Download Physical State Physical State of Media

The Five “I”s: Basic Techniques to Culture Microbes
Tools of the
Microbiology
Laboratory
The Five “I”s: Inoculate
1. Inoculate
2. Incubate
3. Isolate
4. Inspect
5. Identify
Inoculate: Media
Classified according to three properties
1. Physical state
2. Chemical composition
3. Functional types
•Culture: the propagation of microorganisms with various media
•Medium (pl. media): a nutrient used to grow microorganisms outside their natural habitat
•Inoculation: the implantation of microorganisms into or onto culture media Media: Physical State
Physical State of the media is referring to how solidified the media is. The way that we solidify media is by adding agar, a complex polysaccharide. • Liquid media • Semi‐solid media
• Solid media
Physical State of Media: Liquid
• Liquid media has no agar added
• Water‐based solutions that are generally termed broths, milks, or infusions.
• Information you can gain from liquid media: aerobic/anaerobic, clump as they grow, etc.…
• Great way to gather large volumes of bacteria
• Can you isolate bacteria using liquid media?
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Physical State of Media: Semi‐Solid
• Semi‐solid media contain a low percentage of agar usually around 0.4%
• Since this media is very soft motile bacteria can move through it
• For this reason it is used for motility testing
• Solid media contain a high percent (1‐5%) of agar
• This enables the formation of discrete colonies which is useful in both isolation and testing the biochemistry of the microbe
Media: Chemical Composition
The chemical composition of the media is referring to whether or not the exact chemical make‐up of the media is known. • Defined
• Complex
Chemical Composition: Complex
• Complex media contains ingredients that are not chemically defined or pure
• Many complex media contain animal, plant, or yeast extracts
• Example of additives: ground tissue or cells, blood, yeast digests, milk
Physical State of Media: Solid
Chemical Composition: Defined
• Synthetic media
• Media whose compositions are precisely chemically defined
• Contain pure organic and inorganic compounds
• Molecular content specified by an exact formula
Defined media for growth of Staphylococcus aureus
Media: Functional Types
• Functional type of the media is referring to how the media will be used, or the purpose of growing the media.
• General –purpose media
• Differential media
• Selective media
TSA + 5% sheep blood 2
Functional Type: Selective and Differential
Functional Type: General‐Purpose
• General‐purpose media: used to grow a broad spectrum of bacterial species
• Used as a first step in growing up samples from patients and fomites
• Chemicals are added to media for the purpose of:
• testing the biochemistry of the bacteria‐
differential media
• selecting for bacteria that have a specific biochemistry‐ selective media
• More about these in the Identify portion of the Five “I”s
The Five “I”s: Isolate
The Five “I”s: Incubate
Incubator: a chamber where temperature is controlled.
• Usual laboratory propagation temperatures fall between 20°C and 40°C
• Atmospheric gases such as O2
and CO2 may be required for the growth of certain microbes
• During incubation, microbes grow and multiply, producing visible growth in the media
The Five “I”s: Isolate
Three basic methods of isolating bacteria
• Streak Plate
•
•
Based on the concept that if an individual cell is separated from other cells on a nutrient surface, it will form a colony
Colony: a macroscopic cluster of cells appearing on a solid medium arising from the multiplication of a single cell
Isolate: Streak Plate
This method only works if the spreading tool is resterilized
after each of steps 1‐4. • Require the following
– a medium with a firm surface
– a Petri dish
– inoculating tools
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The Five “I”s: Inspect
• During this step the researcher or clinician observes the microbes macroscopically
• Inspect the colonies macroscopically for color size and texture
The Five “I”s: Identify
• Determine the identity of the unknown microbe using various tests such as biochemical, immunological, and DNA analysis.
• We will use biochemical tests for our Mystery Microbe Project
Selective Media: pH
Selecting for bacterial colonies
Inspect: Macroscopically Identify: Differential and Selective Media
• Selective media enables (selects) one type of bacteria to grow by the addition of a chemical that inhibits the growth of particular biochemical traits of a bacteria
• Differential Media: shows different reactions (i.e. colony color or media change) to determine the biochemistry of bacteria
Selective Media: Antibiotic
Selecting for fungal colonies
Selecting for bacteria that carry the ampicillin resistance gene
‐ Ampicillin
pH 7.3
+ Ampicillin
pH 5.6
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Selective Media: Gram‐Positive or Gram‐Negative
This media only allows gram negative bacteria to grow.
This tells us that E. coli is Gram negative and S. aureus is Gram positive.
Identify: Differential and Selective Media
Differential Media: Blood Agar
Alpha‐hemolysis
Beta‐hemolysis
Gamma‐hemolysis
• Blood agar tests whether the bacteria breaks down erythrocytes
• Alpha‐hemolysis is a partial lysis and will look greenish due to reaction with the hemoglobin • Beta‐hemolysis is the complete breakdown of the erythrocyte
• Gamma‐hemolysis indicates an inability for that bacteria to breakdown erythrocytes
Differential and Selective Media: MacConkey Agar Selects for Gram‐negative and indicates if the bacterium ferments lactose
• Some media are both differential and selective giving you even more information
• Examples:
• MacConkey agar • Mannitol salt agar • This tells us that:
• Since E. coli grows and has pink/red colonies it is Gram‐
negative and is able to ferment lactose
• S. aureus is Gram‐positive
• Since S. enterica grows and has colorless colonies it is Gram‐
negative and is not able to utilize lactose
Differential and Selective Media: Mannitol Salt Agar • Mannitol salt agar (MSA) selects for Gram‐positive bacteria by the addition of 7.5% salt • It differentiates between bacteria that are mannitol fermenters and not with the addition of mannitol and a pH S. aureus
S. epidermidis
indicator.
• When mannitol is This tells us that both S. fermented the pH lowers epidermidis and S. aureus are and the media turns Gram‐positive, however only S. yellow
aureus can ferment mannitol
Identify: Staining Bacteria
• Positive stains
– Dye binds to the specimen
Positive differential stain
• Negative stains
– Dye does not bind to the specimen, but rather around the specimen.
• Both positive and negative stains can be:
Negative simple stain
– Simple: 1 stain, or
– Differential: 2 or more stains
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Differential Positive Stains: Gram Stain Most frequently used stain. Used to determine if bacteria are Gram‐positive or Gram‐negative
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Identify: Microscopy
We measure specimens based on the metric system. We will be looking at bacteria ranging from 1 um to 10 um. Identify: Microscopy
• After the bacteria is stained they must be visualized by a microscope
• The progress of microbiology relies heavily on the introduction of new technologies especially in microscopes
• Without a microscope it would be impossible to characterize the morphology of bacteria.
Microscopy: Contrast
• Contrast is the difference between the intensity of light and dark in the microscopic image
• This is effected by:
– staining the sample
– the amount of light that is allowed through the sample
Microscopy: Properties of Light Light has the ability to:
(c) Absorb‐ the darker portions of the sample are absorbing the light.
Microscopy: Properties of Light Resolution can be increased by using immersion oil. This decreases the refraction that occurs when the light hits the working space between the objective and the slide.
(d) Refract‐ as you magnify your specimen more and more this will decrease resolution. That is why we use oil emersion.
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The Five “I”s: Basic Techniques to Culture Microbes
1. Inoculate
2. Incubate
3. Isolate
4. Inspect
5. Identify
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