CN111713486B - Novel dog bone specimen preparation method - Google Patents

Abstract

The invention belongs to the technical field of specimen preparation, and particularly relates to a method for removing soft tissues from a dog bone specimen. The method comprises removing large muscle attached to skeleton, and dividing the skeleton into skull, spine, limbs, sternum and ribs; preparing alkaline protease solutions with the concentrations of 0.3% and 0.5%; putting the skull, the limb bones and the chest ribs into 0.3 percent alkaline protease solution, and respectively soaking for 36 hours, 66 hours and 60 hours in a constant-temperature incubator at 50 ℃; putting spinal bone into 0.5% alkali protease solution, and soaking in a constant temperature incubator at 50 deg.C for 60 h; the bone is taken out and washed clean with water. After soft tissues are removed by the method, the prepared specimen has complete skeleton and little residual grease of the skeleton, and the prepared specimen has white bone.

Description

Novel dog bone specimen preparation method

Technical Field

The invention belongs to the technical field of specimen preparation, and particularly relates to a novel method for preparing a dog bone specimen.

Background

The method for preparing the dog bone specimen mainly comprises the steps of removing soft tissues, degreasing, bleaching and simulating the normal natural posture of animals for concatenation. The soft tissue removal is an important step, the bone is extremely easy to damage in the soft tissue removal process, the residual grease can cause the blackening of the bone in the subsequent preparation, the soft tissue removal degree directly influences the subsequent degreasing time, the bleaching effect and the bone integrity, and the method is the key for preparing the high-quality dog bone specimen. Because the size, the density and the like of bones of different species are different, the methods for removing soft tissues are different, the most common method is to use low-concentration sodium hydroxide to remove the soft tissues and degrease at present, the sodium hydroxide solution is corrosive and easy to damage the bones, the sodium hydroxide solution needs to be continuously observed, irreparable damage can be caused to bone specimens when the concentration and the time are not well mastered, and if the operation is not proper, the skin of people is also damaged, so the method for removing the soft tissues of the dog bone specimens needs to be improved, and on the basis of the improvement, the procedures of degreasing and bleaching are perfected so as to obtain the complete and white bones.

Disclosure of Invention

In order to solve the problems in the prior art, the invention provides a novel method for preparing a dog bone specimen.

In order to realize the purpose, the invention adopts the following technical scheme:

a method of removing soft tissue from a canine bone specimen, the method comprising the steps of:

(1) removing large muscle attached to the skeleton, and dividing the skeleton into a skull, a spine, four limbs, a sternum and ribs;

(2) preparing alkaline protease solutions with the concentrations of 0.3% and 0.5%;

(3) putting the skull, the limb bones and the chest ribs into 0.3 percent alkaline protease solution, and respectively soaking for 36 hours, 66 hours and 60 hours in a constant-temperature incubator at 50 ℃;

(4) putting the spinal bone into 0.5% alkali protease solution, and soaking for 60h in a constant temperature incubator at 50 ℃;

(5) the bone is taken out and washed clean with water.

In the technical scheme, the pH value of the alkaline protease solution is 6.8-8.5.

In another aspect, the present invention provides a method for removing soft tissue from a canine bone specimen, comprising the following steps:

(1) removing soft tissues: a method for removing soft tissue using the aforementioned canine bone specimen;

(2) degreasing: degreasing with 0.02% sodium dodecylbenzenesulfonate (LAS) for 24 hours;

(3) bleaching: bleaching with 4% hydrogen peroxide for 5 h;

(4) and (3) connecting in series: and carrying out series connection installation according to the normal posture of the dog variety.

The invention has the beneficial effects that:

1. the method for removing the soft tissues is simple and easy to implement, 0.3% and 0.5% of alkaline protease solutions are used for soaking, the bones at different parts are selected from the alkaline proteases with different concentrations, the soaking time and the soaking temperature are respectively controlled, the bones are not damaged after the soft tissues are removed, the residual grease of the bones is less, and the subsequent degreasing and bleaching are facilitated.

2. According to the degreasing method, 0.02% sodium dodecyl benzene sulfonate is used for degreasing (LAS) for 24 hours, the LAS has a good degreasing effect in the preparation of bone specimens of dogs, the used degreasing time is short, bone substances are not damaged, and the degreasing method is cheap, efficient, low in toxicity, environment-friendly and not easy to oxidize. The bone preserved after LAS degreasing has better hardness, white color and no oil exudation, and can replace the traditional organic solvent or sodium hydroxide for degreasing.

3. The bleaching method of the invention uses 4% hydrogen peroxide for bleaching for 5h, and the hydrogen peroxide has good bleaching effect and is simple and easy to operate.

Drawings

FIG. 1A canine bone specimen prepared by the present invention;

FIG. 2 is a graph of epiphyseal drop of a limb bone;

FIG. 3 is a skull fracture map;

FIG. 4 pubic dehiscence diagram.

Detailed Description

The invention is further illustrated but is not in any way limited by the following specific examples.

Example 1

A method of removing soft tissue from a canine bone specimen, the method comprising the steps of:

(1) removing large muscle attached to skeleton, and dividing the skeleton into skull, spine, limb bones, sternum and rib;

(2) preparing alkaline protease solutions with concentrations of 0.3% and 0.5%: preparing a protease mixed solution by using alkaline protease (g) and distilled water (ml) according to different concentrations, wherein the pH value of the mixed solution is 6.8-8.5, and the volume of the mixed solution is preferably that the mixed solution is submerged in bones;

(3) putting the skull, the limb bones and the chest ribs into 0.3 percent alkaline protease solution, and respectively soaking for 36 hours, 66 hours and 60 hours in a constant-temperature incubator at 50 ℃;

(4) putting the spinal bone into 0.5% alkali protease solution, and soaking for 60h in a constant temperature incubator at 50 ℃;

(5) the bone is taken out and washed clean with water.

Removing soft tissues, degreasing, and degreasing for 24 hours by using 0.02% sodium dodecyl benzene sulfonate; bleaching after degreasing, and bleaching for 5 hours by using 0.5% hydrogen peroxide; finally, the required dog bone skeleton specimen is obtained by serial connection.

After the method of the invention removes the soft tissue, the skeleton is complete (figure 1), the degreasing time is short, the bleaching is easy, and the prepared specimen skeleton is white and complete.

Comparative example

The skull, spine, limb bones, sternum and rib four parts were soaked in 0.1%, 0.3%, 0.5%, 0.7% and 0.9% alkaline protease solutions, respectively, and the results are shown in tables 1 to 4 below: in the table, a plurality of tissue residues, a local tissue residue, a small amount of tissue residue, a trace amount of tissue residue, no tissue residue, surface bone destruction, and a bone fracture phenomenon are marked.

TABLE 1 Soft tissue residual amount of limbs bones

Concentration of alkaline protease

12h

24h

36h

48h

60h

66h

0.1％

****

***

**

**

**

**

0.3％

****

***

**

Costal cartilage

Costal cartilage detachment

0.5％

***

**

Costal cartilage

Costal cartilage detachment

0.7％

***

**

Costal cartilage detachment

Costal cartilage damage

0.9％

***

**

Costal cartilage detachment

Costal cartilage damage

TABLE 2 Rib and sternum Soft tissue residual amounts

TABLE 3 residual amount of skull soft tissue

Concentration of alkaline protease

12h

24h

36h

48h

54h

60h

66h

0.1％

****

***

***

**

**

*

*

0.3％

****

**

*

o

o

o

o

0.5％

****

**

*

o

o#

o#

0.7％

****

**

*

o

o#

o#※

0.9％

****

**

*

o

o#

o#※

TABLE 4 residual amount of spinal soft tissue

In 0.5% protease decomposition solution, completely decomposing all soft tissues of the spine after 60h, sternum ribs and skull 48h, and causing surface bone destruction to occur in skull 54 h; epiphyseal fracture appeared after 36h of the four limbs bone, but trace connective tissue remained. The remaining trace connective tissue was completely decomposed after 60h and epiphyseal was exfoliated (fig. 2).

In 0.7% and 0.9% protease decomposition solution, the spine completely decomposes all soft tissues after 60h, sternum ribs and skull 48; surface bone destruction appears on the skull for 54h, and bone fracture appears on the skull for 60h (figure 3); epiphyseal fracture appeared 36h after the limbs bone, but trace connective tissue remained, the pubic symphysis cracked 48h later (fig. 4), and the trace connective tissue remained completely decomposed 60h later.

As can be seen from the comparative example and example 1, the bones prepared by the method of the present application had no significant destruction of bone mass, long bones were intact, the suture of the skull was not significantly separated, and the ribs were not fractured.

It will be apparent to those skilled in the art from this disclosure that many changes and modifications can be made, or equivalents modified, in the embodiments of the invention without departing from the scope of the invention. Therefore, any simple modification, equivalent change and modification made to the above embodiments according to the technical essence of the present invention shall still fall within the protection scope of the technical solution of the present invention, unless the contents of the technical solution of the present invention are departed.

Claims (1)

1. A preparation method of a dog bone specimen is characterized by comprising the following steps:

step one, removing soft tissues:

(1) removing large muscle attached to skeleton, and dividing the skeleton into skull, spine, limb bones, sternum and rib;

(2) preparing alkaline protease solutions with the concentrations of 0.3% and 0.5%;

(3) putting skull, limbs bone and chest rib into 0.3% alkaline protease solution, and soaking in 50 deg.C constant temperature incubator for 48 hr, 66 hr and 60 hr respectively;

(4) putting the spinal bone into 0.5% alkali protease solution, and soaking for 60h in a constant temperature incubator at 50 ℃;

(5) taking out the skeleton, and washing with water;

the pH value of the alkaline protease solution is 6.8-8.5;

step two, degreasing: degreasing for 24 hours by using 0.02 percent sodium dodecyl benzene sulfonate;

step three, bleaching: bleaching with 4% hydrogen peroxide for 5 h;

and step four, connecting in series.

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