Micro Lecture Set 4: Microbial Growth PDF

BACTERIAL GROWTH Objectives/ Study guide Define minimal, optimal and maximum temperatures in terms of bacterial growth Define and between each of the thermal preference categories of differentiate bacteria Relate thermal preferences to 1) refrigeration 2) h...

BACTERIAL GROWTH Objectives/ Study guide Define minimal, optimal and maximum temperatures in terms of bacterial growth Define and between each of the thermal preference categories of differentiate bacteria Relate thermal preferences to 1) refrigeration 2) hot springs/thermal vents in nature. Define and differentiate between the pH preference categories of bacteria Define and differentiate between the categories of salt preferences in bacteria Explain how high salt concentration can kill bacteria, and why this has been historically important to humans Define and Relate osmophiles, xenophiles and halophiles  Objectives/ Study guide the chemical requirements for bacteria include explanations for List & Explain elements such as C, N, P, S, and trace elements and the roles each play Explain in general terms why oxygen can be toxic and possibly lethal to bacteria? how bacteria get rid of toxic oxygen compounds including: singlet Describe oxygen/superoxide radicals (enzyme involved in this), Hydrogen peroxide anions Differentiate between the two broad categories of oxygen preferences in bacteria. the subcategories of oxygen preferences: obligate aerobes, Differentiate facultative aerobes, microaerophilic aerobes, aerotolerant anaerobes, and obligate anaerobes. Describe how you test for oxygen preferences in bacteria Define and between the patterns of growth in a stab culture that would help differentiate you identify the oxygen preferences of a bacteria. Objectives/ Study guide Define and differentiate the two groups of bacteria based on growth factor requirements Define combine and the terms : culture media, sterile, liquid, solid, agar, inoculum, inoculation, culture, relate chemically defined media, Minimal, Chemically undefined media, complex media what agar is and why it is useful in making media. What are 3 or 4 qualities that Explain make it a good fit for bacteriological media? Describe the 3 main shapes of solid media Minimal media (chemically defined) and Complex media (chemically undefined). What makes them different. Can you give an example of an ingredient that would Compare and contrast be used in one but not the other Define, combine and the following terms to one another capnophiles, CO2 , reducing media, Anaerobic, relate methylene blue, indicator, candle jar, airlocks, Jar , chamber Explain why we might use an animal or cell cultures for growing bacteria? the differences between and processes of using selective, differentiating, and Explain enriching media, provide an example of each media type. Colony forming unit, bacterial colony, bacterial isolation, mixed culture , pure Describe and relate culture, and plate streaking the steps in order of how you would go from a mixed culture to a pure culture with List individual colonies (suggestion- play the Bacterial isolation labster)  Objectives/ Study guide Describe and Colony forming unit, bacterial colony, bacterial isolation, mixed culture , relate pure culture Differentiate between the items and requirements of the 4 BSL levels. the two methods of long-term storage and why storing bacteria long term Describe is important the generation time(G), number of generation(n), incubation time frame Calculate (t) or number of bacteria given if given at least 2 pieces of information. Describe the 4 stages of bacterial growth Plate counts, Spread plate method, pour plate method, Serial dilution, Explain & CFU What is serial dilution and how is used with plate counts to determine Relate # of bacteria in a culture? If you did a direct plate count and on your 1:1000 dilution plate you got Reason & 50 colonies , what does this mean about your starting (undiluted) Calculate culture? Describe What are the general pros and cons of direct PLATE count. What are the methods for measuring growth indirectly. How is one of Explain these ways related to turbidity and optical density? What do bacteria require? Just like us there are constraints regarding environmental qualities Temperature pH Osmotic stresses As well as nutrients Carbon, Nitrogen, Sulfur, and phosphorous Trace elements Oxygen Organic growth factors Temperature When it comes to temperature bacteria usually have an: Minimum temperature- Optimal temperature where they grow the fastest Maximum temperature Slows growth May kill However, different bacteria have different temperature requirements/preferences.  Three categories of bacteria based on temperature Psychrophi les cold-loving Mesophiles moderate- temperatur e-loving Thermophil es heat-loving We will go through these in detail one at a time Psychrophiles: (mostly archaea) Can grow at 0°C optimum temperature ~ 15°C. found in deep ocean or polar regions. Psychrotrophs: can grow (slowly) even at 0°C optimum temperature of 20-30°C. Some grow slowly inside refrigerator and cause food spoilage. (mostly Bacteria)  Why do we refrigerate food? To stave off spoilage/foodborne illness- which is often caused by bacteria (mostly mesophiles) Most bacteria (even cold loving ones) do not grow well below 4C (~39 F) However, anything above 15C(60F) is Thermophiles Hyperthermophiles (extreme Optimum growth thermophiles) temperature of 50 to 60°C Most are archaea Found in hot springs and Optimum growth temperature > organic compost 80°C many thermophiles cannot Found in hot springs and deep sea grow at temperatures vents below about 45°C. Sulfer is often integral in their metabolism Examples of hyper thermophiles- The MOST extreme Hyperthermophile s in Yellowstone National Park. Immerse a glass microslide into the water (102 °C) and let it sit for a few days and this is what you get: a microcolony of prokaryotes. A hydrothermal vent is a geyser on the seafloor. It continuously spews super-hot, mineral-rich water that helps support a diverse community of organisms. Extreme ends of the spectrum In 1992, Methanococcoides burtonii which lives and grows at -2.5°C, was discovered in Ace Strain 121. (Geogemma Lake, Antarctica. Flexible cell barossii): walls and an ability to produce - An Archaea (isolated from ‘antifreeze’ enable some near deep-sea hydrothermal bacteria to survive a chilling - vents) that survives and 20°C. reproduces at 121°C and 130°C (266 °F) is proven to be only bacteriostatic.  Alkaliphiles: prefer a high pH and are found in regions pH with high carbonate deposits (soda lakes) Most bacteria are neutrophiles however, grow between pH 6.5 and 7.5 Molds and yeasts grow between pH 5 and 6 Acidophiles grow in acidic environments – Many bacteria and archaea that live in lakes near volcanic sites or acidic hot springs  Osmotic pressure Halo= The tonicitysalt of an environment can vary Halophiles rather widely (halo tolerant) Depending on location/time of year. Aside from normal bacteria there are 3 categories of Facultative halophiles Halophiles let’s do a refresher on osmosis before Obligate we talk more Extremehalophiles about these categories  Osmolarity/ Tonicity Hypotonic solution: solute concentration is lower outside than inside the cell; water moves into cell Isotonic solution: solute concentrations equal inside and outside of cell; water is at equilibrium Hypertonic solution: solute concentration is higher outside of cell than inside; water moves out of cell  Osmotic pressure and bacteria Hypertonic environments (higher in solutes than inside the cell) cause cells to plasmolysis However… Some bacteria like it Salty!  Facultative halophiles (= halotolerant) do not require high salt concentrations but can grow at up to 2-15% salt concentrations e.g., Staphylococcus aureus 3 Categories Facultative= they can take or leave it of salt-loving Halophiles bacteria grow optimally in salt concentrations equal to sea (based on water (3% NaCl + many other minerals). e.g., Alivibrio fischeri salt Extreme halophiles (= obligate dependency halophiles) ) have adapted so well to high salt concentrations that they actually require high osmotic pressure for growth. e.g., Halobacterium salinarum Obligate= they are obligated or MUST have this type of environment for survival On the extreme halophile side…. Other osmotic oddities Osmophiles: Organisms that live in high sugar concentrations. Xerophiles: Organisms that live in very dry environments.  pH less than 5 Check for understandingCold temperatures Match up the terms with their high temperatures respective meanings regarding environmental conditions pH between 6.5- A. Halophile _______________ 7.5 than pH greater B. Acidophile _______________ High salt 8conditions C. Alkeliphile _______________ D. Neutraphile _______________ E. Thermophile _______________ F. Pychrophile _______________  Chemical requirements The items that our own bodies require are pretty similar to what bacteria need. Can you think of some of the essential elements for life? Carbon- for various molecules Nitrogen- DNA, RNA proteins Phosphorus- DNA, RNA Sulfur – Amino acids (proteins) Oxygen- various molecules Trace elements. Let’s walk through Carbon! Structural backbone of organic molecules Chemoheterotrophs use organic molecules as energy Autotrophs use CO2 fats CO2 Nitrogen Major component of proteins, DNA, and ATP  Nitrogen Although most bacteria decompose protein material for the nitrogen source… Nitrogen is an essential component of DNA, RNA and Some bacteria use NH4+ or NO3- from organic Sulfur and Phosphorus Sulfur In amino acids, and vitamins such as thiamine and biotin. Most bacteria decompose proteins and some bacteria use SO42– or H2S. Phosphorus In DNA, RNA, ATP, and membranes. PO43– is a source of phosphorus. Check for understanding Explain why atoms like phosphorus are absolutely necessary for bacterial survival when they are not used for energy or needed to make proteins. Phosphorus is a major component of DNA, thus it is required for an organism to remain alive as well as for reproducing Trace elements Inorganic elements required in small amounts Include iron, copper, molybdenum, and zinc These items most often serve as enzyme cofactors Check for understanding What role do micronutrients like zinc and iron play in in the molecular needs of bacteria and other cells? A. These metals hold onto oxygen so it does not leave the cell B. These metals are necessary as they are integral parts of nucleotides that make up the DNA and RNA C. They often serve as cofactors and play a role in enzymatic function D. They are needed as they are integral parts in all amino acids E. They often serve as sources of energy when they are broken down Oxygen Oxygen is very useful and necessary in most cases. Do you remember why Eukaryotic cells need oxygen? What is oxygen’s roll in Oxygen is the final the cell respiration process? Electron acceptor!  Unfortunately, with oxygen too much of a good thing can be deadly especially if you are a microbe Oxygen can form radicals (loose their electrons on accident) Which causes them to go on a rampage and start ripping electrons off of other molecules willy-nilly. This is very bad! Microbes can be classified into 2 broad categories with a total of 5 total distinct categories based on their oxygen preferences  How can oxygen be toxic!? 1. Singlet oxygen (1O2−): normal O2 that has been boosted to a higher-energy state. (i.e. when you split an O2 you make a singlet oxygen) 2. Superoxide radicals or superoxide anions (O2 - ): formed in small amounts when O2 accepts electrons during the respiration. greatly unstable that they steal an electron from a neighboring molecule, leading to a radical chain reaction. All aerobes produce superoxide dismutase (SOD) to convert the superoxide radical into molecular oxygen (O2) and hydrogen peroxide (H2O2). O2 + O2   +  2 SOD + 2HO +HOO  + O + 2 2 2 + 2H2 H2 O 2 + O 2 How can oxygen be toxic!? 3. H2O2 and Peroxide anion (O22– ): hydrogen peroxide produced during normal aerobic respiration. Aerobes produce catalase (produce O2) and peroxidase to neutralize it. These radicals are thwarted by catalase and peroxidase respectively How can oxygen be toxic!? (4th of the 4 ways) 4. Hydroxyl radical (OH ) Probably the most reactive and formed transiently in the cellular cytoplasm by ionizing radiation. It cannot be eliminated by an enzymatic reaction. It can be eliminated by endogenous antioxidants such as glutathione, and dietary Vitamin E. Which is found naturally in many fruits and vegetables How do these radicals form? How are they dealt with? Or it is handled by an antioxidant  Categories of oxygen preference in bacteria Anaerobes- Aerobes- Do NOT need Need O2 O2 Obligate aerobes Aerotolerant absolutely need O2 anaerobes (Streptomyces coelicolor, M. growth same with or tuberculosis..). without O2 (lactic Facultative aerobes acid bacteria..). not needed but grow better (E. coli..). Obligate Microaerophilic aerobes anaerobes killed by O2 needed but at concentrations below those found in the atmosphere (Helicobacter pylori). (Clostridium). Evaluating oxygen preference in bacteria To do this you perform a stab 1. Take a sterile vile containing an nutrient agar mixture 2. Sterilize an inoculation wire 3. Rub the end of the wire on a colony from a plate 4. Flame the opening of the vial with the nutrient agar 5. Use the wire to make 1-3 deep stabs into the agar 6. Incubate the tube under normal oxygen conditions at preferred temperature Let’s look at the different results we can get Effects of oxygen on bacterial growth Expectation/check for understanding Given an image of one of those vials and the pattern of bacterial growth you should be able to identify /categorize the oxygen preference of the bacteria Let’s do a check for understanding What is the oxygen preference of the bacteria grown in this vial: microaerophile  Organic compounds obtained from the environment because they cannot be synthesized Organic Vitamins, Growth amino acids, factors 3 Major Purines/ groups pyrimidines (nitrogenous bases for DNA/RNA) Grouping organisms based on their growth factor requirements 2 groups Prototroph: principal C-source (usually glucose) is enough. Auxotrophs: require secondary organic nutrient (= growth factor) in addition to the principal C-source. trp- auxotroph (mutant) of E. coli requires tryptophan in addition to glucose for survival. Culture media basics Culture media is used in various way to culture/grow bacteria We can enrich or select for specific types of cellular organisms by altering the ingredients suspended in the media of our choice Media may be solid or liquid Before we go too deep, let’s lay a bit of terminology background. Terminology Culture medium: nutrients prepared for microbial growth either liquid or suspended in a matrix of agar Sterile: no living microbes present Inoculum/inoculation: source of microbes to be used / introduction of microbes into a medium Culture: microbes growing in or on a culture medium The Requirements for growing specific organisms 1. it must contain the right nutrients for the microbe we want to grow. 2. it should contain sufficient moisture (free water), a proper pH, suitable level of oxygen. 3. it must initially be sterile (no living microbes) so that the culture will contain only the microbes we add to the medium. 4. it should be incubated at the proper temperature. Agar Complex polysaccharide derived from seaweed used as a solidifying agent in media Is not metabolized by microbes (won’t disintegrate) optimal choice when we desire to grow bacteria on a solid substrate.  Agar Autoclavable- Liquefies at 100°C Solidifies at ~40°C thus it will solidify at room temperature ( ~25° C or 72° F) Oh, and edible by the way… Fun side note Agar is widely popular in Japanese culture Used in many Japanese artistic desserts called wagashi Shapes of Solid media Petri plates slants (1 and 2) (shallow or steep) deeps (3) (for stabs and growing bacteria that are not as tolerant of oxygen)  Microbial growth Media, conditions & cultures When it comes to media The goal is to support microbial growth a medium must provide an energy source as well as sources of C, N, S, P, and.. any organic growth factors the organism cannot synthesize. ( signaling molecules that trigger growth and reproduction) There are two big categories: 1. Minimal/Chemically defined media Minimal /Chemically defined media Chemically defined media (minimal/synthetic media) exact chemical composition is known Fastidious organisms are those that require many growth factors provided in chemically defined media Chemically Defined media Complex media/chemically undefined Complex media/ undefined media: exact chemical composition is not known because at least one ingredient (e.g., beef extract or yeast extract) is chemically undefined. Energy, C, N, and S requirements are provided primarily by proteins that are a partially digested by acids or enzymes to shorter chains of amino acids called peptones. Vitamins and other organic growth factors are provided by meat extracts or yeast extracts. Check for understanding When making various solid media, what ingredient provides the solidity? A. Salts B. Water C. peptone D. Agar E. glucose Check for understanding Which of these ingredient would NEVER be used in a chemically defined media? (select all that apply) Glucose Beef extract NaCl Magnesium Calcium peptone chloride chloride Agar Biotin Water Vitamin A  Anaerobic bacteria – low/no oxygen environment Growing bacteria in Capnophiles – high CO2 special environment environme nts In or on hosts Anaerobic (low or no oxygen) environments To grow more anaerobic bacteria an anaerobic jar can be used Can use the same chemical methods mentioned before but on a larger scale Methylene blue indicator strips are used to verify oxygen free environment they turn blue when oxygen is present Anaerobic conditions Alternatively a special chamber can be used for larger volumes of work with anaerobes. Anaerobic chamber is usually filled with : inert gas (typically about 85% N2, 10% H2, 5% CO2 equipped with air locks to introduce cultures and materials.  Anaerobic conditions Clostridium perfringens Reducing media is used Such as the “oxyplate” Used for the cultivation of anaerobic bacteria Each plate becomes an anaerobic chamber Each plate has oxyrase , O2+ 4H→ 2water Other methods: chemicals (like sodium thioglycolate) that combine to O2 to deplete it Heated to drive off O2 Capnophiles conditions Capnophiles Microbes that require high CO2 conditions Candle jar CO2 packet or CO2 incubator tanks! Candal jar source: https://www.jfmed.uniba.sk/fileadmin/jlf/Pracovi ska/ustav-mikrobiologie-a-imunologie/ In or on a host Mycobacterium leprae is cultured on the feet of mice or on nine banded armadillos due to the inability to culture in vitro. Like viruses, the syphilis spirochete (Treponema pallidum) and the obligate intracellular bacteria (e.g., the Rickettsia and the Chlamydia) Cell infected with Chlamydia CREDIT: Biomedical Imaging can reproduce only in a living Unit, Southampton General Hospital/Science Source  Culture media types overview Type Purpose Chemically Defined Growth of chemoautotrophs and photoautotrophs; microbiological assays Complex Growth of most chemoheterotrophic organisms Reducing Growth of obligate anaerobes Selective Suppression of unwanted microbes; encouraging desired microbes Differential Differentiation of colonies of desired microbes from others Enrichment Similar to selective media but designed to increase numbers of desired microbes to detectable levels Check for understanding We have talked about a special media plate that provides a low oxygen environment without need for a fancy expensive piece of equipment. Which kind of media serves this purpose? A. Oxidation media B. Chemically defined media C. Reducing media D. Complete media E. Minimal media Selective & differential culture media We can detect and identify bacteria through selection and screening using a variety of media in conjunction with biochemical tests. For now we will just focus on the types of media used to grow bacteria.  Selective Media Eosin methylene blue (EMB) Encourages encourages growth of G- growth desired bacteria while suppressing G+ growth, it also microbes, but… differentiates E. coli which Contains produces a metallic green hue S. typhi on bismuth sulfite agar inhibitors to suppress growth bismuth sulfite agar, of unwanted suppresses G- and other bacteria and bacteria from intestine, while encouraging Salmonella typhi encourage the growth growth of the desired microbes. Differential (screening) media This type of media does not inhibit any particular growth It does affect the appearance of some bacteria Allowing for distinguishing colonies of desired species from others on the plate  Differential combined with selective media Isolation of the common bacterium Staphylococcus aureus, found in the nasal passages. S. aureus (as a genus of Staphylococcus) has high NaCl tolerance + ferments mannitol to form acid (lowers pH) We can isolate S. aureus plating onto a Mannitol salt agar Staphylococcus containing 7.5% NaCl Staphylococcus aureus is resistant (to select for high-salt epidermis is to high salt and resistant S. aureus) resistant to high salt ferment mannitol but does not into acids. plus pH indicator ferment mannitol. (phenol red; to screen for mannitol- fermenting S. aureus). Enrichment media Enrichment culture can be used when you want to increase very small numbers of the desired type of microbe to detectable levels. Example: To isolate a microbe from a soil sample that can grow on phenol and is present in much smaller numbers than other species.  Use of enrichment media example Inoculate the soil sample into phenol-containing culture medium (where phenol is the only source of carbon and energy) and incubate for a few days. Transfer 1 ml to another flask of the same but fresh phenol medium, and incubate. After a series of such transfers, the surviving population will consist of bacteria capable of metabolizing phenol. When the last dilution is streaked onto a solid medium of the same composition only those colonies of organisms capable of using phenol should grow. Check for understanding How does selection media differ from differential media? Can these types of media be used together. Provide an example. Differential media will help you tell apart different bacteria, but it does not suppress any particular bacterial type. Selection media DOES suppress the growth of certain bacterial types. You can combine the two to make a selection/differentiation media Example: Mannitol salt agar (high salt only allows salt tolerant bacteria to grow); the pH indicator + mannitol sugar is the differential part- allowing you to see which bacteria can ferment mannitol sugars. Obtaining pure cultures A pure culture contains only one species or strain A colony is a population of cells arising from a single cell or spore or from a group of attached cells Serratia marcescens Obtaining pure cultures A colony is often called a colony-forming unit (CFU) it is also a unit of measure We can dilute bacteria and spread it out over media Each colony was formed by a single bacteria (unit) Allows us to quantify bacteria from samples When we plate we want individual colonies. Whether we are counting Or isolating bacteria Check for understanding and recall If you had a mixed culture, how would you go about obtaining a pure culture. List ALL the steps, from obtaining a liquid sample of mixed bacteria, to the final step of evaluating a plate that has a pure culture. Include all incubations , initial loop sterilization, loop flaming , as well as the steps of streaking out the plate (between the quadrants and flaming in between) You SHOULD be able to list this out in 10-14 steps (be detailed!)  Biosafety levels , bacterial preservation/storage and bacterial growth  Biosafety- BSL4 4 levels BSL4 is the most s BSL3 rou dangerous with the ge n most stringency and da precaution. st Mo BSL2 We will walk through them one at a time. t fes BSL1 Sa  BSL Biosafety- 1 BSL4 BSL-1: no special precautions s BSL3 rou basic teaching labs ge n Many basic research da st labs Mo BSL2 t fes BSL1 Sa  BSL Biosafety- 1 BSL4 s BSL3 rou ge n da st Mo BSL2 t fes BSL1 Sa  BSL Biosafety- 2 BSL4 BSL-2: lab coat, gloves, eye protection s BSL3 May include use of rou ge fume hood (but it is n da not required) st Mo Moderate risk of BSL2 infection. Treatments or cures t fes available BSL1 Sa  BSL Biosafety- 2 BSL4 s BSL3 rou ge n da st Mo BSL2 t fes BSL1 Sa  BSL Biosafety- 3 BSL4 biosafety cabinets required to prevent BSL airborne transmission s For highly infectious rou 3 pathogens such as the ge tuberculosis or HIV. n da The laboratory is st negatively pressurized Mo BSL2 and equipped with air filters to prevent release of the pathogen from the laboratory. All waste materials t fes leaving the lab must be BSL1 Sa autoclaved  BSL Biosafety- 3 BSL4 BSL s rou 3 ge n da st Mo BSL2 t fes BSL1 Sa  BSL Biosafety- BSL 4 4 BSL-3 plus sealing of the room, the exhaust air is filtered twice. s BSL3 rou For easily transmitted ge n viral pathogens causing da fatal hemorrhagic fever st Mo such as Ebola and BSL2 Marburg viruses. Very few BSL 4 labs exist due to the nature of the t fes pathogens dealt with in BSL1 Sa these labs  BSL Biosafety- BSL 4 s 4 BSL3 rou ge n da st Mo BSL2 t fes BSL1 Sa Other Comparison of BSL differences – please review on your own Expectation Explain how each safety level is different from the one above or below it (i.e. What is BSL1 and how is it different from BSL2 , how is BSL2 different form BSL3 etc.) Expectation / Check for understanding Which of the following is true regarding the differences between BSL2 and BSL 4 A. BSL4 is always under negative pressure, this is not required for BSL2 B. There are more BSL4 labs than BSL2 labs in the USA C. BSL4 PPE requires goggles, gloves and lab coats while BSL2 does not require goggles or lab coats (only gloves) D. BSL 2 labs deal with infectious pathogens that often have treatments while BSL4 labs deal with pathogens that are much more pathogenic and often lack treatments. E. BSL2 labs are always under negative pressure while BSL 4 labs are under positive pressure. The 2 Major bacteria 1. Deep-freezing preservatio 2. Lyophiliyzation n (freeze drying) techniques Both are used for long-term storage  Bacteria are prepared in Deep freezing a liquid culture Then mixed with a media that contains glycerol Before being frozen down to and held at −50° to −95°C Deep- freezers Lyophilization (freeze drying) frozen (−54° to −72°C) and dehydrated in a vacuum Why do we use long- term storage Imagine if your experiments relied on bacteria that stayed the same How do you ensure that your bacteria is the same on day 1 of your experiment versus Day 1000 (3 years later) Freezing down your strain then pulling a fresh thaw for a fresh culture regularly is key! Why do we need to store bacteria for long term How do you ensure that your bacteria is the same between day 1 of your experiment vs day 1000? Long term storage of many aliquots Aliquots used to create fresh cultures Check for understanding Given what we just talked about. If a hypothetical disaster occurred on the space station where a hole was ripped open on the station , it would get…very cold and there would be a vacuumed. What would likely happen to any bacterial samples being studied? The bacteria would just go dormant (nothing bad would happen to them), since bacteria can survive at -80 and they are just fine with being freeze dried (dried under a vacuum) they would be just fine  Cell replication : increases numbers but not size of cells Types of replication Conidiospor Fragmentat Binary es Budding ion of fission (actinomyc filaments etes) Binary fission – bacterial replication 1. Replicate genome 2. Elongate cell 3. Form cell wall and membrane barrier Example of binary fission Generation time Time required for a cell to divide 20 minutes to 24 hours Binary fission doubles the number of cells each generation Total number of cells = 2number of generations Growth curves are  Generation time Generation time = the time it takes for a cell to divide 20 minutes to 24 hours Binary fission doubles the number of cells each generation This is why 1 invisible bacteria Turns into a visible colony overnight  G=t/n G = Generation time (time for the cells to divide) t = Time interval in minutes or hours n = Number of generations (number of times the cell population doubles during the time interval) N = N0 x 2n (growth by binary fission) N0 =# of bacteria at the beginning of a time interval N = #of bacteria at the end of the time interval IF you are given the start and end # of bacteria, you can always calculate how many replication cycles.  G=t/n G = Generation time (time for the cells to divide) t = Time interval in minutes or hours n = Number of generations (number of times the cell population doubles during the time interval) I will not ask you to calculate the generation number (the equation from the yellow box) However, I will expect that you can use the equation above, G=t/n As well as : Total number of cells = 2number of generations Generation time n = number of generation (or amount of division cycles) Growth curves are represented logarithmically Simplified equation: 𝑛 ¿ 𝑜𝑓 𝑐𝑒𝑙𝑙𝑠= 2 Generation time Check for understanding Let’s put things in perspective If a bacteria has a generation time of 30 minutes (0.5h). And you start off with 1 cell How many cells do you have after 8 h (hours) 16 𝑡𝑜𝑡𝑎𝑙 ¿ 𝑜𝑓 𝑐𝑒𝑙𝑙𝑠=2 𝑛 ¿ 2 =65 , 536 Check for understanding What if a bacteria had a generation time of 20 minutes (basically times per hour) and an incubation time of 10 hours? How many bacteria will we get at the end of 10 hours? Check for understanding What if a bacteria had a generation time of 20 minutes (basically times per hour) and an incubation time of 10 hours? How many bacteria will we get at the end of 10 hours? 𝑛 𝑡𝑜𝑡𝑎𝑙 ¿ 𝑜𝑓 𝑐𝑒𝑙𝑙𝑠=2  30 ¿ 2 =1 ,073 , 741 , 824 (b) Conversion of the number of cells in a population into the logarithmic expression of this number. To arrive at the numbers in the center column, use the yx key on your calculator. Enter 2 on the calculator; press yx; enter 5; then press the = sign. The calculator will show the number 32. Thus, the fifth-generation population of bacteria will total 32 cells. To Number of bacteria increase arrive at the numbers in the right-hand column, use the log key exponentially, on your calculator. Enter the number 32; then press the log key. The calculator will show, rounded off, that the log10 of 32 is 1.51. So a regular scale mask the amount of growth at the beginning and the y-axis is very odd since we are dealing with very small and very big  Generation time is calculated only for the bacterial culture that is at the stage of Exponential Growth in which cell numbers double within a specific time interval. This is why we call it “log phase” Bacterial growth stages There are 4 distinct phases of bacterial growth Station Lag Log Death ary phase phase phase phase  Phases of bacterial growth Lag phase slow growth as bacteria adapt to their new growth conditions Log phase exponential phase , cell number doubles regularly Stationary phase food running out, equilibrium of new division vs cell death Death phase – rate than they are dividing due to lack of nutrient resources. Cells may or may not lyse. This stage will also be logarithmic Check for understanding Which of these statements are True and which are false F _____ The lag phase is when the bacteria are doubling regularly. T _____ During the stationary phase the rate of cell death is roughly equal to the number of new cells yielded by F binary fission _____ In the log phase (exponential) bacterial death is outpacing the number of new bacterial cells being Measurements of bacterial growth When it comes to Direct measuring bacteria, we have two different ways indirect Measuring bacterial growth  Measuring bacterial growth- Direct methods Plate count – May require counts only live diluting the culture cells Concentrates Direct Filtration the bacteria Count from the sample microbial measuremen Most probable cells Relies on ts number (MPN) statistical probability ( We method are not covering this one) Direct microscopic count Think about these question as we proceed Which of the methods discussed would you use for bacteria that are heat-stable but require an environment with less oxygen to grow well? Which method would you use to quantify bacterial in drinking water (where contaminants are pretty rare but may occur) Describe how the Most probable number is different than the other 3 methods discus (plate counts filtration or direct counting of cells on a slide)  A frequently used method Direct- Plate counts Serial dilution of bacterial culture is made (several dilutions may be tested) Known volume of the total solution is plated Number of resulting CFU (colony forming units) can be counted Must be feasible to count but not too few This may employ either the pour-plate method or spread plate method  Plate count pros and cons Pros cons Measures May not be only viable perfectly cells accurate if cells naturally Is easy and clump or quick to grow in create chains Is a common Takes time method to incubate Pour vs spread plate Colonies will grow 1. within the nutrient agar, & 2. on the surface of the agar. - Heat-sensitive microbes may be damaged, - Colonies formed inside agar may This method not represent the bacterium’s positions all the unique colony morphology. colonies on the surface. Direct- Filtration - When the quantity of bacteria is very small (e.g., when test fecal contamination of water or food), bacteria can be counted by filtration methods. At least 100 ml of water The bacteria are This filter is then transferred are passed through a thin filtered to a nutrient agar plate, membrane filter whose out and retained on where colonies arise from pores are too small to the surface of the the bacteria on the filter’s allow bacteria to pass. filter surface. Direct- Direct microscopic count Volume of a bacterial suspension placed on a slide Average number of bacteria per viewing field is calculated Uses a special Petroff- Hausser cell counter (on right) Measuring bacterial growth- Indirect methods Turbidity Quality of bacterial growth used to estimate cell Metabolic activity number Dry weight indirect i.e. we are not observing bacterial growth directly but rather the collective Effects of their growth  Two indirect methods METABOLIC ACTIVITY DRY WEIGHT— —AMOUNT OF BACTERIA ARE METABOLIC PRODUCT IS FILTERED, DRIED, AND PROPORTIONAL TO THE WEIGHED; USED FOR NUMBER OF BACTERIA FILAMENTOUS ORGANISMS  Third Indirect method - Turbidity Turbidity— measurement of cloudiness with a spectrophotometer Microbes scatter light: More microbes → less light gets through (higher turbidity)  Third Indirect method - Turbidity Spectrophotometer detect measures light getting through a sample and deters how much light was absorbed by the sample This is used to calculate Optical Density (OD). The higher OD value, the more turbid the cell suspension, the more cells there are. OD will increase over time as the bacteria grow in the culture.  microscope Filtratio Turbidit Check for understanding count Serial Spread n y Pour plate dilution+ plate method plate count method Which of the methods discussed would you use for bacteria that are heat-stable but require an environment with less oxygen to grow well? Which method(s) would you use to quantify bacterial in drinking water (where contaminants are pretty rare but may occur) Which method(s) would you use if you had a sample that you thought was heavily contaminated and you wanted to quantify the level of contamination. Coming up next… CONTROL of microbial growth  Supplementary Materials Please review this material on your own as it may aid your studies or show up on the exam.  Question- how Final number of bacteria at Answers to many times per Time interval Number of end of incubation Generation Condition/ hour will the allows for generations that N time problem bacteria divide? incubation occurred Guided notes G t n practice 248 = questions for 4 times per 1 15minutes 12 hours 48 generations hour 2.8* 1014 calculating bacterial growth 220 = 2 30 minutes 2 times 10 hours 20 generations 1.04* 106 3 20 minutes 3 times per 8 hours 24 generations 224 = hour 1.7* 107 2 hours Only ½ per hour (or once every 2 30 hours 15 generations 215 = 4 hours) 32768 Check for understanding Why is sulfur necessary for some bacteria, it is not needed for DNA or for most energy processes, so why is it needed at all? Check for understanding There were three forms of solid media that we mentioned. Do you remember what they are? Which of these is NOT one of them. A. Petri dishes B. spherical C. Slant D. Deep Check for understanding What are the 3 main categories of special conditions that bacteria may need to be grown (provide the name of the bacterial category if it applies) Check for understanding (2/3) Provide two example of how we can provide bacteria with special conditions they need to survive if they require low oxygen or high CO2 (explain how the system you chose provides for the bacteria’s required environment in basic terms) Additional objectives The objectives below should match to the objectives already listed in previous lectures. However, you can use these as an additional check for understanding. Check for understanding Explain what high turbidity is and how it relates to bacterial number. Explain how turbidity (an indirect form of bacterial measurement) differs from any of the direct methods. Turbidity = cloudiness of a liquid culture and is dependent on how many cells are in the suspension. More cells= higher turbidity. We can measure how much light gets through a sample  less light =more bacteria blocking the light and thus higher bacterial number. Check for understanding Which of the methods discussed would you use for bacteria that are heat-stable but require an environment with less oxygen to grow well? Which method would you use to quantify bacterial in drinking water (where contaminants are pretty rare but may occur) Describe how the Most probable number is different than the other 3 methods discus (plate counts filtration or direct counting of cells on a slide) Check for understanding What is the purpose of enrichment media as we have discussed here? How does it achieve this purpose (generally speaking) Can you give an example of what would be an ‘enriching’ component in such media?