3/26/2012. Chap 6 Microbial Growth 1

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1 Chapter 6 Microbial Growth Objectives Lab #9-13, 19 White Book p.33-34, 41-42, , 133, 136, & Black Book p , 39-40, , 132, 136, & Classify microbes into 5 groups on the basis of preferred temperature range. 2. Identify how & why the ph of culture media is controlled. 3. Explain the importance of osmotic pressure to microbial growth and/or lysis, and food preservation. 4. Provide a use for each of the CHONPS needed in large amounts for microbial growth. 5. Identify ways in which aerobes avoid damage by toxic forms of oxygen such as superoxide free radicals and peroxide anion. **Know enzymes & reactions & which organism groups have them. 6. Identify incubation conditions, energy production and relative growth rates for aerobes, obligate anaerobes, aerotolerant anaerobes, microaerophiles, and facultative anaerobes. Objectives Continued 7. Use and explanation of anaerobic generators and pouches 8. Distinguish between chemically defined and complex media. 9. Justify the use of each of the following: anaerobic techniques, living host cells, candle jars. 10. Explain the inoculation, interpretation and uses of the following media types: selective media, differential media, enrichment media, PEA, O-F glucose, starch, PEA, EMB, reducing, thioglycollate, agar, broth, slants, deeps, streak plates. 11. Explain methods to preserve microbes. 12. Define generation time and use it to calculate organism numbers. 13. Compare the phases of a microbial growth curve. 14. Define colony, pure culture, contaminant, binary fission, turbid, hydrolysis & hydrolytic enzymes, dehydration synthesis, anabolism, catabolism-both oxidative & fermentative, carbohydrates, amylase, catalase, indole, oxidase, and biochemical tests. 15. Use and explain aseptic technique. Chapter 6 Microbial Growth 1 Chapter 6 Microbial Growth 2 Requirements for Growth Requirements for Growth 1. Physical 2. Chemical Physical Requirements 1. Temp A. Minimum Growth Temp: B. Maximum Growth Temp: C. Optimum: Temperature Growth Classifications i. Psychrophile ( ):» Grows at» Optimum ii. Psychrotroph:» Grows at» Optimum» iii. Mesophile:»» Chapter 6 Microbial Growth 3 Chapter 6 Microbial Growth 4 Temperature Growth Classifications, Continued iv. Thermophile v. Hyperthermophile/extreme thermophile:»» Producers using D. See Figure 6.1 Growth Rates of Different Groups in Response to Temperature E. See Figure 6.2 & 6.3 Food Spoilage Temperatures ph 2. ph A. Most i. Foods preserved avoid spoilage longer B. Acidophiles C. Buffer: Substance that when acid or base added i. In growth media to neutralize ii. Example: Chapter 6 Microbial Growth 5 Chapter 6 Microbial Growth 6 Chap 6 Microbial Growth 1

2 Osmotic Pressure 3. Osmotic Pressure A. Hypertonic solution i. Plasmolysis occurs: ii. Food preservation with : honey, jam, salted meat iii. Making media-enough water & agar or B. Hypotonic Solution i. limits water intake & prevents See Figure 6.4 Plasmolysis Chapter 6 Microbial Growth 7 Halophiles C. Extreme/obligate halophile i. Requires ii. D. Facultative halophile i. CAN grow w/, but ii. (Facultative always-can, but prefers the opposite) Chapter 6 Microbial Growth 8 Chemical Requirements Chemical Requirements (CHONPS) 1. Carbon 2. Nitrogen: A. B. Nitrogen bases 3. Phosphorus A. 4. Sulfur A. 5. Oxygen A. B. Can be toxic Toxic oxygen forms C. Lysosomes contain O 2 forms toxic to all i. Singlet oxygen: O 2 in state ii. Superoxide free radical: O 2» By-product of D. Steps to break down O 2 & survive: i. Superoxide dismutase (SOD), an enzyme O 2- + O H+ ii. Peroxide ion is toxic iii. Break down w/1 of 2 enzymes:» Catalase:» Peroxidase: Chapter 6 Microbial Growth 9 Chapter 6 Microbial Growth 10 Oxygen Classifications 1. Obligate aerobes: REQUIRE O2 A. Have 2. Facultative anaerobes: CAN grow w/o O2, but grows best ( energy production) w/o2 A. w/o O2; B. Have 3. Obligate anaerobes: Molecular O2 is toxic A. Get oxygen from B. SOD & catalase (reason why O2 toxic) 4. Aerotolerant anaerobes: CAN grow w/o2, but prefer no O2 A. Contain, but 5. Microaerophiles: Require O2, but in lower [ ] than air A. SOD & catalase See Table 6.1 Effect of Oxygen on Various Types of Bacteria Chapter 6 Microbial Growth 11 Graph Example Problems Which line is a facultative anaerobe grown anaerobically? Facultative anaerobe in presence of oxygen? Psychrotroph at room temp? Psychrotroph in the refrigerator? Chapter 6 Microbial Growth 12 Chap 6 Microbial Growth 2

3 Culture Medium Terms 1. Culture medium 2. Agar: A. agent only B. C. Melts at 100C, solidifies 40C 3. Plates 4. Slants 5. Deeps: Tube w/flat surface A. B. at bottom Terms Continued 6. Sterile: Contains no living organisms A. Autoclave: Steam under pressure i. 121C at 15psi for 20 minutes 7. Inoculum 8. Colony 9. Culture 10. Pure Culture 11. Streak Plate A. To obtain colonies See Fig 6.10 Streak Plate Chapter 6 Microbial Growth 13 Chapter 6 Microbial Growth 14 Terms Continued 12. Chemically defined medium A. composition (formulas & amounts) known 13. Complex Media A. Exact composition B. Extracts of C. AKA Nutrient media See Tables 6.2 & 6.4 Chemically Defined & Complex Media Anaerobic Media/Methods 1. Reducing media A. Ingredients 2. Tube media: prior to use 3. Plates: Kept in O2 free environment pre & post inoculation See Figure 6.5 Anaerobic Jar See Figure 6.6 Anaerobic Chamber Chapter 6 Microbial Growth 15 Chapter 6 Microbial Growth 16 Special Techniques 1. CO2 Incubators, candle jars, packets A. Capnophiles: Microbes requiring 2. Living host cells required by Special Tech. Cont: Differential & Selective Media 3. Selective Media A. Only groups grow B. Due to C. PEA (Phenylethyl alcohol) i. Selective for (allows growth) of ii. Dissolves (supposed to kill but may inhibit & have breakthrough growth ) 4. Differential Media A. Appearance of those that grow 1 group from another B. Blood agar C. EMB (Eosin Methylene Blue) i. Purple and/or metallic sheen: ii. Colorless (agar color): Fig 6.7 CO2 Rich Environments See Figure 6.8 Blood Agar Hemolysis See 6.9 Differential Media Chapter 6 Microbial Growth 17 Chapter 6 Microbial Growth 18 Chap 6 Microbial Growth 3

4 Special Tech. Cont: Enrichment & Preserving Bacteria 5. Enrichment Culture A. Selective nutrient liquid used to # of bacteria present in small numbers Fig 6.11 Binary Fission Bacterial Growth ( = Multiplication) 1. Binary Fission See Fig 6.11 Binary Fission Preserving Bacteria 1. Refrigeration ( -term) 2. Deep-freezing 3. Lyophilization ( )- term Chapter 6 Microbial Growth 19 Chapter 6 Microbial Growth 20 Generation Time 1. Time required for a cell or population to divide 2. # organisms = n x 2 x A. n = original # orgs B. x = # generations or doublings 3. Examples: A. If there is 1 cell to begin with, how many are there after 2 generations? B. After 6 doublings? C. If there are 4 cells to begin with, how many are there after 3 generations? See Fig 6.12 Arithmetic Numbers of Cell Division Fig 6.14 Growth Curve Chapter 6 Microbial Growth 21 Chapter 6 Microbial Growth 22 Growth Curve See Fig 6.14 Growth Curve 1. Lag Phase A. 1 st placed in new medium B. 2. Log Phase A. growth (logarithmic) B. C. Most sensitive to 3. Stationary Phase A. B. 4. Death Phase A. decline, # deaths > # new cells Chapter 6 Microbial Growth 23 Example Graph Problems #1 Graph shows growth at 25C 1. If organism has optimum temp of 20C, how would the graph change if: A. Grown at 30C instead? B. Grown at 20C instead? 2. If the organism is a psychrotroph, how would it change if: A. Grown at 5C? Chapter 6 Microbial Growth 24 Chap 6 Microbial Growth 4

5 Example Graph Problems #2 Example Graph Problems #3 Graph shows growth in room air. How would it change if it s a/an 1. Aerotolerant anaerobe grown in: A. Candle jar instead? B. Anaerobic conditions? 2. Facultative anaerobe grown in: A. Candle jar? B. Anaerobic conditions? Graph is on MacConkey s agar which contains lactose. 1. If the organism can utilize lactose & sucrose, similarly to E. coli, how would the graph change A. If sucrose is added? B. Lactose is removed? C. Lactose is doubled? Chapter 6 Microbial Growth 25 Chapter 6 Microbial Growth 26 Growth Pattern Examples-Temperature Groupings 1. Purpose of buffer? Why needed? Review Miscellaneous Which line is a thermophile grown at 4C? Which line is a thermophile grown at 55C? Which line is a psychrotroph grown at 4C? Chapter 6 Microbial Growth Diagram above: A. Type of environment? B. Direction of osmosis? C. Result or effect on cell? 3. Ingredient that indicates complex media? 4. 2 reasons pickles resist spoilage? 5. 5 cells with a generation time of 20 minutes are grown for 2 hours. How many cells now? 6. Lab #10-12 Table 12.1: Complex vs. defined? Chapter 6 Microbial Growth 28 Osmosis Review Review Miscellaneous: Selective vs. Differential Plates What if???? 1. 1% NaCl vs. 2% NaCl 2. 2% NaCl vs. 2% NaCl 3. 2% NaCl vs. 2% Sucrose 4. 1% NaCl vs. 2% Sucrose 5. 2% Glucose vs. 2% Sucrose 6. 2% Lactose vs. 2% Sucrose Which of the following medias are: 1. Selective? 2. Differential? 3. General nutrient? Tryptose Mac MS CNA Staph epi + - Pink + Serratia + Pink - - Salmonella + Clear - - Staph aureus + - Yellow + Chapter 6 Microbial Growth 29 Chapter 6 Microbial Growth 30 Chap 6 Microbial Growth 5

6 Lab #19 Oxygen Aerobic Candle Anaerobic Classification PREDICTED catalase /- - Organisms EXPECTED / Chapter 6 Microbial GrowthChapter 6 Microbial Growth 31 Chap 6 Microbial Growth 6

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