Lab Exam 2

  1. What is media?
    The nutrient preparations used to grow (culture) microorganisms in lab
  2. Broth Cultures
    Used to obtain actively growing populations of bacteria to carry out biochemical tests or observe movement etc.
  3. Solid Medium
    Such as nutrient agar is used to observe colony appearance, to isolate pure cultures , and to carry out certain biochemical tests. May be prepared in a petri plate or in a tube.
  4. Complex Media
    Used for routine lab work as it supports a wide variety of heterotrophic bacteria. This includes nutrient broth and nutrient agar. Contain yeast extract, beef extract or a similar complex material containing proteins, complex carbohydrates and fats.
  5. Chemically defined media
    contain known amounts of specific chemicals. They may be used to grow organisms with special requirements or to exclude certain organisms. Must contain a source of energy, and also sources of carbon, nitrogen, sulfur, phosphorus and any other growth factor that an organism cannot manufacture
  6. Selective Media
    Media designed to supress the growth of unwanted bacteria or other microorganisms while favoring the growth of the target bacterium
  7. Differential media
    Allow you to distinguish visually between 2 bacterial bacterial species growing on the same plate
  8. Enriched Media
    Provide added nutrients to favor growth of terget bacteria. Ex: Blood agar and chocolate agar
  9. Aseptic technique
    methods to help prevent microorganisms from the environment contaminating our experiments, or our experimental bacteria contaminating the environment.
  10. Sterilization
    The complete removal or destruction of all forms of microbial life-is most achieved by physical methods such as autoclaving, falming, filtration or radiation. The method of choice will depend on the substance to be sterilized.
  11. Autoclaving
    Most effective method for sterilizing glassware and media in lab. These items are exposed to steam at high temperature and pressure in a sealed chamber. The heat denatures proteins. All vegetative cells plus bacterial endospores and viruses will be killed in about 15 minutes.
  12. Incineration
    uses dry heat to destroy contaminated material. Also how flame sterilization works.
  13. Filtration
    used to sterilize media that are inactivated by high heat, such as many antibiotic solutions. These media can instead be passed through a filter or screen with pores small enough to exclude microorganisms.
  14. UV Irradiation
    Lethal to many microorganisms because it damages their DNA. A UV lamp can be used to sterilize surfaces, or the air in a room, but it does not penetrate glass or travel far through liquid in a container.
  15. Chemical Sterilization
    Ethylene oxide gas is a chemical method of sterilizing heat-sensitive items such as plastic Petri plates and other plastic equipment. Denatures the proteins of microorganisms, killing both vegetative cells and endospores in 4-18 hours
  16. Disinfectants
    Reduce microbial numbers on surfaces. These DO NOT complete chemcical sterilization. Some examples are vesphene, hand soap or alcohol gel.
  17. Nutrient agar is a complex medium, what does that mean?
    It contains proteins, complex carbohydrates and fats
  18. Give the recipe for nutrient agar?
    200 ml of ultra pure water and 4.6 grams of nutrient agar in powder form
  19. When is nutrient agar used?
    It is used to observe colony appearance, to isolate pure cultures and to carry out certain biochemical tests
  20. List the basic growth requirements for a bacterium and the ingredients in a chemically defined medium that contain each of these requirements
    Must contain a source of energy and also sources of carbon, nitrogen, sulfur, phosphorus and any other growth factor that an organism cannot manufacture for itself.
  21. Why was the nutrient agar autoclaved before use?
    So it doesn't solidify
  22. How does an autoclave achieve sterilization?
    The heat denatures proteins and all vegetative cells plus bcterial endospores and virsues wll be killed.
  23. What is the purpose of a streak plate?
    To observe colony morphology and to isolate single colonies
  24. Colony morphology
    The shape, color, size and general appearance of the bacterial colonies
  25. Basic Categories of Colony Morphology
    Shape, Margin, Elevation, texture, color, optical properties and size
  26. Blood agar
    • Contains animal blood
    • Enriched medium and differential medium (distinguishes between bacteria on the basis of their ability to break down RBCs (hemolysis))
    • Used to culture pathogenic organisms from clinical samples,
  27. Alpha hemolysis
    • partial breakdown of RBCs
    • Results in a production of a greenish discoloration (the pigment biliverdin)
  28. Beta hemolysis
    • complete breakdown of RBCs.
    • Results in a zone of clearing around bacterial colonies
  29. Gamma hemolysis
    no breakdown of RBCs
  30. Chocolate agar
    • Enriched medium
    • Used to culture certain bacteria that require the nutrients in blood in order to grow but cannot lyse RBCs. The blood cells are lysed by heating during the preparation of this medium and the resulting agar is chocolate colored.
  31. Macconkey Agar
    • Selective for Gram negative bacteria.
    • Contains bile salts and crystal violet dye both of which inhibit the growth of Gram positive bacteria.
    • DIfferential as well cuz it distinguishes lactose fermenting bacteria from non lfb. Bacteria that can ferment lactose in the medium produce acid that changes the color of the colonies from whitish to pink. Bacteria that cannot ferment lactose remain white.
  32. Indigenous microflora
    Microorganisms that are permanent residents of the human body, but do not produce disease under normal circumstances 10-100x for abundant than our human cells.
  33. Kirby-Bauer/Disk Diffusion Method
    Used to determine whether a bacterium is resistant or susceptible to a particular drug and helps doctors to make decisions about which drugs to use in treating a patient's infection.
  34. Zone of inhibition
    The area where bacteria were unable to grow.
  35. Catalase Test
    During aerobic respiration toxic oxygen byproducts are formed, including hydrogen peroxide. Aerobic, and some facultative anaerobic bacteria synthesize the enzyme catalase, which breaks down hydrogen persoxide to harmless water and oxygen, but obligate anaerobic bacteria and some facultative anaerobes do not possess this enzyme.
  36. Mannitol Salt Agar
    • Used to isolate and differentiate Staphylcoccus aureus from other bacteria.
    • MSA contains carbohydrate mannitol, plus 7.5% NaCl and the pH indicator phenol red. Phenol red is yellow below pH 6.8, red at pH 7.4 to 8.4 and pink above 8.4.
    • Sa ferments mannitol and produces acid, which turns the pH indicator yellow. Non-pathogenic staphylcocci produce no color change.
  37. What does it mean if the bacterium changes the color of the MSA?
    It ferments it which produces an acidic solution
  38. What causes the color to change with MSA?
    If it is greater than 6.8, it stays pink. If it is lower tjam 6.8, it ferments and turns yellow.
  39. TSI Test
    • Used to distinguish between enteric bacteria. In this test the bacteria are grown on Triple SUgar Iron agar.
    • Has a peptone base and contains the pH indicator phenol red, which is yellow below 6.8.
    • Also contains glucose (0.1%) sucrose and lactose (1%) and FeSO4
  40. TSI Test...If the bacteria can only ferment glucose
    No carbohydrate fermentation means no acid produced so the pH of the agar is not reduced. Aerobic respiration of peptone in the slant produces alkaline products. (No fermentation of sugars-pink slant/red butt)
  41. TSI Test...If the bacteria can only ferment glucose.
    If the bacteria can only ferment glucose, but not the other sugars, the acid produced by fermentation will turn the agar yellow at first. However the bacteria will run out of glucose after about 10 hours and will then catabolize peptone aerobically in the slant. Only glucose fermente-pink slant/yellow butt)
  42. TSI Test...If the bacteria can ferment glucose, and lactose or sucrose
    If the bacteria can ferment lactose or sucrose in addition to glucose, they will continue to produce acid, and as these sugars are present in higher concentrations in the medium, the whole slant will turn yellow. (Yellow slant/yellow butt)
  43. WHat is the TSI test used for?
    To test for glucose, lactose, sucrose and Iron and how it is fermented.
  44. List the ingredients in the agar and explain their relevance to the test
    Peptone base, pH indicator phenol red, glucose (0.1%), sucrose and lactose (1% each) and FeSO4
  45. Explain why there is a lower concentration of glucose in the agar than lactose and sucrose
    Just in case the bacteria can only ferment glucose. Once it is used up, the break down of peptone (proteins) occur
  46. H2S is produced from the breakdown of which sulfur containing amino acid?
  47. What does Coliform mean?
    It means having e coli like characteristics (Gram negative, non spore forming, rod shaped and able to ferment lactose with formation of acid and gas.
  48. Presumptive Test (Coliform Analysis)
    The first part of the coliform analysis. Water samples are inoculated into tubes of lactose broth. The lactose is the sole carbon and energy source in the organic acids and CO2 gas. The gas collects in a small inverted tube, called a Durham tube. Presence of gas in the Durham tube after 48 hours of incubation represents a positive presumptive test.
  49. Confirmed Test (Coliform Analysis)
    If the P Test is a positive 2nd test, the C test is carried out. Other bacteria can also ferment lactose and produce gas, including bacillus and Clostridium species, and these must be ruled out.
  50. Brilliant Green Lactose Bile Broth
    Bacteria from the P test are transfered to a tube of BGLBB. This broth is selective for Gram negative bacteria and again gas production is monitored using a Durham tube. After incubation, gas in the Durham tube provides a positive confirmed test indicating the presence of a Gram negative, lactose-fermenting gas former. Now part 3 of the test, called the Complete test is carried out
  51. Completed Test (Coliform Analysis)
    The bacteria growing in the BGLBB are streaked onto EMB agar plates. The 2 dyes in the agar continue to supress growth of gram positive bacteria and the color of the colonies helps to identify bacterium present. E coli colonies on EMB plates are blueish-black with dark centers when viewed by transmitted light. When viewed by reflected light, they have a distinctive green metalic sheen. These colonies represent a positive completed test.
  52. What is the purpose of the lactose broth (coliform analysis)
    The lactose is the sole carbon and energy source in the broth so only lactose fermenting organisms can grow.
  53. What is the purpose of the Durham tube (coliform analysis)
    It provides a positive confirmed test indicating the presence of gram negative, lactose fermenting gas.
  54. What is the purpose of brilliant green lactose bile broth?
    It is selective for gram negative bacteria
  55. Explain what a positive confirmed test looks like, and what it tells you about the bacteria present in the original water sample?
    It should have a greenish metallic coloring and it proves that there is fecal matter in the water
  56. What is the indicator organism for fecal contamination of water and why is this organism used?
    E Coli is used as a bacterial indicator because it is found in large numbers in human feces
  57. Does a positive presumptive test indicate definitive evidence of fecal contamination of a water sample?
    No it just concludes that gas is formed.
  58. Why did we use BGLBB for the confirmed test
    It indicates the presence of a gram negative, lactose fermenting gas former
  59. What type of medium is EMB agar? WHat is the purpose of each of the infredients listed below?
    Is is both selective and differential. It is selective for gram negative bacteria and gas production is monitored by the Durham tube. After incubation, the Durham tube provides a positive confirmed test indicating the presence of gram negative lactose fermenting gas former.
Card Set
Lab Exam 2
micro lab exam 2