Microbial Growth

The Requirements For Growth

1. The growth of a population is an increase in the number of cells.

2. The requirements for microbial growth are both physical and chemical.

Physical Requirements

1. On the basis of preferred temperature ranges, microbes are classified as psychrophiles (cold-living), mesophiles (moderate-temperature-loving), and thermophiles (heat-loving).

a. Psychrophiles can grow at 0° C but optimum is about 15° C.

b. Psychrotrophs can grow at 0° C also but optimum is 20 - 30° C – important in food spoilage.

c. Mesophiles grow best at moderate around 37° C – many pathogens fall in this category.

d. Thermophiles have a growth optimum at around 60° C.

e. Hyperthermophiles have growth optima of 80° C or higher (archaea).

2. The minimum growth temperature is the lowest temperature at which a species will grow, the optimum growth temperature is the temperature at which it grows best, and the maximum growth temperature is the highest temperature at which growth is possible.

3. Most bacteria grow best at pH value between 6.5 and 7.5.

a. Very few grow below a pH of 4, although acidophiles may grow in pH conditions as low as 1.

4. In a hypertonic solution, most microbes undergo plasmolysis; halophiles can tolerate high salt concentrations and may be obligate or facultative.

Chemical Requirements

1. All organisms require a carbon source; chemoheterotrophs use an organic molecule and autotrophs typically use carbon dioxide.

2. Nitrogen is needed for protein and nucleic acid synthesis. Nitrogen can be obtained from the decomposition of proteins or from NH₄⁺ or NO₃^-; a few bacteria are capable of nitrogen (N₂) fixation.

3. On the basis of oxygen requirements, organisms are classified as obligate aerobes, facultative anaerobes, obligate anaerobes, aerotolerant anaerobes, and microaerophiles.

a. Facultative anaerobes can grow in aerobic or anaerobic conditions although tend to grow better in aerobic conditions.

i. Includes E. coli and some yeasts.

b. Aerotolerant anaerobes don’t use O₂ but tolerate it – have SOD or some similar enzyme.

i. Many ferment carbohydrate to lactate, which inhibits growth of aerobes.

c. Microaerophilic aerobes require low O₂ concentrations; they are sensitive to oxygen-derived free radicals.

4. Aerobes, facultative anaerobes, and aerotolerant anaerobes must have the enzymes superoxide dismutase (2 O₂^-. + 2 H⁺---> O₂ + H₂O₂) and either catalase (2 H₂O₂---> 2 H₂O + O₂) or peroxidase (H₂O₂ + 2 H⁺---> 2 H₂O).

5. Other chemicals required for microbial growth include sulfur, phosphorus, trace elements, and, for some microorganisms, organic growth factors.

Culture Media

1. A culture medium is any material prepared for the growth of bacteria in a laboratory.

2. Microbes that grow and multiply in or on a culture medium are known as a culture.

3. Agar is a common solidifying agent for a culture medium.

a. Seaweed extract (polysaccharide), melts at 100°C, solidifies at 40°C, held at 50°C for plating – won’t kill bacteria if mixed into the media.

b. Most bacteria can’t degrade agar.

4. Broth is also a common culture medium.

Chemically Defined Media

1. A chemically defined medium is one in which the exact chemical composition is known.

Complex Media

1. A complex medium is one in which the exact chemical composition varies slightly from batch to batch.

a. Extracts – beef or yeast.

i. Provides vitamins and minerals.

ii. Large proteins are not used directly by most bacteria; peptones are partially digested proteins (acid or enzymatically digested)

iii. Supplies energy, C, N, and S.

2. May be used as nutrient broth or nutrient agar.

Anaerobic Growth Media and Methods

1. Reducing media chemically remove molecular oxygen (O₂) that might interfere with the growth of anaerobes.

2. Thioglycolate combines with dissolved O₂ to deplete in media.

3. Petri plates can be incubated in an anaerobic jar or anaerobic chamber.

4. Sodium bicarbonate and sodium borohydride are mixed with a small amount of water to produce CO₂ and H⁺.

5. A palladium catalyst in the jar combines with the O₂ in the jar and the H⁺ to remove O₂.

Special Culture Techniques

1. Some parasitic and fastidious bacteria must be cultured in living animals or in cell cultures.

2. CO₂ incubators or candle jars are used to grow bacteria requiring an increased CO₂ concentration (capnophiles).

3. Low O₂ and high CO₂ concentrations mimic conditions found in the intestinal tract.

4. CO₂ incubators allow adjustment of CO₂ concentrations to lower or higher than atmospheric when needed by certain aerobic bacteria.

5. Candle jars increase CO₂ concentrations and still leave some O₂ for aerobic capnophiles.

6. Gas packs can generate CO₂ also and are generally used in place of candle jars.

7. Some bacteria require live cells for culture, won’t grow on artificial media.

a. Mycobacterium leprae is grown in armadillos because of their low temperature.

b. Rickettsias, chlamydias (obligate intracellular pathogens) and Treponema pallidum don’t grow on artificial media.

Selective and Differential Media

1. By inhibiting unwanted organisms with salts, dyes, or other chemicals, selective media allow growth of only the desired microbes.

a. Bismuth sulfite agar inhibits gram-positive and most gram-negative bacteria, used to isolate Salmonella typhi.

b. Brilliant green agar inhibits gram-positive and most gram-negative bacteria and is used to isolate Salmonella species.

c. Sabouraud glucose agar has a pH of 5.6; inhibits most bacteria and is used to isolate fungi.

2. Differential media are used to distinguish among different organisms.

a. Blood agar is used to identify organisms that lyse red blood cells (Streptococcus pyogenes)

3. Selective and differential media may be combined.

a. Mannitol salt agar contains 7.5% NaCl and mannitol plus a pH indicator that changes color if mannitol is fermented to acid.

i. Organisms that tolerate high salt concentrations are selected and those that ferment mannitol to acid are differentially identified – likely to be Staphylococcus aureus.

b. MacConkey agar contains bile salts and crystal violate to inhibit gram-positive bacteria and lactose to indicate lactose fermenters (appear as pink colonies) vs. non-lactose fermenters (colorless colonies).

i. Some gram-negative bacteria can’t grow on lactose, which gives it additional selective properties.

Enrichment Culture

1. An enrichment culture is used to encourage the growth of a particular microorganism in a mixed culture.

2. This is a kind of selection, may encourage certain bacteria that are present in low numbers like in soil and fecal samples

Obtaining Pure Cultures

1. A colony is a visible mass of microbial cells that theoretically arose from one cell.

2. Pure cultures are usually obtained by the streak plate method.

Preserving Bacterial Cultures

1. Microbes can be preserved for long periods of time by deep-freezing or lyophilization (free-drying).

2. Deep freezing is done quickly, from -50° down to -95°C.

3. Lyophilization is a quick freeze in which a vacuum pump removes water in a process called sublimation.

The Growth Of Bacterial Cultures

To review: go to Microbial Growth: Cell Cycles (pp. 170-179)

Bacterial Division

1. The normal reproductive method of bacteria is binary fission, in which a single cell divides into two identical cells.

2. Some bacteria reproduce by budding, aerial spore formation, or fragmentation.

Generation Time

1. The time required for a cell to divide or a population to double is known as the generation time.

2. Most bacteria have a doubling time of 1-3 hours, although some may be greater than 24 hours.

3. E. coli may have a doubling time of 20 minutes; get 20 generations in 7 hours, going from one cell to one million cells.

Logarithmic Representation of Bacterial Populations

1. Bacterial division occurs according to a logarithmic progression (two cells, four cells, eight cells, etc.).

Phases of Growth

1. During the lag phase, there is little or no change in the number of cells, but metabolic activity is high.

a. DNA and enzyme synthesis occurs; may last from 1 hour to several days.

2. During the log phase, the bacteria multiply at the fastest rate possible under the conditions provided.

a. Maintained by use of a chemostat – constant supply of fresh media

3. During the stationary phase, there is equilibrium between cell division and death.

a. Nutrients are exhausted and waste products build up; pH increases.

4. During the death phase, the number of deaths exceeds the number of new cells formed.

Direct Measurement of Microbial Growth

1. A standard plate count reflects the number of viable microbes and assumes that each bacterium grows into a single colony; plate counts are reported as number of colony-forming units (CFU).

2. A plate count may be done by either the pour plate method or the spread plate method.

3. In filtration, bacteria are retained on the surface of a membrane filter and then transferred to a culture medium to grow and subsequently be counted.

4. The most probable number (MPN) method can be used for microbes that will grow in a liquid medium; it is a statistical estimation.

a. Dilution series to no growth.

b. Used for microbes that won’t grow on solid media or are grown in liquid differential media for identification purposes.

5. In a direct microscopic count, the microbes in a measured volume of a bacterial suspension are counted with the use of a specially designed slide.

Estimating Bacterial Numbers by Indirect Methods

1. A spectrophotometer is used to determine turbidity by measuring the amount of light that passed through a suspension of cells.

2. An indirect way of estimating bacterial numbers is measuring the metabolic activity of the population (for example, acid production or oxygen consumption).

3. For filamentous organisms such as fungi, measuring dry weight is a convenient method of growth measurement.