Chapter 5 Microbial Nutrition and Culture cont’d

1. Chapter 5 Microbial Nutrition and Culture cont’d Siti Sarah Jumali (ext 2123) Room 3/14 sarah_jumali84@hotmail.com

2. Microbial Growth Requirements

3. The Requirements for Growth PHYSICAL REQUIREMENTS CHEMICAL REQUIREMENTS (NUTRITIONAL FACTORS) Carbon Nitrogen, sulfur, and phosphorous Trace elements Oxygen Organic growth factor • Temperature • pH • Oxygen • Hydrostatic Pressure • Osmotic pressure

4. 1) pH Physical Factors Required for Bacterial Growth • Optimum pH: the pH at which the microorganism grows best (e.g. pH 7) • Most bacteria grow between pH 6.5 and 7.5 • Molds and yeasts grow between pH 5 and 6 • According to their tolerance for acidity/alkalinity, bacteria are classified as: Acidophiles (acid-loving): grow best at pH 0.1-5.4 Neutrophiles: grow best at pH 5.4 to 8.0 Alkaliphiles (base-loving): grow best at pH 7.0-11.5

5. 2) Temperature • According to their growth temperature range, bacteria can be classified as: Psychrophiles : grow best at 15-20oC Psychrotrophs :grow between 0°C and 20–30°C Mesophiles : grow best at 25-40oC Thermophiles : grow best at 50-60oC • Typical Growth Rates and Temperature • Minimum growth temperature: lowest temp which species can grow • Optimum growth temperature: temp at which the species grow best • Maximum growth temperature: highest temp at which grow is possible

7. Food Preservation Temperatures

8. 3) Oxygen • Aerobes: require oxygen to grow • Obligate aerobes: must have free oxygen for aerobic respiration (e.g. Pseudomonas) • Anaerobes: do not require oxygen to grow • Obligate anaerobes: killed by free oxygen (e.g. Bacteroides) • Microaerophiles: grow best in presence of small amount of free oxygen • Capnophiles: carbon-dioxide loving organisms that thrive under conditions of low oxygen • Facultative anaerobes: carry on aerobic metabolism when oxygen is present, but shift to anaerobic metabolism when oxygen is absent • Aerotolerant anaerobes: can survive in the presence of oxygen but do not use it in their metabolism • Obligate: organism must have specified environmental condition • Facultative: organism is able to adjust to and tolerate environmental condition, but can also live in other conditions

9. Patterns of Oxygen Use

10. 4) Hydrostatic Pressure • Water in oceans and lakes exerts pressure exerted by standing water, in proportion to its depth • Pressure doubles with every 10 meter increase in depth • Barophiles: bacteria that live at high pressures, but die if left in laboratory at standard atmospheric pressure

11. 5) Osmotic Pressure • Environments that contain dissolved substances exert osmotic pressure, and pressure can exceed that exerted by dissolved substances in cells • Hyperosmotic environments: cells lose water and undergo plasmolysis (shrinking of cell) • Hypoosmotic environment: cells gain water and swell and burst

12. Plasmolysis

13. Halophiles • Salt-loving organisms which require moderate to large quantities of salt (sodium chloride) • Membrane transport systems actively transport sodium ions out of cells and concentrate potassium ions inside • Why do halophiles require sodium? 1) Cells need sodium to maintain a high intracellular potassium concentration for enzymatic function 2) Cells need sodium to maintain the integrity of their cell walls

14. Responses to Salt

15. The Great Salt Lake in Utah

16. Chemical Requirement: Nutritional Factors • Carbon sources • Nitrogen sources • Sulfur and phosphorus • Trace elements (e.g. copper, iron, zinc, and cobalt) • Vitamins (e.g. folic acid, vitamin B-12, vitamin K)

17. Chemical Requirements • Carbon • Structural organic molecules, energy source • Chemoheterotrophs use organic carbon sources • Autotrophs use CO2

18. Chemical Requirements • Nitrogen • In amino acids and proteins • Most bacteria decompose proteins • Some bacteria use NH4+ or NO3– • A few bacteria use N2 in nitrogen fixation

19. Chemical Requirements • Sulfur • In amino acids, thiamine, and biotin • Most bacteria decompose proteins • Some bacteria use SO42– or H2S • Phosphorus • In DNA, RNA, ATP, and membranes • PO43– is a source of phosphorus

20. Chemical Requirements • Trace elements • Inorganic elements (mineral) required in small amounts • Usually as enzyme cofactors • Ex: iron, molybdenum, zinc • Buffer • To neutralize acids and maintain proper pH • Peptones and amino acids or phosphate salts may act as buffers

21. Organic Growth Factors • Organic compounds obtained directly from the environment • Ex: Vitamins, amino acids, purines, and pyrimidines

22. Preparation of Culture Media • Culture medium: Nutrients prepared for microbial growth • Sterile: No living microbes • Inoculum: Introduction of microbes into medium • Culture: Microbes growing in/on culture medium

23. Agar • Complex polysaccharide • Used as solidifying agent for culture media in Petri plates, slants, and deeps • Generally not metabolized by microbes • Liquefies at 100°C • Solidifies at ~40°C

24. Types of Culture Media • Natural Media: In nature, many species of microorganisms grow together in oceans, lakes, and soil and on living or dead organic matter • Synthetic medium: A medium prepared in the laboratory from material of precise or reasonably well-defined composition • Complex medium: contains reasonably familiar material but varies slightly in chemical composition from batch to batch (e.g. peptone, a product of enzyme digestion of proteins)

25. Types of Culture Media

26. Culture Media • Chemically defined media: Exact chemical composition is known • Complex media: Extracts and digests of yeasts, meat, or plants • Nutrient broth • Nutrient agar

29. Selective, Differential, and Enrichment Media • Selective medium: encourages growth of some organisms but suppresses growth of others (e.g. antibiotics) • Differential medium: contains a constituent that causes an observable change (e.g. MacConkey agar) • Enrichment medium: contains special nutrients that allow growth of a particular organism that might not otherwise be present in sufficient numbers to allow it to be isolated and identified

30. Selective Media • Suppress unwanted microbes and encourage desired microbes • Ex: Sabouraud’s Dextrose Agar: used to isolate fungi, has a pH of 5.6, outgrow most of bacteria Differential Media • Make it easy to distinguish colonies of different microbes. • Ex: Blood Agar: bacteria that can lysed blood cells causing a clear areas around the colonies.

31. Three species of Candida can be differentiated in mixed culture when grown on CHROMagarCandida plates

32. Identification of urinary tract pathogens with differential media (CHROMagar)

33. Enrichment Media • Encourages growth of desired microbe • Assume a soil sample contains a few phenol-degrading bacteria and thousands of other bacteria • Inoculate phenol-containing culture medium with the soil, and incubate • Transfer 1 ml to another flask of the phenol medium, and incubate • Transfer 1 ml to another flask of the phenol medium, and incubate • Only phenol-metabolizing bacteria will be growing

34. Culturing Bacteria • Culturing of bacteria in the laboratory presents two problems: 1. A pure culture of a single species is needed to study an organism’s characteristics 2. A medium must be found that will support growth of the desired organism

35. Obtaining Pure Cultures • Pure culture: a culture that contains only a single species or strain of organism • A colony is a population of cells arising from a single cell or spore or from a group of attached cells • A colony is often called a colony-forming unit (CFU) • The streak plate method is used to isolate pure cultures

36. The Streak Plate Method uses agar plates to prepare pure cultures

37. The Streak Plate Method Figure 6.11

38. Anaerobic Culture Methods • Reducing media • Contain chemicals (thioglycolate or oxyrase) that combine O2 • Heated to drive off O2

39. Anaerobic Jar Figure 6.6

40. To culture obligate anaerobes, all molecular oxygen must be removed and kept out of medium. Agar plates are incubated in sealed jars containing chemical substances that remove oxygen and generate carbon dioxide or water

41. Anaerobic Transfer

42. An Anaerobic Chamber Figure 6.7

43. Capnophiles • Microbes that require high CO2 conditions • CO2 packet • Candle jar

44. Preserved Cultures • To avoid risk of contamination and to reduce mutation rate, stock culture organisms should be kept in a preserved culture, a culture in which organisms are maintained in a dormant state • Lyophilization (freeze-drying): Frozen (–54° to –72°C) and dehydrated in a vacuum • Deep Freezing: –50° to –95°C • Refrigeration • Reference culture (type culture): a preserved culture that maintains the organisms with characteristics as originally defined

45. CHAPTER 6:MICROBIAL GROWTH

46. Growth and Cell Division • Microbial growth is defined as the increase in the number of cells, which occurs by cell division • Binary fission (equal cell division): A cell duplicates its components and divides into two cells • Septum: A partition that grows between two daughter cells and they separate at this location • Budding (unequal cell division): A small, new cell develops from surface of exisiting cell and subsequently separates from parent cell

47. Binary Fission

48. Binary Fission

49. Thin section of the bacterium Staphylococcus, undergoing binary fission

50. Budding in Yeast