Tools Used in Microbiology

Outline:1. Difference between Light microscopy and electron microscopy

2. Decribe methods for the isolation of microorganisms in pure culture

3. Techniques for studying live bacteria

4. Distinguish between a simple stain and a differential stain and give examples

5. Identify steps in the Gram stain procedure

6. List the major categories of microbial characteristicsused to identify microorganisms. Explain why some of these give more specific info for identification than othrs

Basic techniques that the student of microbiology must learn to use in the laboratory.These techniques are those used in:

a. Growing bacteriab. Isolating them in pure culturec. Observing themd. and finally identifying them

The Microscope

A. Light Microscope- To magnify an object, it uses a system of lenses to manipulate the path a light beam travels between the object being studied and the eye- Produce a maximum useful magnification of about1000 times the original size.- Has three lenses:

1. the condenser(focuses light on the specimen 2. the objective(s)

a. Low-power objectiveb. High-power objectivec. Oil-immersion objective

3. the eyepiece (ocular)

Objective Designation

Objective Magnification

Eyepiece Magnification

Total Magnification

Low Power 10 10 100

High Power (high dry)

40 10 400

Oil Immersion 100 10 1000

Table 2-1

Resolving Power of any microscope-a measure of its ability to discriminate between two adjacentobjects.- is a function of the wavelenght of light and the numerical aperture of the lens system- light microscopes (using visible light) have RP of approximately0.25 um which means that particles of a smaller size cannot be distinguished from one another).

Unit of length

Meter (m) Centimeter (cm)

Millimeter (mm)

Micrometer (um)

Nanometer (nm)

Micrometer (um)

0.000001 10-6

0.0001 10-4

0.001 10-3

1 1000 103

Nanometer (nm)

0.000000001 10-9

0.000000110-7

0.00000110-6

0.00110-3

1

Angstrom (Å)

0.0000000001 10-10

0.0000000110-8

0.000000110-7

0.000110-4

0.110-1

Table 2-2. Metric unit equivalents in expressing microbial cell dimensions

Selected Microorganisms SizePoliovirus 20 nm

Bacteriophage 60 nm

Adenovirus 90 nm

Vaccinia virus 200 nm

Cilia of protozoan 200 nm in width

E. coli 1.5 um in length

Paramecium 200 um in length

Type of Microscopy

Maximum useful magnification

Appearance of specimen

Useful Applications

Bright-field 1000-2000 Specimens stained or unstained; bacteria generally stained and appear color of stain

Gross morphological features of bacteria, yeasts, molds,algae and protozoa

Dark-field 1000-2000 Generally unstained; appears bright or “lighted” in an otherwise dark field

Microorganisms that exhibit some characteristic morphological feature in the living state and in fluid suspension e.g. spirochetes

Fluorescence 1000-2000 Bright and colored; color of the fluorescent dye

Diagnostic techniques where fluorescent dye fixed to organism reveals the organism’s identity

Type of Microscopy

Maximum useful magnification

Appearance of specimen

Useful Applications

Phase-contrast 1000-2000 Varying degrees of “darkness”

Examination of cellular structures in living cells of the larger microorganisms, e.g yeasts, algae, protozoa and some bacteria

a) Flourescent microscope b) flourescent micrograph of chromosomes and mitotic spindle

c) Dark-field photomi-crograph of Mysisd)Phase-contrast micrograph of a cheekcell

B. Electron Microscope-uses a beam of electrons controlled by a system of

magnetic fields -has high resolving power thus greater magnification

(can resolve objects separated by a distance of 0.003 umcompared to 0.25 um of light microscope).

-useful magnification is 200,000 to 400,000-use in the examination of viruses and the ultra-

stucture of microbial cells.- has two types:

a. Scanning electron microscopy (SEM)-employed to study the surface structure

of a specimen(eg. Attachment of bacterial cells toobjects)

b. Transmission electron microscopy (TEM)-used to view subcellular components

(even nucleic acid molecules)

S. aureus E. coli M. tuberculosis

Electron micrograph of:

Pure Culture Techniques

Pure culture – where all cells in the population are identical in the sense that they came from the sameparent cell.

Mixed Culture- many different species occupying the sameenvironment

- where microorganisms normally exist in nature

Isolation and Cultivation of Pure CulturesCulture Media

- nutrient materials where microorganisms are cultivated or grown- maybe in powder form while others are ready-to-use media inPetri dishes or test tubeseg. Nutrient agar (made of meat extract and digested protein (peptone) is one kind of medium

Transfer needle or Inoculation loop- used to streak the sample (e.g. Saliva) across the

surface of the medium so that individual cells become separated from one another.

Inoculum- the material placed in the medium.

During incubation of the inoculated medium, individual cells multiply and produce a large number of cells that together form a Colony.

Isolation of microorganisms- the objective is to thin out the microbial

population so that individual cells are located at a distance from other cells. To isolate a pure a culture, a transfer is made from an individual colony onto a medium in a test tube.

Plate culture techniques for isolation of microorganisms in pure culture

A. Streak-plate method- specimen is streaked

onto the surface of the agar-medium with a loop needle tothin out the population so thaton some regions of the mediumindividual cells will be depicted.

B. Spread-plate method- a drop of diluted sample

of the specimen is placed on thesurface of an agar medium andthis drop is spread over the entire surface using a sterilebent glass rod.

C. Pour-plate method- the inoculum is mixed

Into a melted agar medium that Has been cooled to 45oC and is

poured into a sterile Petri dish; after solidification they are incubated.- in this procedure colonies will grow both on and below the surface

(subsurface colonies) since some cells are trapped within the agar mediumwhen it solidifies.

Preservation of Pure Cultures - if culture is kept for only a short time (days to months),it can be stored at refrigeration temperatures (4 to 10oC) - if for long term storage, cultures are:

a. kept in tanks of liquid nitrogen at -196oC orb. In freezers at -70oC to -120oCc. by lyophilization (also known as freeze-drying)

- cultures are frozen, then dehydrated and sealed undervacuum- widely used in labs- maintains culture viability for many years and is a keyelement in building reference collections ofmicroorganisms

Techniques for Microscopic Study of Bacteria

Techniques for Microscopic Study of Bacteria1. Wet-Mount and Hanging-Drop Techniques

- use to examine living organisms- useful in the study of morphology,internal cell structures, motility or cellchanges

2. Staining Techniques- use dyes (colored organic cpds) tostain microorganisms- used to show the overall structure ofmicroorganisms, to identify their internalstructures, and to help identify and separate similar organisms

Major Steps in preparing a stained microbial specimen for microscopic examination are:

1. Placing a smear (or thin film of specimen), on a glass slide

2. Fixing the dried smear onto the slide with heat to make the microorganisms stick to the glass

3. Staining with one or more dyes

Types of Staining Techniques1. Simple Staining

-film stained with a single dyesolution-shows size, shape andarrangement of cells

2. Differential Staining- two or more reagents used instaining process- Difference observable

between cells or part of cells.

3. Gram staining- one of the most important and

widely used differential staining tech for bacteria

- 1st described in 1884 by Christian Gram of Denmark .

-Primary stain (crystal violet) applied to film and then treated with reagents and counterstained with safranin

-characterizes bacteria in one of two groups:

1. Gram-positive – deep violet2. Gram negative - red

4. Acid-fast-film stained with carbolfuchsin, decolorized, and counterstained with methylene blue-separate acid-fast bacteria, those not decolorized when acid solution is applied(e.g. Mycobacteria), from non-acid –fast bacteria, which are decolorized by acid

5. Giemsa Stain-stain applied to blood smear or film of other

specimens - observation of protozoa in blood smear; rickettsia (small parasitic bacteria) in certain cells of the nucleus; nuclear material in bacteria

6. Endospore- primary stain (malachite green) applied

with heat to penetrate spores; vegetative cells counterstained with safranin-endospores can be seen in Bacillus and

Clostridium species

7. Capsule -smear stained following treatment with

copper sulfate-can be observed as a clear zone

surrounding cells of capsulated microorganisms

Fig. A Acid-fastFig. B Endospore

Fig. C. GiemsaFig. D Capsule

Fig. E. Flagella

8. Flagella- mordant acts to thicken flagella before staining-observe flagella on bacteria

9. Negative staining-specimen mixed with India ink and spread into

thin film.- study morphology; staining procedure and

reagents are very mild in their effect on the microorganism, called a negative stain because microorganism is unstained and is made visible and is made visible because the background is dark.

Information used to characterize microorganisms

1. Morphological characteristic2. Nutritional and cultural charactreristic3. Metabolic char4. Antigenic char5. Pathogenic char6. Genetic char

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