Culturing Microbes: Media, Techniques, and Cell Counting Methods L 19 E 2

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Growth Media and Laboratory Culture

Types of Culture Media

Culturing microbes in the laboratory requires nutrient solutions called culture media. These media must be sterile before use to prevent contamination. There are two broad classes of culture media:

Defined Media: Composed of precise amounts of highly purified inorganic and organic chemicals added to distilled water. The exact chemical composition is known, allowing for reproducibility and control over nutrients.
Complex Media: Made from digests of microbial, animal, or plant products such as casein, beef extract, soybean (tryptic soy broth), and yeast extract. The exact composition is unknown, but these media provide a wide variety of nutrients.

Comparison Table: Defined vs. Complex Media

Feature	Defined Media	Complex Media
Composition	Known, precise chemicals	Unknown, digests/extracts
Reproducibility	High	Variable
Use	Research, physiological studies	Routine culturing, fastidious organisms

Specialized Media Types

Enriched Media: Complex media supplemented with highly nutritious materials (e.g., serum or blood). Used for culturing fastidious (nutritionally demanding) microbes.
Selective Media: Contains compounds that selectively inhibit the growth of some microbes but not others, allowing for the selection of particular microbes.
Differential Media: Contains indicators (usually dyes) that detect specific metabolic reactions, distinguishing different species. Commonly used in diagnostic laboratories.

Nutritional Requirements & Biosynthetic Capabilities

Fastidious Organisms and Media Preparation

Some microbes are fastidious, meaning they have complex nutritional requirements and need additional nutrients. Complex media are often preferred for these organisms because they contain a wide variety of molecules, including trace elements and growth factors.

Different microbes require different nutrients; knowing the requirements of the organism of interest is essential for providing nutrients in the appropriate form and proportion.
Defined media are useful for studying metabolic pathways, while complex media are practical for routine culturing.

Laboratory Culture Techniques

Preparation and Sterilization

After mixing media components, the medium must be sterilized to prevent contamination. The most common method is autoclaving, which uses heat and pressure to kill all living cells, resulting in sterile media.

Pure Culture: A culture containing only a single species or strain of microorganism.
Contaminants: Unwanted organisms in the growth medium.
Aseptic (Sterile) Technique: Procedures used to prevent contamination during culture handling, such as using sterile inoculating loops or needles.
Solid media in Petri plates are essential for maintaining pure cultures by allowing the isolation of individual colonies.

Solidifying Media and Colony Formation

Liquid culture media can be solidified by adding a gelling agent (usually agar or gelatin) at 1-2% concentration.
Heating melts the gelling agent; cooling solidifies it.
Solid media restricts the movement of cells, allowing them to form isolated masses called colonies.
Colonies vary in shape, size, and color depending on media composition, culture conditions, and nutrient availability.
Plates with more than one colony type are considered contaminated.

Microscopic Counts of Microbial Cell Numbers

Microscopic Cell Counting

Total cell counts can be obtained by observing and enumerating cells under a microscope. This can be done with cells dried on slides or in liquid samples using a cell counting chamber (hemocytometer).

Counting chamber has a grid with squares of known area and volume, allowing calculation of cells per milliliter.

Limitations of Microscopic Cell Counts

Cannot distinguish between live and dead cells without special stains.
Precision is difficult; small cells may be overlooked.
Phase-contrast microscope required if stains are not used.
Low-density suspensions (<106 cells/ml) are hard to count.
Motile cells must be immobilized or killed.
Debris can be mistaken for cells.

Applications in Microbial Ecology

Stains such as DAPI (binds DNA) are used for general visualization.
Fluorescent stains can differentiate live and dead cells.
Phylogenetic stains can determine proportions of Bacteria or Archaea.

Viable Counting of Microbial Cell Numbers

Viable Cell Counts (Plate Counts)

A viable cell is one that can divide and form offspring. Viable counting methods rely on the ability of cells to grow and form colonies on agar media. The assumption is that each colony arises from a single cell.

Spread-Plate Method: A volume of diluted culture is spread over the surface of an agar plate and incubated.
Pour-Plate Method: A volume of culture is placed in an empty Petri plate, molten agar is added, and the mixture is swirled before incubation.

Serial Dilutions and Colony Counting

Samples are diluted to achieve a countable number of colonies (preferred range: 30-300 colonies per plate).
Serial dilutions are performed to reach suitable concentrations for plating.

Sources of Error in Plate Counting

Colony number depends on inoculum size, culture viability, medium, and incubation conditions.
Mixed cultures may have different growth rates, affecting colony visibility.
Pure cultures yield more accurate counts.
Errors can arise from inaccurate pipetting, cell clumping, insufficient mixing, or improper agar conditions.

Colony-Forming Units (CFUs)

Results are expressed as colony-forming units (CFUs) rather than actual cell numbers to account for viable cells not forming visible colonies and cell clumps forming single colonies.
Replicate plates are used for accuracy; average counts are reported.

Great Plate Count Anomaly

Microscopic counts often reveal more organisms than are recoverable on plates of any single medium.
Plate counts can be unreliable for assessing total cell numbers in natural samples.
Discrepancies arise because microscopic counts include dead cells and because different organisms have different nutritional and growth requirements.

Turbidimetric Measures of Microbial Cell Numbers

Optical Density (OD) and Turbidity

During exponential growth, all cellular components increase proportionally. Instead of counting cells directly, changes in protein, DNA, or dry weight can be measured. Microbial cells scatter light, allowing estimation of cell numbers based on turbidity (cloudiness) of the suspension.

Turbidity increases with cell number; more cells scatter more light, resulting in less light passing through the sample.
This property is used as a proxy for cell number.

Measuring Turbidity with a Spectrophotometer

Spectrophotometer: Device that measures turbidity by passing light through a suspension and measuring the unscattered light at a specific wavelength (commonly 540 nm).
Optical Density (OD): Unit of turbidity; OD is the amount of light absorbed at a given wavelength.

Using OD to Estimate Cell Numbers

For unicellular organisms, OD is proportional to cell number within certain limits.
OD readings can be used instead of total or viable cell counts only after a standard growth curve is prepared to relate cell number to turbidity.
At high cell concentrations, light scattering can be nonlinear due to multiple scattering events.
Cell size differences mean that the same OD does not always correspond to the same cell number for different cultures.
Once a standard curve is established, OD readings can be used to delineate cell numbers when comparing growth curves.

Advantages and Disadvantages of Turbidimetric Methods

Advantages: Quick and easy, typically does not kill or disturb the sample, and the same sample can be checked repeatedly.
Disadvantages: Can be inaccurate if cells do not grow in uniform suspensions or if cells attach to the sides of the vessel. Shaking helps but is not always effective. OD is not quantitative by itself and must be linked to a quantitative measure.

Key Equation: Relating OD to Cell Number

Once a standard curve is established, the relationship can be expressed as:

where a and b are empirically determined constants for the specific organism and conditions.