Microbial Growth & Culture Media

Microbial Growth and Culture Media

• A culture medium provides nutrients needed for microorganism growth in a lab
• Some bacteria thrive in almost any culture medium
• Some require special media
• Some cannot grow in any nonliving medium
• An inoculum is the microbe introduced into a culture to initiate growth
• Microbes growing/multiplying in/on a medium are a culture

Types of Media

• A wide variety of media exists for lab microorganism growth
• Most are commercially available with premixed components
• They require only water addition and sterilization
• Media are constantly refined for isolation and identification of bacteria
• Agar is often added as a solidifying agent

Agar Component

• Agar which is derived from marine algae has been used in food as a thickener
• Few microbes can degrade agar, it remains solid
• Agar liquefies at 100°C (the boiling point of water)
• At sea level, agar remains liquid until 40°C
• Agar is kept in water baths at 50°C for lab use
• Once solidified, agar can be incubated at temperatures near 100°C before liquefying again
• This is helpful when growing thermophilic bacteria
• Agar media is in test tubes or Petri dishes
• Test tubes with solidified contents at an angle are slants
• Agar solidified in a vertical tube is a deep
• Petri dishes have lids to prevent contamination
• Petri dishes are called Petri plates once filled

Chemically Defined Medium

• Media must provide an energy source, carbon, nitrogen, sulfur, phosphorus, and organic growth factors
• A chemically defined medium has a known chemical composition
• For chemoheterotrophs, it contains organic growth factors
• Glucose is an example ingredient, used for growing chemoheterotrophs like E. coli

Complex Media

• Most heterotrophic bacteria and fungi are grown on complex media
• Complex media have nutrients from yeasts, meat, plants, or digested proteins
• The exact chemical composition varies slightly from batch to batch
• Nutrient broth is a complex medium in liquid form
• Nutrient agar has added agar

Anaerobic Growth Media

• Cultivating anaerobic bacteria is difficult because they can be killed by oxygen exposure
• Reducing media such as sodium thioglycolate is used for these organisms
• Sodium thioglycolate chemically combines with/depletes oxygen in the culture medium.
• Mixing water with sodium bicarbonate and sodium borohydride generates hydrogen and carbon dioxide.
• Hydrogen and oxygen combine to form water using a palladium catalyst and remove oxygen.
• Methylene blue, an anaerobic indicator, turns blue when oxidized and colorless when oxygen is removed

Selective and Differential Media

• Selective and differential media are used to detect specific disease-related microorganisms
• Selective media suppress the growth of unwanted bacteria and promote growth of desired microbes
• Bismuth sulfite agar encourages the growth of Salmonella typhi while suppressing other bacteria

Differential Media

• Differential media distinguishes colonies of desired organisms from others on the same plate
• Blood agar (containing red blood cells) identifies bacteria that destroy red blood cells
• Streptococcus pyogenes creates a clear ring (beta-hemolysis) around colonies, indicating blood cell lysis

Mannitol Salt Agar

• Selective and differential characteristics can be combined
• Media such as 7.5% sodium chloride discourages growth of certain organisms, selects for Staphylococcus aureus
• Mannitol salt agar is a differential medium
• The medium will turn yellow if bacteria ferments mannitol to acid (Staphylococcus aureus)
• High salt concentrations also make this medium selective as it prevents growth for most bacteria

Obtaining Pure Cultures

• Infectious materials (pus, soil, water, food) contain different bacteria
• Plating these materials create exact copies of the original organism in colonies
• Streak plate methods are usually used to obtain pure cultures
• Bacterial growth refers to an increase in bacterial numbers, not individual cell size
• Bacteria normally reproduce by binary fission
• Budding is used by a few bacterial species, which involves a small initial outgrowth enlarging to the parent cell's size

Streak Plate Method

• Streak plate method is the most common isolation method
• A sterile loop is dipped into a mixed culture and streaked across the nutrient medium surface
• The streak plate method is best when the target organism is present in large numbers
• If the target organism is scarce, its numbers need to be increased by selective enrichment

Bacterial Division

• Bacterial growth involves increasing the bacterial cell number not the size of the individual cells
• Bacteria reproduce through binary fission

Generation Time

• Generation time measures how long it takes for a cell to divide doubling the population
• Generation time varies, depending on the organism and the environmental conditions
• Most bacteria have generation times between 1 and 3 hours
• Some bacteria require >24 hours per generation
• Unchecked binary fission can result in massive cell numbers
• Under favorable conditions, E. coli doubles every 20 minutes and over 1 million cells will form after 20 generations

Bacterial Growth Curve

• Lag Phase involves adapting to conditions and no immediate increase in cell numbers
• Log Phase is characterized by exponential increase in population
• Stationary Phase sees population equilibrium resulting from microbial deaths balancing new cell production
• Death Phase refers to a population decrease at a logarithmic rate

Direct Measurement of Microbial Growth

• Serial dilutions are performed, in which the original inoculum is diluted in a series of tubes
• Each tube has one-tenth the microbial cells of the preceding tube.
• Dilutions are used to inoculate Petri plates, on which colonies grow and are counted
• Number of bacteria in the original sample is estimated using this count
• Colony-forming units (CFU) helps count colonies.

Plate Counts

• In serial dilutions, the original inoculum is diluted in a series of dilution tubes
• Each succeeding dilution tube will have only one-tenth the number of microbial cells as the preceding tube
• Then, samples of the dilution are used to inoculate Petri plates, on which colonies grow and can be counted
• This count is then used to estimate the number of bacteria in the original sample
• Colony-forming units (CFU) can then be calculated

Preparing Plates for Plate Counts

• Plate counts use pour plate/spread plate methods
• In either method, 1 ml or 0.1 ml of dilutions are introduced into a Petri dish with nutrient medium in which the agar is kept liquid
• The agar cools at about 50°C
• Gentle plate agitation mixes the medium with the sample
• The plate is incubated once the agar has solidified
• In the pour plate technique, colonies will grow within/on the surface of the agar plate

Direct Microscopic Count

• Petroff-Hausser cell counters can be used to directly count bacteria with a microscope
• Bacterial suspension fills the shallow volume over the squares by capillary action within the cell counter
• The known volume will be calculated by the depth under the cover glass and the area of the squares
• All cells within the large squares are counted to find the numbers are averaged
• The number is then calculated to estimate the number of cells in the original sample.

Turbidity Estimation

• Spectrophotometers can estimate bacterial numbers indirectly
• The amount of light striking the detector is proportional to the number of bacteria under standardized conditions
• The less light transmitted, the more bacteria are in the sample
• Turbidity can be reported as % transmittance or absorbance