1. Elements of Microbial Growth, Nutrition and Environment

2. Do different organisms require specific diets and environments?

3. Why do we care about growth? To Encourage the microbes we want Brewery, winery, food production Vaccine and drug production Microbial fuel cells Bioremediation, Sewage treatment plant, oil spill clean up Resident microbiota-probiotics to aid microbial antagonism and perform other functions To Discourage the microbes we don’t want Pathogens

4. What is Growth? In microbiology, we define growth in relation to the number of cells, not the size of cells. Concentrate on population growth Bacterial cells divide via binary fission, not mitosis.

5. Binary fission The division of a bacterial cell Parental cell enlarges and duplicates its DNA Septum formation divides the cell into two separate chambers Complete division results in two identical cells

6. Generation Time The time required for a complete division cycle (doubling) Length of the generation time is a measure of the growth rate Growth is exponential not arithmetic Dependent on chemical and physical conditions

7. Generation Time Average generation time is 30 – 60 minutes shortest generation times can be 10 – 12 minutes E. coli GT=20 min. Mycobacterium leprae has a generation time of 10 – 30 days 1  1 million cells (20 generations) in 7 hours most pathogens have relatively short generation times

8. Which is bacterial growth curve?

10. Four phases of growth in a bacterial culture (Log)

11. 1. Lag Phase Cells are adjusting, enlarging, and synthesizing critical proteins and metabolites Not doubling at their maximum growth rate

12. 2. Exponential Growth Phase Maximum exponential growth rate of cell division Adequate nutrients Favorable environment Most sensitive to antibiotics. Why?

13. Exponential Growth Phase A person actively shedding bacteria in the early and middle stages of infection is more likely to spread it than a person in the later stages. Why? MRSA

14. 3. Stationary Phase Cell birth and cell death rates are equal Survival mode – depletion in nutrients, released waste can inhibit growth

15. 4. Death Phase A majority of cells begin to die exponentially due to lack of nutrients or build up of waste Slower than the exponential growth phase

16. How do we measure microbial growth? Direct measurement – Standard Plate counts most common, need to DILUTE to get individual, countable colonies – Microscopic Count count with microscope – Filtration when # microbes small, water run thru filter and filter applied to TSA plate and incubated – Coulter Counter Automated cell counter Indirect (Estimation) – Turbidity – more bacteria, more cloudiness – can measure w/ spectrophotometer or eye – Metabolic Activity – assumes amount of metabolic product is proportional to # – Dry Weight – used for filamentous organisms, like molds – Genetic Probing – Real-time PCR

17. Direct: Standard Plate Counts

18. Direct: Microscopic Count Advantages – Easy and fast Disadvantages – Uses special microscope counting slide – Does not differentiate between live and dead bacteria

19. Direct: Membrane Filtration

20. Direct: Coulter Counter Uses an electronic sensor to detect and count the number of cells.

21. The greater the turbidity, the larger the population size. Which culture (left or right) has more bacteria? Indirect: Turbidity Using Spectrometer

22. Indirect: Metabolism Activity The metabolic output or input of a culture may be used to estimate viable count. Examples: Measure how fast gases and/or acids are formed in a culture Or the rate a substrate such as glucose or oxygen is used up

23. Indirect: Dry Weight To calculate the dry weight of cells – cells must be separated from the medium – then dried – the resulting mass is then weighed

24. Indirect: Genetic Methods Use real-time PCR to “count” how many bacterial genes there are in a sample.

25. Which techniques distinguish between live and dead cells? – Standard Plate counts – Direct Microscopic – Filtration – Coulter counter – Turbidity – Metabolic activity – Dry weight – Genetic Probing

26. Which techniques distinguish between live and dead cells? – Standard Plate counts – Direct Microscopic – Filtration – Coulter counter – Turbidity – Metabolic activity – Dry weight – Genetic Probing

27. What are the requirements for microbial growth?

28. Chemical Composition of an Escherichia coli Cell

29. Macronutrients: -carbon, hydrogen, and oxygen -required in relatively large quantities and play principal roles in cell structure and metabolism Micronutrients: -present in much smaller amounts -manganese, zinc, nickel Inorganic nutrients: -Can have carbon OR hydrogen, but not both Organic nutrients: -Contain carbon and hydrogen Microbial Nutrition

30. All cells require the following for metabolism and growth: – Carbon source – Energy source Growth factors (some bacteria are fastidious/picky and require extra supplements)

31. Heterotroph: Organic carbon is carbon source Autotroph: inorganic CO 2 as its carbon source -has the capacity to convert CO 2 into organic compounds -not nutritionally dependent on other living things Phototroph: microbes that photosynthesize Chemotroph: microbes that gain energy from injesting chemical compounds Microbial Nutrition

32. Photoautotrophs: -Photosynthetic -Produce organic molecules using CO 2 -Ex: Cyanobacteria, algea Chemoautotrophs: -Ingest organic compounds for energy -Produce organic molecules using CO 2 Microbial Nutrition: Autotrophs

33. Chemoheterotrophs: -organic compounds for both carbon and energy source -derive both carbon and energy from processing these molecules through respiration or fermentation -The vast majority of microbes causing human disease are chemoheterotrophs -Ex: Most bacteria, all, protists, all fungi, and all animals Microbial Nutrition: Heterotrophs

34. Transport of necessary nutrients occurs across the cell membrane, even in organisms with cell walls Diffusion: Atoms or molecules move in a gradient from an area of higher concentration to lower concentration Diffusion of molecules across the cell membrane is largely determined by the concentration gradient and permeability of the substance Diffusion: Review

35. Osmosis: the diffusion of water through a selectively permeable membrane Isotonic: Equal solutes in cell and in environment -parasites living in host tissues are most likely to be living in isotonic habitats -Hypotonic: More solutes in cell than in environment -A slightly hypotonic environment can be favorable to bacteria cells Hypertonic: Less solutes in cell than in environment hypertonic solutions such as concentrated salt and sugar solutions act as preservatives for food(salted ham is an example) Osmosis: Review

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38. Environmental (Physical) Factors Effecting Bacterial Growth Temperature Gas pH Osmotic pressure Other factors Microbial association Survival in a changing environment is largely a matter of whether the enzyme systems of microorganisms can adapt to alterations in their habitat

39. Environmental Factors: Temperature Effect of temperature on proteins: – Too high, proteins unfold and denature – Too low, do not work efficiently Effect of temperature on membranes of cells and organelles: – Too low, membranes become rigid and fragile – Too high, membranes become too fluid

40. Temperature and Bacterial Growth

41. Five categories of microbes based on temperature ranges for growth Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. 0 Rate of Growth Temperature °C Psychrophile Psychrotroph Mesophile Thermophile Extreme thermophile Optimum Minimum Maximum -20-10102030405060708090100110120130 Which category do human pathogens usually fall into? Why?

42. Environmental Factors: Gases Two gases that most influence microbial growth – Oxygen O 2 has the greatest impact on microbial growth O 2 is an important respiratory gas and a powerful oxidizing agent – Carbon dioxide

43. Oxygen Requirements As oxygen enters cellular reactions, it is transformed into several toxic products – highly reactive and excellent oxidizing agents Resulting oxidation causes irreparable damage to cells by attacking enzymes and proteins

44. Oxygen Requirements As oxygen enters cellular reactions, it is transformed into several toxic products: -singlet oxygen (O) -superoxide ion (O 2 - ) -hydrogen peroxide (H 2 O 2 ) -hydroxyl radicals (OH - ) Most cells have developed enzymes that scavenge and neutralize reactive oxygen byproducts Two-step process requires two enzymes:

45. Catalase Test Oxygen Requirements If bacteria do not have superoxide dismutase or catalase they can not tolerate oxygen.

46. Oxygen Requirements Aerobes Anaerobes Facultative anaerobes Aerotolerant anaerobes Microaerophiles

47. Oxygen Requirements: Obligate Aerobe Requires oxygen for metabolism Have enzymes that neutralize toxic oxygen metabolites Ex. Most fungi, protozoa, and bacteria, such as Bacillus species and Mycobacterium tuberculosis

48. Oxygen Requirements: Facultative Anaerobe Does not require oxygen, but can grow in its presence During minus oxygen states, anaerobic respiration or fermentation occurs Possess superoxide dismutase and catalase Ex. Many Gram-negative pathogens Prefer oxygenated environments because more energy is produced during aerobic respiration compared to anaerobic respiration or fermentation

49. Oxygen Requirements: Obligate Anaerobes Cannot use oxygen for metabolism Do not possess superoxide dismutase and catalase The presence of oxygen is toxic to the cell and will kill it Ex. Many oral bacteria, intestinal bacteria

50. Thioglycollate broth enables the identification of aerobes, facultative anaerobes, and obligate anaerobes. Use of thioglycollate broth to demonstrate oxygen requirements.

51. Anaerobes must grow in an oxygen minus environment, because toxic oxygen metabolites cannot be neutralized. Culturing Technique for Anaerobes

52. Most cells grow best between pH 6-8 – strong acids and bases can be damaging to enzymes and other cellular substances Pathogens like our neutral pH Yeast & Molds like acidic conditions Environmental Factors: pH

53. Acidophiles – thrive in acidic environments. – Ex. Helicobacter pylori Alkalinophiles – thrive in alkaline conditions – Ex. Proteus can create alkaline conditions to neutralize urine and colonize and infect the urinary system Environmental Factors: pH

54. Example of the use of a selective medium for pH Fungal colonies Bacterial colonies pH 7.3pH 5.6

55. Microbes require water to dissolve enzymes and nutrients Water is important reactant in many metabolic reactions Most cells die in absence of water – Some have cell walls that retain water – Endospores and cysts cease most metabolic activity Two physical effects of water – Osmotic pressure – Hydrostatic pressure Environmental Factors: Water

56. Osmotic pressure: Halophiles (Salt lovers) – Requires high salt concentrations – Withstands hypertonic conditions Ex. Halobacterium Facultative halophiles – Can survive high salt conditions but is not required – Ex. Staphylococcus aureus Environmental Factors: Water

57. Other Physical Factors Influencing Microbial Growth Radiation- UV, infrared Barophiles – withstand high pressures Spores and cysts- can survive dry habitats Microbes require different nutrients and different environments specific to survive. They have specialized over the years!

58. Associations Between Organisms – Organisms live in association with different species – Often involve nutritional interactions Antagonistic relationships Synergistic relationships Symbiotic relationships Associations Between Organisms Organisms live in close nutritional relationships; required by one or both members. Mutualism Obligatory, dependent; both members benefit. Commensalism The commensal benefits; other member not harmed. Parasitism Parasite is dependent and benefits; host harmed. Synergism Members cooperate and share nutrients. Antagonism Some members are inhibited or destroyed by others. Organisms are free-living; relationships not required for survival. Symbiotic Non symbiotic

59. Associations Between Organisms Antagonism: free-living species compete -Antibiosis: the production of inhibitory compounds such as antibiotics -The first microbe has a competitive advantage by increasing the space and nutrients available to it -Remember importance of microflora?! A biocontrol agent on the right (a bacteria) is making a material that is keeping the pathogen on the left (a fungus) from growing.

60. Associations Between Organisms Synergism: free-living species benefits together but is not necessary for survival Together the participants cooperate to produce a result that none of them could do alone Gum disease, dental caries, and some bloodstream infections involve mixed infections of bacteria interacting synergistically

61. Associations and Biofilms – Biofilms Complex relationships among numerous species of microorganisms Develop an extracellular matrix – Adheres cells to one another – Allows attachment to a substrate – Sequesters nutrients – May protect individuals in the biofilm Form on surfaces often as a result of quorum sensing Many microorganisms more harmful as part of a biofilm Plaque (biofilm) on a human tooth

62. Quorum sensing: used by bacteria to interact with members of the same species as well as members of other species that are close by Structure of the biofilm -large, complex communities form with different physical and biological characteristics -the bottom may have very different pH and oxygen conditions than the surface -partnership among multiple microbial inhabitants -cannot be eradicated by traditional methods Biofilms: Quorum Sensing

63. Now that you know more about the nutritional needs of bacteria let’s look at using this information to ID bacteria!

64. How to identify bacteria in patient specimens or in samples from nature? -phenotypic: considers macroscopic and microscopic morphology, physiology, and biochemistry -immunologic: serological analysis -genotypic: genetic techniques increasingly being used as a sole resource for identifying bacteria Data from these methods can provide a unique profile for any bacterium Survey of Microbial Diseases

65. Physiological/Biochemical Characteristics Traditional mainstay of bacterial identification Enzyme production and other biochemical properties are reliable ways to ID microbes Dozens of diagnostic tests exist for determining the presence of specific enzymes and to assess nutritional and metabolic activities -fermentation of sugars -capacity to digest complex polymers -production of gas -sensitivity to antibiotics -nutrient sources Survey of Microbial Diseases: Phenotypic Methods

66. Blood agar as a differential medium Beta-hemolysis Alpha-hemolysis No hemolysis (gamma-hemolysis)

67. Tests for fermentation and gas production No fermentation Acid fermentation with gas Durham tube (inverted tube to trap gas) Survey of Microbial Diseases: Phenotypic Methods

68. Direct observation of fresh or stained specimen Stains most often used -Gram stain -acid-fast stain Phenotypic Methods: Direct Examination of Specimen

69. Isolation Media and Morphological Testing -Selective media: encourage the growth of only the suspected pathogen -Differential media: used to identify definitive characteristics and fermentation patterns Survey of Microbial Diseases: Phenotypic Methods

70. MacConkey Agar: Selective and Differential Selects for Gram-negative and tells you if the bacterium ferments lactose

71. Physiological reactions: indirect evidence of enzymes present in a species. If bacteria tests + for superoxide dismutase (an oxidase) what does that tell you? Phenotypic Methods: Biochemical Testing

72. ––+––++––––+–––––––– |||||| ARAAMYMELSACRHASORINOMANGLUINDTDAUREH2SCITVPGELODCLDCADHDNPG Unknown microbe + different substrates Results (+/–) Enzyme-mediated metabolic reactions often visualized by a color change -microbe is cultured in a medium with a special substrate, then tested for a particular end product -microbial expression of the enzyme is made visible by a colored dye Phenotypic Methods: Biochemical Testing

73. Flowchart: We will use this to ID our MM! Gram (–) Cocci Anaerobic, oxidase (–), catalase (–) Veillonella Neisseria Branhamella Moraxella Aerobic, oxidase (+), catalase (+) Catalase (–), pairs, chain arrangement Gram (+) Catalase (+), irregular clusters, tetrads Strictly aerobic Micrococcus Staphylococcus Planococcus Facultative anaerobic Streptococcus

74. -used when morphological and biochemical tests are insufficient. Ex. S. aureus Phage Group I vs. Group II -bacteriophage infect bacteria in a species-specific and strain-specific way, which is useful in identifying some bacteria -a lawn of bacterial cells is inoculated onto agar, mapped off into blocks, and phage are exposed to each block -cleared areas corresponding to lysed cells indicate sensitivity to that phage Phenotypic Methods: Phage Typing

75. Important to rapidly determine if an isolate from a specimen is clinically important or if it is merely a contaminant or normal biota -a few colonies of E. coli in a urine sample can indicate normal biota, but several hundred can mean an active infection -a single colony of a true pathogen such as Mycobacterium tuberculosis in a sputum culture, or an opportunist in a sterile site, is highly suggestive of disease -repeated isolation of a relatively pure culture of any microorganism can mean it is an agent of disease Determining Clinical Significance of Cultures