1. Laboratory Tools in Microbiology
LECTURES IN
MICROBIOLOGY
Laboratory Tools in Microbiology
LESSON 3
Sofronio Agustin
Professor

2. Topics Covered Microscopy Staining Techniques
Methods of Culturing Microbes

3. Units of Measurement 1 µm = 10-6 m = 10-3 mm 1 nm = 10-9 m = 10-6 mm

4. Simple Microscope
A simple microscope has only one lens

5. Parts of a student laboratory microscope
Compound Microscope
A compound microscope has two sets of lenses and is typically used in teaching and research laboratories.
Parts of a student laboratory microscope

6. Optics : Magnification
A specimen is magnified as light passes through the objective and ocular lenses.
Total Magnification =
objective lens X ocular lens
(magnifications)
The pathway of light and two stages of magnification of a compound microscope.

7. Resolution distinguishes magnified objects clearly.
Optics: Resolution
Resolution is the ability of the lenses to distinguish two points.
A microscope with a resolving power of 0.2 um can distinguish between two points > 0.2 um.
Resolution distinguishes magnified objects clearly.

8. Optics: Resolution
Resolution can be increased by using immersion oil and shorter wavelengths of light.

9. Optics: Refraction
Refractive index is the light-bending ability of a medium.
The light may bend in air so much that it misses the small high-magnification lens.
Immersion oil is used to keep light from bending.

10. Brightfield Microscopy
Dark objects are visible against light background.
Used to observe stained or unstained specimens.
Most commonly used in laboratories.

11. Darkfield Microscopy
Light objects are visible against a dark background.
Light reflected off the specimen enters the objective lens.
Used to screen for syphilis agent, Treponema pallidum.

12. Phase-contrast Microscopy
Accentuates diffraction of light that passes through the specimen.
Used to observe internal cellular detail of live specimens.

13. Differential Interference Contrast (DIC) Microscopy
Accentuates diffraction of light that passes through the specimen.
Uses two beams of light to produce more pronounced contrast.

14. Fluorescence Microscopy
Cells are stained with fluorescent dyes called fluorochromes.
Uses UV light as energy source.
Fluorochromes absorb UV light and emit visible light
Fluorescent staining of fresh sample of cheek scrapings

15. Confocal Microscopy Uses fluorochromes and laser light.
The laser illuminates each plane in a specimen to produce a 3-D image.

16. Electron Microscopy Very high magnification of up to 100,000 X.
Transmission electron microscope (TEM)
View internal structures of cells
Scanning electron microscope (SEM)
Three-dimensional images

17. Transmission Electron Microscopy
Transmission Electron Micrograph of (a) a virus and (b) a protozoan.

18. Scanning Electron Microscopy
False-color Scanning Electron Micrograph of Paramecium sp.

19. Summary of Microscope Types

20. Optical and Electron Microscopy: A Comparison

21. TEM and SEM Compared Transmission Electron Microscope
Scanning Electron Microscope

22. Scanning Probe Microscopy
Scanning tunneling microscopy uses a metal probe to scan a specimen.
Resolution 1/100 of an atom.

23. Scanning Probe Microscopy
Atomic force microscopy uses a metal and diamond probe inserted into the specimen.
Produces 3-D images.

24. Preparation of Specimens for Light Microscopy
A thin film of a solution of microbes on a slide is a smear.
A smear is usually fixed to attach the microbes to the slide and to kill the microbes.

25. Stains Positive stains Dye binds to the specimen Negative stains
Dye does not bind to the specimen, but rather around the specimen.

26. Positive and Negative Stains
Positive stains are basic dyes (cationic) that bind to negatively charged cells.
Negative stains are acidic dyes (anionic) that bind the background.

27. Positive Stains Simple - One dye Differential
Two-different colored dyes
Ex. Gram stain
Special
Emphasize certain cell parts
Ex. Capsule stain

28. Bacterial Stain Types

29. Simple Stain Use of a single basic dye is called a simple stain.
A mordant may be used to hold the stain or coat the specimen to enlarge it.

30. Differential Stain: Gram Stain
The Gram stain classifies bacteria into gram-positive and gram-negative.
Gram-positive bacteria tend to be killed by penicillin and detergents.
Gram-negative bacteria are more resistant to antibiotics.

31. Differential Stain: Gram Stain
Color of
Gram + cells
Gram – cells
Primary stain:
Crystal violet
Purple
Mordant:
Iodine
Decolorizing agent:
Alcohol-acetone
Colorless
Counterstain:
Safranin
Red

32. Differential Stain: Gram Stain

33. Differential Stain: Acid Fast Stain
Cells that retain a basic stain in the presence of acid-alcohol are called acid-fast.
Non–acid-fast cells lose the basic stain when rinsed with acid-alcohol, and are usually counterstained (with a different color basic stain) to see them.

34. Culture of Microbes
Five basic techniques
Media
Microbial growth

35. Five Basic Techniques 1. Inoculate 2. Incubate 3. Isolation
4. Inspection
5. Identification

36. Summary of Laboratory Techniques

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

38. Isolation Methods

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

40. Culture Media: Physical State
Liquid media
Semi-solid media
Solid media

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

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

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

44. Culture Media: Chemical Composition
Synthetic or chemically-defined media
Nonsynthetic or complex media

45. Synthetic Media
Synthetic media contain pure organic and inorganic compounds that are chemically defined (i.e. known molecular formula).

46. Complex Media
Complex or enriched media contain ingredients that are not chemically defined or pure (i.e. animal extracts).

47. Culture Media: Functional Types
Enriched media
Selective media
Differential media

48. Enriched Media
Enriched media are used to grow fastidious bacteria.

49. Differential and Selective Media
Selective media enables one type of bacteria to grow
While differential media allows bacteria to show different reactions (i.e. colony color).

50. Differential Media

51. Selective Media
Mannitol Salt Agar
MacConkey Agar

52. Miscellaneous Media
Examples of miscellaneous media are reducing, fermentation and transportation media.

53. Microbial Growth Incubation Inspection Identification
Varied temperatures, atmospheric states
Inspection
Mixed culture
Pure culture
Identification
Microscopic appearance
Maintenance and disposal
Stock cultures
sterilization