4. Methods For Identification Of Bacteria

Question 1

What are classical methods of observation based on?

Answer 1

Direct observation. Morphology and other cell properties are considered

Question 2

What kind of microscopy do we have

Answer 2

Light microscope- gross morphology
Electron microscope-fine morphology

Question 3

What’s light(optical) microscopy based on?

Answer 3

Transmission of light through the microscope and its interaction with observed sample

Question 4

What’s easier method than light(optical) microscopy?

Answer 4

Bright field microscopy- light is transmitted through the sample. Difference in refraction index can be used to highlight structures

Question 5

What’s problem with light microscopy?

Answer 5

Microorganisms are (usually) colorless, and their cytoplasm has a refraction index similiar to water

Question 6

What lenses provide magnification in the light microscope

Answer 6

Objective (closer to the sample)
Ocular (closer to the observer)
Total magnification is given by the product of each lens’ magnification

Question 7

Give example of a variant of light microscope

Answer 7

Inverted microscope

Question 8

What are 3 objectives regularly used in light microscopy

Answer 8

-Low magnification(×10)=whole sample observation
-High magnification(×40)=observation of “great dimensions” microorganisms
-Oil immersion(×100): for bacteria and yeasts observation (and for cells’ and bigger organisms’ particulars). Oil can reduce light dispersion which results in better observation

Question 9

How much can oculars magnify the image?

Answer 9

×10 to ×15

Question 10

What does resolution power depend on in light microscopy

Answer 10

1) light’s wavelength
2) incidence angle of light into objectives

Question 11

What’s solution to a problem of colorless microorganisms

Answer 11

We use strategies to enhance contrast, like:
Dark field microscopy
Phase contrast microscopy
Fluorescence microscopy
Bright field microscopy+stainings

Question 12

What’s dark field microscopy?

Answer 12

By mean of a filter, condensor only allows diffracted light (not direct light) to reach objective lens
Cells will be seen as enlightened on a dark background
Objectives and oculars are the same as the ones used in bright field microscopy

Question 13

What’s phase contrast microscopy?

Answer 13

By mean of two filters, condenser and objectives allow to increase contrast between the cell components with different density (to examine inner details)

Question 14

What’s fluorescence microscopy

Answer 14

As a light source it uses ultraviolet radiation and works on specific wavelength radiation (LED,laser…)

Question 15

Are bacteria fluorescent?

Answer 15

Not usually. Fluorescence is obtained through labelling with fluorescent molecule (fluorophore) which can be fluorescent dye, labelled probe( ex: conjugated antibody) or fluorescent (or conjugated) protein

Question 16

What’s some variant of fluorescence microscopy

Answer 16

Confocal microscopy (also called scanning microscopy)
A spatial pinhole excludes out of focus fluorescence
Focus is serially changes as to obtain 3D tomography of the sample
Resolution and contrast are enhanced

Question 17

What’s electron microscopy and what type of microscope does it use

Answer 17

Instead of visible light and lenses uses electrons and magnets
Types of microscope:
-TEM(transmission electron m.)=e- pass through sample
-SEM( scanning e- m.)-e- hit sample’s surface with definite incidence angle

Question 18

What’s some good sides of electron microscopy?

Answer 18

-enhances resolving power
-obtains higher magnificatuin
-shows ultrastructure
- can be used for observation of viruses

Question 19

What’s biological staining?

Answer 19

Technique used to enhance contrast in bright field microscopy
It’s fixation is preservation of biological materials from decay

Question 20

What does preparation of specimen for staining include?

Answer 20

1) laying a drop of sample on microscope slide
2) evaporation of water by moderate heating until complete drying
3) fixation by heat or methanol (or other fixatives)

Question 21

What kind of (charged)stain we have

Answer 21

-cationic(+ charged)/ alkalyn stains: methylene blue, crystal violet, safranin: binding to negativelly charged structures( cell surface, proteins, nucleic acids)
-Anionic(- charged)/acidic stains: eosin, acidic fuchsin: binding to positively charged structures (cytoplasm)

Question 22

What kind of staining techniques do we have?

Answer 22

-Simple stainings= aqueus or alcoholic solutions of one simple dye. Used only to detect the presence of bacteria, their shape (methylene blue, cristal violet, safranin) and their spatial organization
-Differential stainings= two different dyes are used in different affinity (Gram staining, Ziehl-Neelson staining). The effect can be improved with mordants( chemical) and enhancers(physical)
-Special stainings= negative s.(highlighting bacteria capsule with India ink);spore s.(Alexander’s stain; with malachite green) and flagelli s.(uses mordants to thicken flagelli)

Question 23

What’s gram staining and what kind of dyes does it use

Answer 23

Gram staining is technique based on the differential characteristics of bacterial cell wall
It uses two stains(dyes)
1)Primary stain: Cristal Violet(purple-blue)
2) Secondary stain(counterstain) :Safranin or fuchsine(pink-red)
Lugol’s iodine is used as moderant
Ethanol is used for washing

Question 24

What’s a result of gram staining

Answer 24

1)Bacteria with thicker cell wall will remain cristal violet, looking blue when observed-Gram positive bacteria
2)Bacteria with thinner cell wall will look as pink, since cristal violet will not be retained and will be washed away- Gram negative bacteria
But:
-there are bacteria with variable or undetermined reactivity
-this method isn’t usefull for classification of archaea