Chapter 6: Microbial Growth PDF

Chapter 6: Microbial Growth © 2016 Pearson Education, Inc.  Microbial Growth - increase in cell number, not in cell size. - binary fission - grow fast in a very short period of time - accumulate as colonies © 2016 Pearson Education, Inc.  Microbial g...

Chapter 6: Microbial Growth © 2016 Pearson Education, Inc.  Microbial Growth - increase in cell number, not in cell size. - binary fission - grow fast in a very short period of time - accumulate as colonies © 2016 Pearson Education, Inc.  Microbial growth growth strategies to survive nutrient poor environments - biofilms why do we need to study their growth conditions? 1- control growth - pathogens and food spoilage 2- encourage growth of harmless and useful © 2016 Pearson Education, Inc.  Learning Objectives Microbial requirement for growth: - Physical requirements: temperature, pH range, osmotic pressure. - Chemical requirements carbon, nitrogen, sulfur, and phosphorus, trace elements and oxygen and organic growth factors. Describe the formation of biofilms and their potential for causing infection (assignment 2) © 2016 Pearson Education, Inc.  Learning Objectives (cont.) Culture media Pure cultures Preserving bacterial cultures Growth of bacterial cultures © 2016 Pearson Education, Inc.  Microbial Requirements for Growth 1- Physical requirements Temperature – classify microbes based on temperature requirement Effect of pH on growth Effect of Osmotic pressure on growth © 2016 Pearson Education, Inc.  Microbial Requirements for Growth (cont.) 2- Chemical requirements Carbon Nitrogen, sulfur, and phosphorous Trace elements Oxygen - classify based on oxygen requirements Organic growth factors © 2016 Pearson Education, Inc.  Temperature, range: Minimum growth temperature Optimum growth temperature Maximum growth temperature © 2016 Pearson Education, Inc.  How does temperature affect microbes? What temperature requirements do most bacterial human pathogens have? Hyperthermophiles can survive autoclaving temperatures. Are they a concern in health care? © 2016 Pearson Education, Inc.  Clinical Focus- Resolution The presence of Listeria in Jeni’s blood suggests that her symptoms are due to listeriosis, an infection caused by Listeria monocytogenes. Listeriosis is a serious infection with a 20% mortality rate and is a particular risk to Jeni’s fetus. A sample from the amniotic fluid cultured for the presence of Listeria gave negative results. Because the absence of organisms does not rule out the possibility of infection, a molecular test based on the nucleic acid amplification of the 16S ribosomal RNA of Listeria was performed to confirm that no bacteria crossed the placenta. Fortunately, the results from the molecular test were also negative. © 2016 Pearson Education, Inc.  Jeni was admitted to the hospital for treatment and recovery. She received a high dose of two antibiotics intravenously for 2 weeks. The preferred drugs for the treatment of listeriosis are ampicillin or penicillin G with an aminoglycoside antibiotic. Resistance to common antibiotics is still rare in Listeria and antibiotic treatment is usually successful. She was released to home care after a week and fully recovered from her infection. © 2016 Pearson Education, Inc.  L. monocytogenes is a gram-positive rod bacterium found in soil, water, and food. It is classified as a psychrophile and is halotolerant. Its ability to multiply at refrigeration temperatures (4–10 °C) and its tolerance for high concentrations of salt (up to 10% sodium chloride [NaCl]) make it a frequent source of food poisoning. Because Listeria can infect animals, it often contaminates food such as meat, fish, or dairy products. Contamination of commercial foods can often be traced to persistent biofilms that form on manufacturing equipment that is not sufficiently cleaned. © 2016 Pearson Education, Inc.  The effect of pH  Acidity and alkalinity of a solution © 2016 Pearson Education, Inc.  pH: Acidity inhibits most bacterial growth/food preservation. Alkalinity inhibits growth/not common in food preservation Most bacteria grow best between pH 6.5 and 7.5 (~ neutral pH) Molds and yeasts grow best between pH 5 and 6 © 2016 Pearson Education, Inc.  Classification of microbes based on their pH requirement: Acidophiles (acid loving; pH less than 4) Neutrophiles (pH around neutral pH; 6.5 to 7.5) Alkaliphiles (basic loving; pH 8.5 to 11) © 2016 Pearson Education, Inc.  Peptic ulcers (or stomach ulcers) are painful sores on the stomach lining. Until the 1980s, they were believed to be caused by spicy foods, stress, or a combination of both. Patients were typically advised to eat bland foods, take anti-acid medications, and avoid stress. These remedies were not particularly effective, and the condition often recurred. This all changed dramatically when the real cause of most peptic ulcers was discovered to be a slim, corkscrew- shaped bacterium, Helicobacter pylori. This organism was identified and isolated by Barry Marshall and Robin Warren, whose discovery earned them the Nobel Prize in Medicine in 2005. © 2016 Pearson Education, Inc.  The ability of H. pylori to survive the low pH of the stomach would seem to suggest that it is an extreme acidophile. As it turns out, this is not the case. In fact, H. pylori is a neutrophile. So, how does it survive in the stomach? Remarkably, H. pylori creates a microenvironment in which the pH is nearly neutral. It achieves this by producing large amounts of the enzyme urease, which breaks down urea to form NH4+ and CO2. The ammonium ion raises the pH of the immediate environment. © 2016 Pearson Education, Inc.  Think about and answer these questions: Bacteria grown in Lab produce acids that can interfere with their growth. How to maintain proper pH in media used in lab? Where in our body would we more likely find acidophiles? Where in our body would we more likely find neutrophiles? © 2016 Pearson Education, Inc.  Osmotic pressure Microorganisms require water for growth - cells are 80 - 90% water Osmosis/osmotic pressure - review - effect of movement of water within a cell (cell membrane) © 2016 Pearson Education, Inc.  Osmotic Pressure: © 2016 Pearson Education, Inc. © 2016 Pearson Education, Inc.  High osmotic pressure - hypertonic solutions (environments) result in increased osmotic pressure causing plasmolysis  Adding sugar or salt to food (jelly, honey, condensed milk, or meat and fish)  basis of food preservation - growth of microbial cell is inhibited © 2016 Pearson Education, Inc.  Classification of microbes based on osmotic pressure: Halophiles –moderate to large concentration of salt; ocean water contains 3.5% salt; most bacteria in ocean. Extreme halophiles - very high concentration of salt (20-30%); bacteria in Dead sea (~ 34%). Facultative halophiles - do not require high concentration of salt but can tolerate up to 2% salt Most microorganisms grow at a low salt concentration usually ~ 1.5 % salt. © 2016 Pearson Education, Inc.  Give an example of a bacterium (mentioned in Lab selective and differential media) that grows at 7.5% salt concentration © 2016 Pearson Education, Inc.  Microbial Chemical Requirements: Carbon: - structural backbone of living matter - makes up 50% of dry weight of cell - part of most organic compounds of living organisms. Carbon source: 1. chemoheterotrophs use organic compounds (carbohydrates, proteins, lipids) 2. chemoautotrophs and photoautothrophs use CO2 © 2016 Pearson Education, Inc.  Chemical Requirements (cont.) Other elements needed by microbes: nitrogen, phosphorus and sulfur are component of: Proteins (amino acids) DNA ATP © 2016 Pearson Education, Inc.  Nitrogen (N): - 14% of dry weight of microbial cell - amino acids (proteins), DNA, and RNA and ATP. How do microbes obtain N (sources of Nitrogen)? - most decompose material containing protein to obtain N - from the ammonium (NH4+): found in organic matter - from nitrates - photosynthetic bacteria obtain N directly from atmosphere nitrogen gas (N2) – nitrogen fixation © 2016 Pearson Education, Inc.  Sulfur: - part of amino acids (proteins) and some vitamins (thiamine and biotin). Biotin/ Vit B7 © 2016 Pearson Education, Inc.  Natural sources of sulfur: - most microbes obtain S from sulfur containing amino acids (protein) - other natural sources of S include hydrogen sulfide, sulfates © 2016 Pearson Education, Inc.  Phosphorus: Found in DNA, RNA, and ATP Found in cell membranes: phospholipids Natural source of phosphorus: - Phosphate ion, PO43– © 2016 Pearson Education, Inc.  Other Elements required: potassium, magnesium, and calcium, often required as enzyme cofactors. Trace Elements: many are used as enzyme cofactors: Iron Copper Molybdenum Zinc © 2016 Pearson Education, Inc.  Classification of microbes based on oxygen requirement: Obligate aerobes—require oxygen to live Facultative anaerobes - use oxygen when present; grow well with oxygen. - continue growing when oxygen is not present; grow slower:  produce less energy Obligate anaerobes—unable to use oxygen, harmed by it. © 2016 Pearson Education, Inc.  Oxygen requirement (other groups): Aerotolerant anaerobes—tolerate but cannot use oxygen Microaerophiles—usually obligate aerobes that require lower levels of oxygen than that of atmospheric oxygen (< 21%) © 2016 Pearson Education, Inc.  Based on the knowledge received, label the figure below A ___________ B___________ C___________ D___________ E___________ © 2016 Pearson Education, Inc.  Organic Growth Factors: Most bacteria can synthesize their own vitamins. Some cannot synthesize vitamins but need them as growth factors: - obtain from the environment - essential, but not synthesized by organisms - vitamins, amino acids, purines, and pyrimidines © 2016 Pearson Education, Inc.  The requirements for growth: summary The requirements for microbial growth are both physical and chemical Classify microorganisms based on temperature, pH and osmotic pressure and oxygen requirements. © 2016 Pearson Education, Inc.  Culture Media Specific Learning Objectives: Distinguish chemically defined and complex media. Justify the use of each of the following: anaerobic techniques, living host cells, candle jars, selective and differential media, and enrichment medium. Differentiate biosafety levels 1, 2, 3, and 4 © 2016 Pearson Education, Inc.  Culture media (growth media/discussed in Lab):  material containing nutrients for microbial growth in Lab. Other definitions learned in Lab: inoculum: microbes introduced into a medium for growth culture: microbes growing in or on a growth medium media must initially be sterile : free of living microorganisms. © 2016 Pearson Education, Inc.  Solid growth media: Add agar complex polysaccharide (agarose) solidifying agent only not a source of nutrient not metabolized (degraded) by microbes © 2016 Pearson Education, Inc.  Solid culture Media (cont.) Agar properties (review from Lab): liquefies (melts) at 100C solidifies at ~40C held at about 50C for plating © 2016 Pearson Education, Inc.  Culture Media: To support growth, must offer: - energy source - C, N, S, P sources - growth factors needed but not synthesized by microorganism. © 2016 Pearson Education, Inc.  Culture (Growth) Media: Chemically defined media - exact chemical composition is known - carbon and energy are supplied by organic compounds (such as glucose) - fastidious organisms © 2016 Pearson Education, Inc.  Culture Media: Chemically defined media © 2016 Pearson Education, Inc.  Complex (undefined) media - extracts from yeasts, meat, or plants or digests of proteins. - exact chemical composition is undetermined; varies batch to batch:  nutrient broth  nutrient agar © 2016 Pearson Education, Inc.  Culture Media: nutrient based (cont.) complex (undefined) media © 2016 Pearson Education, Inc.  Complex (undefined) media Add proteins – serve as primary providers of energy, C, N, S, P. Meat or yeast extracts provide vitamins and other organic growth factors  Commonly used in teaching labs © 2016 Pearson Education, Inc.  Anaerobic Growth Media and Methods: Reducing media: - cultivation of anaerobic bacteria - contain sodium thioglycolate - sodium thioglycolate combines with and depletes oxygen - tightly capped tubes; heated to drive off absorbed O2 © 2016 Pearson Education, Inc.  A jar for cultivating anaerobic bacteria on petri plates. Lid with O-ring gasket Clamp with clamp screw Envelope containing inorganic carbonate, activated carbon, CO2 ascorbic acid, H2 and water Anaerobic indicator (methylene blue) Petri plates © 2016 Pearson Education, Inc.  An anaerobic chamber (for labs that have a large volume of work with anaerobes): Air Lock chamber Arm Ports in airtight sleeves © 2016 Pearson Education, Inc.  Special culture techniques Many bacteria have never been grown on artificial media in Lab: 1. Mycobacterium leprae – leprosy 2. Treponema pallidum (spirochete with axial filament ) – syphilis 3. Obligate intracellular bacteria- Rickettsia, Chlamidia obligate intracellular parasites of eukaryotic cells. © 2016 Pearson Education, Inc.  Special Culture Techniques (cont.) 4. Capnophiles - Microbes that require special CO2 concentrations (higher or lower than atmospheric): - Special CO2 incubators (many clinical labs) - Candle jar (lower concentration of oxygen ~ 17 %; increase CO2 to ~3%) - Chemical packets to generate CO2, provide required concentrations of CO2 © 2016 Pearson Education, Inc.  Special Culture Techniques (cont.) Candle jar CO2 generation packet © 2016 Pearson Education, Inc.  Special Culture Techniques (cont.) o Where in our body could we find lower oxygen concentration and higher CO2 conditions favorable for the growth of capnophiles? A. Respiratory tract B. Intestinal tract C. Heart D. Both A & B © 2016 Pearson Education, Inc.  Answer the questions below by filling the blanks: A growth medium that favors the growth of some microorganisms but inhibits the growth of other microorganisms is a __________ medium. A growth medium that distinguishes among different groups of bacteria on the basis of their biological characteristics is called a __________ medium. © 2016 Pearson Education, Inc.  Selective and Differential Media 1. Selective media suppress unwanted microbes and encourage desired microbes contain inhibitors to suppress growth Example 1: bismuth sulfite agar to isolate Salmonella typhi from feces (causes typhoid). Example 2: Sabouraud’s dextrose agar (pH 5.6) to grow fungi. What is an example used in Lab? © 2016 Pearson Education, Inc.  Selective and Differential Media 2. Differential media allow distinguishing of colonies of different microbes on the same plate example: blood agar – contains blood - used to identify bacteria that lyse (destroy) blood cells - Streptococcus pyogenes © 2016 Pearson Education, Inc.  Selective and differential medium. What’s the name of this medium? What makes it selective? What makes it differential? © 2016 Pearson Education, Inc.  Blood Agar: enriched and differential © 2016 Pearson Education, Inc.  Enrichment Culture usually liquid; soil or fecal samples. provides nutrients and environmental conditions to favor growth of some but not others:  encourages growth of a desired microbe by increasing very small numbers of a desired type of organism to detectable levels based on the description above, how would you classify this medium? A. selective media B. differential media © 2016 Pearson Education, Inc.  Culture Media 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 Copyright © 2019 Pearson Education, Inc. All Rights Reserved © 2016 Pearson Education, Inc.  Handling microorganisms that are so dangerous (BSL4) Ebola virus – can only be handled under extreme conditions of containment - commonly known as “hot zone” - sealed rooms within a building - atmosphere under negative pressure - intake and exhaust air are filtered (exhaust is filtered twice with HEPA (high efficiency particulate air) filters - personnel wear space suits connected to air supply © 2016 Pearson Education, Inc.  Biosafety levels (review/ see Lab Safety Guideline): © 2016 Pearson Education, Inc.  Technicians in a biosafety level 4 (BSL-4) laboratory. © 2016 Pearson Education, Inc.  Obtaining Pure Cultures Define colony. Describe how pure cultures are obtained. Copyright © 2019 Pearson Education, Inc. All Rights Reserved © 2016 Pearson Education, Inc.  Obtaining Pure Cultures Most samples (pus, sputum, urine, soil, water etc.) contain a mixture of microorganisms. A pure culture (contains only one species) is usually required for further testing. Which of the following can be used to isolate pure cultures of bacteria from mixtures? A. Spread plates B. Streak plates C. Aseptic technique D. All of the above. © 2016 Pearson Education, Inc.  Streak plate method for isolating pure bacterial cultures. Colonies © 2016 Pearson Education, Inc.  Preserving bacterial cultures For short term storage: Refrigeration For long term storage: Deep freezing (-50 C to -95C) Lyophilization (freeze drying) – frozen and dehydrated in a vacuum. © 2016 Pearson Education, Inc.  The Growth of Bacterial Cultures Specific Learning Objectives Define bacterial growth, including binary fission. Compare the phases of microbial growth, and describe their relation to generation time Copyright © 2019 Pearson Education, Inc. All Rights Reserved © 2016 Pearson Education, Inc.  The Growth of Bacterial Cultures What is bacterial growth? Increase in number of cells, not cell size  binary fission –bacterial division © 2016 Pearson Education, Inc.  Animation: Bacterial Growth: Overview Copyright © 2019 Pearson Education, Inc. All Rights Reserved © 2016 Pearson Education, Inc.  The Growth of Bacterial Cultures Phases of microbial growth Generation time: - time required for a cell (such as bacteria) to divide (or a population to double) - it varies among organisms and with environmental conditions (temperature) © 2016 Pearson Education, Inc.  - most have a generation time of 1 to 3 hours - some have 24 hours/generation - example: E. coli’s generation time is 20 to 30 minutes under favorable conditions.  number of cells double each generation time  total number of cells after several generations 2number of generations © 2016 Pearson Education, Inc.  A tube of TSB (broth) was inoculated with 100 cells of E. coli and incubated over 4 hours. The generation time of E. coli is 30 minutes. 1. Calculate the number of generations A. 16 B. 4 C. 8 D. 24 B. Calculate the final number of bacterial cells after the 4 hours of incubation. A. 256 B. 25600 C. 10000 D. 400000 © 2016 Pearson Education, Inc.  Phases of Growth Lag phase Log phase Stationary phase Bacteria approach the carrying capacity Death phase Copyright © 2019 Pearson Education, Inc. All Rights Reserved © 2016 Pearson Education, Inc.  Growth curves are represented logarithmically: © 2016 Pearson Education, Inc.  Animation: Bacterial Growth Curve Copyright © 2019 Pearson Education, Inc. All Rights Reserved © 2016 Pearson Education, Inc.  Case study: Timothy, an 18-year-old male is transferred to a hospital via air ambulance. His history is significant for having cystic fibrosis. Recently, Tim went to his family physician complaining of fever, malaise, intense chest pain and productive cough. His doctor immediately admitted him into a rural regional hospital. Sputum samples revealed Gram positive cocci predominately in clusters. This finding alone is not sufficient to identify the microbe, so other diagnostic tests are in progress to distinguish between pathogenic and nonpathogenic species. Tim is being transferred since his illness has not resolved despite receiving antibiotic therapy. Further laboratory testing reveals that the organism grows with a yellow colony on Mannitol Salt Agar. © 2016 Pearson Education, Inc.  Based on the case described above, give the scientific name of at least one bacterium that could be causing this illness in patients like Tim. A. Staphylococcus epidermidis B. Staphylococcus aureus C. Streptococcus pyogenes D. Micrococcus luteus Explain how you know this. E. Shape and arrangement of bacterial cells F. MSA test results G. Both A &B H. None of the above © 2016 Pearson Education, Inc.