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Kathy Huschle Northland Community & Technical College

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1 Kathy Huschle Northland Community & Technical College
Prokaryotic Growth Kathy Huschle Northland Community & Technical College

2 Pure Cultures pure culture: population of organisms descended from one organism only approximately 1% of all bacteria can be cultured successfully in the lab Vibro chlorae

3 Pure Culture colony, clone
begins with a single bacterial cell placed on a solid medium such as agar agar provides specific nutrition for bacteria and a medium to grow on Colonies on agar Nutritional Agar

4 Binary Fission method of bacterial reproduction
cell divides exactly in half single cell division reproduction of the entire organism

5 Binary Fission asexual no genetic recombination
the DNA molecule replicates itself when bacterial reproduction takes place E. coli undergoing cell division

6 Bacterial Growth bacterial growth = bacterial cell reproduction
the process of binary fission doubles the population each time binary fission takes place this doubling time demonstrates exponential growth each generation results in a doubling of the population generation time is = to doubling time measure of microbial growth rate

7 Bacterial Growth Curve: laboratory conditions
bacterial growth generally follows a characteristic pattern 5 phases normal growth curve, with optimum environmental and nutritional conditions

8 Bacterial Growth Curve: laboratory conditions
lag phase no increase in cell numbers cells are adapting to the environment cells are preparing for reproduction synthesizing new DNA, etc.

9 Bacterial Growth Curve: laboratory conditions
log phase exponential phase maximal rate for reproduction this happens with a specific set of growth conditions those resources for growth are abundantly available

10 Bacterial Growth Curve: laboratory conditions
stationary growth phase maximum population for the resources available required nutrients become depleted inhibitory end products from cell metabolism accumulate cell growth = cell death

11 Bacterial Growth Curve: laboratory conditions
death phase cell death > new cell formation

12 Bacterial Growth Curve: laboratory conditions
phase of prolonged decline can last from months to years “survival of the fittest”

13 Solid Media on solid media cells do not disperse readily
nutrients become limited in center death phase occurs in the center with exponential phase at periphery of the bacterial colony

14 Bacterial Growth most lab organisms are grown in a batch culture
closed system new materials are not added waste products are not removed under these conditions bacteria populations follow distinct patterns of growth Algae batch cultures

15 Bacterial Growth continuous culture maintained
nutrients must be continually supplied end products must be removed exponential growth phase maintained Continuous culture in lab

16 Natural Chemostat chemostat continuous culture device
A cow, with it’s four stomachs, is natures perfect chemostat; constantly grazing to add nutrients and continually belching and other such mechanics to remove bacterial metabolic end products

17 Environmental Parameters: influencing bacterial growth
not all bacteria favor the same environmental conditions the effects of varying conditions are seen as differences in reproduction (bacterial growth) some environmental conditions that can affect bacterial growth include temperature oxygen salinity pH

18 Environmental Influencing Factors: temperature
ideal temperature for growth varies between organisms specified by the bacterial genome

19 Environmental Influencing Factors: temperature
temperature growth range minimum to maximum temperatures for bacterial growth optimal growth temperature temperature at which the highest rate of reproduction occurs

20 Environmental Influencing Factors: temperature
5 divisions of prokaryotes, based on optimal growth temperature psychrophiles psychrotrophs mesophiles thermophiles hyperthemophiles Psychrophile: Desulfofaba gelida Thermophile: Pyrococcus sp. Hyperthermophile: Thermococcus barophilus

21 Environmental Influencing Factors: temperature
psychrophiles optimum growth temperature: -50C – 150C found in the Arctic and Antarctic regions of the world Bacteria found in melt from a Russian outpost on Lake Vostok Desulfofrigus oceanense

22 Environmental Influencing Factors: temperature
psychotrophs optimum growth temperature: 200C – 300C will grow at lower temperatures most commonly found in refrigerated food spoilage Stemphlium sarcinaeforme

23 Environmental Influencing Factors: temperature
mesophiles optimum growth temperature: 250C – 450C most human pathogens are mesophiles adapted well to growth in the human body, whose normal temperature is around 370C Salmonella

24 Environmental Influencing Factors: temperature
thermophiles optimum temperature: 450C – 700C commonly found in compost heaps and hot springs, water heaters Sulfolobus Thermophile in a hot spring Sulfur pots in Yellowstone

25 Environmental Influencing Factors: temperature
hyperthermophiles optimum growth temperature: 700C – 1100C usually member of the Archae domain found in hydrothermal vents in the depths of the ocean Deep Sea Vent

26 Temperature Ranges psychrophiles -50 C to 150 C psychotrophs
mesophiles 250 C to 450 C thermophile 450 C to 700 C hyperthermophiles 700 C to 1100 C

27 Temperature Considerations
food preservation refrigeration inhibits fast growing mesophiles psychrophiles can still grow in refrigeration, but at a diminished rate freezing destroys microorganisms that require water to grow

28 Temperature Considerations
disease body temperature varies: extremities are usually cooler than 370C some microorganisms can cause disease in certain body parts but not in others due to variations in body temperatures

29 Environmental Influencing Factors: oxygen
oxygen levels vary between environments and within the same environment based on O2 requirements, prokaryotes are separated into the following groups obligate aerobes obligate anaerobes facultative anaerobes microaerophiles aerotolerant anaerobes

30 Environmental Influencing Factors: oxygen
obligate aerobes need oxygen present to multiply Giardia

31 Environmental Influencing Factors: oxygen
obligate anaerobes cannot multiply in the presence of oxygen often killed by traces of oxygen in their environment C. perfringens

32 Environmental Influencing Factors: oxygen
facultative anaerobes grow best with oxygen, but can grow without oxygen respiration if oxygen is available fermentation if no oxygen is present growth is greater in the presence of oxygen due to the production of more ATP (energy source of the cell) Aeromonas hydrophilia on intestinal cells

33 Environmental Influencing Factors: oxygen
microaerophiles require oxygen but have maximal growth at reduced oxygen concentration high concentration of oxygen inhibit growth Helicobacter sp. Helicobacter sp.

34 Environmental Influencing Factors: oxygen
aerotolerant anaerobes indifferent to oxygen S. mutans

35 Environmental Influencing Factors: pH
based on pH of the environment, microorganisms are separated into the following groups neutrophiles acidophiles alkalophiles

36 Environmental Influencing Factors: pH
neutrophiles optimum pH of 7 (neutral) most microorganisms grow best between pH of 5 (acidic) and pH of 8 (alkaline) acidophiles optimal growth, pH of less than 5.5 alkalophiles optimum pH of 8.5 or greater Copper Urinary bacterial infection caused by alkaline urine Copper tolerant acidophile

37 Environmental Influencing Factors: salinity
H2O is required by all microorganisms for growth in some places H2O is hard to come by such as in salt concentrations if a cell is in an environment that has a greater solute concentration than the interior of the cell, then by osmosis the water will leave the cell causing plasmolysis (shrinking of the cell)

38 Environmental Influencing Factors: salinity
halophiles are microorganisms that have adapted to this kind of environment halophiles require high levels of sodium chloride moderate halophiles 3% salt concentration extreme halophiles: Archaea require at least 9% salt solution found in the Dead Sea Dunaliella salina cell, near a salt crystal. 40X Dead Sea

39 Nutritional Influencing Factors
major elements C, O, H, N, S, P, K, MG, Ca Fe essential components of protein, carbohydrates, lipids and nucleic acid needed to synthesize cell components

40 Nutritional/Energy Influencing Factors
heterotrophs utilize organic carbon autotroph utilize inorganic carbon phototrophs harvest the energy of sunlight chemotroph obtain energy by metabolizing chemical compounds Dinoflagellates Myxobacteria Purple Sulfur Bacteria: a chemotroph

41 Nutritional Diversity
prokaryotes are able to use diverse sources of carbon (an essential element) and energy this ability allows them to thrive in virtually and environment Forms of Carbon

42 Nutritional Diversity
photoautotrophs utilize the energy of sunlight obtain carbon from CO2 primary producers of the microbial world 6CO2 + 12H2O C6H12O6 + 6H2O + 6O2 photoheterotrophs obtain carbon from organic compounds Cyanobacteria Rhodobacter sphaeroides

43 Nutritional Diversity
chemolithoautotrophs AKA as chemoautotrophs or chemolithotrophs energy from inorganic compounds such as hydrogen sulfide carbon from CO2 Thiobacillus denitrificans

44 Nutritional Diversity
chemoorganoheterotrophs AKA chemoheterotrophs or chemoorganotrophs utilize organic compounds for energy and as a carbon source most common group of microorganisms associated with humans and animals important organic degraders Brachionus calyciflorus B. vietnamiensis

45 Prokaryotes in the Lab studying microorganisms in their environment, enhances our ability to grow them in the lab lab growth is important for the study of the microbial world and its effect on human life

46 Lab Cultivation of Microbes
complex media used for routine purposes variety of ingredients needed by the microorganism are included in the media nutrient agar, blood agar, PEA agar, Mannitol Salt agar are some examples S. aureus on blood agar

47 Lab Cultivation of Microbes
selective media formulated with ingredients that inhibit the growth of some bacteria, such as an antibiotic, but enhance growth of the target organism ie: MacConkey agar can be used to isolate Gram-negative rods

48 Lab Cultivation of Microbes
differential media includes ingredients, such as chemical indicators, that produce observable differences between species of bacteria ie: ph indicator may be incorporated with the agar medium allowing for the detection of acid producing microorganisms mannitol salt agar: pH indicator turns the agar yellow in the presence of a salt tolerant organism

49 Creating Appropriate Environmental Conditions
to enhance microbial growth in a lab, certain environmental conditions need to be created atmospheric pressure temperature oxygen availability

50 Creating Appropriate Environmental Conditions
atmosphere increase CO2 for some species of microbes Candle jar used in lab to increase CO2 concentration

51 Creating Appropriate Environmental Conditions
anaerobic microorganisms require anaerobic conditions required growth these are some of the most difficult types of microorganisms to culture in the lab, due to the fact that even a brief exposure to oxygen generally results in the death of the organism Anaerobic jars used in labs

52 Creating Appropriate Environmental Conditions
temperature controlled with the use of an incubator allows for setting the optimum temperature for individual microorganisms

53 Bacteria Enumeration lab techniques that monitor bacterial growth
viable plate count direct count most probable number membrane filtration measuring biomass turbidity total weight chemical constituents

54 Bacteria Enumeration viable plate count
measure the number of cells in a sample based on the fact that one cell gives rise to one colony utilizes a series of dilutions in order to calculate the number of viable bacteria in the original sample

55 Bacteria Enumeration direct count
using special equipment capable of making: a direct microscopic count a count of cells suspended in a suspension a count by analyzing the scattering of light as cells pass by a laser

56 Bacteria Enumeration most probable number
a statistical analysis of cell numbers based on the theory of probability

57 Bacteria Enumeration membrane filtration
used when cell numbers are low allows for a concentration of the microbes by filtering before plating Membrane filtration on mEnterococcus agar. The plate at the bottom is uninoculated. The red colonies typical of the Enterococci are clearly visible on the white membrane filters. Membrane filtration equipment

58 Bacteria Enumeration measuring biomass turbidity total weight
chemical constituents turbidity

59 Bacteria Enumeration turbidity
cloudiness, which indicates the presence of microbial growth cell numbers can be measured with a spectrophotometer

60 Bacteria Enumeration total weight tedious work
measure the wet weight, centrifuge and then measure dry weight

61 Bacteria Enumeration chemical constituents
analyzing the quantity of chemical (metabolic byproduct) in a bacterial sample and using that information to calculate biomass Spectroanalysis

62 Bacterial Growth in Nature
similar to a continuous culture nutrients are continually added and byproducts are removed generally multiply more slowly than under lab conditions often the waste of one microorganism is the nutrient of another Microbial mat in Yellowstone

63 Bacterial Growth in Nature
biofilms polysaccharide-encased community slippery rocks, gunk in drains, plaque on teeth, IV’s are all examples of biofilms begins with adherence of a bacterium to a surface bacteria multiplies synthesizes a loose glycocalyx allowing unrelated cell to attach and grow Methanogen biofilm

64 Bacterial Growth in Nature
biofilms medical problems resist antibiotics 65% of human infections involve biofilms often times 100X more resistant to disinfectants Biofilm on endotrachial tube

65 Bacterial Growth in Nature
bioremediation bacteria used to degrade chemicals are enhanced by organisms present in biofilm Acid from an abandoned mine. Microorganisms are introduced to this environment and are successfully able to clean up the “problem”.

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