10/25/2014

Basic techniques needed to study

Bacteria

• 1. Grow Bacteria
Observing Microorganisms • 2. Isolate Bacteria
through a Microscope • 3. Grow Bacteria in pure culture
• 4. Observe Bacteria
• 5. Identify Bacteria

Microscopy – Light Microscopes

Microscope
• Resolving Power - ability to distinguish two
distinct points
– absolute limit of the Resolving Power is about 1/2
the wavelength of light that is used to illuminate
the specimen

Bright-field
Compound
Microscope

PARTS OF MICROSCOPES PARTS OF MICROSCOPES

• Optical or eyepiece – lens near the observer
• Objective – lens near the specimen (LPO, HPO,
OIO)
• Arm – support the body tube and objectives
• Coarse adjustment – bring the object into
vision
• Fine adjustment –finer focusing of the
specimen
• Mirror – collect light from the outside source
• Condenser – provide illuminating cone of light
to fill the aperture of the objective.
• Diaphragm – modify the amount of light

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Microscopy – Light Microscopes

PARTS OF MICROSCOPES Bright-Field Microscope

Light microscope produces a dark image against

• Stage – place where the slide is put brighter background. Ocular lenses

• Stage clip – holds the slide in place

Commonly used to view stained cells.
• Revolving nosepiece – end piece of the microscope
Simple microscopes have single magnifying lens (like
body to which the objectives are attached a magnifying glass).
Objective
Lenses

• Base – supports the whole microscope

Compound microscopes have two sets of lenses for
• Inclination joint – adjust the positioning/inclining of magnification.
the microscope Lens closer to the eye = ocular lens (magnifying power of
10x).

Lenses closer to the object being viewed = objective

lens. (Most light microscopes used in biology have three or
four objective lenses).

OBSERVATION OF MICROORGANISMS

Objective Lenses Objective Lenses

Scanning Objective Lens Low Power Objective Lens
Has yellow band around it.
Has red band around it. Magnifies objects 10x.
Total magnification = 100xTM
Magnifies objects 4x.
Start with this lens to get your
bacterial smear into crisp focus.
Total magnification = 40xTM

You will not see individual bacteria with

This lens is of no use to us in this lens, you are just using it to focus
so that you can move up to the next
looking at bacterial stains.
magnification.

Objective Lenses
High Dry Objective Lens
Has blue band around it.
Oil Immersion Objective Lens
Has black and a white band
around it.

Magnifies objects 40x.

Magnifies objects 100x.

Total magnification = 400xTM Total magnification = 1000xTM

Move up to this lens after focusing Move up to this lens after focusing
your smear at 100xTM. your smear at 400xTM and
covering the 400xTM lens with a
You will not be able to clearly see finger cot.
individual bacteria with this lens. Just
get the image in focus as much as NEVER use coarse focus with high dry
possible. or oil immersion lenses!!!

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Microscopy – Electron Microscopes

Two types:
Staining Reaction
Both huge, expensive
machines.
• Stains - salts composed of a positive and
Transmission Electron negative ion, one of which is colored
Microscope (TEM): 2-D
image: Transmission Electron (chromophore)
Micrograph

A transmission electron micrograph of Escherichia coli (E.coli).

Scanning Electron Microscope • Basic Dyes - chromophore is the positive ion

(SEM):
– dye+ Cl-
3-D image: Scanning Electron
Micrograph • Acid Dyes - chromophore is the negative ion
– Na+ dye-

SEM AIDs virus attacking T4 lymphocyte

Bacteria are slightly negative, so are attracted to

the positive chromophore of the BASIC DYE Preparing smears for staining
• 1. Bacteria on slide
• Common Basic Dyes • 2. Air Dry
– crystal violet • 3. Bacteria are HEAT FIXED to the slide
– methylene blue • 4. Stain is applied
– safranin
– basic fuchsin

Acid Dyes - used for Negative Staining

(background is stained)
Differential Stains
• React differently with different types of
Mordant - intensifies the stain or coats a structure to bacteria
make it thicker and easier to see after it is stained

• 2 Most Common
Example: – Gram Stain
Flagella - can not normally be seen, but a mordant – Acid-Fast Stain
can be used to increase the diameter of the flagella
before it is stained

Salmonella typhosa

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Gram Stain Gram Stain

• 1884 Hans Christian Gram 1. Crystal violet

• most important stain used in Bacteriology

• Divides all Bacteria into 2 groups:

– Gram (+)
– Gram (-)

Gram Stain Gram Stain

2. Grams Iodine (mordant)
3. Alcohol

Gram Stain Results

• Gram (+) Purple
4. Safranin (Counterstain)

• Gram (-) Red

• Difference - due to structure of cell wall

– Gram (+) Thick cell wall
– Gram (-) Thin cell wall

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Identification of a Bacteria Unknown Acid - Fast Stain

• 1. Gram Reaction • Differential Stain - divides bacteria into 2 groups

• 2. Morphology • Acid - Fast

• Non Acid - Fast

• Used to identify organisms in the Genera

Mycobacterium (high lipid and wax content in cell
wall)

2 Important Pathogens Mycobacterium leprae

Mycobacterium tuberculosis

Acid - Fast Stain Special Stains

• 1. Carbolfuchsin (Red) Capsule Stain

• 2. Acid Alcohol
• 3. Counterstain with Methylene Blue

• Acid - Fast Cells Red

• Non Acid - Fast Blue

Klebsiella pneumoniae

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Endospore Stain
Flagella Stain

Spirillum volutans Bacillus cereus

Clostridium botulinum