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

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Presentation on theme: "Laboratory Tools in Microbiology Sofronio Agustin Professor Sofronio Agustin Professor LECTURES IN MICROBIOLOGY LECTURES IN MICROBIOLOGY LESSON 3."— Presentation transcript:

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

3 Topics Covered  Microscopy  Staining Techniques  Methods of Culturing Microbes

4 Units of Measurement  1 µm = m = mm  1 nm = m = mm  1000 nm = 1 µm  µm = 1 nm

5 Simple Microscope  A simple microscope has only one lens

6 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

7 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.

8 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.

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

10 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.

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

12 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.

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

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

15 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

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

17 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

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

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

20 Summary of Microscope Types

21 Optical and Electron Microscopy: A Comparison

22 TEM and SEM Compared Scanning Electron Microscope Transmission Electron Microscope

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

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

25 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.

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

27 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.

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

29 Bacterial Stain Types

30 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.

31 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.

32 Differential Stain: Gram Stain Color of Gram + cells Color of Gram – cells Primary stain: Crystal violet Purple Mordant: Iodine Purple Decolorizing agent: Alcohol-acetone PurpleColorless Counterstain: Safranin PurpleRed

33 Differential Stain: Gram Stain

34 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.

35 Culture of Microbes  Five basic techniques  Media  Microbial growth

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

37 Summary of Laboratory Techniques

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

39 Isolation Methods

40 Culture Media  Classified according to three properties  Physical state  Chemical composition  Functional types

41 Culture Media: Physical State  Liquid media  Semi-solid media  Solid media

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

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

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

45 Culture Media: Chemical Composition  Synthetic or chemically-defined media  Nonsynthetic or complex media

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

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

48 Culture Media: Functional Types  Enriched media  Selective media  Differential media

49 Enriched Media  Enriched media are used to grow fastidious bacteria.

50 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).

51 Differential Media

52 Selective Media  Mannitol Salt Agar  MacConkey Agar

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

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


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