Lab Activity VI

ALIZARIN RED STAINING

Day : Thursday
Date : October 11th, 2018

Name : Pratiwi Kusuma K

Student ID : B1B017007
Group : VII
Subgroup :2
Assistant : Monica Widianti

LABORATORY OF ANIMAL STRUCTURE AND DEVELOPMENT

FACULTY OF BIOLOGY
JENDERAL SOEDIRMAN UNIVERSITY
PURWOKERTO

2018
 I. INTRODUCTION

A. Aims

The aims of this practical class are:

1. To do the procedures of alizarin red staining,

2. To know the process of calcification bone in fish.

B. Benefits

The benefits of this practical class is this practicum gives us the advantage of
making us more focused and having a sense of cooperation between groups.
 II. MATERIALS AND WORK PROCEDURES

A. Materials

The tools that used in this practical class are syringe, and container for specimen.
The materials that used in this practical class are fish, aquadest, KOH 1% and
2%, alcohol 96%, alizarin red, A solution, B solution, and C solution.

B. Work Procedures

The work procedures that used in this practical class are :

1. All tools and materials that will be used are prepared,
2. Fish which has been weakened in ice water is transferred to the container and fill
the container with aquadest for 10 minutes,
3. After 10 minutes, aquadest then replaced using a syringe, and replaced with 96%
alcohol for 12 hours,
4. After 12 hours in 96% alcohol solution, replaced solution with aquadest again for
10 minutes,
5. After 10 minutes, aquadest is replaced with 1% KOH solution using an injection
syringe,
6. After 7 hours, 1% KOH solution is replaced with alizarin red solution,
7. After 10 hours, if the fish muscle is not transparent, alizarin red solution is replaced
with 2% KOH solution,
8. After 30 minutes, 2% KOH solution is replaced with a purifying solution A,
9. After 30minutes, a purifying solution A is replaced with a purifying solution B,
10. After 30minutes, the purifying solution B is replaced with C purifying solution and
is waited until observation,
11. Every transfer of solution, changes seen in fish are observed, recorded, and
photographed for documentation,
12. After various treatments for fish, the stained bone was observed, photographed,
and recorded,
13. The data obtained is compared with other group data.
A. Discussion

Based on the results of practicum that have done, we get the results that bones
of all fish that were used for the experiment of all groups were successfully stained.
Mostly based on entourage data, the portion of bone that has been stained by alizarin
red is the cranial bone and the other parts that are stained show different results. In
groups 1 and 3 the part that stained is cranial, in group 4 the parts that stained are
cranial, eyes cavity, back bone, and fin. The sections that are stained in each group are
different, this is influenced by several factors including the length of coloring time, the
coloring stage, pH, other metal ion compounds, reagent concentrations, alizarin will
be able to bind Cl-ions at a relatively low acidic pH (2.8). Whereas for metal ions such
as Ca2 + will be more effective at alkaline pH (11-12.5) (Tores & Eliana, 2014).
Other reasons for differences in staining are caused by process of bone
calcification occurring in time, so that it does not simultaneously calcified. Differences
in development occur because some of the embryo-bones deposited in the
mesenchyme are not differentiated. Former of ossification is mesenchyme tissue that
differentiated into bone, and endocondral ossification are differentiated into cartilage.
Whereas, in other parts of the body bone formation occurs which is preceded by a
temporary supporting cartilage system. The ossification process of these two things is
basically the same (Mohammed, 2018). For our group, the fish bone that has been
stained by alizarin red are the cranial, operculum, dorsal fin, ventral fin and caudal fin.
Fins of fish can be stained first compared to other parts of the bone due to the narrow
area of the fin, and has a thin diameter so that it can more easily absorb alizarin red
staining (Kessels et al., 2014). For internal factors that can cause differences in
staining, one of them is the process of ossification occurs in 5 periods, namely
embryonic period (mandible, maxilla, humerus, radius, ulna, femur, and fibia), fetal
period (scapula, illium, fibula), (young bones: epiphisis in limbs, carpal, tarsal, and
sesamoids), (juvenile bones: scapula, ribs, hip / waist bones, adult bones. (Soemito,
2002).
In the results of our group's experimental data, it shows that the fish muscles
have been transparent. Transparent of muscle occurs because the influence of addition
KOH 1% and 2% , this is in accordance with Haga et al. (2018), that muscle and
connective tissue are transparent / clear by clearing process with KOH. KOH
transparents lipids and proteins that are in the fish's muscles so that the fish becomes
transparent. Longer incubation (for over 4 weeks or several months) caused the
specimens to become completely transparent without the destruction of tissues, longer
incubation was needed to increase the transparency of the internal organs.
In the staining experiment, the skin and muscle exfolization process is one of
the important processes that can affect the success of staining. This exfolization
process begins at the fixation stage, this fixation process requires a different time for
each specimen. It depends on the species, maturity (embryonic or adult), body size,
and organ/body part made preparation. The more ticker and mature the tissue, the more
times are needed. The process of color formation in fish bones is caused by the dyes
that are given to be bound by calcium in the bone matrix. The main external matrix
component that plays a role in bone hardening process is calcium salt. The process of
calcium salt deposition occurs gradually (Lutfi, 2016).
Based on the results of experiment in our group, the condition of the fish still
intact after being carried out in parallel with the alizarin red staining stage, there was
only damage to the tail of the fish, if the damage occurs in the body of the fish, it can
be caused by the addition of too much or too often KOH. According to Haga et al.
(2018), that long-term immersion in a solution containing KOH at high concentrations
usually lyses tissue and destroys the specimen because KOH have work in making fish
bones soft and transparent.
Alizarin method has the advantage that it is more practical and economical
because the type of chemical used is only a little, can observe the bones in the embryo
or animal as a whole without separating and damaging the shape of the part, and also
can see the shape of bone abnormalities in the embryo. But the weakness of this
method is that only hard bones are colored while cartilage is not stained so it cannot
observe the cartilage that is formed and cannot distinguish cartilage and hard bones in
the embryo, the process takes a long time so it is not time efficient, and easily damaged
because the embryos that are stained with the Alizarin method will be very soft and
easily destroyed if exposed to a vibration that is quite hard (Geneser, 1993).
 A. CONCLUSION AND SUGGESTION

A. Conclusion

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

1. Alizarin red staining procedure on fish begins with the fish is killed by freezed,
then added distilled water for 10 minutes, after that the distilled water is replaced
with 96% alcohol for 12 hours, replaced with distilled water for 10 minutes,
distilled water and 1% KOH for 7 hours , KOH is discarded, and added with
alizarin red for 10 hours, if the muscle has not been transparent added with 2%
KOH for 30 minutes, a purifying solution of A, B, C is inserted alternately for 10
minutes, allowing solution C to remain in the container.

2. The process of calcification occurs in two ways, through the intra-membrane

ossification and endochondral ossification.

B. Suggestion

I suggest that it is better when students has been provided with samples bone of
specimen that has been stained with alizarin red completely, so the students can
observe clearly the parts of bone that has been calsified.
 REFERENCES

Geneser. 1993. Textbook of Histology. Copenhagen: Munksgaard.

Haga, H., Maimi, U., Hiroki, S., Tsuyoshi, T., Mayumi, M., Tomohiro, A., Hiroki, S.
& Toshihisa, H., 2018. A rapid and nondestructive protocol for whole-mount
bone staining of small fish and Xenopus, Scientific Report, 8(7), pp. 1-7.

Kessels, M., Leonie, F., Huitema., Sjef, B., Sander, K, Stefan, S., Johan L. & Sacco,
C., 2014. Proteomics Analysis of the Zebrafish Skeletal Extracellular Matrix.
PLOS ONE, 9(3), pp. 1-13.

Lutfi, M., 2016. Modified Alizarin Red S-Alcian Blue Staining for Reptilian Skeleton.
Biology, Medicine, & Natural Product Chemistry, 5(1), pp. 19-22.

Mohamed, R., 2018. Alizarin Red-S Protocol for Skeletal Staining during Fetal Period
in Rabbit. Academia Anatomica International. 4(1), pp. 41-44.

Soeminto., 2002. Embriologi Vertabrata. Purwokerto: Fakultas Biologi UNSOED.

Tores, M. & Eliana, R., 2014. A New Method for Staining Ligaments and Tendons of
Small Vertebrates. Coepia Journal, 2(3), pp. 708-711.