Microbiology Test 3

What is a broth dilution test? Know how growth is analyzed.

Determine the MIC and minimal bactericidal concentration (MBC) of an antimicrobial drug Test organism is placed into the wells of a tray containing dilutions of a drug; growth is determined Antibiograms Reports that record the susceptibility of organisms encountered clinically

What are the costs of resistance both financially and in public health?

Developing new drugs to replace those that have lost effectiveness is costly. Almost all of these drugs will be more expensive, sometimes priced in a range that makes them difficult to afford even in highly developed countries. In less-developed parts of the world, the costs are simply unaffordable.

Know which tests are performed to determine how effective an antibiotics are against various pathogens.

Disk diffusion, Broth dilution tests.

How are bacteria controlled physically and chemically

Physical agents include such methods of control as high or low temperature, desiccation, osmotic pressure, radiation, and filtration. Control by chemical agents refers to the use of disinfectants, antiseptics, antibiotics, and chemotherapeutic antimicrobial chemicals.

What is the future of chemotherapy?

Target virulence factors Sequester iron, which feeds pathogens Antimicrobial peptides produced by various organisms Phage therapy Bacteriocins: antimicrobial peptides produced by bacteria

Why do antibiotics work on bacterial cells while being relatively harmless to humans?

These two cell types differ substantially in many ways, such as in the presence or absence of cell walls, the structure of their ribosomes, and details of their metabolism. Thus, selective toxicity has numerous targets.

What factors cause hospital infections?

Transmission from healthcare professionals to patients; The presence of patients with weakened immune systems; The presence of a wide range of pathogens in the healthcare environment

Serial dilution tests

In serial dilutions, the original inoculum is diluted in a series of dilution tubes. In our example, each succeeding dilution tube will have only one-tenth the number of microbial cells as the preceding tube. Then, samples of the dilution are used to inoculate Petri plates, on which colonies grow and can be counted. This count is then used to estimate the number of bacteria in the original sample.

How are medical implants sterilized?

Ionizing radiation, especially high-energy electron beams, is used to sterilize pharmaceuticals and disposable dental and medical supplies, such as plastic syringes, surgical gloves, suturing materials, and catheters.

9. What are the different ways bacteria deal with oxygen? What is an obligate anaerobe, facultative anaerobe etc?

Microbes that use molecular oxygen (aerobes) extract more energy from nutrients than microbes that do not use oxygen (anaerobes). Organisms that require oxygen to live are called obligate aerobes -Obligate anaerobe- growth occurs only in absence of oxygen -Obligate aerobe- growth occurs only in presence of oxygen -facultative anaerobe- greater growth in presence of oxygen -Aerotolerant anaerobes- only anaerobic growth but it continues in presence of oxygen -Microaerophiles- only aerobic growth but oxygen required in low concentration

In what ways are antibiotics misused? How does this lead to resistance?

Misuse of antibiotics selected for resistance mutants Misuse includes: Using outdated or weakened antibiotics Using antibiotics for the common cold and other inappropriate conditions Using antibiotics in animal feed Failing to complete the prescribed regimen Using someone else's leftover prescription

How is pH related to bacterial growth? What controls pH in biological systems.

Most bacteria grow best in a narrow pH range near neutrality, between pH 6.5 and 7.5. Very few bacteria grow at an acidic pH below about pH 4. Chemical buffers help to control pH in biological systems

What are the side effects of antibiotics?

most common side effects of antibiotics is on digestive system (vomiting, nausea, diarrhoea, bloating and indigestion, abdominal pain, loss of appetite)

Know the temperature ranges for each type of bacteria. Psychrophiles, mesophiles etc.

psychrophiles (cold-loving microbes), mesophiles (moderate-temperature- loving microbes), and thermophiles (heat-loving microbes) optimum temperature 50-60 C . Most bacteria grow only within a limited range of temperatures, and their maximum and minimum growth temperatures are only about 30°C apart. They grow poorly at the high and low temperature extremes within their range.

What is a biofilm and how can it be dangerous?

thin, slimy layer encasing bacteria that adheres to a surface; Within a biofilm community, the bacteria are able to share nutrients and are sheltered from harmful factors in the environment, such as desiccation, antibiotics, and the body's immune system. The close proximity of microorganisms within a biofilm might also have the advantage of facilitating the transfer of genetic information by, for example, conjugation.

What are the effects of food portion size on the rate at which it achieves a temperature below the spoilage point?

(the pan of rice with a depth of 5 cm (2 in) cooled through the incubation temperature range of the Bacillus cereus in about 1 hour, whereas the pan of rice with a depth of 15 cm (6 in) remained in this temperature range for about 5 hours) if the food portion is larger it will take longer to cool below the spoilage point

Know the history of penicillin and how it works to destroy a bacterial cell.

-Alexander Fleming observed that the growth of the bacterium Staphylococcus aureus was inhibited in the area surrounding the colony of a mold that had contaminated a Petri plate (Figure 1.6). The mold was identified as Penicillium notatum, and its active compound, which was isolated a short time later, was named penicillin -Penicillin kills bacteria through binding of the beta-lactam ring to DD-transpeptidase, inhibiting its cross-linking activity and preventing new cell wall formation. Without a cell wall, a bacterial cell is vulnerable to outside water and molecular pressures, and quickly dies.

What is an antibiotic? What is an antimicrobial compound

-An antibiotic is a type of antimicrobial substance active against bacteria. It is the most important type of antibacterial agent for fighting bacterial infections -antimicrobial drugs act by killing or by interfering with the growth of microorganisms. Unlike disinfectants, however, antimicrobial drugs must act within the host without damaging the host. This is the important principle of selective toxicity.

What elements (C, N etc.) are required for bacterial life and where are those elements utilized in the bacteria cell.

-Carbon is the structural backbone of living matter; it is needed for all the organic compounds that make up a living cell. Half the dry weight of a typical bacterial cell is carbon. Chemoheterotrophs get most of their carbon from the source of their energy—organic materials such as proteins, carbohydrates, and lipids. Chemoautotrophs and photoautotrophs derive their carbon from carbon dioxide. -Organisms use nitrogen primarily to form the amino group of the amino acids of proteins. Many bacteria meet this requirement by decomposing protein-containing material and reincorporating the amino acids into newly synthesized proteins and other nitrogen-containing compounds. Other bacteria use nitrogen from ammonium ions (NH4+), which are already in the reduced form and are usually found in organic cellular material. Still other bacteria are able to derive nitrogen from nitrates (compounds that dissociate to give the nitrate ion, NO3-, in solution). (nitrogen fixing bacteria) -Sulfur is used to synthesize sulfur-containing amino acids and vitamins such as thiamine and biotin. Important natural sources of sulfur include the sulfate ion (SO42-), hydrogen sulfide, and the sulfur-containing amino acids. -Phosphorus is essential for the synthesis of nucleic acids and the phospholipids of cell membranes. Among other places, it is also found in the energy bonds of ATP. A source of phosphorus is the phosphate ion (PO43-). Potassium, magnesium, and calcium are also elements that microorganisms require, often as cofactors for enzymes

What is a fungicide, bacteriocide, virucide, sterilant, germicide?

-Fungicide- a chemical that destroys fungus -virucide- an agent for destroying viruses -sterilant- an agent used to destroy microorganisms\ -germicide- a substance or other agent that destroys harmful microorganisms

What is generation time and how do bacteria grow in optimum conditions?

-Generation time is the time required for a cell to divide -Bacteria grow logarithmically (exponentially) in optimum conditions

What are the major differences between natural penicillin and synthetic penicillin? What are the advantages of each?

-Penicillins extracted from cultures of Penicillium fungi are the so-called natural penicillins. The prototype compound of all the penicillins is penicillin G. It has a narrow but useful spectrum of activity and is often the drug of choice against most staphylococci, streptococci, and several spirochetes. -A large number of semisynthetic penicillins have been developed in attempts to overcome the disadvantages of natural penicillins (Figure 20.6b). Scientists develop these penicillins in either of two ways. First, they can interrupt synthesis of the molecule by Penicillium and obtain only the common penicillin nucleus for use. Second, they can remove the side chains from the completed natural molecules and then chemically add other side chains that make them more resistant to penicillinase, or the scientists can give them an extended spectrum.

What is resistance? What are the methods of resistance used by bacterial cells?

-Persister cells: microbes with genetic characteristics allowing for their survival when exposed to an antibiotic -Superbugs: bacteria that are resistant to large numbers of antibiotics -Resistance genes are often spread horizontally among bacteria on plasmids or transposons via conjugation or transduction

Know what each type of disinfectant is used for. Example CuSO4 - Algicide

-Phenol and Phenolics: injure lipids of plasma membrane causing leakage -Bisphenols: Disrupt plasma membranes -Biguanides: Disrupt plasma membranes (surgical hand scrubs) -Iodine: Impairs protein synthesis and alters membranes -Chlorine: Oxidizing agents; shut down cellular enzyme systems -Alcohols: Denature proteins and dissolves lipids

How do bacterial control agents work?

-Physical agents work by maintaining a temperature that does not allow for the optimal growth of bacteria (if there is growth it will be slow and toxins won't be produced) -chemical agents work by breaking down the bacteria or inhibiting them in some way

Know the various ways that bacteria can be counted. Know how to perform each method.

-Plate counts- measures the number of viable cells. One disadvantage may be that it takes some time, usually 24 hours or more, for visible colonies to form (must go through serial dilution and then pour plate or spread plate done) -filtration- at least 100 ml of water are passed through a thin membrane filter whose pores are too small to allow bacteria to pass. Thus, the bacteria are filtered out and retained on the surface of the filter. This filter is then transferred to a Petri dish containing nutrient medium, where colonies arise from the bacteria on the filter's surface -MPN- This statistical estimating technique is based on the fact that the greater the number of bacteria in a sample, the more dilution is needed to reduce the density to the point at which no bacteria are left to grow in the tubes in a dilution series -Direct Microscopic Count- a measured volume of a bacterial suspension is placed within a defined area on a microscope slide;A 0.01-ml sample is spread over a marked square centimeter of slide, stain is added so that the bacteria can be seen, and the sample is viewed under the oil immersion objective lens. The area of the viewing field of this objective can be determined. Once the number of bacteria has been counted in several different fields, the average number of bacteria per viewing field can be calculated. From these data, the number of bacteria in the square centimeter over which the sample was spread can also be calculated -Turbidity- (indirect) a beam of light is transmitted through a bacterial suspension to a light-sensitive detector (Figure 6.21). As bacterial numbers increase, less light will reach the detector. This change of light will register on the instrument's scale as the percentage of transmission -Metabolic Activity- indirect way to estimate bacterial numbers is to measure a population's metabolic activity -Dry weight- the fungus is removed from the growth medium, filtered to remove extraneous material, and dried in a desiccator. It is then weighed

What is the difference between selective and differential media? Know how each work.

-Selective media are designed to suppress the growth of unwanted bacteria and encourage the growth of the desired microbes (For example, bismuth sulfite agar is one medium used to isolate the typhoid bacterium, the gram-negative Salmonella Typhi (Tī-fē), from feces. Bismuth sulfite inhibits gram-positive bacteria and most gram-negative intestinal bacteria (other than Salmonella Typhi), as well. Sabouraud's dextrose agar, which has a pH of 5.6, is used to isolate fungi that outgrow most bacteria at this pH.) -Differential media make it easier to distinguish colonies of the desired organism from other colonies growing on the same plate. Similarly, pure cultures of microorganisms have identifiable reactions with differential media in tubes or plates. Blood agar (which contains red blood cells) is a medium that microbiologists often use to identify bacterial species that destroy red blood cells. These species, such as Streptococcus pyogenes, the bacterium that causes strep throat, show a clear ring around their colonies where they have lysed the surrounding blood cells

Know the difference between a broad spectrum versus a narrow spectrum antibiotic

-Some drugs have a narrow spectrum of microbial activity, or range of different microbial types they affect. Penicillin G, for example, affects gram-positive bacteria but very few gram-negative bacteria. Antibiotics that affect a broad range of gram-positive or gram-negative bacteria are therefore called broad-spectrum antibiotics -Broad-spectrum antibiotics target many types of bacteria. Both types work well to treat infections. But using broad-spectrum antibiotics when they're not needed can create antibiotic-resistant bacteria that are hard to treat. They may also have side effects, such as diarrhea or rash.

What is a synergist? Antagonist?

-Synergism: the effect of two drugs together is greater than the effect of either alone -Antagonism: the effect of two drugs together is less than the effect of either alone

What are the components of nutrient agar? How is it made? How are bacteria cultures preserved?

-agar: a solidifying agent such as agar is added to the medium. A complex polysaccharide derived from a marine alga, agar has long been used as a thickener in foods -For laboratory use, agar is held in water baths at about 50°C. At this temperature, it does not injure most bacteria when it is poured over them. Once the agar has solidified, it can be incubated at temperatures approaching 100°C before it again liquefies; this property is particularly useful when thermophilic bacteria are being grown. -Refrigeration can be used for the short-term storage of bacterial cultures. Two common methods of preserving microbial cultures for long periods are deep-freezing and lyophilization. Deep-freezing is a process in which a pure culture of microbes is placed in a suspending liquid and quick-frozen at temperatures ranging from -50°C to -95°C. The culture can usually be thawed and cultured even several years later. During lyophilization (freeze-drying), a suspension of microbes is quickly frozen at temperatures ranging from -54°C to -72°C, and the water is removed by a high vacuum (sublimation). While under vacuum, the container is sealed by melting the glass with a high-temperature torch. The remaining powderlike residue that contains the surviving microbes can be stored for years. The organisms can be revived at any time by hydration with a suitable liquid nutrient medium.

What is chemotherapy, when and who discovered it.

-birth of modern chemotherapy is credited to the efforts of Paul Ehrlich in Germany during the early part of the twentieth century -speculated about some "magic bullet" that would selectively find and destroy pathogens but not harm the host. This idea provided the basis for both selective toxicity and chemotherapy

9. When would filtration be a good method of counting bacteria cell numbers?

-when the quantity of bacteria is very small (in lakes and streams) also applied frequently to detection and enumeration of coliform bacteria, which are indicators of fecal contamination of food or water

What is special about ethanol as a disinfectant?

100% ethanol allows for the growth of bacteria, so when it is used in a disinfectant the concentration is 95% or less

Who is responsible for discovering penicillin? Who is the first person to come up with the idea of chemotherapy?

Alexander Fleming discovered penicillin. Paul Ehrlich came up with chemotherapy.

What are the bio-safety levels? What level is out laboratory?

BSL-1: no special precautions; basic teaching labs BSL-2: lab coat, gloves, eye protection BSL-3: biosafety cabinets to prevent airborne transmission BSL-4: sealed, negative pressure; "hot zone" Exhaust air is filtered twice through HEPA filters (our lab would be like a BSL-1)

How do bacteria reproduce?

Bacteria reproduce by binary fission

What is a disk diffusion test? Know how to tell which substance is most effective.

Evaluates efficacy of chemical agents Filter paper disks are soaked in a chemical and placed on a culture Look for zone of inhibition around disks

What is the Disk diffusion method? What is a zone of inhibition?

Evaluates efficacy of chemical agents Filter paper disks are soaked in a chemical and placed on a culture Look for zone of inhibition around disks (area around the chemical agent that is clear)

What are the various ways that food can be preserved? Know the procedure involved with all of them.

Food can be preserved by keeping it out of the danger zone (refrigeration and freezing) and foods can also be canned and chemically preserved (chemical food preservation -Sulfur dioxide prevents wine spoilage Organic acids-Inhibit metabolism; Sorbic acid, benzoic acid, and calcium propionate- prevent molds in acidic foods; Nitrites and nitrates prevent endospore germination)

How does an autoclave work?

Reliable sterilization with moist heat requires temperatures above that of boiling water. These high temperatures are most commonly achieved by steam under pressure in an autoclave. Autoclaving is the preferred method of sterilization in health care environments, unless the material to be sterilized can be damaged by heat or moisture. The higher the pressure in the autoclave, the higher the temperature. For example, when free-flowing steam at a temperature of 100°C is placed under a pressure of 1 atmosphere above sea level pressure—that is, about 15 pounds of pressure per square inch (psi)—the temperature rises to 121°C. Increasing the pressure to 20 psi raises the temperature to 126°C. Sterilization in an autoclave is most effective when the organisms either are contacted by the steam directly or are contained in a small volume of aqueous (primarily water) liquid. Under these conditions, steam at a pressure of about 15 psi (121°C) will kill all organisms (but not prions; see page 198) and their endospores in about 15 minutes.

What is a halophile? What does this allow the bacteria to do?

extreme halophiles, have adapted so well to high salt concentrations that they actually require them for growth. In this case, they may be termed obligate halophiles. Organisms from such saline waters as the Dead Sea often require nearly 30% salt, and the inoculating loop used to transfer them must first be dipped into a saturated salt solution. More common are facultative halophiles, which do not require high salt concentrations but are able to grow at salt concentrations up to 2%, a concentration that inhibits the growth of many other organisms.

Know all of the different ways that antibiotics work to control microbial growth.

inhibit cell wall synthesis, inhibit protein synthesis, injure plasma membrane, inhibiting nucleic acid sequencing, inhibiting synthesis of essential metabolites