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Microbial Nutrition and Growth
“The cause of nutrition and growth resides not in the organism as a whole but in the separate elementary parts—the cells.” ― Theodor Schwann
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Requirements for Growth
Microbe’s gotta eat too: the same elements required for human life, _____________etc., are also required for microbial life
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Classification: Carbon and Energy Acquisition
_______________– can utilize carbon dioxide as its sole source of carbon _______________– must obtain carbon through means other than carbon dioxide _______________– utilizes the sun’s rays as its energy source _______________– must obtain energy through means other than the sun E N R G Y
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Classification: Carbon and Energy Acquisition
Microbiology with Diseases by Body System, 4th Ed., Pearson Education, 2015; Figure 6.1 Four basic groups of organisms based on their carbon and energy sources.
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Classification: Electron Acquisition
_______________— heterotrophs acquire electrons from same organic molecules that provide them carbon __________________________________________________________________________________ _______________— autotrophs acquire electrons from inorganic molecules OH LA
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Are purple non-sulfur bacteria organotrophs or lithotrophs?
Purple non-sulfur bacteria are organisms that utilize light as their energy source Phototroph or chemotroph? Purple non-sulfur bacteria are organisms that utilize carbon sources other than carbon dioxide Autotroph or heterotroph? Are purple non-sulfur bacteria organotrophs or lithotrophs? Genus Sulfolobus are thermoacidophiles that can’t utilize solar energy Genus Sulfolobus are thermoacidophiles that utilize carbon dioxide as their carbon source Are species in genus Sulfolobus organotrophs or lithotrophs?
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Classification: Oxygen Acquisition
_______________– NEED oxygen _______________– require small amounts of oxygen ______________________________– don’t require oxygen but will not be poisoned by oxygen ______________________________– NEED to be WITHOUT oxygen; oxygen is POISON ______________________________– would prefer to have oxygen but can operate without it
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Loose-Fitting Caps 2. 4. 1. 3. 5.
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Temperature and Growth
Microbiology with Diseases by Body System, 4th Ed., Pearson Education, 2015; Figure 6.4 The effects of temperature on microbial growth. Optimum Maximum Minimum 22°C 37°C 55°C
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Classification: Temperature and Growth
Microbiology with Diseases by Body System, 4th Ed., Pearson Education, 2015; Figure 6.5 Four categories of microbes based on temperature ranges for growth. Mésos – Gk. middle Psukhrós – Gk. cold
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Classification: pH and Growth
_______________– grow at acidic pH (appx. 1-2) _______________– grow at neutral pH (appx. 7) _______________– grow at basic pH (appx ) Classification: Salt _______________– require environments with high salt content Háls – Gk. sea Classification: Pressure _______________– require high pressures Barús – Gk. heavy
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Microbial Growth in Biofilms
Collection of various microbes living on a surface (usually inanimate) that form a complex, well-developed community Can form on medical devices and mucous membranes of digestive system pH and oxygen gradients can develop One clinical example you’re all familiar with… What external structure have we learned about might microbes use in order to form biofilms?
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Culturing Microbes _______________– the growing of microorganisms or the microorganisms that have been grown themselves Cultures are grown on _______________ Solid – e.g. traditional Petri dish; main media used for culturing Liquid – growth is exhibited by the formation of a colloidal suspension; used for various parasites and Mycobacterium
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Microbes = Seeds Media = Soil
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Solid Media Prototypical Petri dish: _____________________
Agar: complex polysaccharide derived from certain red algae that cannot be digested by most microbes, thus it provides a solid surface for growth 6 General Types Defined (Synthetic) media Complex media Selective media Differential media Anaerobic media Transport media
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Defined (Synthetic) & Complex Media
Medium in which the ______________________________ is known Fastidious (picky) organisms require the addition of a large number of growth factors, so they are added into defined media Complex Exact chemical composition, who knows? Bacteria will grow on it anyway Used to culture organisms with unknown nutritional needs
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Selective & Differential Media
Selective – _______________; will grow certain bacteria but not others Differential – will grow most all microbes, but will allow for a color change that can help narrow down what type of microbe is present Some media can be both
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Blood Agar (We Don’t Get To Do This One )
Nutrient rich, contains sheep blood Exhibits color changes in the presence of certain bacterial enzymes Alpha-hemolysis – partial lysis of erythrocytes; medium looks bruised with some clear spots on a mostly red medium Beta-hemolysis – complete lysis of erythrocytes; medium turns clear Gamma-hemolysis – no lysis of erythrocytes; medium does not change color
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Blood Agar: Which is Which?
Using the information given to you in the previous slide, is the Blood Agar medium differential, selective, or both?
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MacConkey (MAC) Medium
Composed of bile salts, crystal violet dye, neutral red dye, and lactose Will only grow Gram-negative bacteria Those Gram-negative bacteria species that can ferment lactose will grow in pink colonies Those Gram-negative bacteria species that can’t ferment lactose will grow in yellow colonies
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serotype Choleraesuis
MacConkey (MAC) Medium: Gram-Positive or Gram-Negative, Ferments Lactose? Escherichia coli Escherichia coli Microbiology with Diseases by Body System, 4th Ed., Pearson Education, 2015; Figure 6.15 The use of MacConkey agar as a selective and differential medium. Staphylococcus aureus (no growth) Salmonella enterica serotype Choleraesuis Is the MacConkey medium differential, selective, or both?
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Mannitol Salt (MSA) Medium
Composed of a high salt concentration, mannitol, and phenol red Will only grow halophilic bacteria Those halophilic bacteria that can ferment mannitol will turn the medium yellow Those halophilic bacteria that can’t ferment mannitol will grow in the pink medium
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Mannitol Salt (MSA) Medium: Halophile, Ferments Mannitol?
Is the Mannitol Salt medium differential, selective, or both? Micrococcus luteus (no growth) Staphylococcus epidermidis Staphylococcus aureus
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Anaerobic Media Obligate anaerobes must be cultured in the absence of free oxygen Reducing media contain compounds that combine with free oxygen and remove it from the medium Petri plates are incubated in anaerobic culture vessels Sealable containers that contain reducing chemicals Clamp Airtight lid Chamber Palladium pellets to catalyze reaction removing O2 Methylene blue (anaerobic indicator) Envelope containing chemicals to release CO2 and H2 Microbiology with Diseases by Body System, 4th Ed., Pearson Education, 2015; Figure 6.16 An anaerobic culture system. Petri plates
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Anaerobic Media Airtight chambers with all of the contents, such as the one pictured on the last slide, can be expensive A simpler alternative is just to use a candle jar to consume the oxygen in the environment
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Transport Media Used by hospital personnel to ensure clinical specimens are not contaminated, transported in a timely manner, and to protect people from infection
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Cell Culture Not all microbes will grow on artificial media
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Preserving Cultures Refrigeration Deep-freezing Lyophilization
Stores for short periods of time Deep-freezing Stores for years Lyophilization “Freeze-drying” – freezing the material and subsequently reducing the pressure to allow it to _________________ Stores for decades
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Classification: Colony Morphology
Shape Circular Rhizoid Irregular Filamentous Spindle Microbiology with Diseases by Body System, 4th Ed., Pearson Education, 2015; Figure 6.8 Characteristics of bacterial colonies. Margin Entire Undulate Lobate Curled Filiform Elevation Flat Raised Convex Pulvinate Umbonate Size Punctiform Small Moderate Large Texture Smooth or rough Appearance Glistening (shiny) or dull Colony — aggregation of cells arising from single parent cell Nonpigmented (e.g., cream, tan, white) Pigmented (e.g., purple, red, yellow) Pigmentation Optical property Opaque, translucent, transparent
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Counting Bacteria on Media
Colony Forming Units (CFU’s) Sometimes there are too many colony forming units to count, so the plate will be divided up into sections; one section will be counted and will be used to make an estimate on how many CFU’s are present
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Dilution DILUTED 1. If you had 1 cup of coffee and poured it into 9 cups of water, what is the new total volume of coffee? How strong is this new volume of coffee in comparison to the original undiluted volume? 2. If you had 1 cup of coffee and poured it into 4 cups of water, what is the new total volume of coffee? 3. If you had 1 cup of coffee and poured it into 99 cups of water, what is the new total volume of coffee? 4. You have 1 cup of coffee and pour it into 9 cups of water; you take 1 cup of that diluted mixture and pour it into 9 cups of water; you take 1 cup of that diluted mixture and pour it into 49 cups of water. What is the new total volume of coffee?
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Estimating Bacterial Numbers: Serial Dilution and Viable Plate Counts
1 mL of original culture 1.0 mL 1.0 mL 1.0 mL 1.0 mL 9 mL of broth + 1 mLof original culture = 10 mL total 1:10 dilution (10-1) 1:100 dilution (10-2) 1:1000 dilution (10-3) 1:10,000 dilution (10-4) 1:100,000 dilution (10-5) 0.1 mL of each transferred to a plate 0.1 mL 0.1 mL 0.1 mL 0.1 mL 0.1 mL Microbiology with Diseases by Body System, 4th Ed., Pearson Education, 2015; Figure 6.23 A serial dilution and viable plate count for estimating microbial population size. Incubation period Too numerous to count (TNTC) Y TNTC X 60 colonies 6 colonies 0 colonies
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Dilution: You Try You have 1 mL of bacterial stock solution. You perform a 1/5 dilution 3 times. What is the end dilution factor, i.e. how “strong” is the last diluted solution in comparison to the undiluted 1 mL stock solution? The observed CFU’s at the dilution factor (calculated above) is 227. How many CFU’s were present in the original 1mL stock solution?
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Estimating Bacterial Numbers: Membrane Filtration
Sample to be filtered Membrane transferred to culture medium Membrane filter retains cells To vacuum Incubation Colonies Microbiology with Diseases by Body System, 4th Ed., Pearson Education, 2015; Figure 6.24 The use of membrane filtration to estimate microbial population size.
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Estimating Bacterial Numbers: Most Probable Number (MPN)
1.0 ml 1.0 ml Undiluted 10 mL of original culture 9 mL of broth + 1 mL of original culture = 10 mL total 1:10 dilution (10-1) 1:100 dilution (10-2) Inoculate 1.0 ml into each of 5 tubes Phenol red, pH color indicator, added Incubate Results Microbiology with Diseases by Body System, 4th Ed., Pearson Education, 2015; Figure 6.25 The most probable number (MPN) method for estimating microbial numbers 4 tubes positive 2 tubes positive 1 tube positive
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* *Three dilutions is technically two dilutions plus the original, undiluted solution Microbiology with Diseases by Body System, 4th Ed., Pearson Education, 2015; Table 6.5 Most Probable Number Table (partial)
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Bacterial Growth is Exponential
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…as opposed to Arithmetic
Species growing arithmetically Species growing exponentially Microbiology with Diseases by Body System, 4th Ed., Pearson Education, 2015; Figure 6.18 A comparison of arithmetic and logarithmic growth.
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Bacterial Growth Cycle
Microbiology with Diseases by Body System, 4th Ed., Pearson Education, 2015; Figure 6.20 A typical microbial growth curve.
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Tell Me Why Students transfer some "gunk" from a two-week-old bacterial culture into new media. Why shouldn't they be surprised when this "death-phase" sample grows?
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Obtaining Pure Culture: Streak-Plate Isolation Method
Inoculation After incubation The first 3 streaks have colonies that are too closely packed together to isolate a single pure colony Microbiology with Diseases by Body System, 4th Ed., Pearson Education, 2015; Figure 6.9 The streak-plate method of isolation.
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Obtaining Pure Culture: Pour-Plate Isolation Method
Sequential inoculations 1.0 ml 1.0 ml 1.0 ml 9 ml broth 9 ml broth 9 ml broth Initial sample 1.0 ml to each Petri dish, add 9 ml warm agar, swirl gently to mix Colonies Fewer colonies Microbiology with Diseases by Body System, 4th Ed., Pearson Education, 2015; Figure 6.10 The pour-plate method of isolation.
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