Lecture #14: Growth (Last) to Preferences

Question 1

Batch Culture

Answer 1

A population of microbes growing in a closed culture vessel containing a single batch of medium.

Question 2

Halophiles

Answer 2

Some microbes are adapted to extreme hypertonic environments. These require the presence of NaCl or other salts at a concentration above about 0.2 M. Extreme halophiles have adapted so completely to hypertonic, saline conditions that they require high levels of NaCl to grow.

prefer to live in high salt environments. Modify structure of membranes for hypertonic medium. May stock compatible solutes in the cytoplasm to balance hypertonic medium. Enzymes function in cytoplasm with high concentrations of K.

Question 3

pH Preference

Answer 3

Each species has a definitive pH growth range and pH growth optimum. Acidophiles have their growth optimum between 0 and 5.5; neutrophiles between 5.5 and 8.0; and alkaliphiles (alkalophiles), between 8.0 and 11.5. Extreme alkaliphiles have growth optima at 10 or higher.

Most known bacteria and protists are neutrophiles. Most fungi prefer more acidic surroundings, about 4 to 6; photosynthetic protists also seem to favor slight acidity. Many archaea are acidophiles.

Organic carbon fermenters (sugar) produce acids. Some are more tolerant of acidic conditions. Some, like Streptococcus (yogurt) poison their own environment! Progression of forms.

Some break down proteins to ammonia and thrive in basic conditions of their own making.

Question 4

Yogurt Represents a Progression

Answer 4

Streptococcus species ferment the milk, thicken it as they produce lactic acid. Lowers the pH. Their growth slows as pH drops.

Lactobacillus takes over, as it thrives at lower pH.

One species changes the environment, favoring the growth of another.

Question 5

Psychrophiles

Answer 5

Grow well at 0C and have optimum growth temperature at 15C; the maximum is around 20C. Oceans constitute enormous habitat because 90% of ocean is 5C or colder.

These have adopted to environment in several ways. Their enzymes, transport systems, and protein synthetic machinery function well at low temperatures. The cell membranes of psychrophilic microbes have high levels of unsaturated fatty acids and remain semifluid when cold.

Many begin to leak cellular constituents at 20C+ because of cell membrane disruption.

0 to 16˚C. (Freezing and up). Enzymes, transport systems, metabolism adapted to low temperatures. Cell membranes have different structures to allow fluidity at colder temperatures. Live in Arctic, Antarctic, other cold habitats.

Question 6

Psychrotrophs

Answer 6

optima 20 to 30˚C but tolerate <10 ˚C and spoil food in your refrigerator. Nasty example is
- Listeria monocytogenes (especially dairy products). Current scare.

Question 7

Mesophiles

Answer 7

Microbes that grow in moderate temperatures. They have growth optima around 20-45C and often have a temperature minimum of 15-20C and max of about 45C. Most microbes probably fall within this category. Almost all human pathogens are mesophiles, as might be expected because the human body is a fairly constant 37C.

Question 8

Thermophiles

Answer 8

Grow at temperatures between 55 and 85C. Their growth min is around 45C, and they often have optima between 55 and 65C. The vast majority are members of archaea or bacteria, although few protists and fungi are included. Flourish in many habitats including composts, self-heating hay stacks, hot water lines, and hot springs.

55 ˚C (over 100). Special membrane lipids and enzymes tolerant of heat. Haystacks, compost piles, hot springs.

Question 9

Hyperthermophiles

Answer 9

Growth optima between 85 and about 113C. They usually don’t grow below 55C.

80-100 ˚C. (200 ish). Hot vents, geothermal pools.

Question 10

Oxygen Preference

Answer 10

Importance of oxygen to the growth of an organism correlates with its metabolism-in particular, with the processes it uses to conserve the energy supplied by its energy source. Almost all energy-conserving metabolic processes involve the movement of electrons through a series of membrane-bound electron carriers called the ETC. For chemotrophs, an externally supplied terminal electron acceptor is critical to the functioning of the ETC. The nature of the terminal electron acceptor is related to an organism’s oxygen requirement.

Question 11

Obligate Aerobe

Answer 11

Completely dependent on atmospheric O2 for growth.

Question 12

Microaerophiles

Answer 12

Damaged by the normal atmospheric level of O2 (20%) and require O2 levels in range of 2-10% for growth.

Clostridium (some), Campylobacter.

Question 13

Facultative Anaerobes

Answer 13

Do not require oxygen for growth but grow better in its presence.

Question 14

Aerotolerant Anaerobes

Answer 14

Grow equally well whether O2 is present or not.

Question 15

Obligate Anaerobes

Answer 15

Usually killed in the presence of O2. Employ other methods to generate energy.

Clostridium (some), Archaea.

Question 16

Radiation

Answer 16

Radiation behaves as it it were composed of waves moving through space like waves traveling on the surface of water. The distance between two wave crests or troughs is the wavelength.

As the wavelength of electromagnetic radiation decreases, the energy of the radiation increases; gamma rays and X rays are much more energetic than visible light or infrared waves.

• Shorter wavelengths most damaging, i.e. UV light.
• Low levels, causes mutations in DNA; at high levels, lethal (affects bonds, glumps up molecules = polymerizes them, generates ions).

Question 17

Ionizing Radiation

Answer 17

Radiation of very short wavelength and high energy, which can cause atoms to lose electrons (ionize).

Two major forms of ionizing radiation are X rays (artificially produced) and gamma rays (emitted during radioisotope decay).

Low levels of ionizing radiation may produce mutations and may indirectly result in death, whereas higher levels are directly lethal.

Ionizing radiation causes many changes in cells. It breaks hydrogen bonds, oxidizes double bonds, destroys ring structures, and polymerizes some molecules. Oxygen enhances these destructive effects, probably through generation of hydroxyl radicals (OH.). Although many types of constituents can be affected, the destruction of DNA is probably most important cause of death.

Question 18

Ultraviolet (UV) Radiation

Answer 18

Can kill microbes due to its short wavelength and high energy. Most lethal UV radiation has wavelength of 260 nm, the wavelength most efficiently absorbed by DNA.

The primary mechanism of UV damage is the formation of thymine dimers in DNA, which inhibit DNA replication and function. Thymine dimers are formed when two adjacent thymines in the same DNA are covalently joined.

The damage caused by UV light can be repaired by several DNA repair mechanisms,. Excessive exposure to UV light outstrips the organism’s ability to repair the damage and death results. Longer wavelengths can also harm microbes because they induce the breakdown of the amino acid tryptophan to toxic photoproducts. It appears these toxic photoproducts plus the near-UV radiation itself produces breaks in DNA strands. The precise mechanism is not known, although it’s different from that seen with 260 nm UV.

Question 19

UV destroys DNA, use it to kill microbes

Answer 19

260 nm most lethal, targets DNA specifically. Forms thymine dimers. Skin cancer.

Closer to 400 nm, leads to breaks in DNA.

As dose increases (length of time), effects more extensive. Some dimers missed.

In lab, use a UV light. Keep skin covered. Do not look at the light.

Glass, even plastic, are effective shields. Use that to stop the dose.

Repair mechanisms in visible light; photoreactivation.

Question 20

Radiation from Radioactive Sources

Answer 20

Radiation which does not penetrate well is used in surface sterilization. Beta.

Gamma radiation has good penetration. Effective in wiping out bacteria, used to eliminate the possibility of pathogens in food. Not indicated for viruses.

Question 21

Sterilization

Answer 21

Process by which all living cells, spores, and acellular entities (viruses, viroids, virusoids, and prions) are either destroyed or removed from an object or habitat. A sterile object is totally free of viable organisms, spores, and other infectious agents. When sterilization is achieved by a chemical agent, the chemical is called a sterilant.

Question 22

Disinfection

Answer 22

The killing, inhibition, or removal of microbes that may cause disease; disinfection is the substantial reduction of the total microbial population and the destruction of potential pathogens.

Disinfectants are agents, usually chemical, used to carry out disinfection and normally used only on inanimate objects. A disinfectant does not necessarily sterilize an object because viable spores and a few microbes may remain.

Question 23

Antisepsis

Answer 23

The destruction or inhibition of microbes on a living tissue; it’s the prevention of infection or sepsis.

Antiseptics are chemical agents applied to tissue to prevent infection by killing or inhibiting pathogen growth; they also reduce the total microbial population. Because they must not destroy too much host tissue, antiseptics are generally not as toxic as disinfectants. The exposure of microbes to increasing biocide concentrations decreases the number of viable organisms.

Question 24

Chemotherapy

Answer 24

The use of chemical agents to kill or inhibit the growth of microbes within host tissue.

Question 25

Kill vs. inhibit growth

Answer 25

• cide. Kills, as in bactericide (bacteriocide) or germicide, fungicide.
• iostatic (stasis). Arrests growth, as in bacteriostatic, fungistatic. By extension, bacteriostatin is a drug that inhibits bacterial growth. My- or nystatin inhibits fungi.

Target may be pathogens, but effects not limited to pathogenic forms. Can affect all.

Question 26

Conditions Influencing the Efficiency of Antimicrobial Agent: Population Size

Answer 26

Because an equal fraction of a microbial population is killed during each interval, a larger population requires a longer time die than a smaller one.

Death is logarithmic or exponential, larger population requires longer time to die.

Question 27

Conditions Influencing the Efficiency of Antimicrobial Agent: Population Composition

Answer 27

The effectiveness of an agent varies greatly with nature of organisms being treated because microbes differ markedly in susceptibility. Bacterial spores are much more resistant to most antimicrobial agents than are vegetative forms, and younger cells are usually more readily destroyed than mature organisms. Some species are able to withstand adverse conditions better than others.

Endospores, capsules, pigments, & reinforcements alter susceptibilities.

Question 28

Conditions Influencing the Efficiency of Antimicrobial Agent: Concentration or Intensity of an Antimicrobial Agent

Answer 28

Often, but not always, the more concentrated a chemical agent or intense a physical agent, the more rapidly microbes are destroyed. However, agent effectiveness usually is not directly related to concentration or intensity. Over a short range, a small increase in concentration leads to an exponential rise in effectiveness; beyond a certain point, increases may not raise the killing rate much at all. Sometimes, an agent is more effective at lower concentrations.

Question 29

Conditions Influencing the Efficiency of Antimicrobial Agent: Contact Time

Answer 29

The longer a population is exposed to a microbial agent, the more organisms are killed. To achieve sterilization, contact time should be long enough to reduce the probability of survival by at least 6 logs.

(single hit, repeated, continuous).

Question 30

Conditions Influencing the Efficiency of Antimicrobial Agent: Temperature

Answer 30

An increase in temperature at which a chemical acts often enhances its activity. Frequently a lower concentration of disinfectant or sterilizing agent can be used at a higher temperature.

Interaction; higher temp., lower dose.

Question 31

Conditions Influencing the Efficiency of Antimicrobial Agent: Local Environment

Answer 31

The population to be controlled is not isolated but surrounded by environmental factors that may either offer protection or aid in its destruction. For example, because heat kills more readily at an acidic pH, acidic foods and beverages such as fruits and tomatoes are easier to pasteruize than more alkaline foods such as milk.

A second important environmental factor is organic matter, which can protect microbes against physical and chemical disinfecting agents. Biofilms are a good example. The organic matter in a biofilm protects the biofilm’s microbes. Furthermore, bacteria in biofilms are altered physiologically, and this makes them less susceptible to many antimicrobial agents. Because of the impact of organic matter, it may be necessary to clean objects before they are disinfected or sterilized.

pH, slime, grease or oil, particulates.

Question 32

Moist Heat

Answer 32

Moist heat readily destroys viruses, bacteria, and fungi. Kills by degrading nucleic acids and denaturing enzymes and other essential proteins. It also disrupts cell membranes. Exposure to boiling water for 10 min is sufficient to destroy vegetative cells and eukaryotic spores, but not bacterial spores. Boiling can be used for disinfection of drinking water and objects not harmed by water, but does not sterilize.

steam is effective against endospores , so autoclave. Because sterilization is achieved at 12 minutes at 121˚C (15 pounds of pressure), use 15 minutes as standard cycle. Margin of error. Checks – heat and pressure sensitive tape (turns color), spore strips or ampules every 30 hours. Geobacillus stearothermophilus endospores, place in bag and test growth.

Question 33

Autoclave

Answer 33

To destroy bacterial endospores, moist heat sterilization must be carried out at temperatures above 100C, and this requires use of saturated steam under pressure.

Question 34

Dry Heat

Answer 34

Sterilized in absence of water. Some items are sterilized by incineration. For instance, inoculating loops can be sterilized in a small, bench-top incinerator. Other items are sterilized in an oven at 160-170C for 2-3 hours. Microbial death results from oxidation of cell constituents and denaturation of proteins. Dry air heat is less efficient than moist heat.

(not always reliable), 2-3 hours at 160˚C or more. Glass, metal. Routine is 200 ˚C overnight.

Question 35

Pasteurization

Answer 35

Many heat-sensitive substances are treated with controlled heating at temperatures well below boiling, a process known as pasteurization.

Use of heat to discourage growth of organisms present in liquids (milk, wine) and retard spoilage.

Pathogens most susceptible. Temperatures below boiling. As temperature goes up, time goes down.
63˚C for 30 minutes vs. 72˚C for 15 seconds.

Now, ultra-high temperature sterilization at 150˚C for 1-3 seconds. Above boiling but very brief.

Question 36

Low Temperature

Answer 36

Freezers . Don’t kill, just inhibit growth.

Used for storage. Freeze cells in glycerine solution to prevent damage by ice formation.