L6a - Methods of microbiology - culturing microorganisms
Culturing Techniques
Overview of Five Basic Techniques
Inoculate
Introduce a sample into a growth medium to produce a culture.
Incubate
Maintain the inoculated sample under specific environmental conditions to encourage growth.
Isolation
Separate one species from another to obtain pure cultures.
Inspection
Examine the cultures for characteristics or colonies.
Identification
Utilize biochemical tests and other techniques to determine the specific type of microorganism.
Inoculation and Isolation
Inoculation involves introducing a microorganism into a nutrient medium to produce observable growth, called a culture.
Isolation refers to the separation of individual microbial cells, allowing for the formation of distinct colonies from a single bacterium.
Methods of Isolation
Streak Plate Method
Inoculation loop is used to streak a sample across the medium surface to achieve isolation of individual cells.
Pour Plate Method
Sample is diluted serially in liquid agar and poured into Petri dishes to solidify, allowing colonies to develop within the medium.
Spread Plate Method
A diluted sample is spread over the surface of the agar using a sterile tool to separate cells evenly.
Comparison of Methods
Pour Plate vs. Spread Plate
Pour Plate: Colonies can grow both on the surface and within the medium.
Spread Plate: Colonies grow only on the surface of the medium
Media: Nutrients in the Laboratory
Culture Medium Requirements
Must be sterile and contain suitable nutrients for microbial growth.
Incubation conditions must be controlled (temperature, pH).
Classification of Media
By Physical State
Liquid
Broths that remain liquid. do not solidify at temperatures above freezing, flow freely when container is tilted
Semisolid
Contain less than 1% agar; used for motility tests.
Solid
Contain 1-5% agar; allows for colony formation. Liquefiable and nonliquefiable
Liquefiable: Contain agar that can be melted and solidified, enabling the medium to be re-used or modified for different tests.
Nonliquefiable: Maintain a consistent solid state, providing a stable environment for long-term culture storage.
By Chemical Content
Synthetic Media
Compositions are precisely defined.
Complex Media
At least one component is not chemically definable.
Widely used for heterotrophic microorganisms.
Two forms of complex media:
Nutrient broth: Liquid media
Nutrient agar: Solid media
Function of Media
General Purpose Media
Support a variety of organisms (e.g. nutrient agar).
To grow as broad a spectrum of microbes as possible
Enriched Media
Contain additional nutrients to support fastidious bacteria (e.g. blood agar).
Selective Media
Inhibits the growth of certain microbes while promoting others.
Saboraud’s Dextrose Agar: pH of 5.6 discourages bacterial growth. Used to isolate fungi.
Brilliant Green Agar: Green dye selectively inhibits gram-positive bacteria. Used to isolate gram-negative Salmonella.
Bismuth Sulfite Agar: Used to isolate Salmonella typhii. Inhibits growth of most other bacteria.
Differential Media
Distinguish between different types of microorganisms based on visible characteristics (e.g. MacConkey agar).
Blood Agar: Used to distinguish bacteria that destroy red blood cells (hemolysis). Hemolysis appears as an area of clearing around colony.
Both Selective and Differential Media
Used both to distinguish colonies of a desired organism, and inhibit the growth of other microbes.
Mannitol Salt Agar: Selectively isolates staphylococci and differentiates Staphylococcus aureus, which ferments mannitol, producing a yellow zone around the colonies.
MacConkey Agar: used to used for the isolation of gram-negative enteric bacteria and the differentiation of lactose fermenting from lactose non-fermenting gram-negative bacteria. e.g. Salmonella.
Miscellaneous Media
Reducing Media: To grow anaerobes by excluding oxygen.
Carbohydrate fermentation media: contain sugars that can be fermented and a pH indicator; useful for identification of microorganisms
Transport Media: Maintain specimen viability during transfer.
Assay media: used to test the effectiveness of antibiotics, disinfectants, antiseptics, etc
Enumeration Media: Count microbial populations in samples.
Cultivation of Anaerobes
Techniques to culture anaerobic organisms by excluding oxygen using various methods (e.g. reducing agents, anaerobic chambers).
Reducing agents such as thioglycollate or cystein must be added in the growth media. Medium is boiled during preparation to dissolve the components and drive-off the O2
Using anaerobic chamber – most of the air is removed with a vacuum pump and purges with nitrogen. In the presence of palladium catalyst, the hydrogen and last remaining molecules of O2 react, to form water creating anoxic environment.
Using GasPak jar – which also uses hydrogen and palladium catalyst to remove O2.
Using plastic bag/pouches containing calcium carbonate and a catalyst, which produce an anoxic, CO2-rich atmosphere
Microbial growth: Incubation
Incubation: an inoculated sample is placed in an incubator to encourage growth.
Usually the temperature for incubation varies between 20 °C and 40 °C.
Can control atmospheric gases as well.
Can visually recognize growth as cloudiness in liquid media and colonies on solid media.
Microbial growth: Inspection and identification
Using appearance as well as metabolism (biochemical tests) and sometimes genetic analysis or immunologic testing to identify the organisms in a culture.
Pure culture = growth of only a single known species (also called axenic) • Usually created by subculture
Mixed culture = holds two or more identified species
Contaminated culture = includes unwanted microorganisms of uncertain identity, or contaminants.
Measurement of Microbial Growth
Can measure changes in number of cells in a population and in mass of population
Direct Cell Count Methods
Direct microscopic counts, electronic counters, and flow cytometry.
Counting Chambers: Easy, inexpensive, and quick • Useful for counting both eukaryotes and prokaryotes
Direct Microscopic Count:
A specific volume of a bacterial suspension (0.01 mL) is placed on a microscope slide with a special grid.
Advantages: No incubation time required.
Disadvantages: Cannot always distinguish between live/dead bacteria. • Motile bacteria are difficult to count.
Membrane Filters: Cells filtered through special membrane that provides dark background for observing cells • Cells are stained with fluorescent dyes
Flow Cytometry: Microbial suspension forced through small orifice with a laser light beam.
• Specific antibodies can be used to determine size and internal complexity 52 Direct Measurement of Cell
Viable Counting Methods
Spread Plate and Pour Plate: Count colonies to estimate the number of viable cells.
Results expressed as CFU.
Advantages:• Measures viable cells
Disadvantages:• Takes 24 hours or more for visible colonies to appear. • Only counts between 25 and 250 colonies are accurate.
Membrane filter technique: bacteria from aquatic samples are trapped on membranes
membrane soaked in culture media
colonies grow on membrane
Most Probable Number (MPN): Used mainly to measure bacteria that will not grow on solid medium.
Dilute a sample repeatedly and inoculate several broth tubes for each dilution point.
Count the number of positive tubes in each set.
Statistical method: Determines 95% probability that a bacterial population falls within a certain range.
Indirect Measurement Methods
Turbidity
Measure cloudiness to estimate population size.
Metabolic Activity
Assess metabolic products produced by growing bacteria.
Dry Weight
Weigh the cellular biomass produced in culture.
Maintenance and Disposal of Cultures
Stock cultures are used for long-term storage, while unused cultures must be properly sterilized and disposed of.