FORMAT OF ANIMAL DEVELOPMENT PRACTICAL REPORT:

Practical report is typed writting using Times New Roman font, font size 12,
spacing 1.5, margin 4 cm left, 3 cm top, right and bottom on Quarto Paper (A4).
- The report consists of:
Cover Page
I. INTRODUCTION
a. Aims
b. Benefits
II. MATERIALS AND METHODS
a. Materials
b. Methods
III. RESULT AND DISCUSSION
a. Result
b. Discussion
IV. CONCLUSION AND SUGGESTION
a. Conclusion
b. Suggestion
REFERENCES
Terms for references:
- Indonesian or international journal min 5
- Indonesian or international text book min 5
- Please attach one international journal, it should be citated in the
discussion and marked in the attachment
- Figure or data from the practical class should be attached
 ALIZARIN RED STAINING

By:
Name : Refhany Afidha Syafillina
Student ID : B1B016010
Group : VII
Subgroup :2
Assistant : Monica Widianti

PRACTICAL REPORT OF ANIMAL DEVELOPMENT

MINISTRY OF RESEARCH, TECHNOLOGY, AND HIGHER EDUCATION

JENDERAL SOEDIRMAN UNIVERSITY
FACULTY OF BIOLOGY
PURWOKERTO
2017
 I. INTRODUCTION
A. Aims
The aims of this practical class are student are able to do the Alizarin
staining and explained bone calcification in embryo.

B. Benefits
The benefits of this practical class are can know the ordinances order
alizarin red staining, knowing how alizarin red can color the fish's body, and can find
any solution that is used.
 II. MATERIALS AND METHODS

A. Materials
The tools that used in this practical class are surgical instrument, watch
glass, specimen bottle or bowl, and dropper pipette.
The materials that used in this practical class are nilem fish, alcohol 96%,
alizarin red stain, A solution, B solution, C solution, KOH 1% solution, glycerin
solution, and aquades

B. Methods

The method used in this practical class are:

1. Nilem fish was prepared, died use ice.

2. Put to aquades 10 minutes
3. Fixed use 96% alcohol 12 hours
4. Put aquades 10 minutes
5. Put 1% KOH 12 hours
6. Put Alizarin red 7 hours
7. 2% KOH 1 ml each 30 minutes (max 5 times) was dropped
8. A solution was put 15 minutes
9. B solution was put 15 minutes
10. C solution was put until group observation
 III. RESULT AND DISCUSSION

A. Result

Figure 3.1 Fish with aquadest Figure 3.2 Fish with alcohol 96%
12 hours

Figure 3.3 Fish with KOH 1% Figure 3.4 Fish with Alizarin red +
KOH 2%

Figure 3.5 Fish with A solution 15

minutes

Figure 3.6 Fish with B solution 15

minutes
 Figure 3.7 Fish with C solution
untill group observation

Table 3.1 Procedure of Alizarin Red Staining

TIME SOLUTION DESCRIPTION
10:55 Aquadest The fish is fresh
11:08 Alcohol 96% The fish is pale
23:07 Aquadest The fish is starting to disaseas
11: KOH 1% scales on the dorsal part began to peel and fish flesh began
to look
15: Alzarin red The scaless fish is going pale, skin pale, the caudal was
broken some part was coloring
7:04 A solution The caudal was broken, the head was transparent, digestive
system was seen, the ribs was seen
7:19 B solution Digestive was clearly seen transparent, the bone and ribs
clearly seen transparent, fins are broken.
7:35 C solution All body was transparent, some part of the body are staining
by alzarin red, but some body are no

Table 3.2 Stained Bone Data Group VII

Subgroup Stained Bone
1 Maxila, Neurocranium, Angular, and all
the bone we can see but not staining
2 Neurocranium, Operculum, Vertebrata a
half, The ribs are seen but not staining,
the fin are seen but not staining
3 Neurocranium, angular, maxila, pelvic
fin girdle, articular
 B. Discussion

Based on our results, our group get stained bone is neurocranium, operculum,
Vertebrate a half, the ribs are seen but not staining, the fins are seen but not staining.
Group 1 get stained bone is Maxila, neurocranium, Angular, and all the bone we can
see but not staining. And the last group, the third group is neurocranium, angular,
maxila, fin pelvic girdles, articular. There are some similarities in the bone stained
from 3 three groups. According to Ville (1988) bone staining techniques with
Alizarin Red method, there are several sections stained bone is the spine, the gill
cavity, skull, eye socket, the ribs, the dorsal fin, pelvic fins, pectoral fins, tail fins and
dorsal fin. It is not in accordance with the observations.
Based on the above results, it can be seen that not all bones can be stained by
Alizarin Red solution, this is because the process of bone calcification occurs in each
time so as not calcified simultaneously. Similarly, the basic cranial bone and the
entire cranial bones showed discoloration if done Alizarin Red staining. The change
in color appearance varies from red to deep red depending on its growth (Vilmann,
1969). The differences in development occur because some of the embryonic bones
are deposited in undifferentiated mesenchyma. Whereas, in other parts of the body
there is bone formation which is preceded by a temporary liver cartilage system. The
ossification process of these two things is essentially the same (Djuhanda, 1983).
The bones have calcified will be dark red. According Soeminto (2000) were
not stained bones will be colored transparent. Bones are not stained or may not
calcified, when the colored solution may not be absorbed into all parts, the calcium
content of a little, old sertaterlalu in 1% aqueous KOH immersion that causes the fish
be destroyed. KOH transfigures lipids and proteins that reside in the fish muscles so
that the fish becomes transparent. The process of color formation in fish bones is due
to the dyestuff that is bound by the calcium in the bone matrix. The main external
matrix component which plays a role in the process of hardening of the bone is a
calcium salt. Calcium salt deposition processes occur gradually.
The colored part of each group varies, this is influenced by several factors
such as the length of time of staining, the staining stage, internal factors and external
factors (Sufyani, 2009). Changes in the appearance of the color varies from red to
dark red depending on the growth (Vilmann, 1969). The different results in Alizarin
Red staining caused by several factors, namely:
1. The amount of calcium mineral content in the bone matrix.
According to Fisher et al., (2003) bone calcification will determine bone
quality, bone quality will determine bone maturity.
2.The bone has not been calcified,
So that Alizarin red dye can not bind calcium in the matrix if calcification has
not occurred in a bone matrix called osteocytes (Kalthoff, 1996).
3. Soak the fish in solution.
The use of 2% KOH solution in some groups, allows many bones to be
colored because the fish becomes more transparent and the skeleton is more
pronounced with increasing KOH concentrations (Jasin, 1989).
According Puchtler (1969), Alizarin Red dye solution, causing the coloring
process skeleton to scarlet or purple indicating that the bones had been stained with
the color of the process of calcification has occurred in the test animals used. Factors
affecting alizarin red staining, namely:
1. Foods is influential in the calcification process. This is particularly true of the
supply and availability of minerals such as calcium and phosphorus, which are the
major organic components of bone.
2. Parathyroid hormone, calcitonin, and vitamin D are responsible for normal blood
levels of calcium, which will affect the calcification process. Calcitonin has an
action in lowering blood calcium levels and inhibits bone resorption so that it
affects the calcification process.
According to Chuang, et al., (2007), alizarin red staining reveals the
accumulation of calcium from time-to-time. Alizarin Red very clearly describes the
progress of mineralization in bone formation. Factors that influence the success of
alizarin red staining are:
1.The time of fish immersion
2.The timeliness of solution removal
3. Concentration of solution used
4. Age of fish used, the more age of the animal is used then the calcified bone more
and more.
Factors affecting calcification are foods that have an effect on the
calcification process. This applies particularly to the adequacy of supplies and the
availability of minerals such as calcium and phosphorus which are the major organic
components of bone. Lack of calcium or phosphorus in the diet results in bone
fragility. When a situation where calcium is adequate but vitamin D is lacking there
is a disruption in mineral absorption. Calcitonin, parathyroid hormone, and vitamin
D are responsible for normal blood levels of calcium that will affect the calcification
process. Calcitonin is a hormone derived from the follicular cells of the thyroid
gland. The hormone has an action in lowering blood calcium levels that inhibit bone
resorption affecting the calcification process (Jasin, 1989).
Factors that may affect alizarin staining are pH, other metal ion compounds,
and reagent concentrations. Alizarin Red will be able to bind Cl- ions at a relatively
low pH (2,8). For metallic ions such as Ca2 + will be more effective at alkaline pH
(11-12.5). Therefore, before Alizarin Red added substrate was added 1% KOH
solution to facilitate the condition of alkaline atmosphere in fish (Somasundaran,
1986).
Factors that influence the success of Alizarin red staining according to
Junqueira (1982), they are :
1. Nutrition, Bone sensitive to several nutritional factors. Insufficiency of protein in
the diet causes a deficiency of the amino acid and vitamin C is necessary for
collagen synthesis by the osteoblasts.
2. Age, fish that are too young yet visible presence of calcium so that staining can
not be identified.
3. Long time, the time interval required for soaking the fish against alizarin red is
very influential on the identification of embryonic bone.
4. Backup calcium, calcium deficiency causes imperfect organic matrix of bone
calcification, therefore alizarin red can not bind the calcium if the calcium needs
less.
The KOH mechanism of muscle transfers is that KOH will degrade lipids,
leading to LDL (low-density lipoprotein) in muscle. LDL will lead to tissue damage
and repair occurs and eventually lead to plaque growth. The process of endothelial
dysfunction in the muscle associated with nitric oxide (NO). NO not only plays a role
in relaxation of smooth muscle cells but also inhibits activation, adhesion, platelet
aggregation and prevention of vascular smooth muscle cell proliferation and
leukocyte adhesion in the endothelium lining. In endothelial dysfunction, the
vascular injury results in a series of maladaptive phenomena resulting in an
unfavorable vascular response. As a result the lipid content was disrupted. The
muscles will look transparent in the presence of this disturbance process (Ge et al.,
1995).
 KOH plays a role to transcend muscle and skeleton so that the bones in it can
be seen after Alizarin Red staining. Yang et al. (2015) has conducted research on Ca2
+ active sites against Alizarin Red solution. KOH helps coordinate / bind calcium
ions with hydroxyl chains from Alizarin Red (deprotonated) solution and form a red
Ca-ARS complex. In addition, Konishi et al. (2010) in experiments with dried fish
specimens show that KOH performs muscle and skeleton digestion, causing muscle
and skeleton to become transparent. So the bones in it can be seen clearly and
colored when added Alizarin Red solution.
Specimens were destroyed can be caused due to wrong treatment. Example
is the addition of KOH, KOH which can make the muscles become transparent, if
excessive in marinated it will make her muscles were destroyed. in addition to the
KOH soaking too long, spesiment be destroyed because the size is too small, so the
specimens are easily destroyed when KOH soaked too long. KOH solution is a
strong alkaline solution which is hydrophilic and corrosive if in solid form. This
solution also reacts with fat and can cause irreversible damage if it comes in contact
with skin. KOH solution at alizarin red staining is used to make the transparency of
the body of the fish so that internal organs including bone in his body can be seen.
This relates to the nature of KOH compounds that can be used for the saponification
reaction, the saponification process of the fatty acid and the hydrophilic nature so as
expected according to the circumstances osmotic muscle cells of fish. The fatty acids
found in fish muscle tissue reacts with KOH to form compounds carboxylate salt and
water molecules. The effect of the reaction that causes the muscle tissue of fish
becomes transparent (Hill & Finster, 2010).
 IV. CONCLUSION AND SUGGESTION

A. Conclusion

Based on the result and discussion, it can be concluded that:

The Alizarin red staining procedure was performed by immersion in a 10-
minute distilled aqueous solution, 96% alcohol for 12 hours, distilled for 10 minutes,
KOH 1% for 12 hours, Alizarin dye solution for 5.5 hours, A purifying solution for
15 minutes, B for 15 minutes, and a solution of C purifier to observation. Alizarin red
staining is used to detect bone calcification processes. The bone may be stained
because the Alizarin red dye is bound by calcium in the bone matrix. Calculated or
colored bone fragments are neurocranium, operculum, vertebrates a half, The ribs are
seen but not staining.
B. Suggestion

Practikan should be more accurate and timely in the pouring of solution,

soaking, until the replacement of the solution so that the results obtained maximal
and not destroyed and be careful when bringing fish because the fish more and more
easily destroyed.
 REFERENCES

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Djuhanda. 1983. Embriologi Perbandingan. Bandung: Armico.

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Developmental Biology 264: 64-76.

Ge T, Hughes H., Junquero D.C., Wu K.K., Vanhoutte P.M., and Boulanger C.M.
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Calcitat-Apatite System. International Journal of Mineral Precessing, 18,
pp 287-296.
Sufyani, Fahrullah. 2009. Pengaruh Ion Pengganggu Al (Iii) Dan Fe (Iii) Pada
Penentuan Zn (Ii) Dengan Alizarin Red S (Ars) Secara Spektrofotometri.
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Villee, C. A., W. F. Walker, and R. D. Barnes. 1988. Zoologi Umum. Jakarta :

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Yang, Jin-Xiang, Yan-Bin H., Li-Na L., Jun-Bo L., & Xiao-Liang S. 2015.
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