CN1369699A - Section staining method for tooth of odontoceti - Google Patents

Abstract

The present ivnention has opened a tooth slice dyeing method of tooth whale to the public. The method works as steps of decalcification as the first including to wash tooth soaked in formalin with water, to remove methanal, to decalcify the tooth by putting it into nitric acid solution of 5%, to wash it with water after decalficicatino, to soak it in distrilled water to remove acid liquid; and slicing to be the second including, the dewater tooth by ascending akohol after it having been soaked in distilled water, xylene transparency, soft paraffin soaking, hard paraffin embeding, slicing slice annexing and roasting; dyeing to be the third including the tooth slide annexed on the glass carrier to be dewaxed with xylene, deascending alkohol, dyeing with Mayrian logwood fine dyeing fluid, ammonia water blackeite, washing, ascending alkohol, xylene transparency and mounting with neutral gum.

Description

The method of Odontoceti tooth slice dyeing

Technical field

The present invention relates to animal feeding, conservation of resources and field, aquarium, more specifically relate to a kind of method of Odontoceti tooth slice dyeing.

Background technology

Since the 1980s, along with the development of cetacean zooscopy, moving the popularizing of raising of aquarium cetacean, and more and more higher to the cry of cetacean animal protection, small-sized Odontoceti age authentication method and technology have also had development fast.The tooth of small-sized Odontoceti is a homodont, does not move back throughout one's life and does not change.Being deposited in calcareous in the tooth can not be absorbed again.Along with the growth of individuality, tooth matter is sealed up to pulp cavity to the growth of pulp cavity direction.Cementum is to outgrowth, until throughout one's life.Both all can form level, are the foundations of identifying at the age.Tooth is a sclerous tissues, and before the 1980s, external adopt worn into certain thickness thin slice to tooth more, observes, counts the method for the ages in the tooth by anatomical lens.Afterwards, developed into gradually with special-purpose Buddha's warrior attendant saw tooth was sawn into thin slice, passed through processes such as polishing, decalcification, dyeing again, made the tooth thin slice.After also useful special-purpose Buddha's warrior attendant saw amputated half to tooth, decalcification with freezing microtome section, dyeing, was made tooth slice.But the sheet thickness that the saw blade polishing is made is wayward, and a tooth only can obtain a tooth thin slice, and fragile cementum level.Freezing microtome costs an arm and a leg.And two kinds of methods all need special-purpose Buddha's warrior attendant saw, and this saw price is expensive and be difficult for buying.

Summary of the invention

The object of the present invention is to provide a kind of method of Odontoceti tooth slice dyeing.This method can be carried out Odontoceti tooth slice, dyeing under the general histology laboratory condition.Method is easy, and cost is low, and is effective.

In order to achieve the above object, the present invention adopts following technical measures: with the salpeter solution decalcification of complete tooth, the tooth after the decalcification is paraffin embedding after depickling, dehydration, manually histotome section, slice thickness 10～20 μ m.A tooth can obtain several to the uniform tooth slice of tens of Zhang Houdu.Through dewaxing, the Mei Shi haematine dye liquor dyeing course of improvement can obtain the tooth slice of clear layer again.

The steps include:

1, decalcification

The tooth of dipped into formalin was washed 12-20 hour at least with tap water, remove removing residue formaldehyde as far as possible.Then, complete tooth is put into 5% salpeter solution, decalcification 24-30 hour.The requirement decalcification is just right, and decalcification is not enough can cut into slices in influence, and decalcification excessively can destroy the growth level.Judge that the decalcification degree can just can pierce through tooth as the standard of judging that decalcification is finished with syringe needle by small size injection needle puncture exploration.But the puncture exploration can influence the section effect, the appearance change of tooth after therefore the probable ranges that can sound out a definite decalcification time with puncturing earlier and decalcification are finished.Can find that by visual inspection it is translucent that the tooth that decalcification is finished is milk yellow, but roll over bending with light finger.The tooth that decalcification is finished is used distilled water immersion 24-30 hour, to remove acid solution remaining in the tooth as far as possible again with tap water flowing water flushing 24-30 hour.

2, section

Tooth behind the distilled water immersion is used the essence of serving a round of liquor to the guests (by low concentration to high concentration) dehydration step by step, dimethylbenzene is transparent, places 50 ℃ of soft waxs to soak 3-4 hour transparent tooth, puts into 58-62 ℃ hard wax 38-42 minute then, the hard paraffin embedding.820Rotary Microtome histotome hand section with the production of AO (American Optical, U.S.'s optics) company.Longitudinal section, slice thickness 10-20 μ m.Choose of 10-12 the section of every tooth middle part, be attached to 3-4 and open on the microslide, for dyeing near axis portion.

3, dyeing

Be attached to the tooth slice on the microslide, through dimethylbenzene dewaxing, descending alcohol (by high concentration to low concentration), with the Mei Shi haematine dye liquor dyeing of improvement.The Mei Shi haematine dye liquor of improvement is on the basis of standard recipe (promptly in 1000 ml distilled waters, dissolving haematine 1.0 restrains), and haematine concentration is doubled to 2.0 grams, color contrast to improve grown layer.Dyeing time 2-3 hour, 1% ammoniacal liquor oil blackeite, flushing, up alcohol, dimethylbenzene are transparent, use the neutral gum mounting at last.

The present invention compared with prior art, have the following advantages and effect: method is easy, can carry out under the general histology laboratory condition, compares with the frozen section method with Buddha's warrior attendant saw saw blade, abrasive disc, and is with low cost, effective, the efficient height.The present invention also may be used for the section of other sclerous tissueses except that being used for the Odontoceti tooth slice.Have obvious society and economic benefit.

Embodiment

Embodiment:

A, decalcification:

With the former dolphin tooth slice dyeing of spot is example.To place with the tooth of dipped into formalin under the tap water and wash 12 hours, to remove the formaldehyde in the tooth as far as possible.Then, complete tooth sample is put into 5% salpeter solution, the volume of solution is generally 5～10 times of tooth volume.Decalcification was generally 24～30 hours, because the difference at age, so the used time of decalcification also can change thereupon, was entirely the best with decalcification just.Small size injection needle puncture on probation is soundd out during beginning, with the probable ranges of determining required time and the variation that tooth was taken place.The tooth of the former dolphin of spot is milk yellow after decalcification is finished translucent, flexible with the light finger folding.The tooth that decalcification is finished places under the tap water and washed 24 hours, soaks 24 hours in distilled water then, as far as possible the acid solution in the tooth is eliminated.

B, section:

Getting the tooth that distilled water immersion crosses dewaters step by step by up alcohol, then by dimethylbenzene and 1: 1 mixed liquor of anhydrous alcohol, enter carry out in the dimethylbenzene transparent, notice that clearing time can not be long, just transparent fully with tooth is the best, time, oversize meeting was seriously shunk tooth, the influence section.Place 50 ℃ soft wax to soak 3 hours with finishing transparent tooth, the hard wax of putting into 60 ℃ then takes out the tooth hard wax embedding of having soaked wax about 40 minutes at last.Embedded wax stone just can be cut into slices after must solidifying fully, and embedded wax stone is placed section again more than 12 hours in room temperature (below 25 ℃).820 Rotary Microtome histotome hand sections with the production of AO (American Optical, U.S.'s optics) company.Along the longitudinal axis profile section of tooth, slice thickness 10-20 μ m.The 10-12 that chooses every close axis portion in tooth middle part opens section, is attached to the 3-4 that scribbles protein adhesive and opens on the microslide, and the prescription of protein adhesive is prepared in strict accordance with standard recipe.The microslide that will post section is then put into 58-62 ℃ roasting sheet 15-20 hour of constant temperature oven.

C, dyeing:

The slide that is loaded with tooth slice was put into dimethylbenzene (I), dimethylbenzene (II) each 1.5 hours, the wax in the tooth slice is taken off totally, make slide then by dimethylbenzene and 1: 1 mixed liquor of water.Behind descending alcohol, also need in distilled water, soak 2 minutes.The Mei Shi haematine dye liquor of then slide being put into improvement dyeed 2 hours.The Mei Shi haematine dye liquor of improvement is on the basis of standard Mei Shi haematine formula for dye liquor (promptly in 1000 ml distilled waters, dissolving haematine 1.0 grams), and haematine concentration is doubled to prepare, and color contrast with the raising grown layer.After dyeing finishes, dye good slide after the tap water flushing, immersed in 1% the ammonia spirit oil blackeite 5-10 minute, wash with tap water subsequently, by up alcohol, wherein in 100% alcohol, answer the proper extension soak time step by step, so that the moisture in the section takes off totally.Then, make slide, make section transparent successively by dimethylbenzene and 1: 1 mixed liquor of anhydrous alcohol, dimethylbenzene (I), dimethylbenzene (II).The time that needs proper extension in dimethylbenzene, to soak, but soak time is unsuitable long, and the two temporal summation should not be above 20 minutes.At last, use the neutral gum mounting.Pack into before the section box, neutral gum is parched, lucifuge is dried in the shade.

Claims (1)

1, a kind of method of Odontoceti tooth slice dyeing comprises the following steps:

A, decalcification, the tooth of dipped into formalin was placed under the tap water flushing 12-20 hour, remove removing residue formaldehyde, then complete tooth is put into 5% salpeter solution, decalcification 24-30 hour, the tooth that decalcification is finished places under the tap water and washed 24-30 hour, uses distilled water immersion 24-30 hour again, removes remaining acid solution in the tooth;

B, section, tooth behind the distilled water immersion, use that the essence of serving a round of liquor to the guests is dewatered step by step, dimethylbenzene is transparent, place 50 ℃ of soft waxs to soak 3-4 hour transparent tooth, put into 58-62 ℃ hard wax 38-42 minute, the hard paraffin embedding, with the microtome section, slice thickness 10～20 μ m choose 10-12 the section at tooth middle part, be attached to 3-4 and open on the microslide, put into 58-62 ℃ constant temperature oven then and baked sheet 15-20 hour;

C, dyeing are attached to the tooth slice on the microslide, through dimethylbenzene dewaxing, descending alcohol, with the Mei Shi haematine dye liquor dyeing of improvement, dyes 2-3 hour, and 1% ammoniacal liquor oil blackeite is washed, and up alcohol, dimethylbenzene are transparent, use the neutral gum mounting at last.