BACTERIAL GROWTH

The Biologist, Stephen J. Gould, makes a compelling argument in his book, "Full House" (which you can read for extra credit), that we live in the AGE of the BACTERIA. He feels that it has always been and always will be thus. If he is correct, then it behooves us to learn how this came about and how the bacteria maintain their dominance on Earth. He supports this conclusion by the fact that bacteria flourish in prodigious numbers in almost every environment on Earth where free-water is available, including the deepest parts of the ocean, at temperatures of <0oC to >120oC, in acid and alkaline waters, in the presence of toxic substances, and even bathed in the radiation's emanating from atomic reactors. Bacteria metabolize everything from gasoline, antibiotics and plastics to elemental sulfur and manganese. They even live deep in the bowels of the earth. Recently a bacterium, Shewanella putrefaciens, has been found that uses manganese rather than oxygen as an electron acceptor; that is, it is "a manganese breather".

A major advantage the bacteria have is that the vast majority of them are capable of rapid growth rates. Many bacteria can divide, that is, produce a NEW GENERATION, every 20 to 30 minutes under optimal environmental and nutrient conditions. Whereas, the human generation time is ~25 years. All forms of life are capable of what is called exponential or logarithmic growth in which the numbers of a species double each generation (e.g. 1, 2, 4, 8, 16, 32, 64 etc.). When this data is plotted as the numbers of a species vs. time it produces a classical growth curve that looks like this:


Figure 1. Typical bacterial exponential Growth Curve. In a rich culture medium bacteria, grown under aerobic conditions, achieve a final concentration of 2-5 x 109 cells per ml in about 12-18 hours. Although plotted on a different time scale the human growth curve looks the same; the human population at similar points on the growth curve are shown in red.

In the case of bacteria the timeline could, in theory, start with a single cell and end 24 hours later (assuming no death) with a bacterial-mass near the weight of the earth. It is interesting to note that the human population growth curve looks exactly the same; the population reached 6 billion people this month and it may double again by the time you are my current age. Another way to look at this is that a single bacterium can produce, in about 12 hours, ~5 billion offspring in two milliliters of medium; something it has taken the human 3.5 billion years to do. Unhindered growth, of course, requires unlimited nutrient supply and an unchanging environment; however, in real life this is impossible. At some point, usually sooner rather than later, something becomes unfavorable. Usually a critical nutrient becomes limiting. For example, the supply of the energy source may run out, or a trace element may become limiting or a predator may appear or waste will become toxic or a disease will take hold; any of these inhibit and eventually stop growth. The norm in nature is for a catastrophic crash to occur following a period of rapid, vigorous reproduction. In such cases the population usually undergoes a rapid die-off and the organism may even become extinct in a local region. The cycles of population increase, followed by a rapid population decline are typical of the natural world. One cannot help but wonder if such a fate may await the human population.

A major advantage of the rapid reproduction time, and one that makes the bacteria so successful is that they are able to evolve so much faster. Evolution occurs through mutations that produce variations in the offspring of organisms. Then the environment determines which, if any mutations are advantageous. Those organisms with the advantageous mutations tend to survive and live on. Because of their faster generation times bacteria can test billions of mutations for survival while a single human may take 25 or more years to test a given mutation.

BACTERIAL ENVIRONMENTAL REQUIREMENTS

Bacteria live in the most diverse range of environments of any forms of life. There are bacteria that live in salt water below freezing in melted ice pockets in the Antarctic, in saturated salt solutions, in boiling hot springs, in acid waters at pH <2.0, around the core of atomic reactors, in the fuel tanks of airplanes, at the bottom of the ocean at >110oC (Click here to view a video of life around volcanic vents), and hundreds of feet below the surface of the ground. A basic principle of microbial growth is that every microbe has a PREFERRED ENVIRONMENT and it is a challenge to the investigator to tease out the unique set of conditions, called the OPTIMAL GROWTH CONDITIONS that suits each microbe best.

The most common environmental conditions that a microbiologist considers are temperature, pH, oxygen, light, salt/sugar concentration and special nutrients. Each bacterium has an optimum range of these conditions within which it grows at a maximum rate. In some cases this may be a fairly broad range such as bacteria that can grow maximally over a five to 10 degree temperature span. In other cases it may be quite narrow; the temperature allowing growth spanning only a few degrees. To determine the optimum environmental conditions a scientist considers the environment where the bacterium is NORMALLY FOUND. In the laboratory exercise #12 you will see why some bacteria are grown at room temperature and others at 37oC and why others won't grow on the surface of petri dishes.

Growth requirements

Temperature (optimal and range) There is a lot of variation in each category

o mesophiles (20-45C) -- body and room temp

o psychrophiles (0-15C) -- will not grow at room temp

o thermophiles (>45 C ) -- important in compost piles

o hyperthermophiles (>80C) - thermal vents

o psychrotrophs (0-30C)- food spoilage

 

pH (optimal and range)

o importance of buffers in media

o acidophiles - yogurt, sauerkraut, Lactobacillus in vagina

Osmotic pressure

o plasmolysis (in hypertonic solution -- sugar, salt)

o halophiles - some are obligate (Dead Sea etc.)

Light requirements

o photosynthetic microbes require (sometimes a specific wavelength)

o some microbes are light sensitive

oxygen requirements

o obligate aerobe

o obligate anaerobe,

o microaerophiles

o facultative anaerobes

o aerotolerant

water

BACTERIAL NUTRITION

All life has the same BASIC NUTRITIONAL REQUIREMENTS, which include:

A SOURCE OF ENERGY. This may be light (the sun or lamps) or inorganic substances like sulfur, carbon monoxide or ammonia, or preformed organic matter like sugar, protein, fats etc. Without energy life can not exist and quickly dies or becomes inactive.

A SOURCE OF NITROGEN. This may be nitrogen gas, ammonia, nitrate/nitrite, or a nitrogenous organic compound like protein or nucleic acid.

A SOURCE OF CARBON. This can be carbon dioxide or monoxide, methane, carbon monoxide, or complex organic material

A SOURCE OF OXYGEN. All cells use oxygen in a bound form and many require gaseous oxygen (air), but oxygen is lethal to many microbes.

A SOURCE OF PHOSPHOROUS, SULFUR, MAGNESIUM, POTASSIUM & SODIUM.

A SOURCE OF CALCIUM. Most cells require calcium in significant quantities, but some seem to only need it in trace amounts.

A SOURCE OF MINERALS LIKE IRON, ZINC, COBALT ETC. These are called TRACE metals that are required by some enzymes to function.

The sources of these various requirements DEFINE AN ORGANISM, so a description of every organism should include this information. Many bacteria can synthesize every complex molecule they need from the BASIC MINERALS, but others, said to be FASTIDIOUS, require PREFORMED organic molecules like vitamins, amino acids, nucleic acids, carbohydrates; humans are fastidious. In general bacterial pathogens need more PREFORMED ORGANIC MOLECULES than do nonpathogens, but that is not always true. For example some bacteria that are found in milk hardly make any of their own basic organic molecules, which they let the cow make these things for them. A simple rule of thumb is "if humans can use something for food, many microbes will also love it". The reverse is not always as true as microbes can "digest" some very strange substances including cellulose, sulfur, some plastics, turkey feathers and asphalt, to name just a few.

Types of Media Used in the Laboratory

The media used in the laboratory have to be chosen to suit the nutritional requirements of the species of organism to be grown. Isolation from a mixture can sometimes be facilitated by the use of media designed for a special purpose.

Growth Media: Media designed specifically to allow for the growth of microorganisms.

Differential Media: These are media in which some metabolic activity of an organism can be detected by inspection of the growth of the organism on the medium. This is often accomplished by observing changes in the color of a pH indicator. Examples include Triple Sugar Iron agar, Simmon's citrate agar, urea agar, carbohydrate broth tubes, amino acid decarboxylase or dihydrolase tubes, MIO medium (for motility, indole, and ornithine decarboxylase), and MacConkey agar.  Note: Some media can be both selective and differential.

Selective Media: In the broadest sense, all media are selective, in that there is no universal medium on which all species of bacteria can grow. This term, however, is generally restricted to situations where an ingredient is added which allow the growth of a particular organism, while inhibiting to a considerable extent the growth of other organisms, which might be found in the same environment. Inhibitors such as dyes in low concentration, bile salts, high NaCl concentration and other substances such as phenylethyl alcohol are often used. Examples include PEA agar (phenylethyl alcohol), which inhibits the growth of gram-negative enteric bacilli and facilitates the isolation of gram-positive organisms such as staphylococci in aerobic cultures. In anaerobic culture, it is additionally selective for certain gram-negative anaerobic bacilli such as Bacteroides spp.. MacConkey agar, containing bile salts and dyes, inhibits gram-positive organisms and Thayer-Martin agar, containing small quantities of the antimicrobial agents vancomycin, colistin, and nystatin, inhibits the common microbiota of the genital area, while selecting for Neisseria spp.  Note: Some media can be both selective and differential.

SPECIFIC TYPES OF MEDIA AND TESTS

Tryptic Soy Agar (TSA) - general-purpose medium used for the isolation of wide variety of bacteria. Contains tryptone, soy (source of B vitamins and amino acids)

Nutrient Agar (NA) - General-purpose medium used for the isolation of wide variety of bacteria.

Blood Agar Plate (BAP) - enriched with blood to enhance the growth of fastidious bacteria usually pathogenic. Groups of bacteria determined by hemolysis of red blood cells by bacterium. Alpha (a) hemolysis - bacterium partially hemolyses RBC's with a partial clearing zone around bacterial growth; sometimes with greenish pigment caused by reduction of hemoglobin. Beta (b) hemolysis - complete lysis of RBC's with total clearing zone around bacterial growth. Gamma (g) hemolysis - no lysis of RBC's with noclearing zone around bacterial growth.

MacConkey's Agar (MAC) - selects and differentiates members of Enterobacteriaciae. Differentiates genera Salmonella & Shigella from other Enterobacteriaciae. Contains bile salts and crystal violet that inhibits growth of gram positive bacteria. Differentiates non coliforms from coliforms of Enterobacteriaciae based on ability to ferment lactose. Lactose fermenting bacterial colonies appear red or pink. Non lactose fermenting bacteria appear transparent or non colored.

Triple Sugar Iron Agar (TSI) - characterizes members of Enterobacteriaciae on basis of glucose, sucrose, & lactose fermentation, gas production, and hydrogen sulfide production. Contains pH indicator phenol red which changes from red to yellow color if fermentation occurs. Gas production by fermentors indicated by bubbles or cracks in medium or butt pushed up in test tube. Hydrogen sulfide production indicated by black precipitant.

Simmon's Citrate Agar Slant (SC) - characterizes members of family Enterobacteriaciae based on ability to use citrate as carbon energy source and inorganic ammonium salts as source of nitrogen. Differentiates members of genus Escherichia from genera Enterobacter and Klebsiella. If organism can utilize citrate & ammonium salts then growth alkalizes medium. Brom thymol blue pH indicator in medium. Positive results indicated by green slant turning blue. Negative result indicated by no color change in agar slant.


Carbohydrate Fermentation Tubes
- characterizes bacteria on based on ability to ferment a specific carbohydrate (sugar) such as lactose, sucrose, or glucose. Carbohydrate fermentation tube contains one type sugar and phenol red pH indicator. In the fermentation tube, a small inverted test tube (Durham) has been placed. Bacterium that can ferment the sugar is indicated by a color change in the medium. The medium will go from a red to a yellow color. This is a positive result. If the bacterium can not ferment that particular sugar, then no color change is seen. This is a negative result. The Durham (fermentation) tube is inverted to trap gas released during the fermentation process. Bubbles in the Durham tube is a positive result for gas production. No bubbles in the Durham tube indicates a negative gas production result. Some microorganisms may ferment the sugar resulting in an alcohol end product. This will result in a color change from red to bright red or fuchsia color. Gas may also be produced. This would be a negative acid production result but a positive gas result.

Catalase Test - used to characterize bacteria that have the intracellular enzyme catalase present. Differentiates between aerobic bacteria such as Staphylococcus and anaerobic bacteria such as Streptococcus. Aerobic respiring bacteria produce Hydrogen Peroxide in presence of oxygen. Build up of H2O2 becomes toxic to the cell but with the enzyme catalase, bacterium can break down toxic H2O2 into H2O and O2. When hydrogen peroxide is added to bacteria that contain catalase, bubbling from the release of water and oxygen can be seen. This is a positive catalase test result. If no bubbling is seen, this is a negative catalase test result.

Starch Agar Plate (SA) - used to differentiate organisms that break down (hydrolyze) starch into smaller molecules of sugar by the enzyme amylase. After inoculation and growth, plate is flooded with IKI (Iodine) which is a reagent that test for the presence of starch. When the reagent is placed onto the SA plate, a color change in the surrounding agar (turns blue/black in the presence of iodine) indicates that the bacterium did not hydrolyze the starch thus this is a starch negative result. If the iodine does not change color (remains brown) and there is a clear zone surrounding the bacteria, this indicates a starch positive result.