Micro Lab Quiz 7
enrichment cultures
bacteria can colonize vastly different niches due to their adaptability and vast metabolic diversity, which allows them to utilize a wide variety of molecules for energy generation. This diversity is why less than 1% of the bacterial species on the planet are culturable (to be culturable is based on their specific metabolic and environmental needs)
define enrichment:
changing conditions to select for specific species from a mixed culture
Obligate aerobes, obligate anaerobes, aerotolerant anaerobes, and facultative anaerobes are all present at different places on/in the human body.
true
Drive workflow efficiency of bacterial testing with anoxomat system:
the Anoxomat III anaerobic jar system drives workflow efficiency in three key areas: 1.reducing time to diagnosis by up to 50% due to faster growth of bacteria 2. increasing productivity with more hands-off tech time and the ability to create environments ideal for bacterial growth in minutes 3.saves money - within 2 yrs or less and saves 10,000$ or more in technologists time savings alone Steps: 1.load petri dish samples into petri dish holders 2.load holders into jars 3.seal jars 4.attach connection to system 5.place into incubator
What might explain the reason why obligate anaerobes cannot grow at all in the presence of oxygen?
they lack enzymes to degrade toxic forms of oxygen
1. E. coli grows throughout an FTM tube. What would be its oxygen preference classification? 2. Clostridium, a known obligate anaerobe, does not grow in the GasPak jar. What might be the reason for this lack of growth? 3. If an organism grew on both plates in this experiment, what is its classification?
1. facultative anaerobe 2. The chemical reaction failed to remove all of the oxygen in the jar 3. facultative anaerobe
1. The spectrophotometer measures the transmittance of growth. More light transmittance would mean ________ growth in the sample tube. 2. What is the variable in these tubes? 3. What is the purpose of the sterile broth tube? 4. The results indicate that the organism prefers a pH of
1. less 2. pH levels 3. used to calibrate the spectrophotometer 4. 7
what environmental factors affect microbial growth?
1. osmotic pressure - cell membranes are freely permeable to water - "osmosis" = the diffusion of water through a semipermeable membrane from high concentration to low concentration - *water will move in the direction necessary to equilibrate the cell's solute concentration to that of the surrounding environment - ISOTONIC environments: NO movement of water because [solute]0 = [solute]i (0 = outside, i = inside). cell membrane is attached to cell wall - HYPOTONIC environments: water moves into the cell because [solute]0 < [solute]i. causes cell to become larger, which could lead to lysing - HYPERTONIC environments: water moves outside the cell because [solute]0 > [solute]i. causes cell to shrivel/"crenation", results in cell membrane detachment from cell wall, "plasmolysis" 2. pH - pH = -log[H+] - scale ranges from 0 - 14 - each pH unit represents a tenfold change in hydrogen ion concentration (pH 0 = 10,000,000. pH 7 = 1. pH 14 = 1/10,000,000) - indicates whether bacteria is fermenting or alkalizing. ex. mannitol salt agar plate (staphylococcus aureus ferments, giving yellow color because of phenol red which turns yellow when pH is below 7) & (staphylococcus epidermis is pink) ex. urea test; negative/neutral pH = orange/yellow & positive/basic pH = pink --> pH indicator = methyl red, methyl orange, or methyl yellow ex. green vial/neutral pH vs blue on top(alkalizing), green on bottom vial = bromothymal blue - Each organism has 3 cardinal growth pH (graph is a perfect n) ~~1.optimum growth pH: the most favorable pH for the growth of an organism, the peak, grows most quickly ~~2.minimum growth pH: the lowest pH value that the organism can tolerate, beginning of graph ~~3.maximum growth pH: the highest pH value that an organism can tolerate, end of the graph -Organisms can be divided into three groups based on their pH preferences ~~1.Acidophiles (pH<5) ~~2.Neutrophiles (pH5-8) ~~3.Alkaliphiles (pH>8) 3. temperature -each organism has three cardinal growth temperatures (graph is an "n" with a steeper down on the right) ~~1.Optimum growth temperature: the most favorable temp for the growth of an organism, peak ~~2.Minimum growth temperature: lowest temp that an organism can tolerate, beginning of graph with longer line ~~3.Maximum growth temperature: highest temp that an organism can tolerate, end of graph with shorter line -Organisms can be divided into five groups based on their temperature preferences ~~1. Psychrophiles (cold lovers: optimum </=15C and a growth range of -20-20C) ~~2. Psychrotrophs (cold tolerant: optimum >/=16C and a growth range of 0-35C) ~~3. Mesophiles (optimum ~37C and a growth range of 20-45C) ~~4. Thermophiles (optimum ~60C and a growth range of 45-80C) ~~5. Hyperthermophiles (optimum ~90C and a growth range of 80-110C) 4. oxygen - Many microorganisms can grow in the presence of molecular oxygen (O2). Some even use oxygen as a terminal electron acceptor (TEA) in the electron transport chain (known as AEROBIC RESPIRATION) - In the absence of oxygen, microorganisms can engage in ANAEROBIC RESPIRATION / FERMENTATION - Oxygen has its own risks and benefits: ~~Oxygen benefits aerobes, which are organisms that can use it as a TEA to extract energy from nutrients ~~Oxygen is toxic to all cells that do not have enzymes capable of efficiently destroying the reactive oxygen species (ROS). *catalase and peroxidase are important here to get rid of oxygen radicals. ie. anaerobes - Microorganisms can be divided into five groups based on oxygen requirements ~~1. Obligate/strict aerobes: can only grow in oxygen. ie Neisseria spp. ~~2. Microaerophiles: grow only at lower O2 levels. ie Lactobacillus spp. ~~3. Obligate/strict anaerobes: die in the least bit of oxygen. ie Clostridium spp. ~~4. Aerotolerant anaerobes: grow in oxygen but do not use it in their metabolism ~~5. Facultative anaerobes: can live with or without oxygen. posses both the ability for fermentative metabolism and respiration (anaerobic and aerobic). ie Escherichia coli, staphylococcus aureus - Three main oxygen-removing techniques ~~1. Special reducing agents (such as thioglycollate) or enzyme systems (such as oxyrase) can be added to ordinary liquid (agar) media 2. Gas pak anaerobe jar. oxygen is removed by a reaction catalyzed by palladium, gas pak releases hydrogen and carbon dioxide. as well as a pH indicator that goes colorless when there is no oxygen 3. an anaerobic chamber with glove ports. oxygen is removed by vacuum and replaced with N2 and CO2
enrichment and detection of clostridium perfringens toxinotypes (B and D - produce the epsilon toxin) in retail food samples w/o the use of chambers or incubators
1. prepare RPM according to directions and autoclave mixture at 121 C for 15 mins. 2. allow to cool to 40C 3. Add D-cycloserine 4. Transfer RPM to tubes 5.store 6.warm medium 7.Add food being tested into tubes 8.tightly seal tubes to produce an anaerobic environment 9.incubate tubes overnight at 37C TSC sandwich plating: 1.prepare tryptose sulfite cycloserine (TSC agar) 2. inoculate overnight with RPM cultures 3.transfer molten TSC agar into petri dishes 4.maintain remaining TSC agar at 40C 5.transfer overnight RPM culture onto agar base and spread with glass beads or cell spreader 6.After 10 min, transfer molten TSC agar on top 7. allow to solidify, then incubate at 37C sub-culturing of sulfite-reducing colonies 1.examine plates for bacterial growth -aerobic bacteria may be present at the surface, while anaerobic bacteria may be embedded in the agar -sulfite reducing bacteria will turn agar black -C perfringens colonies will be black and embedded in agar (an eyedropper can be used to penetrate agar and pluck out colonies) 2.transfer colonies to fresh RPM to a tube 3. tightly secure, wrap with paraffin film, and incubate overnight at 37C DNA extraction and PCR genotyping: *C perfringens is a BSL-2 organism 1.invert RPM culture 2.transfer to a microcentrifuge tube and pellet bacteria through centrifugation 3.wash pellet with PBS 4.aspirate gelatin and PBS and re-suspend 5.perform DNA extraction using specialized kit for gram positive bacteria 6.Run PCR 7. analyze using gel electrophoresis
How could you distinguish between Escherichia coli, staphylococcus aureus, staphylococcus epidermis, and proteus vulgaris?
By using an MSA plate, an EMB plate, and two TSA (control) plates for each bacterial species. To distinguish which organisms can survive in low oxygen, place one TSA plate from each species into an anaerobic chamber (all should grow but faintly, because they are not obligate anaerobes) MSA = Staphylococcus aureus (yellow bc ferments mannitol) and staphylococcus epidermis (clear but still prevalent) EMB =
to further enrich for an organism of interest, some media types contain indicators
Gives insight into the metabolism of the organism Examples include: - Mannitol Salt Agar (MSA). ~~inhibits the growth of organisms sensitive to high salt ~~toxic to most gram negative species ~~favorable for gram positive staphylococcus genus, ~~indicative of organisms that are able to ferment mannitol because the acid byproducts of fermentation *will turn the methyl red indicator in the media to a bright yellow* ex. staphylococcus aureus (yellow bc ferments mannitol) and staphylococcus epidermis (clear but still prevalent) - Eosin Methylene Blue Agar (EMB). ~~Contains eosin and methylene blue dyes that are toxic to gram positive organisms (outer membrane of gram negative bacteria prevents the toxic dyes from entering the cells) ~~contains lactose that some bacteria can ferment and produce acids that lower the pH encouraging dye absorption leading to a *dark/metallic color of the colony* ~~selective for gram negative organisms ex. Eschrechia coli (turns dark purple. a green metallic sheen indicates lactose fermentation) ex. proteus vulgaris (grows on medium but is light pink/light purple /clear because does not ferment lactose)
Why can jams and honey be stored at room temperature for long periods of time?
The high sugar content causes plasmolysis of bacterial cells
Many bacteria that cause urinary tract infections must be able to withstand the conversion of urea to ammonia in the environment. Therefore, they must be at least tolerant of ________ conditions.
alkaline
What type of environment is created in the GasPak jar?
anaerobic
Where would you expect obligate anaerobes to grow in an FTM culture tube?
at the bottom
Which parts of a cell are affected by temperature?
enzymes, membrane fluidity, ribosomes
The organism inoculated onto the plates was likely a facultative anaerobe
false
Microbial species can be defined as _____, ______, or _____
generalists (able to tolerate a wide variety of states or environments) fastidious (culturable but only in specific conditions. ie. neisseria or haemophilus species require media that has partially broken down red blood cells and a high carbon dioxide concentration) extremophiles (lives in extreme conditions. ie. very low or very high temperatures, reduced or oxygen absent conditions, high salt concentrations)
What class of organisms is likely to inhabit the human body?
neutrophiles
Which of the following is the likely classification for the organism in the FTM tube?
obligate aerobe
Which type of organism requires oxygen as its final electron acceptor in the electron transport chain?
obligate aerobe
How to use a gas pak anaerobic jar
used to cultivate anaerobic and microphilic bacteria easily and without expensive equipment. As well as it can be used to identify unknown microorganisms to determine oxygen requirements needed for growth bsl-2 requirements necessary Steps: 1. gather gas pack and anaerobic jar with lid, a gas generator packet (sodium borohydride and sodium bicarbonate), an indicator strip (pink in the presence of oxygen), loading tray for organizing plates in gas pack container, and a TSA plate (or brewers plate - blue. protects anaerobes and microphiles from oxidizing prior to being placed in jar) 2.place plate upside down into loading tray, then into jar. followed by indicator strip (once white, there is no oxygen) and the gas generator packet 3. seal jar tightly and incubate at 30 C Results: no growth = obligate aerobe growth = obligate anaerobes, facultative anaerobes, or aerotolerant anaerobes
