Microscopy and Specimen
Preparation
Prepared by: DR. RICHARD D. PASCUA
Instructor
Microorganisms
The sizes of bacteria and protozoa are usually expressed in terms of
micrometers. For example, a typical spherical bacterium is approximately 1µm
in a diameter. About seven cocci could fit side-by-side across a human red
blood cell.
Microscopes
An optical instrument that is used to observe tiny objects, often objects that can not be seen at all
with the unaided human eye. Each optical instrument has a limit as to what can be seen using
that instrument. This limit is referred to as the resolving power or resolution of the instrument.
Magnification is the process of enlarging something only in appearance, not in physical size. ...
Optical magnification is sometimes referred to as "power" (for example "10× power"),
although this can lead to confusion with optical power.
Simple Miroscopes
A simple microscope is defined as a microscope containing only one magnifying lens.
Actually, a simple magnifying glass could be considered as a simple microscope.
During the 1600’s, Anton Van Leeuwonhoek made use of simple microscope to observe
microorganisms.
His microscope had a maximum magnification of about 300x.
Compound Microscopes
A compound microscope that contains more than one magnifying lens. Compound light
microscopes usually magnify objects about 1,000 times. Photographs taken through the lens
system of compound microscopes are called photomicrographs.
Because visible light is used as the source of illumination, the compound microscope is also
referred to as a compound light microscope.
Stereo Microscope
Stereo Microscope (dissecting microscope): These microscopes
magnify up to about maximum 100x and supply a 3-dimensional
view of the specimen. They are useful for observing opaque objects.
Darkfield Microscope
Dark field optics are a low cost alternative to phase contrast optics. The contrast and resolution
obtained with inexpensive dark field equipment may be superior to what you have with student
grade phase contrast equipment. Dark field illumination is most readily set up at low
magnifications (up to 100x), although it can be used with any dry objective lens. Any time you
wish to view everything in a liquid sample, debris and all, dark field is best. Even tiny dust particles
are obvious. Dark field is especially useful for finding cells in suspension. Dark field makes it easy
to obtain the correct focal plane at low magnification for small, low contrast specimens. Use dark
field for
Fluorescence Microscopes
A fluorescence microscope is an optical microscope that
uses fluorescence and phosphorescence instead of, or in addition to,
reflection and absorption to study properties of organic or inorganic
substances.
Electron Microscopes
Modern electron microscopes can magnify up to 2 million times. This is possible, because the
wavelength of high energy electrons is very small. At the same time, the high energy electrons are
pretty tough on the sample being observed. It may take a long time to completely dehydrate and
prepare the specimen. Some biological specimens also need to be coated with a very thin layer of a
metal before they can be observed.
Transmission electron microscopy (TEM): In this case, the electron beam is passed through the
sample. The result is a two dimensional image.
Scanning electron microscopy (SEM): Here the electron beam is projected on the sample. The
electrons do not go through the sample but bounce off. This way it is possible to visualize the
surface structure of the specimen. The image appears 3 dimensional.
Electron Microscopes
Staining Techniques
Staining is an auxiliary technique used in microscopy to enhance contrast in
the microscopic image. Stains and dyes are frequently used in biology and
medicine to highlight structures in biological tissues for viewing, often with
the aid of different microscopes.
Staining Techniques
Bacteria are microscopic organisms. They are also colorless for the
most part. In order to visualize them to study their structure, shape
and other structural characteristics, it becomes necessary to make
them more easily visible.
This means that the structures have to be contrasted from their
environment so that they can be seen easily.
Types of Stain used for microscopy
ACIDIC: Negatively charged acid radicals imparts color in eosin, acid fuchsine,
malachite green, nigrosin, Indian ink.
BASIC: Positively charged basic radicals combines with negatively charged
particles in cytoplasm and gives color.
Ex: Haematoxillin, methylene blue, crystal violet, gention violet.
NEUTRAL: Both positively and negatively charged imparts different colors to
different components.
Ex: Geimsa’s stain, Leishman’s stain, Wright’s stain.
Staining Techniques
Gram’s Staining
The Gram stain was devised by the Danish physician, Hans Christian
Joachim Gram, while working in Berlin in 1883. He later published this
procedure in 1884. At the time, Dr. Gram was studying lung tissue sections
from patients who had died of pneumonia.
Gram’s Staining
The most widely used staining procedure in microbiology is the Gram stain, discovered by the Danish
scientist and physician Hans Christian Joachim Gram in 1884. Gram staining is a differential staining
technique that differentiates bacteria into two groups: gram-positives and gram-negatives. The
procedure is based on the ability of microorganisms to retain color of the stains used during the gram stain
reaction.
BACTERIAL SMEAR PREPARATION:
Smear - is a distribution of bacterial cells on a slide for the purpose of viewing
them under the microscope.
Method:
-Aseptically a small sample of the culture is spread over a slide surface.
-This is then allowed to air dry.
-The next step is heat fixation to help the cells adhere to the slide surface.
-The smear is now ready for staining.
BACTERIAL SMEAR PREPARATION:
Smear Fixation
1) Heat fixation
a) Pass air-dried smears through a flame two or three
times. Do not overheat.
b) Allow slide to cool before staining.
2) Methanol fixation
a) Place air-dried smears in a coplin jar with methanol for
one minute. Alternatively, flood smear with methanol for 1
minute.
b) Drain slides and allow to dry before staining.
