Experiment one
Examination of Bacteria
Visualizing Bacteria
• Staining is required to properly visualize
bacteria
Microscopy
Bright Field Microscopy
Microscopy
Bright Field Microscopy
• Three different objective lenses are commonly
•
•
•
•
used
10x: To scan the slide for specimens
40x: To view parasites, filamentous fungi
100x: To observe single cells
Total magnification= (objective lens
magnification) X (ocular lens magnification)
Microscopy
Bright Field Microscopy
Note how light scatters
via refraction
Refractive index of air is
lower than that of glass
Loss of refracted light is
minimized by applying
mineral oil (same RI as
glass)
Microscopy
Dark Field Microscopy
• Creates contrast between the object and the surrounding
•
•
•
•
field. Background is dark and the object is bright.
An annular stop ring permits light coming from the
outside of the beam.
When light from the stop is deflected and deviated by
the object can it be seen.
Advantageous for viewing thin bacteria
(Ie. Treponema pallidum)
Disadvantage: internal structure is not as clearly visable
compared to bright field
Microscopy
Dark Field Microscopy
Light Field
vs
Dark Field
Microscopy
Phase-Contrast Microscopy
• Most of the detail of living cells is undetectable
in bright field microscopy
– Little contrast exists between structures with similar
transparency
– Insufficient natural pigmentation.
• Organelles show wide variation in refractive
•
index (the tendency of the materials to bend
light) providing an opportunity to distinguish
them with phase contrast mircoscopy
Internal features are more easily viewed
Microscopy
Phase-Contrast Microscopy
Microscopy
Fluorescent Microscopy
• Organisms are stained with fluorescent dies
•
•
•
•
(fluorochromes) and then viewed.
Fluorescent microscopes emit a shorter
wavelength of light than in bright field
microscopy.
The short wavelength excites the fluorochromes,
and they fluoresce.
Allows for easier low magnification scanning.
Bright object occurs against a dark background.
Microscopy
Fluorescent Microscopy
Fluorescent vs
Dark Field
Microscopy
Fluorescent Microscopy
• Different fluorescent
stains can bind to
different targets
• Digital merging with
differential stains
Electron Microscopy
• Uses magnetic coils to direct a beam of
electrons through the specimen onto a
screen
• Uses a very short wavelength, thus
magnification and resolution is improved
• Samples are stained/coated with metal
ions to create contrast.
Electron Microscopy
• Transmission electron microscope
– Electrons pass through the specimen
Electron Microscopy
• Scanning Electron Microscope
– Electrons bounce off the surface of a
specimen and create a 3D image.
How to use the oil immersion lens of microscopy
The substage condenser to raise to the highest position
The iris diaphragm fully opened
To adjust the light entering the lens with low-power lens
The oil immersion lens to be rotated into position
The specimen to be put on the center of stage
A drop of oil to be placed on the slide directly over
the area to be viewed
Up the stage with the coarse adjustment knob, to let
oil lens into the oil
Looking into the ocular lens and down the stage
slowly with the coarse adjustment knob until the
specimen comes into focus
Using the fine adjustment knob, to bring the
specimen into sharp focus
The oil immersion lens should be cleaned with lens
paper after experiment
Examination Methods:
Direct Examination
India Ink
• Darkens the background rather than
•
the cell
Useful in detecting Cryptococcus
capsules
– Capsule excludes ink
Morphological Observation Of
Bacterial Cells
• S. aureus
Morphological Observation Of
Bacterial Cells
• E. coli
Morphological Observation Of
Bacterial Cells
• V. choleriae
• Gram -ve
Morphological Observation Of
Bacterial Cells
• Streptococcus pneumoniae
Morphological Observation Of
Bacterial Cells
• Salmonella typhi
Morphological Observation Of
Bacterial Cells
• Clostridium tetani
• Club shape is due to endospore production
at one termini of the cell.
Staining of Bacteria
• PURPOSE :
•
To make bacteria more easily observable
To acquaint you with Gram stain
MATERIALS:
– Simple stain
– Gram stain
– Acid-fast stain
– Special stain
• Spore stain
• Capsule stain
• Flagella stain
• Metachromatic granules stain.
Gram stain
• purpose:
differentiating bacteria
• MATERIALS :
– Slant cultures of and Escherichia coli and
S.aureus (18 to 24 hours old)
– Crystal violet, iodine solution, 95%
alcohol, safranin
– Microscope slides
Gram stain
• PROCEDURE:
– Smear: size of a dime to form
a thin film
– Dry : air dry
– Fix: through the warm air
above the flame two or three
times.
Process of Gram’s Stain
Crystal violet
(primary staining)
1min
Washing
Lugol’s iodine
(mordant staining)
Washing
Fuchsion red
(counterstaining)
30s
30s
1min
95%ethyl alcohol
(decolorization)
Washing
Washing
Blot dry with bibulous papers
Observation with the oil immersion lens
Results :Gram –positive blue color Gram-negative red color
Gram Staining
• In gram-positive bacteria, the crystal violet and
•
•
•
iodine combine to form a larger molecule that
precipitates out within the cell.
Gram +ve bacteria have low lipid content
Lipid is dissolved by alcohol
The alcohol/acetone mixture then causes
dehydration of the multilayered peptidoglycan
– Thus causing the cell wall to trap the crystal violetiodine complex within the cell.
Gram Staining
• Gram-negative bacteria have higher lipid
•
•
contents
The alcohol/acetone mixture, being a lipid
solvent, dissolves the outer membrane of the
cell wall and may also damage the cytoplasmic
membrane to which the peptidoglycan is
attached.
The single thin layer of peptidoglycan is unable
to retain the crystal violet-iodine complex and
the cell is decolorized.
Gram Staining: Common Errors
• There are several factors that could result in a grampositive organism staining gram-negatively:
– The method and techniques used. Overheating during heat
fixation, over decolorization with alcohol, and even too much
washing with water between steps may result in gram-positive
bacteria losing the crystal violet-iodine complex.
– The age of the culture. Cultures more than 24 hours old may
lose their ability to retain the crystal violet-iodine complex.
– The organism itself. Some gram-positive bacteria are more able
to retain the crystal violet-iodine complex than others.
• Therefore, one must use very precise techniques in gram
staining and interpret the results with discretion.