PAPER
OF MICROBIOLOGY
“Microscope and Staining”
By
:
1. Nur
Meili Zakiyah (110210153004)
2. Wontin
Muyassaroh (110210153005)
BIOLOGY EDUCATION STUDY
PROGRAME
MATHEMATIC AND SCIENCE
DEPARTMENT
TEACHER TRAINING AND
EDUCATION FACULTY
JEMBER UNIVERSITY
2013
PRINCIPLES
OF MICROSCOPY
The
microscope is one of the important tools in science laboratory activities,
especially biology. The microscope is a tool that enables us to observe objects
that are very small (microscopic). This helps solve the human problems of small
organisms. To know it is to know microscopy microscope parts, kinds of
microscopes, use and maintenance.
1. Microscope components
a. foot
Sustain and strengthen the functioning leg position microscope. In
the attached foot arm with a kind of hinge, the simple microscope (model
student).
b. arm 2
With the hinge between the legs and arms, the arm can be enforced
or recline. Arm also used to hold the microscope when moving the microscope.
c. Mirror.
The mirror has two sides, the flat mirror and the concave mirror,
serves to reflect light and light sources. Flat mirror is used when the light
source is quite bright, and the concave mirror is used when the light source is
less. Mirror can be detached and replaced with a light source of the lamp. In
the new model microscope, it no longer fitted mirror, because there is a light
source mounted on the bottom (foot).
d. condenser
The condenser is composed of the combined lens serves to collect
light.
f. table preparations
Preparations is a laid table objects (preparations) to be seen.
Objects placed on the table with the clamped by the clamp. Section of the table
there is a sleeve for dilewat rays. In certain types of microscopes, the
position of the table can not be raised or lowered. In some microscopes, especially
the latest models, hair preparations can be hung upside down.
g. Tube.
At the top of the tube attached to the ocular lens, with a
particular magnification (15X,
10X, and 15X). Bottom of the tube there is a device called a
revolver. In these revolvers are the objective lens.
h. objective lens
The objective lens work in the formation of the first shadow. these
lenses
determine the structure and the trace will appear on the final
image. Another important feature is the objective lens by the magnification of
an object shadow enlarge assortment according to the model and the
manufacturer, for example, 10X, 40X, and 100X and have value Apertura (NA).
Apertura is a measure of the value of separating an objective lens that will
determine the split specimen, so as to demonstrate the structure of adjacent
microscopic as two separate objects.
i. ocular lens
Microscope lens that sits at the top end of the tube, adjacent to
the eye of the observer. This lens serves to magnify the image produced by the
objective lens. Magnification images forming ranges between 4-25 times.
j. Regulatory Rough and Smooth
This component is located on the arm and serves to adjust the
position of the objective lens to the object to be seen. In the microscope with
a tube straight / straight, coarse and fine control for the dip tube once objective lens. In the microscope with oblique tube,
coarse and fine control to turn the tide table preparations.
Procedure Principle
Total
magnification is obtained by multiplying the objective magnification eyepiece
with magnification. For example, the total magnification is obtained from the
objective and eyepiece 40 times 10 times is 40 x 10 = 400 times. Magnification
of 100 times while using the objective, condenser iris diaphragm should be used
in the fully open state, because objective with a hight magnification need more light.
Objective magnification of 100 times also have to use oil immersion. It aims to
prevent the loss of light caused by the differences in refraction (refractive)
between glass and air. The refractive index of air 1, while the glass 1:56 and
immersion oil refractive index of glass is equal to 1.56. Note the picture is
to show the process of diffraction.
Objective lens serves for the formation of the first
shadow and determine the structure and microscopic section will look at the
final image to enlarge and develop the shadow object so that it can have a
value of "Apertura" which is a measure of separating an objective
lens that will determine the split specimen, so as to demonstrate adjacent
microscopic structure as two separate objects.
Ocular lens, lens microscope is located at the top
end of the tube adjacent to the eye of the observer, and serves to magnify the
image produced by the objective lens ranges from 4 to 25 times.
Condenser lens, is a lens that serves to support the
creation of light on the object to be seen so that the appropriate arrangements
will be obtained maximal separation power. If the separation is less maximum
power then the two objects will appear to be one and zoom in be less than optimal.
LIGHT MICROSCOPE
A
light microscope can magnify our vision to be 1,000 times. Thus lead to an
enlarged diameter of 0.2 micrometers objects we can see. Light microscope has
two types, namely Ojective and eyepiece lenses, the system works in a way
assisted penetrating reflections observed objects and shadows can enlarge
objects up to 1000x.
Although
it's been 300 years passed since the microscope was found, to date remains the
standard light microscope based on optical principles. Microscopes are you
using now is as good as that used by Schleiden, Schwann, and Virchow, the
inventor of the cell theory in the mid-19th century. The microscope is much
better than the first microscope used by Robert Hooke, who first used the term
cell.
Types of light microscopes
The bright field microscope is best known to students and is most likely to
be found in a classroom. Better equipped classrooms and labs may have dark
field and/or phase contrast optics. Differential interference contrast,
Nomarski, Hoffman modulation contrast and variations produce considerable depth
of resolution and a three dimensional effect. Fluorescence and confocal
microscopes are specialized instruments, used for research, clinical, and
industrial applications.
Other than the compound microscope, a simpler instrument for low
magnification use may also be found in the laboratory. The stereo microscope,
or dissecting microscope usually has a binocular eyepiece tube, a long working
distance, and a range of magnifications typically from 5x to 35 or 40x. Some
instruments supply lenses for higher magnifications, but there is no
improvement in resolution. Such "false magnification" is rarely worth
the expense.
This type of microscope is
composed of an objective lens, an ocular lens, a stage, a lightsource,
a condenser, a tube, an arm to support the tube, and a focusing system. The
specimen is set on the stage, a platform usually equipped with metal arms to
hold the specimen or slide in place. The light bulb
is situated beneath the stage so that the light shines
up through the specimen. The tube focuses down on the stage so that the ocular
lens, or eyepiece, is at the far end of the tube and the objective lens is at
the end closer to the specimen.
How
to Use Light Microscope
Before doing
lab work using light microscopy then consider the following steps:
1.
Put
the microscope on the table by holding the arm microscope so that the
microscope is directly in front of the user.
2.
Rotate
the revolver so weak objective lens with a magnification in the position of the
shaft with marked ocular lens click on the revolver
3.
Set
up a mirror and diaphragm to see the power of incoming light, until of the
ocular lens looks bright round (field of view).
4.
Place
the preparation on the table right thing at the hole and clamp with clamp
preparations object / object.
5.
Set
the focus to clarify the picture by rotating the object rough player, while
views of the ocular lens. To sharpen the players rotate fine.
6.
If
the shadow of an object is found, replace it to enlarge the size of the
objective lens 10x, 40x or 100x, by turning a revolver to a click.
7.
When
finished using, clean the microscope and store in a place that is not damp.
Rules
For Use Of The Microscope
1.
Always
carry the microscope in a straight upright position with one hand around the
arm and the other hand under the base.
The eyepieces are not attached and will fall out if the microscope is
carried at an angle or upside down.
2.
Check
out the microscope to make sure all the lenses are clean and the mechanical
parts are in working order. Report any
malfunction to the instructor so that it may be remedied.
3.
Keep
the microscope clean. When anything is
spilled or otherwise gets on the microscope, clean it up immediately.
4.
When
using the microscope start with the low power lens and work up to the desired
magnification. These microscopes are
parfocal, which means that all powers should be in focus when the turret is
rotated.
5.
Never
move the stage upwards with the coarse adjustment while viewing through the
eyepieces. Get the lens close to the
slide while viewing from the side to make sure that they never touch. Then move the stage downward with the coarse
adjustment while viewing through the lense.
This will prevent the possibility of ramming the lens into the slide,
thereby ruining a slide you have just made and, quite possibly, damaging the
lens.
6.
Moist,
living or preserved materials must be observed through a coverslip. This protects the lens as well as tends to
make the object under view optically flat.
Be sure to maintain a safe distance between the coverslip and the
objective lenses.
7.
Clean
the lenses with lens paper only. Don’t
clean the lenses with handkerchiefs, facial tissues, paper towels, etc.
8.
If
you cannot obtain clear focus or good lighting, or if your microscope seems not
to be working properly, immediately call your instructor. He/she can either
assist you or see that the microscope is repaired.
9.
Return
your scope to the cabinet with light cord wrapped around its base and with the
lowest power objective lens in position.
ELECTRON
MICROSCOPE
Electron
microscopy is basically utilized to study structures which are unvisible to the
naked eye, or are too small to be well revealed with a light microscope.
Electron have bigger resolution than light electron. Light can achieve 200 nm
only, while electron microscope can achieve until 0,1 – 0,2 nm. Besides that,
with use electron we can get some reflection that has benefit to characterizing.
If electron hit an object, from the object will rise 2 reflections, that are
elastic reflection and non-elastic reflection. The picture show below
Much
like the traditional Scanning Electron Microscope, the Field Emission Scanning
Electron Microscope uses electrons to illuminate a sample, instead of visible
light as is used in optical microscopy.
To see objects smaller than 200
nanometers, needed a microscope
with a shorter wavelength. From this idea, in 1932
electron microscope was born. As the name suggests, electron
microscopes use beams of electrons
that shorter wavelengths
of light. Therefore,
the electron microscope has a magnification ability of the object (resolution) is higher than the optical microscope.
Actually, the function
of the object magnification,
electron microscopy also use the lens,
but not from the type
of glass, as in an optical microscope, but of the magnet. The nature of the
magnetic field can control and
influence through which electrons, so that it can function replaces the lens
on a microscope optical properties. Another particularity
of this electron
microscope observations of the object is under
vacuum (vacuum). This
is done because the electron beam
is inhibited when mashing the flow of
molecules that exist in normal
air. By making observations of
objects unconditioned space vacuum, electron-molecule collisions can
be avoided.
PARTS
OF ELECTRON MICROSCOPE
- Illumination
Source
A tungsten
filament is heated to 2,700OC, in a vacuum to produce a beam of electrons (LM
uses photons). Electrons behave in the same manner as light when in a vacuum.
The entire microscope column is under vacuum otherwise the electrons would
collide with air molecules and be absorbed.
- Condenser
Lens
Makes the light
source into a parallel beam and focuses it onto to the specimen (TEM has 2 or 3
condenser lenses) All TEM lenses are electro magnetic lenses whereas LM has
glass lenses. Electric and magnetic fields have the same effect on electrons as
glass lenses and mirrors have on visible light.
- Objective
Lens
Strong lens.
Focuses. 1st magnifying lens. Brings electrons which have passed through the
specimen to focus.
- Specimen
Stage
Specimen
Injector Rod inserts the specimen inside the Objective Lens. Although the
specimen is thin there is information at various depths and these can be viewed
by tilting the specimen using a goniometer.
- Intermediate
Lens
Magnifies the
image.
- Prejector
Lens
Projects the image
onto yellow/green phosphorescent screen.
There
are some type of Electron microscope based on the function, that are :
a.
Transmission electron microscope (TEM)
The
original form of electron microscope, the transmission electron microscope (TEM)
uses a high
voltage electron
beam to create an image. The electron beam is
produced by an electron
gun, commonly fitted with a tungsten
filament cathode as the
electron source. The electron beam is accelerated by an anode
typically at +100 keV (40 to
400 keV) with respect to the cathode, focused by electrostatic and electromagnetic lenses,
and transmitted through the specimen that is in part transparent to electrons
and in part scatters them
out of the beam. When it emerges from the specimen, the electron beam carries
information about the structure of the specimen that is magnified by the objective lens system
of the microscope. The spatial variation in this information (the
"image") may be viewed by projecting the magnified electron image
onto a fluorescent viewing screen coated with a phosphor or scintillator
material such as zinc sulfide. Alternatively, the image can be photographically
recorded by exposing a photographic
film or plate
directly to the electron beam, or a high-resolution phosphor may be coupled by
means of a lens optical system or a fibre
optic light-guide to the sensor of a CCD (charge-coupled device)
camera. The image detected by the CCD may be displayed on a monitor or
computer.
Resolution
of the TEM is limited primarily by spherical aberration, but a
new generation of aberration correctors have been able to partially overcome
spherical aberration to increase resolution. Hardware correction of spherical
aberration for the high-resolution
transmission electron microscopy (HRTEM)
has allowed the production of images with resolution below 0.5 angstrom (50 picometres) and
magnifications above 50 million times. The ability to determine the positions
of atoms within materials has made the HRTEM an important tool for
nano-technologies research and development.
An
important mode of TEM utilization is electron diffraction. The
advantages of electron diffraction over X-ray crystallography are that the
specimen need not be a single crystal or even a polycrystalline powder, and
also that the Fourier transform reconstruction of the object's magnified
structure occurs physically and thus avoids the need for solving the phase
problem faced by the X-ray crystallographers after obtaining their X-ray
diffraction patterns of a single crystal or polycrystalline powder. The major
disadvantage of the transmission electron microscope is the need for extremely
thin sections of the specimens, typically about 100 nanometers. Biological
specimens typically require to be chemically fixed, dehydrated and embedded in
a polymer resin to stabilize them sufficiently to allow ultrathin sectioning.
Sections of biological specimens, organic polymers and similar materials may
require special `staining' with heavy atom labels in order to achieve the
required image contrast.
In a TEM the object (for example a cluster of cells) is usually
previously cut in very thin sections (ultrasection <100nm) and pre-treated
with heavy metals which by preference bind ("stain") to certain
characteristic structures, like membranes, proteins and DNA (see preparation TEM). Sometimes the objects (e.g. virussen or polymeric aggregates)
are thin enough to be partly permeant to the electron beam. In such cases
hardly any pretreatment is necessary. Once in the TEM the object is bombarded
by a beam of electrons,the so-called primary electrons. In areas in the object
where these electrons encounter atoms with a large (heavy) atomic nucleus (e.g.
the nuclei of the heavy metals of the pretreatment), they rebound. Electrons
are also repulsed (or absorbed) in areas where the material is relatively
condense or thick. However, in regions where the material consists of lighter
atoms or where the specimen is thinner or less concentrated, the electron are
able to pass through. Eventually the traversing electrons (transmission) reach
the scintillator plate at the base of the column of the microscope. The scintillator
contains material (e.g. phosphor compounds) that can absorb the energy ot the
strucking incoming electrons and convert it to light flashes. The contrasted
image that is formed on this plate corresponds with the selective pattern of
reflection or permission of electrons, depending on the local properties of the
object. Thus, one can see for example where cytoskeletal elements and membranes
are located because the corresponding area remain dark,whereas the cytosol
around these structures appears as light (see example; G= Golgi, AF = Actin filaments, Mt = Mitochondrion). In practice
the bombarding electrons are focussed to a bundle onto the object. The fine
pattern of exiting electrons leaving the object is then greatly enlarged by
electromagnetic lenses: a many times enlarged projection image is the result.
Transmission electron microscopes produce images by recording the
electron beam after it has passed through a thin slice of specimen. The
specimen is placed on a copper wire grid and subjected to an electron beam,
normally generated by running high voltage across a tungsten filament. The
electron beam travels through a condenser lens, strikes the specimen and continues
through objective and projective lenses before being collected onto a phosphor
screen. As with all forms of electron microscopy, the target specimen must be
dehydrated and isolated in a vacuum to avoid water vapor contamination, which
can cause unwanted electron scattering. TEMs produce the highest magnification
of all electron microscopes.
1: Electron cannon in the upper part of the column. 2
Electro-magnetic lenses to direct and focus the electron beam inside the
column. 3: Vacuum pumps system. 4: Opening to insert a grid with samples
into the high-vacuum chamber for observation. 5: Operation panels (left for
alignment; right for magnification and focussing; arrows for positioning
the object inside the chamber). 6: Screen for menu and image display. 7:
Water supply to cool the instrument.
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b.
Scanning
Transmission Electron Microscopy (STEM)
Is the result of development from Transmission Electron Microscopy
(TEM). In STEM, electron will pierce to the specimen and similar with working
principle of SEM. electron optics
Focused directly on a tight angle with scanning the area
where the object scanned from side to side and produce rows
of dots that make up an image as generated by the CRT TV / monitor.
Scanning transmission electron microscopes, like traditional TEMs,
pass an electron beam through a thin slice of specimen. Instead of focusing the
electron beam after passing through the sample, a STEM focuses the beam
beforehand and constructs the image through raster scanning. Scanning
transmission electron microscopes are well suited for analytical mapping
techniques such as electron energy loss spectroscopy and annular dark-field
microscopy.
c.
Scanning
Electron Microscopy (SEM)
Scanning Electron Microscopy (SEM) used to observe
the details of the cell surface
or other microscopic
structures, and is
able to display three-dimensional objects.
Formatting image of SEM is different
with formatting image of TEM. In the SEM, the image is made
based on the detection of new electrons (secondary electrons) or reflection electron that arise from the surface of the sample when the
sample surface is scanned by
an electron beam. Secondary electrons or reflection electron further that was detected then powered the signal, and then the amplitude
is shown in shades of darkness on the screen CRT
(cathode ray tube). In the CRT screen, the image that has been magnified object structure can be seen. In the process of operation, the SEM does not require a
diluted sample, which can be used to view objects from three-dimensional viewpoint.
In SEM the object is bombarded by primary electrons from the source
according to a scanning pattern. These strucking electrons cause the emission
of secondary electrons (to some extend one could compare the effect with that
of a pool balls, in which the striking balls pushes the receptive one away). In
SEM an image of the surface of the object is made. The height and slope of the
surface of the object in a particular area co-determines the number of
generated secondary electrons per time unit and the velocity of those
electrons. The secondary electrons are attracted by the Corona and strike the a
scintillator disc. Like in the TEM, this disc contains sun=bstances which can
convert the energy of the striking electrons into photons (light). The more
secondary electrons reach the scintillator, the brighter the signal is at that
point. This luminescence signal is further amplified by a photomultiplier tube
and transduced to a video signal that is fed to a cathode ray tube in synchrony
with the scan movement of the electron beam. Nowadays the analog image is
converted to a monochromatic (gray shades) computer image that can be further
digitally processed. The intensity pattern in the final image reflects the
levels in the sample and looks like a shadow-cast view (see an Ambrosia pollen
grain as an example).
In Cryo-SEM
microscopes frozen material, e.g. cells, can be fractured in order to obtain a
view of the surface of the structures inside the broken volume. In microscopes
equipped with EDS
(Energy Dispersed Spectroscopy) or EDAX (Energy-Dispersed Analysis of X-rays)
detectors which collect the energy of directly reflected electrons (back
scattered electrons) and X-ray, it is possible to map which lements are present
in the outermost surface layer.
1: Electron
cannon in the upper part of the column (here a so-called field-emission
source). 2 Electro-magnetic lenses to direct and focus the electron beam
inside the column. 3: Vacuum pumps system. 4: Opening to insert the object
into the high-vacuum observation chamber in conventional SEM mode. 5:
Operation panel with focus, alignment and magnification tools and a
joystick for positioning of the sample. 6: Screen for menu and image
display. 7: Cryo-unit to prepare (break, coat and sublimate) frozen
material before insertion in the observation chamber in Cryo-SEM mode. 8:
Electronics stored in cupboards under the desk. 9: Technicians Mieke
Wolters-Arts and Geert-Jan Janssen discussing a view.
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Scanning electron microscopes, along
with transmission electron microscopes, are the most widely used. Unlike the
TEMs, scanning electron microscopes produce images by collecting the secondary
or inelastically scattered electrons that bounce off the surface of a specimen.
The primary electron beam travels through several condenser lenses, scan coils
and an objective lens before striking the surface of the specimen. The electron
beam is scattered upon hitting the specimen and a secondary electron detector collects
the scattered electrons. The electron data is then raster-scanned to produce
surface images with considerable depth of field.
d.
Reflection
Electron Microscope (REM),
REM is an electron microscope that has a similar way of working with the workings of TEM, but the detection system uses the reflection of electrons on the surface of the object. This technique is specifically used by combining it with the technique of reflection high-energy electron diffraction (Reflection High Energy Electron Diffraction) and release techniques Reflection high-energy spectrum (reflection high-energy loss spectrum - RHELS).
Reflection electron microscopes operate very similar to SEMs in
terms of structure. REMs, however, collect the backscattered or elastically scattered
electrons after the primary electron beam strikes the specimen surface.
Reflection electron microscopes are most commonly coupled with spin-polarized
low-energy electron microscopy to image the magnetic domain signature of
specimen surfaces in computer circuitry construction.
As
we all know that electron microscope have ability more than Light microscope.
Besides that, there are some differences, that are:
Light Microscope:
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Electron Microscope:
|
1.
The radiation source (source of illumination)
is light,
2.
wavelength 400-700 nm.
3.
Lens is made of glass.
4.
Not affected by magnetic field.
5.
Maximum magnification 1500-2000
times.
6.
Resolving power 0.1-0.2 nm.
7.
Image is colored (natural color of
object is seen), enable the viewer to watch living cells.
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1.
Radiation source is electrons,
focused by magnetic lenses.
2.
wavelength about 0.005nm.
3.
Lens is electromagnetic.
4.
Affected by magnetic field.
5.
Maximum magnification 1, 60,000 to
2, 50,000 times.
6.
Resolving power 200-300 nm.
7.
Image is black and white. image of
the shadows cast by atoms of heavy metals used as stains; the living tissue
is destroyed by the intense beam of electrons.
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STAINING
Microorganisms that exist in nature has
a morphology, structure and
unique properties, as well as bacteria. Bacteria
that live almost colorless and the contrast with
water, in which bacterial cells
are suspended. One
way to observe the bacterial
cell shape is so
easy to identify with the method of
painting or staining.
It also serves to determine the nature of the physiological reactions that determine
the bacterial cell wall through a series
of painting.
Staining method was first discovered by Christian
Gram in 1884. With this method,
Bacteria can be grouped into two, that are:
gram-positive and gram-negative bacteria that based on
the reaction or the nature of the bacteria to the paint. Reaction Or the bachteria characteristic determined
by the composition of the bacterial cell wall, so the staining of gram
can’t doing in microorganism without cell wall like Mycoplasma sp.
(Waluyo, 2004).
Success or failure of a coloring is determined
by the timing of the color and culture of the colored age (age of culture that good is 24 hours). Commonly, dyes that used is salts that are built by the ions that have positive and negative charge ions which one of them is colored. The dye grouped into two, namely
the acidic and bases dyes. If the ion that containing the
color is a positive ion dye, so called dye
bases. And if the color is ion-containing negative ions then dye called
negative dye (Hadiutomo. 1990).
The dye used
in coloring is base and acidic. In base dye, the part that role in cleaning color called chromosphores and have a positive charge. Conversely, the acid dyes instrumental sections provide dyes having negatively charge dye base more widely used because of the negative charge is found on the walls of cells, and cell membrane staining process cytoplasm positive charge on the dye bases will be associated with a negative charge inside the cell, thus microorganisms more clearly visible (Dwidjoseputro.1998).
1.
simple staining
Using one kind of dye (methylene blue /
water fukhsin) the goal is only to see the
shape of the cell. Simple
Staining is the most commonly used. Various morphological
types of bacteria (cocci, bacilli, spirilum,
etc.) can be distinguished using a
simple Staining, the bacterial cells staining only use one kind of
dye. Most bacteria react easily with simple dyes because cytoplasm
is basophilic (this
will base) while
the dyes were used to simple staining
is generally alkaline (chromophoric components is positively charged).
The
dye used consists of only one substance that dissolve in a solvent. Simple
staining is a
quick way to see the morphology of the bacteria in general. Some examples
of commonly used dye is methylene blue (30-60
seconds), crystal violet (10 seconds)
and fukhsin-carbolic
(5 seconds).
In simple staining used only one kind of dye to enhance the contrast between microorganisms and their surroundings. Commonly, this staining procedure using alkaline dyes such as crystal violet, methylene blue, basic fuchsin carbolic acid, safranin or green malakit. Sometimes negative dyes used for coloring is simple: acid dyes are frequently used nigrosin and red kongo (Lay.1994).
Simple
Staining procedure is easy and fast, so the coloring is often used to look at the shape, size and arrangement of the bacteria. In the bacteria known some shape : round (coccus), rod (bacillus), and spiral. With a simple staining also can be seen arrangement of bacteria. In coccus can be seen coloring like chain (stertococcus), grapes (stafilococcus), pairs (diplococcus), cube shape consisting of 4 or 8 (saranae) (Lay.1994).
Some microbes difficult colored with dyes that are alkaline, but easily visible with negative staining, the method of microbial mixed with Indian ink or nigrosin, then rubbed on glass object. Color substance will not stain the bacteria, but will color the surrounding environment of bachteria. With microbes microscope will appear colorless with black background (Lay.1994).
2.
Differential staining divided
into gram staining and acid-fast stain
Staining of bacteria that use more than one such dye staining, like acid-fast and gram staining. Explanation as
follows:
a.
Gram staining
Gram method or Gram staining is a method of differentiating species of bacterial into two large groups, the gram-positive and gram-negative, based on the chemical and physical properties of
their cell walls. The
method is named after its discoverer, Danish scientist Hans Christian Gram (1853-1938) who developed the technique in 1884 to distinguish between pneumococcal and Klebsiella pneumoniae bacteria.
With
the Gram staining method, the bacteria can be grouped into two, namely Gram positive and Gram negative bacteria based on the reaction or the nature of the paint. Or the nature of the reaction is determined by the composition of the bacterial cell wall. Therefore, Gram could not be done on microorganisms that do not have cell walls of bacteria such as Mycoplasma sp. Examples belonging acid resistant bacteria, which is of the genus Mycobacterium and some species of the genus Nocardia. Bacteria of both genera are known have a large number of substances lipodial (fat) in the cell wall, causing the cell wall is relatively impermeable to substances common color that bacterial cells are not stained by the usual staining methods, such as simple or Gram staining.
In gram staining, reagents that required are :
•
The main dye (crystal violet)
•
Mordant (iodine solution) is a compound which is used to intensify the main color.
•
Washer / laxative dyes (alcohol / acetone) the organic solvents used to fade main dye.
•
The second dye / paint cover (safranin) is used to color back the cells that has lost the main cat after treatment with alcohol.
Gram-negative bacteria are bacteria that do not retain methyl violet dye in the Gram staining method. Gram-positive bacteria will retain the dark purple dye methyl after washing with alcohol, while gram-negative bacteria. In the Gram stain test, a dye (counterstain) added after methyl violet, which makes all the gram-negative bacteria become red or pink. Testing is useful to classify these two types of bacteria based on differences in their cell wall structure.
Characteristic gram-negative bacteria:
-
The structure of the
cell wall is thin, about 10-45mm, three-or multi-layer coated
-
the cell Wall contains more fat (11-22%), peptidoglycan layer found in rigid, next to the small amount of 10% of the dry weight, not lactic acid.
-
Less susceptible
to penicillin compounds.
-
Not resistant
to physical disturbance
The characteristics of gram-positive
bacteria:
- The
structure of thick walls
- The
walls of his cell containing a more normal lipid
- Equity more susceptible to penicillin compounds
- Growth is inhibited significantly by dyes such as crystal violet
- Composition required more complex
- More resistant to physical disturbance.
Painting gram carried out in 4 stages. Namely
a. Provision
of primary colored paint (liquid crystal violet) color purple
b. Pengintensifan paint color with the addition of a solution of Mordant
c. Laundering (dekolarisasi) with acid alcohol solution
d. Giving your opponent the paint color paint safranin
Positive
gram Bacteria Negative
gram Bacteria
b.
Staining Acid Resistant
Staining was directed against bacteria
that contain high concentrations of
fat (difficult to absorb the dye),
but if the
bacteria were given special
dyes such karbolfukhsin
through the heating process, it will
absorb the dye and
will hold up without being able to laxative by the strongest laxative such
as acid-alcohol.
Because of these bacteria called acid-resistant bacteria
(AFB).
Staining techniques can be used to diagnose the presence of the bacteria that causes tuberculosis is Mycobacterium tuberculosis. There is several ways acid-fast stain, but
the most is the way by Ziehl-Neelsen.
Acid Resistant Bacteria (pink)
and the bacteria are
resistant acid (blue).
3.
Special
coloring to see
certain structures: flagella staining, spore staining,
capsule staining.
a. Spore Tint
Bacterial spores (endospores) cannot be colored with ordinary staining, special
staining techniques are required. Coloring Klein is
the most widely used spores. Endospores is Difficult
stained with Gram's method. For staining endspores,
needs to heating so that the malachite green paint
get into the spores, as well as in Basil coloring
where the carbol tint fuschsin should be heated
to pierce candle membrane of myolic acid of Mycobacterium.
Working principle:
Bacteria spores have thick walls so that the necessary heating for enlarged pores and the fuchsin dye can enter.
With washing, pores shrink back that causing
fuchsin dye cannot
be removed or pass away with acid
alcohol, while in the body of bactheria, the color of fuchsin are
released and take the blue color
from methylen blue.
How It Works:
•
Created the bacteria suspension, added with carbol and fuchsin
(the volume is same).
•
heated on low heat for 6 minutes
or in 80oC
water bath for 10 minutes.
•
Made preparations and dried.
•
Entered into H2SO4 1% for 2 seconds
•
Entered into alcohol so there
is no more red that flowing.
•
The preparation washed by water.
•
Colored with methylen blue for 1
minute and then washed and dried.
•
observe under a microscope.
a.
Flagellum staining
Flagellum staining with gives colloidal suspension
of Tannates acid salts that unstable, so it will formed thick precipitates
in the cell wall and flagella.
i.
Capsule staining
this staining use a
solution of heat violet crystal and solution of copper sulphate
as rinsing solvent and will produces pale blue
color in capsule. If we use water as rinsing solvent, the capsules can dissolved.
Copper salts also give color to the background, were colored dark blue.
4. Special coloring to look at other components and bacteria:
•
Neisser staining (granular volutin)
•
iodine staining (glycogen granules).
5. negative staining
Purpose : to
observe the morphology of organisms which are difficult to colored by the simple staining. Bacteria are
not colored, but the background is colored. This method is for bacteria that
difficult to colored like Spirochaeta.
The
way of negative staining :
Negative
staining, this method
is not bacteria coloring but to color background
become dark black. In this staining microorganisms
appear transparent (see-through). This technique is useful to determine the morphology and size of the
cell. In this staining, the spread did not heated or hard treatment with chemicals compounds, then the depreciation and
one form that is less so the cells can be obtained
by determining more precisely.
This method uses china
paints or inks nigrosin.
Negative
staining requires acidic dyes
such as eosin or
acid negrosin. Acidic dyes has negative charge of chromogen, it will not penetrate into the cells because the negative charge just
present on the bacterial surface. Therefore,
colorless cells easily
seen with a colored background.
REFERENCES
Dwidjoseputro,
D.1998.Dasar-Dasar Mikrobiologi, Malang : Djambatan
Hadiutomo.
1990. Mikrobiologi Dasar Jilid I. Jakarta: Erlangga
Lay,
Bibiana.W.1994.Analisis Mikroba di Laboratorium.Jakarta : Rajawali
[17th of
February 2013]
Waluyo,lud.
2010. Buku
Petunjuk Praktikum Mikrobiologi Umum. UMM. Malang
