1. Methods of Culturing Microorganisms

2. Specimen Collection

3. Five basic techniques 1. Inoculate 2. Incubate 3. Isolation
4. Inspection
5. Identification

4. Inoculation
Culture: the propagation of microorganisms with various media
Medium (pl. media): a nutrient used to grow microorganisms outside their natural habitat
Inoculation: the implantation of microorganisms into or onto culture media 4

5. Incubation
Incubator: media containing inoculants are placed in temperature-controlled chambers
Usual laboratory propagation temperatures fall between 20°C and 40°C
Atmospheric gases such as O2 and CO2 may be required for the growth of certain microbes
During incubation, microbes grow and multiply, producing visible growth in the media 5

6. Isolation
Based on the concept that if an individual cell is separated from other cells on a nutrient surface, it will form a colony
Colony: a macroscopic cluster of cells appearing on a solid medium arising from the multiplication of a single cell
Requires the following
a medium with a firm surface
a Petri dish
inoculating tools
Discussed in more detail later in notes 6

7. Inspection 7

8. Identification 8

9. Isolation: A single visible colony represents a pure culture or single type of bacterium isolated from a mixed culture

10. Isolation: Various Conditions of Cultures
Pure Culture
Mixed Culture
Contaminated Culture
(a)
(b)
(c)
One type of microbe
More than one type of microbe
Unwanted microbe present

11. Question
Can you isolate a colony using liquid culture?

12. Three basic methods of isolating bacteria
Streak Plate
Loop Dilution
Spread Plate

13. Isolation: Streak Plate

14. Isolation: Loop Dilution14

15. Isolation: Spread Plate15

16. Media Classified according to three properties Physical state
Chemical composition
Functional types

17. Physical State 1. Liquid media (I.e. tryptic soy broth=TSB)
2. Semi-solid media
3. Solid media (I.e. tryptic soy agar=TSA)

18. Liquid media are water-based solutions that are generally termed broths, milks and infusions.

19. Semi-solid media contain a low percentage (<1%) of agar, which can be used for motility testing.

20. Solid media contain a high percent (1-5%) of agar, which enables the formation of discrete colonies.

21. Chemical content
Synthetic media
Nonsynthetic or complex media

22. Chemically defined media
media whose compositions are precisely chemically defined
contain pure organic and inorganic compounds that vary little from one source to another
molecular content specified by an exact formula
Minimal media
contain nothing more than a few essential compounds such as salts and amino acids
some contain a variety of defined organic and inorganic chemicals

23. Complex media contain ingredients that are not chemically defined or pure (i.e. animal extracts).
TSA + 5% sheep blood
(Do we know the exact percentages of chemicals in the sheep blood?)

24. Functional types of growth media
Selective media
Differential media

25. Comparison of Selective and Differential Media
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
Mixed
sample
Mixed
sample
General-purpose
nonselective medium
(All species grow.)
General-purpose
nondifferential medium
(All species have a similar
appearance.)
Selective medium
(One species grows.)
Differential medium
(All 3 species grow but may
show different reactions.)
(a)
(b)
Selective media enables (selects) one type of bacteria to grow, while differential media allows bacteria to show different reactions (i.e. colony color)

26. Selective media enables (selects) one type of bacteria to grow, while differential media allows bacteria to show different reactions (i.e. colony color)

27. Differential media we will use in class
Mannitol salt agar (MSA) contains 7.5% salt which selects for gram positive and also contains mannitol (carbohydrate) plus a pH indicator to indicate when mannitol has been fermented to an acid…media turns from pink to yellow

28. Differential media we will use in class
MacConkey agar contains bile salts and dyes that inhibit gram-positive bacteria and hence selects for gram negative. Also turns dark color when lactose is fermented 28 28

29. Maintenance and Disposal of Cultures
Cultures and specimens constitute a potential hazard
Prompt disposal is required
Stock cultures represent a “living catalog” for study and experimentation
The American Type Culture Collection (ATCC) in Manassas, VA is the largest culture collection in the U.S.

30. Microscopy

31. Science relies on technology
The progress of microbiology and many other disciplines relies heavily on the introduction of new technologies
Technologies such as new types of media or microscopes lead to advances in microbiology
Without a microscope it would be impossible to characterize the morphology of bacteria.

32. History of the Microscope
Development of the microscope originated in 1676 by Anton van Leeuwenhoek
Luckily, he was a skilled observer and artist and produced many fine illustrations of microbes (“animalcules”)
Unfortunately, he was not willing to share his microscopes or knowledge with other scientists until much later in life to help the progress of microbiology

33. We measure specimens based on the metric system
We measure specimens based on the metric system. We will be looking at bacteria ranging from 1 um to 10 um.

34. Principles of Light Microscopy
Magnification
objective lens: closest to the specimen, forms the initial image called the real image
ocular lens: forms the second image called the virtual image that will be received by the eye and converted to the retinal and visual image
Total magnification
Power of Objective
Usual power of ocular
Total magnification
4x scanning objective
10x
40x
10x low power objective
100x
40x high dry objective
400x
100x oil immersion objective
1000x

35. Magnification is useless without good resolution.
Resolution (resolving power)
the capacity of an optical system to distinguish or separate two adjacent points or objects from one another
the human eye can resolve two objects that are no closer than 0.2 mm apart
The shorter the wavelength, the better the resolution (the ability to see 2 separate units)

36. The Effect of Wavelength on Resolution
(b)
Low resolution
High resolution
High resolution distinguishes magnified objects clearly

37. Wavelength and Resolution
Is (a) or (b) resolved?
Which resolves images better, gamma rays or visible light?

38. Properties of Light
A, B, & C D
Light has the ability to a) reflect, b) transmit (pass through), c) absorb, d) refract and

39. Resolution can be increased by using immersion oil.

40. A compound microscope is typically used in teaching and research laboratories

41. One objective lens can hold up to a dozen small lenses inside

42. Optical microscopes All have a maximum magnification of 1000x--2000X
Bright-field
Dark-field
Phase-contrast
Differential interference
Fluorescent
Confocal

43. Types of Optical Microscopes
Bright-field
Most commonly used in laboratories
Observe live or preserved stained specimens

44. Types of Optical Microscopes
Dark-field
Observe live unstained specimens
View an outline of the specimens 44

45. Types of Optical Microscopes
Phase-contrast
Observe live specimens
View internal cellular detail 45

46. Fluorescent Microscopy
Fluorescence stain or dye
UV radiation causes emission of visible light from dye
Diagnostic tool

47. Fluorescent staining on a fresh sample of cheek scrapings from the oral cavity. Fuzzy cells are cheek cells, distinct rods and cocci are bacteria. Red=dead, green=live

48. Example of a confocal microscope
Fluorescence or unstained specimen images are combined to form a three-dimensional image.

49. Electron microscopy Very high magnification (100,000-1,000,000X)
Transmission electron microscope (TEM)
View internal structures of cells
Scanning electron microscope (SEM)
Three-dimensional images

50. Transmission Electron Microscopy (TEM)

51. Example of False-color Scanning Electron Microscopy (SEM)

53. Comparison of optical and electron microscopes

54. What does an electron microscope use to visualize specimens
What does an electron microscope use to visualize specimens? Which type of microscope has better resolving power, bright-field or electron?

55. In lab, we use Bright-Field Light Microscopy
We will perform the following light microscopy techniques:
Wet mounts
Wet mount and hanging drop methods
Smears
Spread, air-dry, heat fix
Simple Staining
Cheek cells with methylene blue (BI231)
Differential Staining
Gram stain
Acid-fast (time permitting)
Special Staining
Capsule stain
Flagellar stain (prepared slides since this is a difficult technique)
Endospore stain (time permitting)

56. Wet mounts
We will use the hanging drop technique to investigate bacterial motility:
Advantages of the hanging drop:
Easy to prepare
Bacteria are live so we can see motility
Disadvantages of the hanging drop:
Requires special depression slide
Difficult to visualize since microbes are not stained
Bacteria are live and therefore slides must be disinfected

57. Smears 1. Smear bacteria onto slide from broth or agar 2. Air dry
Performing quality smears takes practice. There are many steps in the preparation of a smear that must be performed correctly. (Think of Goldilocks that wants her porridge “just right”)
1. Smear bacteria onto slide from broth or agar
Too thick and you can’t see through specimen, too thin and you can’t find any bacteria
2. Air dry
Again, you do not want too large a smear or it will take a long time for the smear to completely air dry.
3. Heat Fix (see next slide)

58. Smears (cont.) 3. Heat Fix Pass slide through flame quickly 3-4 times
Heat fix too little and organisms may wash off slide
Heat fix too much and organisms may be distorted
Heat fixation:
1. kills organisms
2. adheres specimen to slide
3. promotes stainability of specimen

59. Stains Positive stains Negative stains Dye binds to the specimen
Dye does not bind to the specimen, but rather around the specimen.
Which type of stain do we usually use?
Why do positive stains bind to the specimen?
What makes a specimen negatively charged?

60. Positive stains are basic dyes (positive charge) that bind negative charge cells, and negative stains are acidic dyes (negative charge) that bind the background.
Molecular structure of methylene blue
(a positive stain attracted to ?)

61. Positive stains Simple stain
One dye
Ex. Methylene blue, crystal violet
In BI231, you applied one stain (methylene blue) to visualize your cheek (buccal) cells.

62. Positive stains Differential stain
Two-different colored dyes
Ex. Gram stain
Allow us to differentiate between types of bacteria.
Gram Stain
Gram-positive vs. Gram-negative
See next slide
Acid-Fast Stain
Used to identify acid-fast bacteria with a waxy, lipid cell wall.
Detects Mycobacteria like M. tuberculosis and M. leprosae
Most of the bacteria in our lab in Acid-Fast negative 62

63. Positive Stains: Gram Stain
Most frequently used stain
Dictates which type of antibiotic treatment
Gram-Positive
Purple
Penicillin
Thick Peptidoglycan
4 P’s (Positive, Purple, Penicillin, Peptidoglycan)
1 plasma membrane
Gram-Negative
Red
Streptomycin
Thin peptidoglycan
2 plasma membranes

64. Positive stains Special stain Emphasize certain cell parts
Ex. Capsule stain 64

65. Special Stains Capsule Stain Flagellar Stain Endospore Stain
We use these special stains to identify special structures that some bacteria possess
Capsule Stain
Capsule does not stain so we stain the background
Can not heat-fix or capsule will be destroyed
Flagellar Stain
Very difficult stain, we will look at prepared slides
Endospore Stain
The endospore is difficult to stain since it is made of a thick, hard to penetrate spore wall. We will boil the stain to help it penetrate the spore coat.