MICROBIAL GROWTH {ST1}

MICROBIAL GROWTH {ST1} BY RANJEET RAMAN

The term microbial growth refers to the growth of a population (or an increase in the number of cells), not to an increase in the size of the individual cell. Cell division leads to the growth of cells in the population.

Two Types of Asexual Reproduction in Microbes: 1.) Binary Fission - Bacterial reproduction occurs through fission, a primitive form of cell division that does not employ a spindle fiber apparatus. [A spindle fiber apparatus made of protein filaments is responsible for moving the chromosomes around during cell division (mitosis & meiosis) in most eukaryotic cells.

Bacteria do not have these structures.] The bacterial cell doubles in size and replicates its chromosome. Following DNA replication, the two chromosomes attach to separate sites on the plasma membrane, and the cell wall is laid down between them, producing two daughter cells.

2.) Budding - A few bacteria and some eukaryotes (including yeasts) may also replicate by budding, forming a bubble-like growth that enlarges and separates from the parent cell.

I. Microbial Growth A. Phases of Growth - A microbial lab culture typically passes through 4 distinct, sequential phases of growth that form the standard bacterial growth curve: (Not all growth phases occur in all cultures).

1. Lag Phase - In the lag phase, the number of cells doesn't increase. However, considerable metabolic activity is occurring as the cells prepare to grow. (This phase may not occur, if the cells used to inoculate a new culture are in the log phase & provided conditions are the same).

2. Log Phase (logarithmic or exponential phase) - cell numbers increase exponentially; during each generation time, the number of cells in the population increases by a factor of two). The number of microbes in an exponentially increasing population increases slowly at first, then extremely rapidly.

Organisms in a tube of culture medium can maintain log growth for only a limited time, as nutrients are used up, metabolic wastes accumulate, microbes suffer from oxygen depletion.

3. Stationary Phase - The number of cells doesn't increase, but changes in cells occur: cell become smaller and synthesize components to help them survive longer periods without growing (some may even produce endospores); the signal to enter this phase may have to do with overcrowding (accumulation of metabolic byproducts, depletion of nutrients, etc.).

4. Death Phase - In this phase, cells begin to die out. Death occurs exponentially, but at a low rate. Death occurs because cell have depleted intracellular ATP reserves. Not all cells necessarily die during this phase!

B. Continuous Culture of Microbes In the lab, cultures usually pass through all growth phases - not in nature. In nature, nutrients continuously enter the cell's environment at low concentrations, and populations grow continually at a low but steady rate.

The growth rate is set by the concentration of the scarcest or limiting nutrient, not by the accumulation of metabolic byproducts - in nature there is always some other microbe that can use these metabolic byproducts for their own metabolism. In the lab, we have to continually replace the media.

II. Measuring Numbers of Microbe A. Indirect Measurements (measure a property of the mass of cells and then ESTIMATE the number of microbes) 1. Turbidity – Can hold tube up to the light and look for cloudiness as evidence of growth (difficult to detect slight growth). A spectrophotometer can measure how much light a solution of microbial cell transmits;

the greater the mass of cells in the culture, the greater its turbidity (cloudiness) and the less light that will be transmitted. Disadvantages: Not sensitive in terms of numbers of bacterial cells & not useful for detecting minor contamination.

2. Metabolic Activity - 3 ways: a. The rate of formation of metabolic products, such as gases or acids, that a culture produces. b. The rate of utilization of a substrate, such as oxygen or glucose. c. The rate of reduction of certain dyes. Ex. methylene blue becomes colorless when reduced.

B. Direct Measurements - Give more accurate measurements of numbers of microbes. 1. Direct Counts - Coulter Counter - electronic counter; rapid & accurate only if bacterial cells are the only particles present in the solution. [gives a total count - live & dead cells].

3. Plate Count – Bacterial colonies are viewed through the magnifying glass against a colony-counting grid; called a Quebec colony counter. [gives a viable count]

4. Filtration - A known volume of liquid or air is drawn through a membrane filter by vacuum. The pores in the filter are too small for microbial cells to pass through. Then the filter is placed on an appropriate solid medium and incubated.

The number of colonies that develop is the number of viable microbial cell in the volume of liquid that was filtered. This technique is great for concentrating a sample, ex. a swimming pool, where small populations may go undetected using some other methods. [gives a viable count]

III. Growth Factors - Microbes can exist in a great many environments because they are small, easily dispersed, need only small quantities of nutrients, are diverse in their nutritional requirements.

A. Physical Factors 1. pH – bacteria can classified as: a. acidophiles (acid-loving) – grow best at a pH of 1 to 5.4; Ex. Lactobacilllus (ferments milk) b. neutrophiles – exist from pH to 5.4 to 8.5; most bacteria that cause human disease are in this category. c. alkaliphiles (base loving) – exist from pH to 7.0 to 11.5; ex. Vibrio cholerae (causes cholera)

2. Temperature – bacteria can be classified as: a. Psychrophiles (cold-loving) 15oC to 20oC; some can grow at 0oC. b. Mesophiles - grow best between 25oC and 40 C; human body temp is 37oC. c. Thermophiles (heat-loving) – 50oC to 60oC; found in compost heaps and in boiling hot springs.

3. Moisture – only the spores of sport-forming bacteria can exist in a dormant state in a dry environment.

4. Hydrostatic pressure – pressure exerted by standing water (ex. lakes, oceans, etc.); some bacteria can only survive in high hydrostatic pressure environments (ex. ocean valleys in excess of 7000 meters); the high pressure is necessary to keep their enzymes in the proper 3-D shape; without it, the enzymes lose their shape and denature and the cell dies.

5. Tonicity (hypotonic, hypertonic, isotonic) – The use of salt as a preservative in curing meats and the use of sugar in making jellies is based on the fact that a hypertonic environment kills or inhibits microbial growth. Halophiles (salt lovers) inhabit the oceans.

6. Radiation – UV rays and gamma rays can cause mutations in DNA and even kill microorganisms. Some bacteria have enzyme systems that can repair some mutations. B. Oxygen Requirements 1. strict or obligate anaerobes – oxygen kills the bacteria; ex. Clostridium tetani

2. strict or obligate aerobes – lack of oxygen kills the bacteria; ex. Pseudomonas 3. facultative anaerobes – can shift their metabolism (anaerobic if oxygen is absent or aerobic if oxygen is present); ex. E. coli, Staphylococcus

4. aerotolerant – the bacteria don’t use oxygen, but oxygen doesn’t harm them; ex. Lactobacillus 5. microaerophiles – like low oxygen concentrations and higher carbon dioxide concentrations; ex. Campylobacter

C. Nutritional (Biochemical) Factors – Nutrients needed by microorganisms include: Carbon – carbon containing compounds are needed as an energy source (ex. glucose) and for building blocks. Nitrogen - needed for amino acids and nucleotides; some can synthesize all 20 amino acids; others have to have some provided in their medium.

Sulfur – needed for amino acids, coenzymes, Phosphorus – needed for ATP, phospholipids, and nucleotides Vitamins – a vitamin is an organic substance that an organism requires in small amounts and that is typically used as a coenzyme; many bacteria make their own, but some are required in the medium;

microbes living in the human intestine manufacture vitamin K, needed for blood clotting, and some of the B vitamins, thus benefiting their host. Certain trace elements – ex. copper, iron, zinc, sodium, chloride, potassium, calcium, etc.; often serve as cofactors in enzymatic reactions.

III. CULTURING BACTERIA A. Methods of Obtaining Pure Cultures (a culture that contains only 1 species of organism) 1. The Streak Plate Method – Bacteria are picked up on a sterile wire loop, and the wire is moved lightly along the agar surface, depositing streaks of bacteria on the surface.

The loop is flamed and a few bacteria are picked up from the region already deposited and streaked onto a new region. Fewer and fewer bacteria are deposited as the streaking continues, and the loop is flamed after each streaking. Individual organisms (individual cells) are deposited in the region streaked last.

After the plate is incubated at a suitable growth temperature for the organism, small colonies (each derived from a single bacterial cell) appear. The loop is used to pick up a portion of an isolated colony and transfer it to another medium for study. The use of aseptic technique assures that the new medium will contain organisms of a single species.

IV. CULTURE MEDIA A. Types of Media 1. Synthetic medium – prepared in the lab from materials of precise or reasonably well-defined composition. 2. Complex medium – contains certain reasonably familiar materials but varies slightly in chemical composition from batch to batch (contains extracts from beef, yeasts, blood); ex. nutrient agar, nutrient broth

B. Selective & Differential Media (we will learn about these in detail in lab!) 1. Selective – one that encourages the growth of some bacteria but suppresses the growth of others. 2. Differential – has an ingredient that causes an observable change in the medium when a particular biochemical reaction occurs (ex. a color or pH change).

C. Controlling Oxygen Content of Media 1. Candle jars – the inoculated tube or plate is placed in a jar; a candle is lit before the jar is sealed; the burning candle uses the oxygen in the jar and adds carbon dioxide to it; when the carbon dioxide extinguishes the flame, condition are optimum for the growth of microorganisms that require small amounts of carbon dioxide (ex. Neisseria gonorrhoeae)

2. Thioglycollate medium – oxygen-binding agent added to the medium to prevent oxygen from exerting toxic effects on anaerobes; media is usually dispensed in sealed screw-cap tubes. 3. Anaerobic Chamber (Brewer Jar) – A catalyst is added to a reservoir in the lid of the jar. Water is added to the gas- pak.

Water is converted into hydrogen gas and carbon dioxide. The hydrogen gas can then bind with any oxygen in the jar to form water. A methylene blue test strip is included in the jar to ensure that anaerobic conditions are reached. When oxidized (oxygen is present) the strip is blue; when reduced (no oxygen), the strip is clear.

H2O -----------> CO2 + H2 H2 + O2 -----------> H2O

MICROBIAL GROWTH {ST1}