REQUIREMENTS FOR MICROBIAL GROWTH!

There is a useful learning device to keep in mind for the needs of all living organisms to survive and reproduce:

F: Food

A: Acidity (pH requirements)

T: Time

T: Temperature

O: Oxygen (Gaseous) requirements

M: Moisture

Step back for a moment and think of each of these requirements. We all need food to eat. Our cells function within a certain physiological pH and temperature range. We need to "breathe" and we need time to grow. Our cells also have to remain "wet" and maintain a proper salt/electrolyte balance. Why should microbes be fundamentally different?! We will consider each of these needs in more detail.

Food

"Food" is a very general and vague term with respect to microbial growth requirements. We will consider two broad meanings to the word "food."

• Chemical requirements
• Culture media used to grow microbes under laboratory conditions

Chemical requirements

The majority of living cells need over 20 chemical elements in order to function. Six elements in particular are critical for building biological macromolecules and maintaining their structure and function.

• Carbon is needed to build all organic compounds. All organisms require a carbon source for this purpose and many microbes also use some form of carbon as their energy source (review class notes on microbial metabolism).
• Hydrogen occurs in organic hydrocarbon molecules and in inorganic molecules such as water. A major role of hydrogen in biological systems is to maintain structure and conformation of macromolecules. Hydrogen bonds contribute to base-pairing between complementary DNA strands and to folding of proteins.
• Oxygen or gaseous requirements of microbes will be discussed under a separate subheading of this lecture.
• Nitrogen is used in protein/ amino acid synthesis and nucleic acid synthesis. Nitrogen-containing bases help spell out information contained within the genetic code. A nitrogenous base ( adenine) is a building block of the energy-rich molecule, ATP. Peptidoglycan is also a nitrogen-rich substance.
• Phosphorus is essential for nucleic acid synthesis and formation of phospholipids that contribute tto the integrity of cell membranes
• Sulfur occurs in certain amino acids such as cysteine and methionine . Disulfide bonds help maintain tertiary and quaternary levels of protein structure and are crucial in maintaining the function of many proteins.

Culture Media

A culture medium is any material prepared for growth of an organism in a laboratory setting. Microbes that can be cultured on a petri-plate or in a test-tube containing media are said to grow under in vitro conditions ("within-glass".)

It was not until the era of Robert Koch and his coworkers that Agar was introduced as a a common medium for bacterial growth. Agar is a complex polysaccharide derived from a marine red algae. Few bacteria possess enzymes capable of digesting agar and therefore it is useful as a solidifying agent and for isolating microbes in pure culture. Prior to the advent of agar, gelatin was used as a growth medium. Unfortunately, many bacteria possess enzymes that liquify gelatin and therefore this medium is not useful for isolating pure cultures. However, gelatin liquefaction is one among a series of biochemical tests that helps differentiate species of bacteria.

What is a PURE CULTURE?

• A pure culture represents a single species (clonal in nature) of microorganisms
• A clone is a genetically identical population of microbes that have descended from a single parent cell
• Colonies are visible clones that have grown on solid media and represent millions of bacterial cells
• Distinctive characteristics of colonies should be noted such as:
• pigmentation
• odor
• elevation
• margin (border of the colony)
• consistency, such as mucoid, irridescence, filamentous, etc.

Media vary in their chemical composition . In turn, the composition of the media determines microbial growth and the type of microbes that will grow.

Chemically defined media : exact chemical composition is known. Such media is often commercially prepared.

Selective media .Contain chemicals which encourage growth of certain types of microbes but inhibits the growth of others.

Differential media allows different microbes to be distinguished on the basis of various biochemical reactions. Fermentation reactions involving the catabolism of various sugars are particularly useful biochemical tests

Many media are both selective and differential, such as MacConkey agar and Mannitol Salt agar.

Enrichment media contains a rich supply of nutrients to encourage the encourage growth of microorganisms. A commonly used enrichment medium is blood agar. This medium is also differential and it permits detection of differnt patterns of hemolysis.

FASTIDIOUS ORGANISMS; "Picky eaters"

Many microbes can not be simply grown in a test tube or by streaking them onto a petri plate! Scientists cannot always determine the exact growth requirements of all microbes in order to culture them under in vitro conditions. Such "picky-eaters" are known as fastidious microbes and these generally need to be grown either in tissue culture (using living cells) or by using an animal (living) model. Microbes that grow in living cells or organisms are said to grow under in vivo conditions. This aspect of scientific research is very controversial and emotional for many people. No one enjoys using animals for research ( at least no credible scientist enjoys this). However, humans have to weigh the odds of a necessary scientific breakthrough against the taking of an animal life. There are no easy answers here!

The pickiest-eaters are viruses. ALL VIRUSES NEED LIVING CELLS in order to survive. It is NOT possible to study viruses without living cells, and in many cases an animal model.

Many bacteria are also intracellular parasites such as species of Chlamydia and the Rickettsias

Our current understanding of deadly picky-eaters such as HIV and malaria parasites would not be possible without in vivo studies.

Acidity (pH requirements)

• The majority of bacteria that we are concerned with from a medical standpoint are those that grow on and in the human body. Such bacteria (including pathogens) grow best at neutral or biological pH which is typically between pH 6.8 to pH 7.4. Extreme shifts in pH can damage cells by denaturing proteins and enzymes and by interfering with transport of ions across cell membranes.
• Foods that are acidic such as vinegars and pickles generaly are not favorable for microbial growth.
• Fungi such as yeasts and molds prefer slightly more acidic conditions and grow best between pH 5 to pH 6. This is about the pH of normal human skin.
• Certain normal microbiota such as Lactobacilli ( an acidophile) help establish pH conditions that are not favorable for the growth of pathogens. Lactobacilli of the vagina help maintain a naturally acidic pH between pH 3 to pH 4. Interestingly, semen neutralizes the vaginal pH in order to favor survival of sperm. If an STD agent is prsent in semen it may be given a window of opportunity to establish an infection under altered pH conditions.
• The pathogen Helicobacter pylori is able to survive the hostile pH of the stomach by producing urease. This enzyme is responsible for cleaving the nitrogenous waste product urea into carbon dioxide and ammonia. In turn ammonia raises the pH toward more basic conditions. Helicobacter is thus shielded from stomach acid and is able to produce inflammation and ulcers.
• Some acidophiles such as those that metabolize sulfur can tolerate extremes as low as pH 1.
• On the other extreme, bacteria that prefer alkaline (basic) conditions) are known as alkaliphiles. Examples are Vibrio cholerae (prefers pH 9) and the soil bacterium Agrobacterium grows in soil with a pH of 12!

pH, Acids, Bases, Salts and pH

The pH scale is a measure of the concentration of hydrogen ions (H+) in a solution. The more hydrogen ions ion the more acidic a solution and the and lower the pH solution. pH values below pH 7 are considered acidic. pH values above pH 7 are considered alkaline.

pH is calculated by using the Henderson-Hasselbach equation:

pH = - log₁₀ (H+)

The pH scale is logarithmic. For example, stomach acid with a pH of 2 has 10,000 times (10⁴) more hydrogen ions than urine with a pH of 6.

An ACID can be described as a hydrogen donor. An acid dissociates in water into hydrogen (H+) ions and negative ions (anions) . For example, HCl dissociates into H + and Cl- ions

A BASE can be described as a hydrogen acceptor. A base dissociates in water into one or more cations and and hydroxyl (OH-) ions . For example, NaOH dissociates into Na+ and OH- ions

A SALT is an ionic compound that dissociates in water into anions and cations, neither of which is a hydroxyl group or hydrogen ion. For exmple, NaCl dissociates into Na+ and Cl- ions.

When bacteria are cultured in a laboratory most will produce acidic metabolic wastes that would eventually build up and interfere with their own growth. For this reason, many culture media contain physiological buffers to stabilize the pH (a commonly used buffer is phosphate-buffered saline or PBS).

Many culture media also contain pH indicators. Often these chemicals are dyes that change color when a shift in pH occurs. For example, phenol red is used in mannitol salt agar. This dye appears red above pH 6.8 but as microbes ferment the sugar in the medium and produce acids the dye turns yellow. Thus, shifts in pH can help determine the products of a biochemical reaction and/or determine if media is contaminated.

Time required for microbial growth

Asexual reproduction in Bacteria

Most bacteria divide by a process known as binary fission. Prokaryotes lack a mitotic apparatus. DNA is replicated bidirectionally and then is allocated into two new, genetically identical daughter cells. Cells may remain attached forming characteristic packets, chains or filaments.

Bacterial cell division is logarithmic

Phases of Bacterial Growth

• Lag phase: At this point there is little or no change in cell numbers but the cells are metabolically active. This period preceeds cell replication as it takes some time for bacteria to adjust to their environment. The lag phase varies with the cells involved but in general lasts a few hours.
• Log phase: Once bacteria have adapted to their environment they multiply at the fastest rate possible under the conditions provided. A generation time is the time required for cell to divide or population to double. For most bacteria generation times are typically less than 1 hour. E.coli doubles every 20 minutes under optimal conditions.

In order to treat bacterial infections appropriately with antibiotics such as penicillins, drugs have to target bacteria in the log phase. This enables the drugs to interfere with peptidoglycan synthesis.

Mycobacteria, such as those that cause tuberculosis and leprosy have some of the slowest generation times known.

• Stationary phase: an equilibrium occurs between the number of new cells produced and cell death
• Death phase: cells use up their nutrients and toxic waste products accumulate in the medium. Deaths exceed number of new cells formed. Dying cells often become irregular in form and are difficult to identify. Spore-forming bacteria may survive these condtions.

Temperature requirements

All microbes have an optimum temperature range for growth

Gaseous requirements

There is great diversity among microbes with respect to their gaseous requirements.

• Obligate organisms absolutely require an environmental factor for their metabolism
• Faculatative organisms are adaptable and can switch their metabolism depending on their environment.

Moisture

All living cells, including bacteria, require an aqueous environment . However certain structures formed by microbes are environmentally resistant and can withstand dessication for long periods. These include:

• bacterial endospores
• protozoan cysts
• helminth ova (worm eggs)

A hypertonic environment has a high solute concentration with respect to the inside of a cell. Consequently there is more water inside the cell with respect to the exterior and water tends to diffuse out of the cell. The result is the cell shrinks because of plasmolysis .

Salting can be used to preserve foods and takes advantage of this important principle.

The higher the water contant of a food the more it is said to be "potentially hazardous." That is the high moisture content favors bacterial growth and is the food is prone to spoilage.

Archaebacteria known as halophiles are salt-loving bacteria found in oceans and salt marshes that actively pump salt into their cells.