Microbiology Lab (Exercises 1-20)

Outline the procedure for preparing a smear

1. A small portion of a solid culture is mixed with a drop of water and spread over the surface of a glass slide and air-dried. a. or a loopful of liquid bacterial culture can be spread over the surface of a glass slide and air dried. 2. Only a small drop of water should be mixed with a portion of a bacterial colony. 3. When mixing bacteria with water, avoid over mixing because this can lead to inaccurate bacterial cell patterns. 4. Once the smear is prepared the slide must be allowed to air-dry to completion. 5. Once smear is air-dried, heat-fix the slide: passed through flame 2-3 times. Allows for slide to accept stain.

Distinguish between the five classifications of organisms based on their oxygen requirements.

1. Aerobes: these organisms require oxygen as the final electron acceptor in aerobic respiration, and their survival is dependent upon the presence of oxygen in the atmosphere. 2. Obligate anaerobes : do not require oxygen as an final electron acceptor, and some are even killed by oxygen. They undergo fermentation or anaerobic respiration, and oxygen is not required for either process. Lack both superoxide dismutase and catalase or peroxidase, so toxic derivatives of oxygen could not be destroyed. 3. Facultative anaerobes: these organisms undergo aerobic respiration, anaerobic respiration, or fermentation, depending upon the conditions that exist in their particular niche. Because they can utilize oxygen, they contain both superoxide dismutase and catalase or peroxidase to detoxify the by-products of its metabolism. 4. Aerotolerant anaerobes: these organisms undergo fermentation, so oxygen is not required. However, these organisms do contain superoxide dismutase and catalase or peroxidase to detoxify the by-products of its metabolism. 5. Microaerophiles: require oxygen, but at a concentration lower than that found in the Earth's atmosphere. Undergo aerobic respiration and contain superoxide dismutase and either catalse or peroxidase.

Compare and contrast the four forms of an agar medium that can be used in the laboratory.

1. Agar slant: liquefied medium allowed to solidify in a test tube at a slanted position. 2. Agar deep: liquefied medium allowed to solidify with the tube remaining upright. 3. Agar pour: liquefied medium is poured into a large test tube and is intended for pouring into a Petri Plate at a later date. 4. Agar plate: liquefied medium poured into a petri plate in order to create a solid surface for growing bacteria

List the five modes of action of antibiotics.

1. Alcohol group - only ethyl and isoprophyl alcohol are used to control microbial growth. - One hundred percent alcohol simply inhibits growth of microbes by dehydration - Diluted solutions of alcohol can denture proteins and can dissolve lipids within the cytoplasmic membrane, disrupting the surface tension of the microbe. 2. Halogen group - common disinfectants that are microbicidal and, with prolonged exposure, sporocidal. - halogens act by oxidizing proteins or by denaturing proteins by covalently binding to them. - common household bleach 3. Oxidizing agents - comprised of the perozides, ozone, and peracetic acid, are powerful oxidizing agents that can sterilize materials when used under controlled circumstances. - human cells contain the enzyme catalase, which neutralizes hydrogen peroxide by breaking it down into water and oxygen - used as an antiseptic on closed wounds 4. Surfactants - surface-active agents such as soaps and detergents. - help break through oily layer on the skin, process = emulsification - positively charged nature of these detergents, these compounds function to disrupt the cytoplasmic membrane of microbes. - quat 50 in lab is this compound -highly effective against Gram positive bacteria, fungi, and viruses 5. Heavy metals - are a class of antimicrobial, they are not commonly used because they can be quite toxic to humans if ingested and serious pollution to the environment - cannot be classified as sterilants 6. Aldehydes - the chemical responsible for the intense vapors that often emanate from such preserved specimens. - attach to functional groups of proteins and nucleic acids 7. Gaseous agents - Ethylene oxide gas is a sterilizing agents which destroys all life forms and has great penetrating capabilities. Dentures proteins and nucleic acids by binding to organic function groups

Discuss some of the problems encountered when preparing a smear and list several ways to minimize these problems.

1. During heat-fixation the purpose is to kill cells. a. could heat for too long and incinerate cells which won't take the stain b. could not heat long enough and cells will not be killed and won't take stains. i. hold slide for a few seconds. 2. heat-fixing an incompletely air-dried smear boils the organisms and so the cells will not adhere to the slide a. PRACTICE!

Compare and contrast the three domains of life

1. Eukarya- Unicellular and Multicellular-May consist of one or more cells. Eukaryotic-Cells which contain a nucleus and internal complexity. 2. Bacteria- Unicellular-Consists of only one cell. Prokaryotic-Cells which have no nucleus and lack internal complexity. 3. Archaea- Unicellular-Consists of only one cell. Prokaryotic-Cells which contain no nulcleus and lack internal complexity. (Hint: this domain is closest to bacteria)

Compare and contrast hypotonic, hypertonic, and isotonic solutions.

1. Hypotonic: the cytoplasm of the cell has a lower water activity than the environment. Once the cell is placed into the solution water will rush into the cell to equilibrate the solute concentrations. The cell will eventually swell and rupture (a process known as plasmoptysis). 2. Hypertonic: the environment can have a lower water activity than the cell's interior. Once the cell is placed in the solution the cell loses water and the cytoplasmic membrane draws inward and away from the cell wall, which causes the cell to shrink (a process known as plasmolysis). 3. Isotonic: (Osmosis) the passage of water from an area of high concentration (low solute concentration, high aw) to low concentration (high solute concentration, low aw). The water can pass both in and out of the cell.

Explain the difference between ionizing and non ionizing radiation

1. Ionizing radiation: these types of rays have short wavelengths and are said to be "ionizing" because of their ability to eject electrons from atoms or molecules. These electrons directly damage cellular DNA and cause peroxides, also harmful to the cell, to form (ex. X-rays and gamma rays). 2. Nonionizing radiation: the wavelength of light in the nonionizing range are actually essential for biochemical processes. However, even these wavelengths can be harmful to some cells, including bacteria and viruses (ex. ultraviolet radiation).

Distinguish between the three major temperature classifications of organisms based on their preferred growth temperature.

1. Psychrophiles Minimum: 0 C Optimum: 10-15 C Maximum: 20 C 2. Psychotrophs Minimum: 0 C Optimum: 15-30 C Maximum: 25 3. Mesophiles Minimum: 15-30 C Optimum 30-40 C Maximum: 45 C 4. Thermophiles Minimum: 45 C Optimum: 50-85 C Maximum: 100 C

Identify all parts of a compound light microscope and describe the function of each.

1. Resolving power - the ability of an optical device to produce separate images, focuses on the object. 2. Magnifying power - how much larger a lens can make the object appear, zooms in on the object.

Explain the differences between non selective (all-purpose), selective, and selective and differential media

1. Selective media: these types of media contain compounds that inhibit the growth of some organisms, while allowing the growth of other organisms 2. Differential media: these types of media contain substances that are utilized differently by microorganisms and allow the differentiation between organisms based on their particular metabolic capabilities. 3. Selective and differential media: select for specific organisms, and then differentiate between the organisms capable of growing under those selective conditions. 4. Complex all-purpose growth medium: these media do not contain selective agents and allow for the growth of most microbes under standard incubation conditions.

Discuss the difference between a simple stain and a differential stain

1. Simple stains: consist of a single dye. A. Used when information about cell shape and size, as well as the arrangement of the bacterial cells, is wanted. Because all bacteria take up the stain, difference between cell components cannot be discerned. Common simple stains are Loeffler's alkaline methylen blue, safranin, Kinyoun's carbonfuschsin, and crystal violet. 2. Differential stains: composed of two or more dyes. A. Can differentiate between organisms' cellular components. This type of staining highlights the differences between bacteria and can be used as a classification tool. The most common type of differential stain is the gram stain. Others are the acid-fast stain and the endospore stain.

Explain why different types of soil can contain different numbers and types of microorganisms.

1. TSA pH 7.3: the growth of most fungi, which prefer more acidic conditions, will be inhibited. soil bacteria will be the predominant organism found in TSA. 2. SDA pH 5.6: encourages the growth of fungi, discourages the growth of bacteria that prefer a more neutral growth medium 3. GYE: useful for the isolation and enumeration of bacteria in samples that contain larger numbers of fungi. The antibiotic cycloheximide prevents the synthesis of eukaryotic proteins by blocking the translocation step in translation. Used for isolation and enumeration of actinomycetes.

Describe the proper procedures for caring for, transporting, and storing a compound light microscope

1. Transport - grip the microscope arm with one hand and support the base with the other 2. Caring - clean each objective lens and ocular lens before and after using. Use kimwipe moistened with small amount of 70% ethanol.

Differentiate between the three types of helminths

1. Trematodes are bilaterally, symmetrical leaf-shaped flat, and have both male and female reproductive organs (hermaphroditic). Range in size from a few milometers to a few centimeters. Most have complicated life cycles which includes one or more intermediate hosts. 2. Cestodes are commonly associated with meat and fish, but they can be transmitted to humans by eating uncooked meat. Tapeworm-parasitizes the intestines of animals and can grow up to 30 feet long. 3. Roundworms have cylindrical bodies that are tapered at both ends - bilateral symmetry.

Explain the differences between the agar media being used (in exercise 7).

1. Tryptic soy agar (TSA): can be used for the isolation of most microbes. Contains digests of casein (a milk protein) and soybean meal as sources of carbon and energy, glucose as an energy source, and various salts. Allows for the growth of a wide variety of microorganisms. Differentiate between colony shapes, edges, and coloration.

Explain the differences between the agar media being used (in exercise 7). Part 2

2. Columbia CNA agar with 5% sheep blood: used for the isolation and differentiation of Gram positive organisms. This medium is selective due to the inclusion of the antimicrobials colistin (C) and nalidixic acid (NA). Colistin disrupts the cytoplasmic membrane of Gram negative bacteria, while the nalidixic acid prevents DNA replication in susceptible Gram negative organisms. The 5% sheeps blood allows for the differentiation of organisms based upond their heolysis. b. beta-hemolysis: coloonies are surrounded by a clear zone in which few or o intact red blood cells are present. a. alpha-hemolysis: colonies are surrounded by a zone of intact but discolored red blood cells. g: gamma-hemolysis: no change is observed in the medium around these coloines.

Compare and contrast the terms broad spectrum and narrow spectrum as they apply to antimicrobial action.

A broad spectrum antibiotic to control the infection. This is not often the best choice, however, because broad spectrum drugs can affect the normal flora of the host and may allow for overgrowth of other organisms. It is generally best to receive a drug with a more narrow spectrum of activity, so that such side effects can be prevented. Nonetheless, this option is not always practical because it takes several days to identify the causative agent of an infection.

Compare and contrast the methods of aseptically obtaining organisms from different forms of media.

A. Removing organisms from a liquid culture: 1. shake broth tubes to re-suspend organism 2. flame inoculating loop over Bunsen burner 3. Remove cap, flame neck (2-3 times) 4. Insert cooled inoculating tube. Should see liquid in looped portion of the instrument upon removal 5. re-flame neck, place cap, return, re-flame loop completely

Provide a definition of a virus, including how it is different from other types of microbes.

All viruses are obligate intracellular parasites. This description indicates that viruses multiply within living host cells in a type of symbiosis in which the virus benefits at the expense of the host. Viruses are incapable of independent replication and must use the enzymes and metabolic machinery of the host to construct new viruses. Viruses contain only a single type of nucleic acid, either DNA or RNA, but never in order for cells to multiply. Also, the viral nucleic acid may be linear, composed of several segments, circular, double-stranded, or single-stranded. Those viruses that infect bacteria are known as bacteriophages or phages and it is this type that will serve as a model virus in this exercise.

Differentiate between superoxide dismutase, catalase, and peroxidase.

Any microbe that lives in the presences of oxygen must contain the enzyme superoxide dismutase which prevents the accumulation of the superoxide radical. Unfortunately, the result of this reaction generates hydrogen peroxide, another toxic oxygen product. However, most aerobic organisms contain the enzyme catalase which react with hydrogen peroxide to form water, thereby decomposing this radical. Alternately, those organisms that grow in the presence of oxygen may contain the enzyme peroxidase, which also detoxifies hydrogen peroxide.

Explain how aseptic technique is used to minimize contamination in the laboratory.

Aseptic technique (sterile technique): how to preserve the purity of objects used in a microbiology lab a. Commonly maintained through the use of a Bunsen burner flame b. When an object such as the top of a test tube or an inoculating loop or inoculating needle is passed slowly through the flame heat will incinerate any organisms that may be present and will sterilize the object.

Compare and contrast the methods of aseptically obtaining organisms from different forms of media (cont. 2)

B. Removing organism from a slant culture: 1. flame inoculating loop and mouth of test tube 2. insert inoculating loop and scrape some of the bacteria growing on the medium. Try not to break the surface of the agar 3. re-flame neck and tube. Transfer the inoculum to fresh medium 4. re-flame loop completely.

List some methods of microbial control that make use of ultraviolet light.

Because ultraviolet radiation can ultimately be lethal to microorganisms, this type of radiation is routinely used to control microbial growth in the air, hospital rooms, nurseries, operating rooms, and cafeteria.

Compare and contrast the methods of aseptically obtaining organisms from different forms of media (cont. 3)

C. Removing organisms from an Agar plate culture: 1. examine agar plate carefully 2. flame inoculating loop 3. lift lid of petri dish at 45 angle 4. insert loop, gently scrape small amount of growth from the age without gouging the agar 5. replace lid. Transfer inoculum to fresh medium 6. re-flame loop completely

Compare and contrast a disinfectant, a chemotherapeutic agent, and an antiseptic.

Chemical factors used to control microbial growth include disinfectants, antiseptics, and chemotherapeutic agents. These chemical agents control growth of microbes on living tissues and inanimate objects. Disinfectants are chemical agents used on inanimate objects to lower the level or number of microbes of the surface. Antiseptics are chemicals used on living tissues to decrease the number of microbes. Chemotherapeutic agents are chemical agents that are used internally and may be natural or synthetic.

Define the term contamination and explain how materials may become contaminated.

Contamination refers to the non-intended or accidental introduction of microbes These items can become contaminated in many different ways because microorganisms are everywhere - in the atmosphere, on your hands, in the air you exhale, and on inanimate objects. The more exposure = the higher possible contamination.

Compare and contrast the methods of aseptically obtaining organisms from different forms of media (cont. 4)

D. Inoculation of fresh media: 1. remove cap from the tube of medium. Flame neck of tube a. inoculating broth medium: dip the loop into the broth and gently move back and forth to transfer organisms from loop to new medium. b. inoculating agar slant: gently streak loop along surface of the slant to transfer organisms c. inoculating agar deep: stab loop 3/4 of the way to the bottom of the deep and withdraw 2. agar plate: lift lid 45 and streak loop along surface of agar to transfer organisms a. sport inoculation: a concentrated area of organisms about the size of a quarter is placed on plate b. performing streak plate technique, the bacteria are diluted across surface of medium in attempt to isolate individual colonies of bacteria c. replace lid on plate 3. flame loop completely

Define an indicator organism and describe why these organisms are important.

E. coli is an indicator organism, one whose presence in a sample suggest the presence of other, potentially pathogenic fecal organisms.

List the characteristics of a coliform.

E. coli is the most commonly used indicator organism, but any of the coliform bacteria can be used as indicator organisms. Coliforms are aerobic or facultatively anaerobic, Gram negative, nonsporing rods that ferment lactose with acid and gas formation within 24-48 hours. The presence of a significant number of coliforms in a water sample indicates fecal contamination has occurred and that the water may not be safe for recreation or drinking.

Distinguish between the three pH classifications of microorganisms.

Each microbial species has a definitive pH range (outside) and pH optimum (inside). 1. Neutrophiles: have pH optima between pH 5.5 and 8.5, but ideally prefer values of 6.6-7.0. Most bacteria and protozoa. 2. Acidophiles: have pH optima between pH 1.0 and 5.5. Strong acidophiles need a strong concentration of hydrogen ions is necessary to maintain the stability of the cytoplasmic membranes. Thus, strong acidphiles lyse when the pH is raised to near neutral pH values. This is shown in the preservation of foods: pickles, tomatoes. 3. Alkaliphiles: have pH optima between pH 8.5 and 11.5. Used as laundry detergents. These enzymes function well at alkaline pH values and thus function in the alkaline environments created by detergents in a washing machine. The proteases (degrade proteins) used as supplements, but the sue of amylases (which degrade starch) and lipases (which degrade fats) is increasing.

Compare and contrast the cardinal temperature points all microorganisms have.

Each microbial species has its own set of cardinal temperature points - the minimum, maximum, and optimum growth temperatures. 1. Minimum growth temperature: lowest temperature at which the organism will grow. If the temperature extends below this temperature, enzyme activity is inhibited and the organisms become metabolically inactive, growth is nonexistent. 2. Maximum growth temperature: highest temperature at which an growth occurs. At temperatures above this level, enzymes will denature and organisms will die. 3. Optimum growth temperature: microbial growth is most rapid here. Organisms are found growing in an environment that supports the optimum growth temperature requirements.

List several important contributions of microbes to food production.

Fermentation is a means by which microbial growth causes a desirable change in a product. The microorganisms causing this change may be present naturally or may be added exogenously as starter cultures. Lactic acid-producing bacteria aid in the production of all types of cheese. When yeast is added to a mixture of flour, salt, sugar, shortening, and water, they ferment the carbohydrates from flour and sugar to produce carbon dioxide, alcohols, and acidic end-products. Carbon dioxide becomes trapped in the dough and causes the dough to rise. This increases the size of the load and also produces the light texture. The alcohols and acidic end-products help produce some of the flavors within the bread. Wines result from the anaerobic fermentation of fruits, normally grapes, by yeasts.

Explain the role of lequminous plants in nitrogen fixation.

Free-living nitrogen-fixing bacteria that are found in high concentrations within the rhizosphere. The second and more important type of nitrogen-fixing bacteria are the symbiotic nitrogen-fixing bacteria. These bacteria infect the roots of leguminous plants, such as soybeans, beans, peas, alfalfa, clover, and peanuts, and form root nodules, or growths on the plant roots. Nitrogen is fixed in a symbiotic process between the plant and the bacteria. The bacteria contained within the root nodules fix nitrogen for the plant, which the plant harbors the organisms and provides nutrients in the form of plant sap.

Why are fungi larger than bacteria?

Fungi are eukaryotes which are 10 micrometers, or greater, because they have cell organelles, nucleus, and larger chromosomes in their cells. Where as bacteria, a prokaryote is only one or few micrometers because they lack what the eukaryotes have.

Explain what halophiles are and where they can be found.

Halophiles are "salt-loving" organisms. Mild halophiles require between 1-6% sodium chloride for growth, moderate halophiles require between 6-15% sodium chloride for growth, and, amazingly, extreme halophiles require between 15-30% sodium chloride for growth.

Compare and contrast foodborne infection and microbial intoxication.

In a foodborne infection a person ingests food contaminated by microorganisms. Organisms must first multiply in the gut and invade the intestinal tissues prior to the initiation of signs and symptoms. Several hours to days following ingestion of contaminated food, diarrhea, cramps, nausea, vomiting, and fever commence. In an intoxication microorganisms gwoa and multiply in a food source and produce toxins that remain in the food and which are not destroyed by heating. The ingestion of these preformed toxins produces the illness. The toxins cause the signs and symptoms, so onset of signs and symptoms occurs within minutes to hours. Affected individuals experience diarrhea, cramps, nausea, and vomiting, but fever is generally absent.

Compare and contrast the lytic cycle with the lysogenic cycle.

In the lytic cycle of replication, the host cell is destroyed once new viruses have been synthesized. The replication cycles of bacteriophages occurs in five steps: attachment, entry, synthesis, assembly, and release. In the lysogenic cyle of replication, the virus lives in harmony with its host by inserting its genetic material into the host's chromosome. These viruses begin their invective cycle just as lytic viruses do, but do not proceed through the synthesis, assembly, and release steps.

Differentiate between the types of organisms isolated on m-Endo agar and KF streptococcus agar.

KF steptococcus agar detects fecal streptococci. These organisms are also present in the intestinal tracts of humans and animals and are indicative of fecal contamination. If both fecal coliforms and fecal streptococci are present in a water sample, officials can reasonably certain that fecal contamination of the water has occurred.

Distinguish between defined and complex media

Laboratory personnel often prepare synthetic media (defined media; nutrient preparations used for microbial growth) for the growth of microorganisms. Commonly used for fastidious organisms microbiologists prepare complex, or chemically non-defined media for routine growth of microbial cultures. These types of media (complex) contain the same elements as defined media, but the amount of each particular nutrient may vary from recipe to recipe. Three typical components of complex media are: beef extract, yeast extract, and peptones.

Compare and contrast flash pasteurization with ultrahigh temperature sterilization.

Louis Pasteur discovered that mild heating of beer and wine helped prevent spoilage, but did not seriously alter the taste of these products. This process later became known as pasteurization and is now principally associated with milk. Today, milk is pasteurized by the flash method, in which milk is heated to 72 C for at least 15 seconds followed by rapid cooling. The process by which milk is sterilized is known as the ultrahigh temperature (UHT) processing procedure. The temperature of milk is raised from 74 C to 140 C and then dropped back to 74 C in less than five seconds.

Explain the differences between the agar media being used (in exercise 7). Part 3

MacConkey agar: both a selective and differential medium. The crystal violet and bile salts are inhibitory for Gram positive bacteria. Consequently this medium is selective for Gram negative organisms. Is differential on the basis of lactose fermentation. The neutral red indicator serves to contrast lactose-fermenting from lactose-nonfermenting organisms. If an organism ferments lactose the drop in pH causes the colony to absorb the indicator, appearing pink to brick-red. If the organism does not ferment lactose the bacterial colonies remain translucent.

Explain the differences between the agar media being used (in exercise 7). Part 4

Mannitol salt agar: selective and differential medium. Contains 7.5% sodium chloride, which results in the partial or complete inhibition of microbial species other than halophiles (salt loving). It is differential on the basis of mannitol (a carbohydrate) fermentation, and the indicator phenol red is incorporated to identify mannitol-fermenting organisms. Colonies of bacteria that ferment mannitol will turn yellow. Colonies of bacteria that cannot ferment mannitol will remain translucent.

Explain the value of microbes in our environment.

Microbes recycle carob, oxygen, and nitrogen which is detrimental to our existence.

Explain the differences between microbicidal and microbistatic agents.

Microbicidal agents are those agents that result in microbial death. Microbistatic agents are those agents that cause a temporary inhibition in microbial growth.

Define the concept of a pure culture.

Microbiologist generally want to study a single species of microorganism without interference from other microorganisms. In order to study a single type of organism, a pure culture of the organism must be obtained. This is simply a culture of one type of organism.

Describe how the physical requirement of pH affects microbial growth.

Most microbes are capable of growth over a range of three pH units, with the most vigorous growth occurring near the midpoint. This range reflects the effect of the changing hydrogen ion concentrations on enzymes and the reactions they catalyze. The specific pH requirements of microorganisms reflect the organisms' adaptation to their particular niche. It is important to remember tat pH values represent the pH of the extracellular environment. The environment inside the cell must always remain at or near neutral in order to prevent the destruction of important cellular macromolecules.

Differentiate between the nutritional classifications of organisms and list the carbon and energy sources found in each category.

Nutritionally speaking, microbes are classified based upon both the source of carbon utilized and the source of energy needed for enzymatic reactions. 1. Heterotrophs: organisms that utilize organic carbon as a carbon source 2. Autotrophs: obtain carbon by incorporating the carbon present in atmospheric carbon dioxide 3. Chemotrophs: obtain their energy from the oxidation of organic chemicals Lithotrophs: obtain energy from the oxidation of inorganic chemicals 4. Phototrophs: use radiant energy in the form of sing light as their source of energy Thus, with respect to both carbon and energy sources, four basic groups of organisms exist: (1) chemoheteroptrophs: use organic chemicals as a sources of both carbon and energy (2) chemoautotrophs: obtain their energy from organic sources and obtain carbon from carbon dioxide (3) photoheterotrops: utilize sunlight as an energy source and organic chemicals as a carbon source (4) photoautotrops: obtain energy from sunlight and carbon dioxide Lithotrophic species are either 1-lithoautotrophs: obtain energy from inorganic compounds and carbon from carbon dioxide, or 2-lithoheterotrophs: utilize organic chemicals for carbon, while utilizing inorganic compounds as a source of energy.

Why is oil necessary when using the 100X objective

Oil has the same refractive index as glass, the loss of light passing through the lens is minimized

Discuss the role of algae play in the production of atmospheric oxygen and why they are known as primary producers.

Oxygen is a by product of photosynthetic metabolism, and the algae, specifically the marine phytoplankton, are a major source of the world's oxygen. Additionally, their photosynthetic metabolism enables them to incorporate atmospheric carbon dioxide into organic compounds, and they are responsible for nearly half of the photosynthetic production of organic carbon globally. As such, they are often referred to as primary producers.

Discuss the methods by which obligatory anaerobic microorganisms can be grown in the laboratory.

Reducing media contain chemicals that combine with oxygen to eliminate it from the atmosphere. This medium contains the chemical sodium thioglycollate that reduces the eoxidation-reduction potential of the medium. The indicator resazurin shows if the medium has been oxidized. This indicator causes the medium to turn pink where oxidation has occurred and remains colorless where anaerobic conditions remain.

Discuss the life cycle of the Plasmodium protozoan

Refer to page 27 in lab manual.

List the properties of the growth medium used to grow the fungi (in exercise 2)

Sabouraud's Dextrose Agar - this growth medium has a high sugar concentrations and a slightly acididc pH of 5.6 that make it unsuitable for the growth of bacteria.

Discuss the mechanisms by which materials can be sterilized.

Sterile: free of all microorganisms and life forms prior to use. 1. An autoclave is routinely used in microbiology laboratories for sterilizing both media and instruments because it is both quick and dependable. a. This device sterilizes materials using steam (moist heat) heated to 121C at 15 pounds per square inch of pressure for 20 minutes. 2. Dry heat sterilization is most often used for glassware. a. consists of placing items in a hot air oven at 170C for 90 minutes. 3. Filtration is used for media additives that cannot withstand heat sterilization methods. a. liquid is passed through a filter that traps microorganism while allowing the liquid to continue through the filter into a sterile container. 4. Ultraviolet radiation is often used in hospitals to sterilize the air or surfaces in a room. 5. Ethylene oxide gas is used to sterilize heat sensitive materials, such as plastic petri dishes, cotton packing, and syringes.

Explain the importance of bacteria in the biogeochemical cycling of nitrogen.

The conversion of inorganic components from one from to another is biogeochemical cycling. Nitrogen (N2) is an essential element required by all organisms, including humans, for the synthesis of proteins and nuclei acids. Although nitrogen is the most abundant gas in teh atmosphere (approximately 79%), few organisms can use it as a source of nitrogen. Most living organisms utilize nitrogen in the form of ammonia (NH3) and nitrogen fixation is the process by which certain microorganisms convert atmospheric nitrogen into ammonia.

Define the concept of dilution.

The dilution of a particular bottle or tube is equal to the ratio of the volume added to the total volume following the addition. For example: Adding 1 ml of culture to a 99 ml dilution blank results in a total volume of 100 ml. The dilution is 1:100 or 10^-2. Adding 1 ml of a 10^-2 dilution to a second 99 ml blank results in a 1:10,000 or 10^-4 dilution. Plating 1 lm of the 10^-4 dilution does not change the dilution because we are trying to determine the number of microbes per milliliter. Plating 0.1 ml of the 10^-4 dilution results in an additional 10-fold dilution for a total dilution of 10^-5.

Describe why lactophenol cotton blue stain is used when viewing a fungal specimen under a microscope

The lactic acid and phenol components of this stain help to kill and preserve the organism, while cotton blue stains the chitin present in the cell wall

Explain how microbial contamination of foods can occur.

The mass production of foods along with large food processing plants that provide ideal environment for cross contamination of large volumes of food with microorganisms leads to foodborne illnesses.

Explain how m-Endo agar is both selective and differential.

The medium usually employed for coliform detection is m-Endo medium, which is both a selective and differential medium. Sodium lauryl sulfate and sodium desoxycholate are included to inhibit the growth of Gram positive organisms, while lactose serves to differentiate between coliforms and noncoliforms.

Identify the different types of organisms found in the soil.

The most prevalent type of soil in terrestrial environments is known as mineral soil, while organic soil forms from sediment that accumulates in bogs and marshes. 1. Rhizophere: develops around the plants' roots. It contains dense populations of microbes growing on the surfaces of the soil particles. 2. Fungi : usually the primary decomposeres within their habitat, and break down highly complex substrates such as cellulose, starch, and lignins. 3. Actinomycetes: a group of Gram positive filamentous and/or branching bacteria, are also involved in the decomposition of complex organic substances, and are responsible for giving soil its characteristics earthy smell. Play a vital role in decomposition of compounds that are less easily degraded.

Calculate a phenol coefficient for a disinfectant.

The phenol coefficient (PC) is used to measure the effectiveness of a phenol-based antimicrobial agent with respect to phenol. To determine the PC of a compound, the highest dilution of the test compound that kills all organisms in ten minutes is divided by the highest dilution of phenol that kills all organisms in ten minutes.

Describe the purpose, importance, and applications of the pour plate technique.

The pour plate technique is another method for isolating bacterial colonies. A small volume from each dilution is pipetted into an empty petri plate. Melted agar medium is then poured into the petri plate and solidifies before incubation. Colonies will grow within and on the agar.

Discuss the concept of osmotic pressure and how it relates to water activity.

The pressure that develops on this membrane by solutions of different solute concentrations is called the osmotic pressure. (Think osmosis and the semi-permeable membrane)

Describe the purpose and importance of the spread plate technique.

The spread pate technique is a third method for achievein gisolated colonies. A small volume of broth culture of microbes is spread over the surface of an agar medium. A sterile, L-shaped glass rod known as a glass spreader or a hockey stick is used to spread this volume over the surface of the agar. To sterilize the rod dip it in a beaker of 95% ethanol and pas it through the Bunsen burner flame. Once the flame is burned out, the stick it ready for use. This technique can be used to estimate the total number of bacteria in a broth culture.

Differentiate between a standard set and an action limit.

The standard set for drinking water is a limit of one coliform per 100 ml of water. The action limit for drinking water, the limit at which action must be taken, is four coliforms per 100 ml of water.

Define water activity.

The water activity (aw) of a substance is defined as the vapor pressure of water in a substance divided by that of pure water at the same time. The aw is the relative availability of water within a substance. The aw is also a function of the concentration of solutes dissolved in the water because solutes have an affinity for water.

Discuss the widespread nature of microorganisms

The widespread nature of microbes is based on three major properties: (1) their small size allows for easy dissemination by air and water, (2) microbes demonstrate an incredible versatility and flexibility with regards to their metabolism, and (3) microbes have a unique ability to tolerate unfavorable environmental conditions.

Explain why toxic oxygen species are harmful to cells.

The will oxidize key cellular components if they are not neutralized. Thus, the presence and distribution of enzymes that react with oxygen and its derivatives dictate an organism's sensitivity to oxygen.

List several characteristics of viruses.

They are composed of a protein coat known as a capsid which surrounds the nucleic acid. Some viruses may also contain a phospholipid envelope which surrounds the capsid. Viruses may be helical, polyhedral, or complex in shape. This shape is dictated by the arrangement and types of capsid subunits, known as capsomeres, present in the virus.

Describe qualities of eukaryotic microorganisms.

They have a nuclear membrane around their DNA. Their DNA is wound around histones. They have a complex membrane-bound organelles.

Explain how ultraviolet affects cells.

Ultraviolet radiation exerts its effects on the DNA of all types of cells and causes covalent bonds to form between adjacent thymine bases. During DNA replication, new nucleotides on the daughter strand of DNA cannot complementary base pair with a thymine-thymine dimer, and replication is terminated.

Discuss how a plaque assay allows for the quantification of viruses in a sample.

Virologists use a method known as the plaque assay in order to enumerate bacteriophage. In this method dilutions of the bacteriophage are prepared. Next, a sample of each bacteriophage dilution is mixed with host bacteria and a small volume of melted agar. This mixture is then poured over a layer of solidified medium in a petri plate. When the thing layer of bacteriophage, bacteria, and agar solidifies, a layer of bacteria approximately one cell thick results.

Explain how water activity influences microbial growth.

Water makes up between 50-90% of the mass of microbes and they require water in order to grow. When appropriate nutrients and physical conditions, along with an abundance of water, exist, microbes will grow and reproduce - that is, they will grow.

Discuss how organisms can overcome the effects of ultraviolet light.

When bacteria containing thymine-thymine dimers are incubated in the presence of visible light, transcription and trasnlation of the enzyme DNA photolyase is activated. This enzyme acts to split the thymine-thymine dimers in a reaction known as light repair or photoreactivation. DNA replication can then proceed normally. Dark repair mechanisms and each involves a different enzymes. Both base excision and mismatch repair are activated when there is only slight damage to the DNA molecules. In each other repair mechanisms. an enzyme cuts the damages DNA from the double-stranded molecule. Nucleotides are added to the resulting gap by DNA polymerase, and the ends are sealed by DNA ligase. In the case of significant DNA damage SOS repair is activated. Again, the damaged DNA is removed, but a novel DNA plymearse is responsible for nucleotide addition. This new DNA polymerase does not preserve the complementary base pairing between the parent and the daughter strands of DNA. It simply adds nucleotides in an almost random fashion. This may result in an organism with a mutation in one or more proteins, but it may also result in a potentially fatal mutation.

Describe how water can become contaminated with microorganisms.

When pathogens are present in water, their numbers are generally low and they would probably be missed during the identification process.

Define what microorganisms are.

living organisms that are too small to be observed using the naked eye