Chapter 3 & 7
Review Sheets
REQUIREMENTS FOR MICROBIAL GROWTH
"Food" is a very general and vague term with respect to microbial growth requirements. We will consider two broad
meanings to the word "food."
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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.
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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 ("withinglass".)
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?
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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:
o pigmentation
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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 cannot 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)
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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.
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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 (10 4) 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
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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.
o 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.
o 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
Group
Temperature Optima
Cold-loving organisms grow best at a temperature
of 15 C but some others can grow between 0 and
Psychrophiles
20 C. NOTE: BACTERIA ARE NOT KILLED
BY FREEZING, they are merely suppressed.
Mesophiles
Grow at optima between 25 C and 40 C.
Example(s)
Most live in cold water and soil. No
psychrophiles can grow on or in the human
body! Some can cause food spoilage as they
are capable of growing at refrigerator
temperature (4 C).
Normal microbiota and most pathogens grow at
or near human body temp of 37 C. Mesophiles
are responsible for most food spoilage
(heat-loving)
utilized in organic compost
Thermophiles
Optima between 50 and 60 C
Extreme
thermophiles
Archaebacteria which favor optima of 80 C or
more
found in volcanic hot springs and hydrothermal
deep-sea vents
Gaseous requirements
There is great diversity among microbes with respect to their gaseous requirements.
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Obligate organisms absolutely require an environmental factor for their metabolism
Faculatative organisms are adaptable and can switch their metabolism depending on their environment.
Group
Requirements
Strict/Obligate
Molecular oxygen required
Aerobe
for aerobic respiration
Microaerophilic
Also known as "Capnophilic" or carbon-dioxide loving.
Only use low concentrations of oxygen
Example(s)
Pseudomonas aeruginosa
Campylobacter jejuni
Helicobacter pylori
Escherichia coli and the large
family of gram negative
enteric rods
Facultative
anaerobes
Adaptable orgaisma that use oxygen when present but can
switch to anaerobic pathways in its absence
Strict or obligate
Anaerobes
Molecular oxygen is toxic to these organisms. Lack enzymes Bacteriodes
catalase and superoxide dismutase (SOD) to neutralize
hydrogen peroxide and free radicals, respectively
Clostridium
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:
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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.