7. Culturing Microorganism
Pure Culture
- one consisting of a single type microorganism derived from a single cell
- exist rarely in nature
- Mixed culture : many different microorganisms living together
Obtaining a pure culture
1. Sterilization
2. Isolation and cultivation of single microbial cell (production of a clone)

8. The 5 I’s of culturing microbes
Inoculation – introduction of a sample into a container of media
Incubation – under conditions that allow growth
Isolation –separating one species from another
Inspection
Identification

9. Media – providing nutrients in the laboratory
Most commonly used:
nutrient broth – liquid medium containing beef extract & peptone
nutrient agar – solid media containing beef extract, peptone & agar
agar is a complex polysaccharide isolated from red algae
solid at room temp, liquefies at boiling (100oC), does not resolidify until it cools to 42oC
provides framework to hold moisture & nutrients
not digestible for most microbes

10. Growing a Pure Culture Types of culture media
1. Defined media – prepared from pure chemicals, its exact chemical composition is known
– has just enough ingredients to support growth is called a minimal medium
2. Complex media – made from extracts of natural materials
(beef, blood, casein, yeast, and soybeans)
– precise chemical composition is not known
– broth : liquid complex medium
cf) casein – common component of complex medium
– hydrolyze with enzymes or acid to make it more soluble
particially hydrolysis : protein peptide (peptone)
complete hydrolysis : protein aminoacid (casein hydrolysate)

11. change of neutral red colors)
3. Selective media – favor the growth of particular microorganisms
– used to isolate or detect the favored species in a complex mixture of other
microorganisms
– some media contain toxic chemicals
(sodium azide, potassium tellurite, or crystall violet)
– employ an extream pH value, unusual carbon source
4. Differential media – used to identify microbes by the appearance of their colonies
– In blood agar : Streptococcus pyogenes
 surrounded by a clear zone (lysis by red blood cells)
5. Selective-Differential media
MacConkey agar – used to detect strains of Salmonella and Shigella
– selective : crystal violet and bile salts (Killing of other microbes)
– differential : Caliform bacteria (lactose  acid 
change of neutral red colors)
6. Enrichment culture – used to isolate a particular microbes or type of
micobes from a large, complex natural population
ex) endospore-forming bacteria (boiling)
nitrogen-fixing bacteria (nitrogen-free medium)

12. Table 3.4 Ingredients of a Minimal Medium Suitable for Cultivating Escherichia coli
Table 3.5 Ingredients of a Minimal Medium Suitable for Cultivating Leuconostoc citrovorum

13. Table 3.6 Ingredients of Nutrient Broth, a Complex Medium Suitable for Cultivating Many Species of Bacteria

14. Types of media
synthetic – contains pure organic & inorganic compounds in an exact chemical formula
complex or nonsynthetic – contains at least one ingredient that is not chemically definable
general purpose media- grows a broad range of microbes, usually nonsynthetic
enriched media- contains complex organic substances such as blood, serum, hemoglobin or special growth factors required by fastidious microbes

15. Enriched media

16. selective media- contains one or more agents that inhibit growth of some microbes and encourage growth of the desired microbes
differential media – allows growth of several types of microbes and displays visible differences among desired and undesired microbes

17. selective & differential media

21. Differential media

25. Miscellaneous media
reducing medium – contains a substance that absorbs oxygen or slows penetration of oxygen into medium; used for growing anaerobic bacteria

26. The Requirements for Growth: Physical Requirements
Figure 6.4

27. The Requirements for Growth: Chemical Requirements
Carbon
Structural organic molecules, energy source
Chemoheterotrophs use organic carbon sources
Autotrophs use CO2

28. The Requirements for Growth: 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
Sulfur
In amino acids, thiamine and biotin
Some bacteria use SO42– or H2S
Phosphorus
In DNA, RNA, ATP, and membranes
PO43– is a source of phosphorus

29. The Requirements for Growth: Chemical Requirements
Trace elements
Inorganic elements required in small amounts
Usually as enzyme cofactors

30. The Requirements for Growth: Chemical Requirements
Organic growth factors
Organic compounds obtained from the environment
Vitamins, amino acids, purines, and pyrimidines

31. 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

32. 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 ~40°C

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

34. Anaerobic Culture Methods
Reducing media
Contain chemicals (thioglycollate or oxyrase) that combine O2
Heated to drive off O2

35. Anaerobic Culture Methods
Anaerobic jar
Figure 6.5

36. Anaerobic Culture Methods
Anaerobic chamber
Figure 6.6

37. Capnophiles Require High CO2
Candle jar
CO2-packet
Figure 6.7

38. Selective Media
Suppress unwanted microbes and encourage desired microbes.
Figure 6.9b–c

39. Selective Media
Contains one or more agents that inhibit the growth of a certain microbe and thereby encourages, or selects, a specific microbe.
Example: Mannitol Salt Agar encourages the growth of S. aureus.

42. Differential Media
Make it easy to distinguish colonies of different microbes.
Figure 6.9a

43. Differential Media
Differential shows up as visible changes or variations in colony size or color, in media color changes, or in the formation of gas bubbles and precipitates.
Example: Spirit Blue Agar to detect the digestion of fats by lipase enzyme. Positive digestion (hydrolysis) is indicated by the dark blue color that develops in the colonies.

45. Growth of Staphylococcus aureus on Manitol Salt Agar results in a color change in the media from pink to yellow.

48. Enrichment Media
Is used to encourage the growth of a particular microorganism in a mixed culture.
contains complex organic substances such as blood, serum, hemoglobin or special growth factors required by fastidious microbes
Ex. Manitol Salt Agar for S. aureus

49. Enriched media

50. Preserving Bacteria Cultures
Deep-freezing: –50°to –95°C
Lyophilization (freeze-drying): Frozen (–54° to –72°C) and dehydrated in a vacuum

51. Culture media Plate Broth Slant Deep

52. Some vocabulary for colonies morphology
Shape: round, irregular, punctiform (tiny dot)
Elevation: convex, umbonate, flat, raised
Color: translucent, shiny, dull, white
Texture: moist, mucoid, dry (or rough)

53. Colonial morphology Margin- edge

54. FYI

55. Bacteria motility
Non motile bacteria will only be found at the site of inoculation
Motile bacteria  swim around go everywhere

56. Objective 3:c How to inoculate a slant
Provide a solid growth surface in a tube format (take less space)
Inoculate as you did for the petri plate
One streak in the middle of the surface do not dig/ nor stab. Only on the surface.

57. Slant observation

58. Broth observation

59. Uses Broth Slant Plate Deep High concentration of bacteria
Space saving solid culture
Plate
Individual colonies
Can be used to count bacteria
Deep
Look at motility & oxygen requirement