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By :

: Nurul Inas Tsabitah

: B1K014024
: IV
: Atina Istiqomah




A. Background
Bone is always formed within the framework of connective tissue (connective
tissue) that have been there before. Differences in development occurs because the
embryo some of the bones were deposited in the mesenchyme which have not
been differentiated (bone formation intra-membrane), whereas in other parts of the
body occurs bone formation was preceded by a system of cartilage leverage the
temporary (Setyawati 2011 ).
The detection process of calcification in the bones of fish embryos can be
followed its development with alizarin red staining method, this method can be
carried out in sequence at different ages nilem fish embryos in order to obtain
information about the process of calcification. Practicum alizarin red staining was
used embryos aged 10-20 days (Yatim, 1983). Alizarin red is the process of bone
staining with alizarin dyes help to determine the extent of calcified bone, so it can
be seen the age and maturity level of an animal (Soeminto, 2004).
Alizarin red is a method to determine bone formation in embryos or method
for detecting bone calcification process in the embryo. Embryonic bone that
stained by alizarin red will look dark red. The red color comes as a given dye is
bound by calcium in the bone matrix. Giving alizarin red can be done in stages at
various age levels of the embryos, generally occurring intra bone ossify
membrane faster than bone formed endrokondral (Mardanung, 1985) .Alizarin is a
compound soluble in water, synthesize shape dAPT water soluble than an
indicator, Alizarin Red S-CTAB an ion binder liphophilic which makes the sensor
can be used in PVC (Gupta et al., 2009).
Intramembranous bone ossify formed faster than in endokondral. Ossification
occurs more rapidly in bone formed intra membrane. The establishment of order
in the fish embryo begins on the fifth day of incubation marked by events
condensing mesenchyme pre cartilage. Condrification occurred on the eighth day
of incubation and ossification on the ninth day (Jasin, 1989).

The reason why wearing preparations nilem small fish, because fish nilem
easily observed. Nilem fish embryos used due to the limited number of fetal mice.
The price is cheap, easy to obtain, and the time required for staining too little. The
whole is the reason for using fish nilem as the preparation.
B. Function
Alizarin Red practicum goal is to equip students to work on alizarin red
staining procedures and explain the process of calcification of the bones.


A. Materials
The tools used in the lab Alizarin Red Is a watch glass, tissue, bath
preparations, injection syringes, and paper labels.
The materials used in fish Redadalah Alizarin prektikum nilem (Osteochilus
hasselti) aged 10-15 days, a solution of 96% alcohol, alizarin red dye solution,
KOH solution of 1% and 2%, purifying solution A, B and C, a solution of pure
glycerine , physiological saline and distilled water.
A. Methods
The method that used in this lab are as follows:
1. Fish killed by placed it on ice.
2. Nilem fish moved to physiological NaCl solution.
3. The fish put into a clear tube which already contains 96% alcohol solution
and left for 12 hours.
4. After 12 hours, the fish were transferred to distilled water for 10 minutes and
subsequently replaced with 1% KOH solution for 1 hour 15 minutes.
5. After that, the fish were transferred to a solution of alizarin red and allowed to
stand for 5 hours.
6. If the fish has been stained, then the fish moved to 2% KOH solution.
7. After 10 minutes, the fish was moved again to the purifying solution A for
one hour, then continued by purifying B for one hour, and the last purifier C
until the time of observation.
8. After a period of observation, fish bones stained sections were observed and
identified each part.


A Results
A. Observations Data of Alizarin Red Staining Solution

Description :

Figure A: Fish before it is treated

Figure B: Fish after being put on alcohol 96%
Figure C: Fish after being put on distilled water
Figure D: Fish after being put on KOH 1%
Figure E: Fish after being put on alizarin red

6. Figure F: Fish once incorporated on purifying solution A

7. Figure G: Fish once incorporated purifying solution B
8. Figure F: Fish once incorporated purifying solution C (observation day)
A. Table 1. Data Pengamatan Pemindahan Larutan Alizarin Red



Preparation Condition


10.00 - 22.00
22.00 - 22.10

Alkohol 96%

22.10 23.40

KOH 1%

23.40 - 04 .40

Alizarin Red

04.45 - 05.45

A Purifiers

05.45 06.45

B Purifiers

06.45 ~

C Purifiers

Pale, stiff, intact, and curved

Pale, stiff, intact, and curved
The muscle isnt transparent yet and the
fish body started to soften
The bone and muscle started to shown
The bone looked more transparent and
the body also became more transparent
Intact and the body looked more
The on still clearly shown or transparant

B. Table 2. Observation Data of Stained Bone





Stained Bone
Operculum, ceratobranchial, parietal bone, frontal bone,
dorsal fin, anal fin, and pectoral fin
Ceratohyal, parietal bone, otolith, caudal vertebrae, frontal
bone, ceratobranchional, parasphenoid, rib, and caudal
Abdomal fin, anal fin, frontal bone, ceratobranchial, tail
bone, and dorsal fin bone
Dorsal fin and abdominal fin

B. Disscussion
Results obtained from the lab Alizarin Red staining on fish for group 1 is that
the stained sections are the operculum, ceratobranchial, parietal bone, frontal
bone, dorsal fin, anal fin, and pectoral fin. There are differences in staining results
between each group in the same group with the same specimen. Group 2 the
sections that stained are on the part of the skull Ceratohyal, parietal bone, the
otolith, caudal vertebrae, frontal bone, ceratobranchional, parasphenoid, rib, and
caudal vertebrae. Group 3 were stained in Ceratohyal, parietal bone, the otolith,
caudal vertebrae, frontal bone, ceratobranchional, parasphenoid, rib, and caudal
vertebrae. Group 4 none of the bones that stained. According to members of the
group this can happen due to lack of Alizarin Red concentration that given. 5
groups were stained on the dorsal fin and abdominal fin. The bones that have the
appearance of red, from bright red to dark red. This is consistent with the
explanation Villee et al., (1988), that are characterized by bone relieve alizarin
crimson, if the bone has calcified. According to Huffman et al (2007), bone is
uniquely experienced vascular tissue mineralization as part of its development
process. Bone mineral has an important role on the function of the spine,
including structural support, a reversible storage of calcium and phosphorus, and a
place to store the content of metal and carbon.
Alizarin Red staining process used on specimens of fish in some groups
experience the difference. Differences may occur due to several factors, including
the age difference of fish that used, the difference in treatment when using various
solutions, and fault practitioner. But the main in answering the differences is to
understand the concept of Alizarin Red staining itself. Alizarin Red staining
technique used to determine the process of calcification or ossification of the
bone. Which has calcified bone will be known after Alizarin Red staining method.
Interpretation of calcified bone has to be red to dark red. Calcification on the bone
is influenced by the age of development. Mineral calcium is deposited in bones
would be more comparable with the growth of age. As stated Anat (1969), that
changes the appearance of the color varies from red to dark red depending on
growth. Differences in the development of the case because some of the bones

were deposited in the mesenchyme embryos that have not differentiated.

Meanwhile, in other parts of the body that preceded the formation of bone by
cartilage leverage the system temporarily. Ossification process two things are
basically the same (Djuhanda, 1983).
Practicum Alizarin Red this time using a solution of 96% alcohol, distilled
water, 1% KOH, 2% KOH, alizarin red, aquades, purifying solution B and
purifying solution C. The first step Alizarin Red staining method by immersing
the fish to a solution of alcohol 96% for 12 hours. Giving alcohol serves as a
fixative in order to turn off the muscle cells of the fish without damaging the cell
structure. The next step is the provision of clean water for 10-15 minutes which
serves to rinse and neutralize the fish of a solution of 96% alcohol. Furthermore,
clean water is replaced with KOH 1% for 1 hours, 1% KOH solution serves to
create muscle tissue in fish be transparent or translucent. The next step is the
provision of Alizarin Red solution for 4-5 hours, dye alizarin here serves as a dye
in the bone that has calcified and attached to the bone so the bone matrix stained.
2% KOH solution used flexibly if found parts of fish which have not been looked
transparent. Furthermore, given the purifying solution A, solution B and solution
C respectively for 1 hour which aims to remove excess solution of Alizarin Red
on bone test animals. This is in accordance with the opinion of Partridge et al.,
(2009) which states that alizarin red is used to look for calcium deposits.
Fish bones can be stained because of the calcium in the bones do bond with
alizarin red dye which has a red color, so that calcium has a cationic charge of the
electron 2 binds to the anion part of alizarin (Daniela et al., 2012). The process of
growth and development of bone tissue that is highly dependent on extracellular
matrix mineralization. According to Huffman (2007), Bone is a vascular tissue
that becomes mineralized during the development program. Calcium and
phosphorus play an important role in the process of bone growth, because the
bones are dynamic continuously formed and resorbed (Sutekyet al., 2006). Factors
that affect the calcification is a major extracellular matrix component which plays
a role in bone hardening process of which is the calcium salt. The process of









intramembranous bone ossify faster than the bone formed endokondral (Jasin,
1989). Another factor is a factor that affects soaking with solutions such as KOH

and alcohol must be considered the timing of each of the soaking solution.
Another factor is the age of the specimens used, the older the specimen used the
more stained the specimen anyway. Collagen degradation as by the activity of
bone-forming material bromelain may also inhibit calcification of cartilage in the
joint metacarpus and metatarsus fetus (Setyawati et al., 2011). Fish bones can be
stained because of the calcium in the bones do bond with alizarin red dye which
has a red color, so that calcium has a cationic charge of the electron 2 binds to the
anion part of alizarin (Daniela et al., 2012).


A. Conclusions
Based on the objectives and discussion, it can be concluded as follows:
1. Alizarin red dyes are used to detect the presence of bone calcification.
2. The parts of calcified bone in the observation group 3 is the pectoral fins,
dorsal fin and tail fin.
1. Factors that influence the success of Alizarin Red staining is the presence or
absence of calcified bone and treatment using a solution.
B. Suggestion
Suggestions for this lab is to utilize the free time to discuss things that are
poorly understood in the event lab.


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