1. 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

2. Fig. 3.1a

3. Fig. 3.1b

4. Fig. 3.1c

5. Isolation
If an individual bacterial cell is separated from other cells & has space on a nutrient surface, it will grow into a mound of cells- a colony
A colony consists of one species

6. Isolation technique

7. Fig. 3.3

8. Fig. 3.1d

10. 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

11. 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

12. Enriched media
Blood agar
Chocolate agar

13. 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

15. Selective media
Mannitol salt agar
MacConkey agar

16. Differential media
TSIA
CHROMagar Orientation

17. Miscellaneous media
reducing medium – contains a substance that absorbs oxygen or slows penetration of oxygen into medium; used for growing anaerobic bacteria
carbohydrate fermentation medium- contains sugars that can be fermented, converted to acids, and a pH indicator to show the reaction; basis for identifying bacteria and fungi
transport media

18. Carbohydrate fermentation media

19. Fig. 3.12

20. Cultures Pure culture- only grows one type of microbe
Mixed culture- contains 2 or more microbes
Contaninated culture- was once pure, but unwanted microbes became introduced
Axenic- free of other living things except the one being studied

21. Maintenance and disposal of cultures
Catalogues of microbes are maintained at the American Type Culture Collection
Microbes are disposed of by various forms of sterilization

22. Why use animals?
You can test the effects of drugs, etc without risking the lives of humans
To evaluate disease, or study the process and cause of disease
Source of antibodies, antitoxins
May be used to determine the pathogenicity or toxicity of certain bacteria
To grow microbes which cannot be grown on artificial media

23. compound light microscope

24. magnification – ability to enlarge objects
resolving power – ability to show detail
refraction- bending ability of light

25. Pathway of light

26. Total Magnification Ocular power Objective power Total magnification
10x 4x
Scanning
40x
Low
100x
High and dry
400x
Oil immersion
1000x

27. resolution The human eye can only resolve objects about 0.2mm apart
Oil immersion allows resolution to about 0.2um
RP= wavelength of light (nm)/2 x NA
NA is the cone of light entering the lens

28. Effect of wavelength on resolution

29. Oil immersion lens

30. Other constraints for a clear image
Other things needed for a clear image are
Quality of the lens
Quality of the light source
Lack of contrast in the specimen

31. Effect of magnification

32. Types of light microscopes
Bright-field – most widely used, specimen is darker than surrounding field
Dark-field – brightly illuminated specimens surrounded by dark field
Phase-contrast – transforms subtle changes in light waves passing through the specimen into differences in light intensity, best for observing intracellular structures

33. 3 views of a cell
Brightfield 400x
Darkfield 400x
Phase contrast 400x

34. Types of light microscopes
Differential interference contrast- uses light waves and 2 prisms which produce contrasting colors; well defined, colored, 3D images

35. Phase contrast 600x
Fig. 3.21
DIC 160x

36. Fluorescence Microscope
Modified compound microscope with an ultraviolet radiation source and a filter that protects the viewer’s eye
Uses dyes that emit visible light when bombarded with shorter uv rays.
Useful in diagnosing infections

37. Cheek cells and streptococci

38. Light Microscopes
Confocal- uses a laser to scan sections of the specimen

39. Electron microscopy
Forms an image with a beam of electrons that can be made to travel in wavelike patterns when accelerated to high speeds.
Electron waves are 100,000X shorter than the waves of visible light.
Electrons have tremendous power to resolve minute structures because resolving power is a function of wavelength.
Magnification between 5,000X and 1,000,000X

41. 2 types of electron microscopes
Transmission electron microscopes (TEM) – transmits electrons through the specimen; darker areas represent thicker, denser parts and lighter areas indicate more transparent, less dense parts
Scanning electron microscopes (SEM)– provides detailed three-dimensional view. SEM bombards surface of a whole, metal-coated specimen with electrons while scanning back and forth over it.

42. Scanning Electron Micrograph
paramecium

43. Table 3.6

44. Table 3.5

45. Specimen Preparation The way a slide is prepared depends upon
The condition of the specimen
The needs of the examiner
The type of microscope available

46. Specimen preparation
wet mounts & hanging drop mounts – allow examination of characteristics of live cells: motility, shape, & arrangement
Wet mount- a drop of culture is placed on a slide with a cover glass
Quick and easy to prepare
Cover glass may damage larger cells
Slide is susceptible to drying and can contaiminate the handler’s fingers

47. Specimen preparation
Hanging drop- uses a special slide with a depression; sample is placed in the depression and vaseline is placed around it; coverslip overlies the sample

48. Specimen preparation
fixed mounts are made by drying & heating a film of specimen. This smear is stained using dyes to permit visualization of cells or cell parts.
Kills the microbe
Allows adherence to the slide
Preserves cellular components in a natural state with minimal distortion

49. Staining
cationic dyes - basic, with positive charges on the chromophore
anionic dyes - acidic, with negative charges on the chromophore
surfaces of microbes are negatively charged and attract basic dyes – positive staining.
negative staining – microbe repels dye & it stains the background
Simple
Reduced shrinkage or distortion of cells
Can observe capsules

51. Staining simple stains –one dye is used
differential stains – use a primary stain and a counterstain to distinguish cell types or parts. examples: Gram stain, acid-fast stain and endospore stain
special stains: capsule and flagellar stains

53. Table 3.7