Bacterial Cell Staining
Preliminary
The introduction of microbial forms (morphology), except microalgae must be
carried out in advance to be clearly observed. The purpose of coloring is:
1. Facilitate observation of cell shapes of microorganisms (especially bacteria).
2. Clarify the size of the body
3. Can observe the outer structure and structure of the microbial cells.
4. Seeing the reaction of the bodies of the dye given, so that the physical
properties, the chemistry of the body can be known. Thus, we can use coloring
as one way for bacteria classification. The success or whether or not the
coloring is very determined by the time of giving colors and the age of the tinged
(good culture age is 24 hours).
Various kinds of coloring methods
Direct coloring with base dyes
Generally dye substances are salts built by positive or negative-charged ions,
where one of these ions is colored. If a bacterial cell is a relatively negative cell
wall with a positively charged ion from a coloring, it causes the bacterial cells to
be colored. Coloring can be grouped into two groups, namely alkaline dyes and
acid dyes. If the positive ion of the coloring substance contains the color, the
dye is a base dye. And if the color is in a negatively charged ion, the dye is an
acid dye.
Tools
a. Bacterial culture is masted in previous practices and some pure cultures.
breakfast. Methylene Blue Dyes
C.. Violet crystal dye.
d. Carbolic fuhsin
Hey. Clean glass object and glass cover.
How it works.
a. Place an object of fat-free object on the work desk.
breakfast. Drop a drop of water in the middle of the glass of the object.
C.. By using the OSE needle that has been spent, take a little bacterial culture
that you are isolated in the previous lab or from pure bacterial culture provided
by your assistant.
d. Make a bacterial medan on the water placed on the glass of the object by
swifting the OSE needle that contains bacterial culture, so that a thin and even
mixed mixture is obtained.
Hey. Fixation over the Bunsen API with a distance of about 30 cm. From the
flame. In the
This fix is not too hot because it can damage the cell shape.
f. Drop one of the dye solutions provided by the assistant to a fixed preparation.
Methylene blu dyes will color cells in 30-60 seconds, crysty violet in 10 seconds,
and carbolic fuhsin in 5 seconds.
g. Wash with running water.
h. Dry the preparation by placing it between filter paper.
I. Observe the microscope by using an objective lens with a magnification of 100
times, and immersion oil.
a. Record and draw cell shapes and color microorganisms that you see.
Question:
1. Is the culture that your sister isolation is a pure culture, why?
2. What do you mean fixing the preparation?
Negative coloring or indirect coloring
One of the acid dyes is nigrosin or Chinese ink. Because the coloring power of
this substance is in negative ions and does not react with other negative ions of
bacterial cells, this dye does not color cells. In this case, what is colored is the
environment around the cell. In this way you can observe the forms of bacterial
cells and also clearly.
Tools
a. Bacterial culture isolation or culture results provided by the assistant.
breakfast. Nigrosin liquid or Chinese ink
C.. Glass fat free object.
Work
a. Drop sati fluid drops of nigosin or Chinese ink on the edge of the windshield.
breakfast. By using the OSE needle that has been spent, take a little bacterial
culture and suspend the droplets of nigrosin on the glass surface of the object.
C.. Flatten the bacterial suspension in nigrosin on the surface of the glass of this
object using other objects.
d. Let it dry at room temperature, and observe with a microscope (100 x
objective lens magnification), and use the oil immersion.
Hey. Picture of a bacterial shape you see.
The following ways of coloring are differential coloring techniques, as
One way in classifying bacteria.
Gram coloring
Gram coloring is a very common coloring in the field of bacteriology. With this
coloring, the bacterial group can be divided into two, namely groups of gram
positive bacteria and gram negative bacteria. There are many modifications of
this coloring technique, but all these techniques are based on the same
principle, namely:
1. Coloring microorganisms with basic dyes, namely with violet crystals or violet
gentians.
2. Color fixation, which is to strengthen the basic color attachment, for example,
it is done with iodine salts (modification of the Lugol solution).
3. Washing or removal of basic colors with alcohol, acetone, or a mixture of
alcohol with acetone.
4. Return coloring with comparative dyes or contrast different from basic dyes,
namely to color the cells that have been lost in color by color removal. For
example in this case saffranin or carbolic fuhsin ..
Bakeri which after being colored by the basic coloring color is not erased by
alcohol will be violeted because it is colored by Violet crystals, and no longer
absorbs contrast dyes. The bacterial group that has this trait is grouped into
gram positive bacteria. Meanwhile, bacterial groups that have been colored with
basic dyes and colors are erased after being treated with alcohol, it will absorb
saffranin dye or carbolic fuhsin used as a contrast dye, so that in the
preparation it will look red (color Safranin or carbolic fuhsin). Such bacterial
groups are called gram negative bacteria.
Tools
a. Young culture isolated or provided by the assistant.
breakfast. Violet crystal solution
C.. Iodine saline solution.
d. Alcohol 95%.
Hey. Safranin solution
f. Glass object
Work
a. Make bacterial apusabn on dry and clean glass objects.
breakfast. Fixation on the flame of the bunsen or in the air.
C.. Coloring with violet crystal solution for 1 - 1.5 minutes.
d. Wash with distilled water.
Hey. Tetesi with Iodine salt solution, and leave for 1 minute.
f. Wash with a 95% alcohol solution until the color is erased, usually for 0.5
minutes (30 seconds).
g. Wash with water.
h. Coloring with saffranin or carbolic Fuhsin for 2 minutes.
I. Wash with water, and exhaust excess water using suction paper, without
rubbing the preparation.
a. Dry in the air or on the flame of the bunsen.
k. Observe under a microscope with a magnification of 1000 times. (Remember
the use of immersion oil).
l. Write down your observation.
namely groups of gram positive bacteria and gram negative bacteria. There are
many modifications of this coloring technique, but all these techniques are
based on the same principle, namely:
1. Coloring microorganisms with basic dyes, namely with violet crystals or violet
gentians.
2. Color fixation, which is to strengthen the basic color attachment, for example,
it is done with iodine salts (modification of the Lugol solution).
3. Washing or removal of basic colors with alcohol, acetone, or a mixture of
alcohol with acetone.
4. Return coloring with comparative dyes or contrast different from basic dyes,
namely to color the cells that have been lost in color by color removal. For
example in this case saffranin or carbolic fuhsin ..
Bakeri which after being colored by the basic coloring color is not erased by
alcohol will be violeted because it is colored by Violet crystals, and no longer
absorbs contrast dyes. The group of bacteria that have this trait is grouped into
gram positive exhibits. Meanwhile, bacterial groups that have been colored with
basic dyes and colors are erased after being treated with alcohol, it will absorb
saffranin dye or carbolic fuhsin used as a contrast dye, so that in the
preparation it will look red (color Safranin or carbolic fuhsin). Such bacterial
groups are called gram negative bacteria.
Tools
a. Young culture isolated or provided by the assistant.
breakfast. Violet crystal solution
C.. Iodine saline solution.
d. Alcohol 95%.
Hey. Safranin solution
f. Glass object
Work
a. Make bacterial apusabn on dry and clean glass objects.
breakfast. Fixation on the flame of the bunsen or in the air.
C.. Coloring with violet crystal solution for 1 - 1.5 minutes.
d. Wash with distilled water.
Hey. Tetesi with Iodine salt solution, and leave for 1 minute.
f. Wash with a 95% alcohol solution until the color is erased, usually for 0.5
minutes (30 seconds).
g. Wash with water.
h. Coloring with saffranin or carbolic Fuhsin for 2 minutes.
I. Wash with water, and exhaust excess water using suction paper, without
rubbing the preparation.
a. Dry in the air or on the flame of the bunsen.
k. Observe under a microscope with a magnification of 1000 times. (Remember
the use of immersion oil).
l. Write down your observation.
Tools
a. Culture mycobacterium and E. coli
breakfast. Fuhsin carbolic solution.
C.. Alcohol solution of acid (64 ml of HCl in 1000 ml of 95% ethanol solution)
d. Blue methylene solution.
Hey. Water bath with platform
f. Glass object
Work
a. Make a ply of mycobacterium and E. coli on the object's glass and fixation.
breakfast. Place the preparation on the wire plate on the water bath.
C.. Place a piece of suction paper on the smear and wet with the carbolic
coloring dye, and keep your staff dry down by continuing to give dyes. Avoid
giving excessive colors. Do this for 5-10 minutes.
d. Wash with water.
Hey. Wash with acid alcohol for 10-30 seconds.
f. Color with Methylene Blue for 30 seconds without heating.
g. Wash with water and dry it.
h. Observe with a microscope with a magnification of 1000 times. (Remember
the immersion oil). The acid-resistant bacteria will be red and that can not be
acid resistant will be blue.
Coloring structure
To color parts of the cell, a special coloring technique is needed, among others:
Endospora coloring and capsule coloring.
Endospora coloring
Bacillus species and clostridium can produce endospores
Resistance, which can live for a long time though in a poor state of environment,
such as the heat and the presence of toxic chemicals. The controller is resistant
to coloring, and once tiled will be washed. There are several methods that can
be used to color endospores, namely Dorner methods and Schaefer and Fulton
methods.
A. Dorner Method
Tools
a. Distilled water.
breakfast. Bacillus cereus bacteria and clostridium sporogenes.
C.. Carbolic fuhsin
d. Nigrosin
Work
a. Place 5 drops of distilled water in each 1 tube reaction and make a
concentrated suspension of B. Cereus and CL. Sporogenes.
breakfast. Give in the same amount of carbolic Fuhsin in these suspension.
C.. Place it in a water bath for 10 minutes.
d. Make nigrosin staining.
Hey. Observe with a microscope and record the results.
B. Method of Schaeffer and Fulton
Tools
a. Culture B. Cereus and CL. sporogenes
breakfast. Malap
C.. Safranin solution.
Work
a. Make Bacterial B. Cereus and Cl. Sporogenes and fixation.
breakfast. Coloring with green malap on the RAM wire on a water bath for 5
minutes. (Coated with suction paper).
C.. Wash with distilled water.
d. Color with Safranin for 30 seconds.
Hey. Wash with water and dry it.
f. Observe with a microscope, record and image results. Endospora will be
green and other parts of the cell will be pink.
Capsule coloring
This staining function is to see if the bacteria are observed in capsules or not.
Tools
a. Baisan bacteria Alkalets Viscous, or aerobacter aerogenes in milk or so
breakfast. Glacial acid acid
C.. Carbolic fuhsin
d. CUSO4 20%
Hey. Violet 1% crystal solution
f. Physiological saline solution
g. Lid
How it works (Welch method)
a. Place 5-6 loop loops in milk on the glass object.
breakfast. Cover with glacial acetic acid, and let no more than 10 seconds.
C.. Wash by pouring carbol fuel, and let the dyes flow.
d. Wash Fuhsin Fuhbol with a physiological salt solution; Don't use water.
Hey. Cover with glass cover, and observe with a microscope with a
magnification of 1000 times (remember using the oil immersion). The capsules
will be pink and dark red cells.
How to Work (Anthony Method)
a. Make the astony as follows: If you use milk culture, share one loop on the
glass object and let it dry. If you use Dextrose broth, or culture so that it is used,
place a drop of serum on the glass object and suspect the culture into it, and
spread it to form a thin layer and let it dry in the air.
breakfast. Color with Violet crystal 1% for 2 minutes and do not heat it.
C.. Wash with a 20% CUSO4 solution.
d. Dry and observe with a microscope with a magnification of 1000 times with
immersion oil.
