CHAPTER 6  MICROBIAL GROWTH

 

Growth in microbes refers to an increase in the number of cells, not the size of cells.

 

 

REQUIREMENTS FOR GROWTH—PHYSICAL REQUIREMENTS

 

TEMPERATURE

Every bacterial species has an optimum (ideal) growth temperature, at which it grows best and numbers increase at the fastest possible rate.   

 

Minimum growth temperature—the lowest temperature at which the species can grow at all

Maximum growth temperature –the highest temperature at which the species can grow at all

 

The optimum growth temperature is usually very close to the maximum.

 

Microbes are classified into 3 groups, based on preferred growth temperature:

   1. Psychrophiles (cold-loving microbes)—these can grow at  0o C.  However, they vary in their optimum growth temperature.

        a. One group prefers 15o C and cannot grow in temperatures much higher than that.

        b. Another group prefers 20 - 30o C. These are called psychrotrophs or facultative psychrophiles and often cause food spoilage in refrigerators over days of time. Because many bacteria grow very slowly at cool temperatures, refrigeration is a common method of food preservation. Even psychrophiles grow slowly in cool temperatures, and only a few pathogenic bacteria are able to grow at all.     

 

   2. Mesophiles (moderate temperatures)--these grow best at temperatures of 25 - 40o C. This is the most common type of microbe and includes all microbes of medical importance. Human body temperature is 37o C and disease-causing microbes must be able to grow well at that temperature.

 

   3. Thermophiles (heat-loving microbes) --optimum growth temperatures of 50 - 60o C. Many of these cannot grow below 45o C. They are found in locations such as compost piles and hot springs.

 

Some archaea have an optimum growth temperature of 80o C or more. These are called extreme thermophiles or hyperthermophiles. They are found in volcanic hot springs and near deep-sea thermal vents.

 

 

pH

Most bacteria grow best at a pH of 6.5 - 7.5 (neutral or near neutral). Most bacteria do not grow at all below a pH of about 4 but a few acidophiles do tolerate acidity.

 

Molds and yeasts prefer a pH of 5 - 6, but tend to grow at least some over a wide range of pH.

 

Acid foods such as pickles and sauerkraut usually do not undergo bacterial spoilage. Alkalinity could also be used to preserve food, but high pH tends to make foods bitter and slimy, so this method of preservation is not desirable.

 

As bacterial cultures grow in a closed container, they frequently produce acids as waste products, so microbiological lab media usually contain buffers to prevent drastic shifts in pH.

 

OSMOTIC PRESSURE

 

If bacteria are surrounded by a solution with a high osmotic pressure (high solute concentration), water tends to leave the bacterial cell (plasmolysis). This can slow growth or even kill the cell, so most bacteria must be grown in media with high water and low solute content. High levels of salt or sugar can be used for food preservation. There are some exceptions to this:

   Extreme halophiles—tolerate very high salt concentrations

      Obligate halophiles—actually require an extremely high salt concentration for growth, possibly up to 30% salt

      Facultative halophiles—can grow at salt concentrations up to 2%, but do not require them. A few of these can even tolerate 15% salt.

 

Although bacteria frequently find themselves in surroundings with a lower osmotic pressure than that inside the cell, the cell wall usually prevents excessive water from entering the cell. If osmotic pressure of a solution is extremely low, such as pure distilled water, some microbes might be damaged.

 

 

REQUIREMENTS FOR GROWTH—CHEMICAL REQUIREMENTS

 

CARBON

All microbes require carbon for growth. Carbon is needed for synthesis of all organic compounds. Half the dry weight of a bacterial cell is carbon. 

     Chemoautotrophs and photoautotrophs get their carbon from CO2

     Chemoheterotrophs must get theirs from organic compounds, which they also use for energy

 

 

NITROGEN, SULFUR, PHOSPHORUS

 

NITROGEN

Nitrogen makes up about 14% of the dry weight of a bacterial cell. The main use of nitrogen is to form amino acids for protein synthesis. It is also required for synthesis of DNA, RNA, and ATP. Various ways organisms obtain nitrogen:

    1. Break down proteins and recycle the amino acids into new proteins and other nitrogenous compounds.

   2. Use nitrogen from ammonium ions (NH4+)

   3. Use nitrogen from nitrates (compounds that dissociate to form nitrate ions  NO3 -

   4. Use gaseous nitrogen from the atmosphere. Bacteria able to do this are called nitrogen-fixing bacteria. These may live free in the soil, but often they are found within nodules on the roots of legumes—plants such as clover, soybeans, etc. This adds nitrogen to the soil—like free fertilizer from the air.  Rhizobium and Bradyrhizobium are the most important genera.

 

SULFUR

 

Used to synthesize sulfur-containing amino acids and vitamins. Sources include sulfate ions (SO42-), hydrogen sulfide, and the sulfur-containing amino acids.

 

PHOSPHORUS

Required for the synthesis of nucleic acids and the phospholipids of cell membranes as well as ATP. The phosphate ion (PO43-) is an important source.

 

Smaller amounts of potassium, magnesium,  and calcium may also be required. These mostly function as cofactors for enzymes.

 

TRACE ELEMENTS

 

Microbes require very small amounts of iron, copper, zinc, and molybdenum. These are called trace elements. They also often act as cofactors.

 

OXYGEN

 

We think of oxygen as essential to life—which it is for many organisms—but it also at the same time is a toxic gas. Toxicity:

   1. Singlet oxygen—normal molecular oxygen (O2) that has been boosted to a higher energy state and is extremely reactive. It is used in phagocytic cells to help eliminate ingested bacteria.

 

   2. Superoxide free radicals (O2 -)—very unstable and steal electrons from other molecules, destroying cellular components. These free radicals are formed during normal metabolism if oxygen is present. Organisms must produce special enzymes to neutralize them or suffer damage or death to cells. One of the main protective enzymes is superoxide dismutase (SOD).

 

O2 -  +  O2 -  + 2 H+   SUPEROXIDE DISMUTASE      H2O2  +  O2

Organisms that cannot produce this enzyme may be killed by the presence of oxygen.

 

   3. The hydrogen peroxide produced in the above reaction is also toxic. Enzymes to neutralize peroxide are also needed. Many microbes produce one called catalase.

 

  2 H2O2  à   2 H2O  +  O2

 

Another peroxide-neutralizing enzyme is peroxidase:

 

     H2O2  +  2H+  à 2 H2O

 

   4. Hydroxyl radical (OH-)—another intermediate form of oxygen produced in very small amounts during normal aerobic metabolism and in much greater amounts by ionizing radiation.

 

OXYGEN REQUREMENTS OF MICROBES

   1. Obligate aerobes (aerobic microbes)—these require oxygen to live and grow (this description also fits us!). They must produce enzymes that counteract the toxic effects of oxygen. The advantage of using oxygen is that aerobic cellular metabolism is the most efficient way to produce energy.

 

   2. Facultative anaerobes—can live and grow well either with or without oxygen. However, energy production is more efficient with oxygen present. These must produce protective enzymes. 

                   With oxygen available--aerobic cellular respiration

                   Without oxygen--fermentation

 

   3. Obligate anaerobes (strict anaerobes)—do not use oxygen in their metabolic reactions and cannot live in its presence. These do not carry the genes for making the protective enzymes. Energy is produced by fermentation or anaerobic cellular respiration.

 

   4. Aerotolerant microbes—do not use oxygen but can at least partly neutralize its toxic effects, so they can grow fairly well to very well in its presence. Energy is produced mainly by fermentation.

 

   5. Microaerophiles—must have oxygen but in very low levels. These require a little in at least some of their metabolic reactions but cannot tolerate normal atmospheric levels.  Energy is produced by fermentation.

 

The technique we use in lab to show these differing requirements is a special growth medium called fluid thioglycolate medium. This is placed in a deep test tube. It contains a very small concentration of agar to thicken it slightly, plus a chemical called sodium thioglycolate to tie up free oxygen. It is described as a reducing medium.

 

ORGANIC GROWTH FACTORS

 

These are essential organic compounds an organism cannot synthesize. Vitamins are one example. These function as coenzymes and are required for essential metabolic reactions.

 

Many bacteria can synthesize all their own vitamins (humans can make only a few), but some do require that certain vitamins be supplied. Other organic growth factors for bacteria include amino acids, purines,  and pyrimidines.

 

 

CULTURE MEDIA

 

A nutrient material prepared for the growth of microbes in the lab is called a culture medium. The microbes that grow are called a culture. Bacteria vary in their requirements:

   Some grow on almost any medium

   Some have special, unusual requirements

   Some do not grow on any nonliving medium

 

Requirements for a culture medium:

   1. Must have the right nutrients for the microbe.

   2. Must supply sufficient moisture.

   3. Must have the proper pH, and often a buffer to maintain it.

   4. Must supply suitable level of oxygen.

   5. Medium must be sterile before intended microbes are added.

   6. Must be incubated at the proper temperature.

 

Modern media are purchased in a dried form. For use, the proper amount of water is added and the mixture is sterilized.

 

Media can be used in a liquid form—this is called a broth. This type is generally used in a test tube.

 

Media can also be solid. Usually a polysaccharide called agar, derived from seaweed, is added to solidify a medium. Agar is chosen because:

 

   1. Microbes do not break it down, so the medium remains solid as microbes grow.

 

   2. Once solidified, agar does not melt until it reaches 100o C (boiling). This means it remains solid in all normal incubation temperatures.

 

   3. Once melted, agar remains liquid until the temperature reaches about 40o C. Bacteria can be added to liquid agar which has been held at a temperature slightly above 40o C. After adding the bacteria, the agar can be quickly cooled and most bacteria are not harmed.

 

Agar media are used in test tubes or Petri dishes. If the agar in a test tube is solidified while the tube is tilted, the tube is called a slant. This gives a larger surface area for growth. If the tube is left vertical while the agar sets, this is called an agar deep.

 

A Petri dish containing agar is called a plate.

 

GENERAL LAB MEDIA

 

Used for most routine work, these media are prepared with a variety of ingredients such as yeasts, meat, plant products, and sometimes partly digested proteins from these sources are included. Other ingredients sometimes include blood and milk. All of this provides an energy source as well as carbon, nitrogen, sulfur, vitamins, and other growth factors, and will support growth of the great majority of microbes. Relatively large amounts of protein and fragments of proteins (peptones) are included .

 

This general growth medium is called nutrient broth if left liquid and nutrient agar if agar is added to solidify it.

 

Requirements of some organisms are very simple. Escherichia coli, is a good example of a chemoheterotroph with very simple requirements. Glucose is the only organic growth factor needed. With it plus some electrolytes and water, E. coli  can synthesize everything else it needs.   TABLE 6.2  P. 168

 

The other extreme is microbes that are described as fastidious. These require many organic growth factors and sometimes have unusual needs. An example of one of these, Neisseria gonorrhoeae  is shown in TABLE 6.3  P. 169

 

 

ANAEROBIC GROWTH MEDIA AND METHODS

 

Remember, strict or obligate anaerobes do not tolerate the presence of oxygen at normal atmospheric levels, although aerotolerant anaerobes can.  Special media and techniques are required for strict anaerobes.

 

   1. Special reducing media such as fluid thioglycolate medium can be used. Although this medium is slightly thickened with a small amount of agar, it basically is a liquid medium in tubes. Individual colonies cannot be seen.

 

   2. If solid media are required, the plates are inoculated and quickly put into a special jar. Modern technique involves then activating a packet of chemicals and sealing the jar.  The chemical reactions occurring in the sealed jar use up all the oxygen and leave anaerobic conditions.

 

   3. In large labs, an anaerobic glove box chamber is used. This is a sealed transparent chamber which has all room air pumped out and replaced with inert gases after all microbes and necessary supplies have been placed inside. From this point on, work is done by using rubber gloves built in to the walls of the chamber with air-tight seals. Worker insert their hands into the gloves and work without opening the chamber to room air. This means that the microbes do not get any exposure to oxygen at all, which is ideal.

 

4. A new method being used in clinical labs involves adding an enzyme called oxyrase to solid growth medium in a special Petri dish called an OxyPlate. This enzyme breaks down reduces oxygen to water and provides an anaerobic environment.

 

 

SPECIAL CULTURE TECHNIQUES

 

     1. Some bacteria have never been successfully grown on artificial media. These must be grown in cultures of living cells called tissue cultures, or in living lab animals.

                        Mycobacterium leprae

                     Pathogenic strains of Treponema pallidum

                       Rickettsias

                       Chlamydias

                       All viruses

 

   2. Some microbes require higher than normal levels of carbon dioxide. They are called capnophiles. For these, special carbon dioxide incubators, candle jars, or jars or bags with special carbon dioxide-producing chemical packets can be used. These produce conditions similar to those of the intestinal tract, for example.

                        Campylobacter

                       Neisseria

 

 

SELECTIVE AND DIFFERENTIAL MEDIA

 

   1. Selective media—suppress the growth of unwanted bacteria and encourage the growth of a desired microbe or group of microbes.

          Bismuth sulfite agar---Salmonella typhi

          Sabouraud’s dextrose agar—fungus organisms

          Brilliant green agar---Salmonella and other gram-negative bacteria

 

   2. Differential media---these media cause colonies of certain microbes to grow with a characteristic appearance. Just by observing growth, the presence of a certain type of microbe can be determined.

           Streptococcus pyogenes---blood agar (a clear ring forms around colonies)

 

   3. Some media combine the two types and are both selective and differential.

          Mannitol salt agar---Staphylococcus aureus

          Levine's EMB agar

          McConkey's agar

          Desoxycholate agar

 

 

More about selective media for fungi. These media in general contain a higher level of sugar (4%) and a lower pH (3.8 - 5.6) than bacterial media. These conditions discourage growth of bacteria, which we need to do because fungi often grow very slowly and bacteria will overun them if we don't intervene. To make the medium even more selective for fungi, an antibacterial antibiotic an be added.

 

 

OBTAINING PURE CULTURES

 

Most samples from natural conditions contain many different species of bacteria, and often other types of microbes as well. Each microbe placed on the surface of a solid medium grows and forms a visible colony. Colonies formed by different microbes may have different appearances. In obtaining, a pure culture, one that contains only one type of microbe, the goal is to thin out the original microbes so that the resulting colonies are separate from each other. This can be done in several ways:

     1. Streak plate

     2. Pour plate

     3. Spread plate

     4. Micromanipulator

 

METHODS OF PRESERVING BACTERIAL CULTURES

 

1. Refrigeration—short-term

 

2. Deep-freezing in liquid suspending medium--  temperature quickly lowered to  -50o C to -95o C. Years.

 

3. Lyophilization (freeze-drying)—microbes are quickly frozen while the water content is removed at the same time (formation of ice crystals is damaging to cells). The result is a dry powder sealed in a vacuum which can be stored for decades.

 

GROWTH OF BACTERIAL CULTURES

 

Growth of bacteria is an increase in the number of cells, not in the size of cells.

 

 Bacteria normally reproduce by binary fission:

   1. Cell elongates and chromosome is replicated. One copy of the chromosome moves to each end of the cell.

   2. Cell wall and cell membrane begin to grow inward between the 2 chromosomes.

   3. Cell walls meet, forming 2 separate cells, each identical to parent.

 

A few bacterial species reproduce by other means:

   1. Budding—a small outgrowth forms and splits off the parent cell

   2. Spores--some filamentous bacteria produce chains of reproductive spores at the tips of the filaments. These are reproductive spores, different from endospores.

   3. Fragmentation—a few filamentous bacteria fragment—break into pieces and each piece forms a new cell

 

 

GENERATION TIME

 

This is the time required for a cell to divide and its population to double. This counts the time beginning when a cell is newly produced by binary fission and ending when that cell itself divides. Generation time varies due to:

     1. Species

     2. Conditions

Under ideal conditions, most bacteria have a generation time of 1 - 3 hours, although this can vary, with the shortest being about 15 - 20 minutes and the longest being over 24 hours. Each individual species has its own generation time in ideal conditions, but when conditions are less than ideal, generation time becomes longer.

 

Numbers increase very rapidly. One cell, after 20 generations, could produce over a million bacteria. With a generation time of 20 minutes, this could occur in about 7 hours. 30 generations (10 hours) would bring the population to 1 billion. After that, numbers become so large that logarithmic scales are used.

 

BACTERIAL GROWTH CURVE

 

This is a graph that shows the growth of a population of cells over time:

 

1. Lag phase—when cells are placed in a new medium, there is a period of little or no division that may last from 1 hour to several days. Cells are metabolically active, adjusting to the new surroundings, synthesizing new enzymes, replicating DNA, etc. but not yet actually dividing.

 

2. Log phase—cells begin to divide and enter a phase of rapid growth called the log phase or the exponential growth phase. Generation time is at a minimum and cells are most active. During this phase, microbes are most sensitive to adverse conditions, such as antimicrobial drugs.

 

3. Stationary phase---growth slows because nutrients become exhausted, toxic products accumulate, pH may change, etc. Some cells are still dividing, although it may be less frequently, while other cells are dying. During this period, the number of cells remains stable.

 

If the microbes are being used in industry to produce a product, the ideal is to keep the culture in the log phase, keeping all the microbes healthy, active and at maximum production. This is called continuous culture. It is achieved by monitoring the amount of a certain key nutrient present in the culture medium and keeping it constant. Used medium is frequently drained off (and then purified to recover the product) while fresh medium is added. The apparatus that does this is called a chemostat.

 

4. Death phase---eventually the number of deaths exceeds the number of new cells. This is called the death phase or the decline phase. It continues until all or almost all of the cells are dead.

 

The time required for a culture to move through all 4 phases varies considerably—from a few days to much longer. Some species may have a few surviving cells almost indefinitely.

 

DIRECT MEASUREMENT OF MICROBIAL GROWTH

 

Liquid cultures are usually used and population numbers are recorded as the number of cells per milliliter of the culture medium. Usually numbers in a very small sample are counted and then the number per milliliter (ml) is calculated.

 

To get the small sample, a series of dilutions is performed. This is done because bacterial numbers are usually so large that actually counting all the bacteria in 1 ml is difficult or impossible. The original sample is usually diluted by taking 1 ml from it and putting it into sterile liquid--either 1 ml into 9 ml (which gives a 1:10 dilution) or 1 ml into 99 ml (which gives a 1:100 dilution). If necessary, the first dilution can be mixed and then 1 ml of this transferred to 9 or 99 ml  again, and so on. When a “countable” dilution is achieved, the original number per ml can be calculated.

 

1. PLATE COUNTS—this is the most frequently used method, because it counts only viable cells (living cells capable of reproduction), which is usually what we need to know. It does take time, because colonies must be allowed time to grow before they can be counted. For a plate count, we assume:

   a. Each bacterium grows and establishes a single colony

   b. Original sample is homogeneous—the same number of bacteria are present in all parts of the sample

   c. No aggregates of cells are present (cells stuck together)

 

Unless a sample which contains extremely small numbers of bacteria is being examined, serial dilutions are done. The ideal for a  plate count is a dilution that would contain 25 - 250 bacteria per ml. This produces a plate that is readily counted.

 

EXAMPLE: an original sample contains 3,000,000 bacteria per ml (actually this is a very small number as bacterial growth goes). We could never accurately count 3,000,000 colonies on a plate. Of course, we start out not knowing the number of bacteria—that is what we are trying to determine. What is normally done is to make an educated “guesstimate” of the possible number and then make a series of dilutions that should be in the ball park. Each dilution must be plated, and then plates with too many and too few are discarded.

 

     a. POUR PLATES---To make a pour plate, generally either 1 ml or 0.1 ml of the dilution is placed in a sterile empty Petri dish. Nutrient agar which has been held in liquid form at about 45o C is added to the Petri dish and carefully mixed. The agar quickly solidifies and bacteria grow spread out in the agar. Bacteria are exposed to the 45o C temperature for only a brief time. Most species are not harmed by this brief exposure, but some of the most heat-sensitive types may be damaged. Also colonies may not have their characteristic appearance when they grow below the surface of the agar.

 

     b. SPREAD PLATES-----A spread plate begins with a Petri dish with solid agar already in place.  .1 ml of a liquid sample is poured onto the surface of the agar and spread evenly over the entire surface with a sterile implement. This method does not expose the microbes to heat and puts them all on the surface of the agar.

 

     c. FILTRATION-----If the original sample contains very small numbers of bacteria, a sample of 1 ml may not be large enough. Water samples would be an example. A larger quantity of the sample, often 100 ml, is poured through a sterile membrane filter, which has pores small enough to trap bacteria. The filter, which is round and just slightly smaller than the inside of a Petri dish, is smoothed onto the surface of an agar plate and incubated. Trapped bacteria grow into visible colonies, which can be counted.

 

                                                  

2. DIRECT MICROSCOPIC COUNT-----A measured volume of the sample is placed on a special microscope slide. The slide, which is marked off in squares, allows a direct count of bacterial cells in a measured area of the slide. In counting bacteria in milk, for example,  .01 ml of milk is spread over 1 square centimeter (cm2) of the slide. The cm2 is subdivided into smaller squares that fit into one microscopic field of view. Every small square need not be counted, only enough to get a representative number. The number of bacteria per  .01 ml is determined this way and then multiplied by 100 to get the number of bacteria per ml.

 

Another direct count method uses a Petroff-Hausser cell counter slide. A shallow well which holds a known volume of liquid is indented in the surface of the slide. The well area has lines marking it off into squares. Bacteria in a set number of squares are counted and a multiplication factor gives the number per ml.

 

Drawbacks to direct microscopic count:

 

·         Works well only when there are relatively large numbers of bacteria per ml of sample.

·         Motile bacteria may be hard to count.

·         Both live and dead bacteria are counted

 

Advantage: No incubation time

 

4. ELECTRONIC ENUMERATION-----In a method similar to counting blood cells, electronic cell counters (Coulter counters) may be used to count bacteria. In these, a measured volume of a liquid sample is placed in one chamber of the machine and passed into a second chamber through a tiny opening with an electronic sensor, which counts the number of particles passing through.

 

Both direct microscopic count and electronic count have the advantage of not having to wait for incubation (growth of bacteria, which can take 24 - 48 hours). The main drawback to these is that dead cells are counted just like live ones.

 

 

ESTIMATING BACTERIAL NUMBERS BY INDIRECT METHODS

 

Sometimes, an exact count is not required. A good estimate of numbers is often more quickly and easily obtained, so one of these methods will be chosen in many cases.

 

1. TURBIDITY-----Sterile liquid media are clear. As bacterial numbers increase, the medium becomes more cloudy (turbid). An instrument called a spectrophotometer or colorimeter is used. A beam of light is passed through a sample of the culture which has been placed in  a special test tube, and the percentage of the light which reaches a photoelectric cell on the other side is measured (percentage of transmission).

 

Visible turbidity barely begins when the number of cells reaches one million per ml. The number needs to be 10 million per ml for the spectrophotometer to work well.

 

To use this method, a culture of bacteria is grown in the same culture medium and conditions that will be used in this particular lab work. The turbidity is frequently measured as growth occurs, and a direct count of bacteria is also made at the same time. This is then used to relate a certain degree of turbidity to an actual count of bacteria. Having established these values once, from that point on turbidity alone can be used.