Microbio Chapt 6 Learning Objectives
Why is it difficult to measure realistically the growth of a filamentous mold isolate by the plate count method?
*A plate count would not measure the increase in filamentous mass. In plate counts of actinomycetes and molds, it is mostly the number of asexual spores that is counted instead. This is not a good measure of growth.
Classify microbes into five groups on the basis of the preferred temperature range.
1). Psychrophiles: cold-loving, -15 and 20C 2) Psychrotrophs: cold-tolerant, survives at 0, but prefers temps in the mesophiles zone. 10 to 50C, cause food spoilage 3) Mesophiles: moderate temp loving, 10 to 50C 4) Thermophiles: heat-loving, Optimum growth temperature of 50 to 60°C, found in hot springs 5) Hyperthermophiles: greater than 80C
If there is no good method for analyzing a product for its vitamin content, what is a feasible method of determining the vitamin content?
A feasible method of determining the vitamin content is by measuring the metabolic activity. Where acid production is used to determine amounts of vitamins. Bacterial growth in a culture medium is reflected by the amount of lactic acid produced, as it will be proportional to the amount of vitamin in the test substance. The more lactic acid the more vitamin is present.
Define colony.
A visible mass of microbial cells arising from one cell or from a group of the same microbes.
Justify the use of each of the following: anaerobic techniques, living host cells, candle jars, selective and differential media, enrichment medium.
Anaerobic Techniques (Methods used to cultivate anaerobic microorganisms): To routinely grow and maintain pure cultures of obligate anaerobes, microbiologists use reducing media (Media that contains ingredients that deplete the oxygen in the culture medium) These media are heated shortly before use to drive off absorbed oxygen. Many techniques are used. Living host cells: Some bacteria have never been successfully grown on artificial laboratory media. And like viruses, they can only reproduce in a living host cell. Candle jars: Used to create the conditions needed to grow bacterias that require an increased concentration of carbon dioxide and decreased concentration of oxygen. A sealed jar with the lit candle inside, the flame will consume most of the oxygen in the jar while producing an elevated level of carbon dioxide Selective media: Suppress unwanted microbes and encourage desired microbes Differential media: Allow distinguishing of colonies of different microbes on the same plate Enrichment medium: Similar to selective media but designed to increase numbers of desired microbes to detectable levels. Bacteria in small numbers can be missed, especially if other bacteria are present in much larger numbers
Could Louis Pasteur, in the 1800s, have grown rabies viruses in cell culture instead of in living animals?
No, as viruses can only reproduce in a living host cell
What BSL is your laboratory?
BLS-1 a teaching lab
Differentiate biosafety levels 1, 2, 3, and 4.
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
Define bacterial growth, including binary fission.
Bacterial growth refers to an increase in bacterial numbers, not an increase in the size of the individual cells. Bacteria normally reproduce by binary fission, it doubles the number of cells each generation Binary fission is Prokaryotic cell reproduction by division into two daughter cells: 1) cell elongates and DNA is replicated 2) Plasma membrane begin to constrict and new wall is made 3) cross-wall forms, completely separating the two DNA copies 4) cells separate = 2 identical daughter cells Bacterial grow is the action, binary fission is the method used to complete said action.
Describe the formation of biofilms and their potential for causing infection.
Biofilms are microbial communities that form a slime that adheres to surfaces. It acts as a protective barrier from antibiotics, the environment, and host defenses. It also aids in the ability to share nutrition. This closeness allows for cell to cell communication, like the transfer of genetic information. Formation: A biofilm usually begins to form when a free-swimming bacterium attaches to a surface. They often form pillar-like structures with channels between them, to allow incoming nutrients and outgoing wastes to travel through. Potential: Due to its structure, the microbes in biofilms are more resistant to microbicides, and other defenses. Blocking antibiotics as well as immune responses. Biofilms are involved in about 70% of human bacterial infections
Distinguish chemically defined and complex media.
Chemically defined media: The exact chemical composition is known (recipe of nutrients is needed to grow culture) It is usually reserved for laboratory experimental work or for the growth of autotrophic bacteria. Complex media: The exact chemical composition is not known. It is made up of nutrients including extracts from yeasts, meat, or plants, or digests of proteins from these and other sources. Chemical composition varies from batch to batch.
Explain how microorganisms are preserved by deep-freezing and lyophilization(freeze-drying).
Deep-freezing: A pure culture of microbes is placed in a suspending liquid and quickly frozen at temperatures ranging from -50 to -95C. They can be thawed and culture years later Lyophilization (freeze-drying): Quickly frozen at -54 to -72C, the water is removed by a high vacuum (sublimation), or dehydrated in a vacuum. 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.
Can you think of any reason why a colony does not grow to an infinite size, or at least fill the confines of the Petri plate?
Due to lack of/running out of nutrients
Direct methods usually require an incubation time for a colony. Why is this not always feasible for analyzing foods?
Incubation time requires time. For example, the plate count method can take 24 hours or more for visible colonies to form. This creates a quality control issue, which is why direct methods are not always feasible for analyzing foods.
Differentiate direct and indirect methods of measuring cell growth.
Indirect methods do not count microbial cells to estimate their numbers. Instead, it's a method of measuring their metabolic activity. Direct methods do involve counting microbial cells. Indirect methods include, estimating turbidity (the cloudiness of a suspension) measuring a population's metabolic activity, and by measuring the growth of filamentous organisms by dry weight.
Explain the importance of osmotic pressure to microbial growth.
Microorganisms obtain almost all their nutrients in solution from the surrounding water. Thus, they require water for growth. Osmotic pressure prevents the inward flow of water (osmosis) High osmotic pressures have the effect of removing necessary water from a cell, and the growth of the cell is inhibited. The addition of salts (or other solutes) to a solution, and the resulting increase in osmotic pressure, can be used to preserve foods. Extra: Hypertonic environments (higher in solutes outside than inside the cell) cause plasmolysis (Loss of water from a cell in a hypertonic environment) due to high osmotic pressure Extreme or obligate halophiles require high osmotic pressure (high salt) Facultative halophiles tolerate high osmotic pressure
Identify how and why the pH of culture media is controlled.
Most bacteria grow between pH 6.5 and 7.5, a range near neutrality. When bacteria are cultured in the laboratory, they often produce acids that eventually interfere with their own growth. To neutralize the acids and maintain the proper pH, chemical buffers are included in the growth medium. The peptones and amino acids in some media act as buffers, and many media also contain phosphate salts. Phosphate salts have the advantage of exhibiting their buffering effect in the pH growth range of most bacteria. They are also nontoxic; in fact, they provide phosphorus, an essential nutrient.
Identify a way in which pathogens find it advantageous to form biofilms.
Pathogens, like every organism, will try to find a way to grow and survive. Being part of a community biofilm allows pathogens to do just that by providing protection and nutrients.
Explain four direct methods of measuring cell growth.
Plate Count: A method of determining the number of bacteria in a sample by counting the number of colony-forming units on a solid culture medium. The most frequently used method of measuring bacterial populations. Advantage- It measures the number of viable cells. Disadvantage- takes usually 24 hours or more for visible colonies to form. Reported as colony-forming units (CFU - Visible bacterial colonies on solid media) Serial Dilutions: The process of diluting a sample several times. This ensures that some colony counts will be within the preferred plate range of 30-300 colonies. Counts are performed on bacteria mixed into a dish with agar (pour plate method) or spread on the surface of a plate (spread plate method) Filtration: The passage of a liquid or gas through a screen-like material, filter that removes most bacteria. The bacteria are filtered out and retained on the surface of the filer. This filter is then transferred to a Petri dish containing nutrient medium, where colonies arise from the bacteria on the filter's surface. This method is used in the detection and enumeration of coliform bacteria, which indicates fecal contamination of food or water. Most probable number method (MPN): A statistical determination of the number of coliforms per 100ml of water or 100g of food. This 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: Enumeration of cells by observation through a microscope. A measured volume of a bacterial suspension is placed within a defined area on a microscope slide. Because of time considerations, this method is often used to count the number of bacteria in milk.
If bacterial cells were given a sulfur source containing radioactive sulfur (35S) in their culture media, in what molecules would the 35S be found in the cells?
Protein (Sulfur is used to synthesize sulfur-containing amino acids, amino acids make protein) Bacterial cells exposed to radioactive sulphur (35S) will contain radioactive protein, with 35S being the only source of sulphur it will incorporate into the protein.
How would one determine whether a microbe is a strict anaerobe?
The microbe would only grow where there is no oxygen, as the presence of oxygen would kill it. In a lab setting, an anaerobe would only grow at the bottom of a test tube where no oxygen is present.
Compare the phases of microbial growth, and describe their relation to generation time.
The phases of growth allow us to understand what is happening to the bacterial population during generation time. This understanding helps in the areas of infectious diseases, food preservation, and more The time required for a cell to divide (20min to 24hrs) and its population to double is called the generation time Microbial growth phases: Lag phase: Intense activity as bacteria prepare for population growth, but no increase of population Log phase: Logarithmic or exponential increase in population due to reproduction by binary fission (bacteria) or mitosis Stationary phase: Period of equilibrium (number of deaths equal number of new cells) Death phase: Population is decreasing at a logarithmic rate (number of deaths exceeds number of new cells formed)
Describe how pure cultures can be isolated by using the streak plate method.
The streak plate method is the most common method used when isolating pure cultures, the process is as follows. A sterile inoculating loop is dipped into a mixed culture that contains more than one type of microbe and is streaked in a pattern over the surface of the nutrient medium. As the pattern is traced, bacteria are rubbed off the loop onto the medium. The last cells to be rubbed off the loop are far enough apart to grow into isolated colonies. These colonies can be picked up with an inoculating loop and transferred to a test tube of nutrient medium to form a pure culture containing only one type of bacterium.
Oxygen is so pervasive in the environment that it would be very difficult for a microbe to always avoid physical contact with it. What, therefore, is the most obvious way for a microbe to avoid damage?
They adapt and produce enzymes that neutralize toxins. For example, Aerotolerant anaerobes are organisms that do not use molecular O2 but are not affected by it either. These bacteria can tolerate oxygen because they possess SOD (Superoxide dismutase - a neutralizing enzyme) or an equivalent system that neutralizes the toxic forms of oxygen. Example: lactobacilli are used in the production of many acidic fermented foods, such as pickles and cheese. In the laboratory, they are handled and grown much like any other bacteria, but they make no use of the oxygen in the air.
Explain three indirect methods of measuring cell growth.
Turbidity- As bacteria multiply in liquid medium, the medium becomes turbid or cloudy with cells. In the spectrophotometer (instrument used to measure turbidity), a beam of light is transmitted through a bacterial suspension to a light sensitive detector. As bacterial numbers increase, less light will reach the detector. The less light transmitted, the more bacteria in the sample. Turbidity is not a useful measure of contamination of liquids by relatively small numbers of bacteria. Metabolic Activity-This method assumes that the amount of a certain metabolic product such as acid or CO2, is in direct proportion to the number of bacteria present Dry Weight- The usual measuring methods are less satisfactory for filamentous beacteria and molds. For the dry weight method, fungus is removed from the growth medium, filtered to remove extraneous material, and dried in a desiccator then weighed. Bacteria follows the same procedure.
Could a pure culture of bacteria be obtained by the streak plate method if there were only one desired microbe in a bacterial suspension of billions?
Yes, if selective media is used to kill off unwanted colonies first followed by the use of the streak method, a pure culture could be obtained.
Could humans exist on chemically defined media, at least under laboratory conditions?
Yes, under laboratory conditions the chemically defined medium (Composition is known) must contain organic compounds that serve as a source of carbon and energy. Chemoheterotrophs (An organism deriving energy from organic compounds and is incapable of creating on its own) include humans
Mannitol salt agar has a high salt concentration (7.5% NaCl) which inhibits the growth of many organisms other than Staphylococci. It also contains a pH indicator to detect fermentation of the mannitol which is a characteristic of Staphylococci aureus. This plate would be considered a)Enrichment media. b)Selective media. c)Differential media. d)Both selective and differential. e)None of the above.
d)Both selective and differential Selective because the high salt concentration prevents (Inhibits) the growth of most bacteria but not Staphlylococcus Differential because the pH indicates the difference between bacteria that can ferment mannitol and those that cannot
