History of microbiology/Microscopy

Question 1

How has Robert Hooke contributed to microbiology?

Answer 1

R.H. was the first microscopist that was able to have microscopic observation (mold) which was described in “Microphia”

Question 2

How has Antoni van Leewenhoek contributed to microbiology?

Answer 2

A.v.L. was the first person to observe microbial life (bacteria) with a microscope; he used a very simple light microscope.

Question 3

What is the structure of a light microscope? What variable affect its magnification?

Answer 3

STRUCTURE
- three lenses: (1) condenser lens (the one that focus all the light to the specimen) and (2) objective lens (the interchangeable one) and (3) ocular lens (fixed in the microscope). The total magnification of the microscope is given by the product of the (2) magnification and (3) magnification

VARIABLES

• magnification: capacity to enlarge an image
• resolution: capacity to distinguish two adjacent objects as distinct and separate. The smaller it is, the better and more microbes can be seen
• numerical aperture: light-gathering ability of objective lens

To increase magnification, oil-immersion is used, since the oil’s reflective index is very similar to glass’s, causing light to bend less

Question 4

What types of LM there are?

Answer 4

1. Bright field LM (BFLM)
2. Phase-contrast LM (PCLM)
3. Differential interference contrast (DIC)
4. Dark-field LM (DFLM)
5. Fluorescent LM (FLM)

Question 5

How can contrast can be increased?

Answer 5

1. Simple staining in BFLM: stains are added to dried samples to colour specific cellular material (basic stains stain DNA since it is negatively charged)
2. Differential staining in BFLM: stains makes different kinds of cells of different colors. Bacteria are divide in gram-positive (thicker cell wall and single cell membrane, purple color) and gram-negative (thinner cell wall and multiple layers, pink color)
3. Phase-contrast microscopy: this technique enhance contrast based on the different refractive indexes each cell material has
4. Dark-field microscopy: this technique enhance contract by scattering light and enlightening the sample from the sides
5. Fluorescent microscopy: this technique allow seeing specimens that emit light (either autofluorescent or stained)

Question 6

What are the different staining process?

Answer 6

1. preparation of smear: spread of the sample over a thin glass layer and air dried
2. heat fixing and staining: the glass is passed through the flame to heat fix it and then the stain is added and rinsed
3. microscopy: the glass is place on the stage and oil is put to allow high magnification

Question 7

How can sample be seen in three dimesion?

Answer 7

1. Differential interference contrast: use of two beam of polarised light
2. Confocal scanning light: computer controlled microscope that couples a laser to a fluorescent microscope