. the spores can be stained by using a strong dye such as malachite green and heating (mordant) to drive the primary dye in. spores appear green and the vegetative cells are countered stained with safranin and appear pink/red
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