Inoculate: Media. Physical State of Media: Liquid. The Five I s: Basic Techniques to Culture Microbes Tools of the Microbiology Laboratory

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1 The Five I s: Basic Techniques to Culture Microbes Tools of the Microbiology Laboratory 1. Inoculate 2. Incubate 3. Isolate 4. Inspect 5. Identify The Five I s: Inoculate Inoculate: Media Classified according to three properties Culture: the propagation of microorganisms with various media 1. Physical state 2. Chemical composition 3. Functional types 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? 1

2 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 Physical State of Media: Solid 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: 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 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 TSA + 5% sheep blood 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 2

3 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 Functional Type: Selective and Differential Chemicals are added to media for the purpose of: testing the biochemistry of the bacteriadifferential media selecting for bacteria that have a specific biochemistry selective media More about these in the Identify portion of the Five I s Incubator: a chamber where temperature is controlled. The Five I s: Incubate Usual laboratory propagation temperatures fall between 20 C and 40 C Atmospheric gases such as O 2 and CO 2 may be required for the growth of certain microbes During incubation, microbes grow and multiply, producing visible growth in the media 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 The Five I s: Isolate The Five I s: Isolate Three basic methods of isolating bacteria Streak Plate 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 3

4 The Five I s: Inspect During this step the researcher or clinician observes the microbes macroscopically Inspect: 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 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: ph Selective Media: Antibiotic Selecting for bacterial colonies Selecting for fungal colonies Selecting for bacteria that carry the ampicillin resistance gene Ampicillin + Ampicillin ph 7.3 ph 5.6 4

5 Selective Media: Gram Positive or Gram Negative Differential Media: Blood Agar This media only allows gram negative bacteria to grow. This tells us that E. coli is Gram negative and S. aureus is Gram positive. Alpha hemolysis Gamma hemolysis Beta 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 Identify: Differential and Selective Media 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 Gramnegative and is able to ferment lactose S. aureus is Gram positive Since S. enterica grows and has colorless colonies it is Gramnegative 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 indicator. When mannitol is fermented the ph lowers and the media turns yellow S. epidermidis S. aureus This tells us that both S. epidermidis and S. aureus are Gram positive, however only S. aureus can ferment mannitol Identify: Staining Bacteria Positive stains Dye binds to the specimen Negative stains Dye does not bind to the specimen, but rather around the specimen. Both positive and negative stains can be: Simple: 1 stain, or Differential: 2 or more stains Positive differential stain Negative simple stain 5

6 Differential Positive Stains: Gram Stain Most frequently used stain. Used to determine if bacteria are Gram positive or Gram negative 31 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. Identify: Microscopy We measure specimens based on the metric system. We will be looking at bacteria ranging from 1 um to 10 um. 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. 6

7 The Five I s: Basic Techniques to Culture Microbes 1. Inoculate 2. Incubate 3. Isolate 4. Inspect 5. Identify 7

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