CN108130279A - A kind of mold-proof method of marine organism specimen - Google Patents
A kind of mold-proof method of marine organism specimen Download PDFInfo
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- CN108130279A CN108130279A CN201810013825.5A CN201810013825A CN108130279A CN 108130279 A CN108130279 A CN 108130279A CN 201810013825 A CN201810013825 A CN 201810013825A CN 108130279 A CN108130279 A CN 108130279A
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/14—Fungi; Culture media therefor
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N1/00—Preservation of bodies of humans or animals, or parts thereof
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N47/00—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom not being member of a ring and having no bond to a carbon or hydrogen atom, e.g. derivatives of carbonic acid
- A01N47/08—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom not being member of a ring and having no bond to a carbon or hydrogen atom, e.g. derivatives of carbonic acid the carbon atom having one or more single bonds to nitrogen atoms
- A01N47/10—Carbamic acid derivatives, i.e. containing the group —O—CO—N<; Thio analogues thereof
- A01N47/18—Carbamic acid derivatives, i.e. containing the group —O—CO—N<; Thio analogues thereof containing a —O—CO—N< group, or a thio analogue thereof, directly attached to a heterocyclic or cycloaliphatic ring
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- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
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- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
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- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
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Abstract
The present invention relates to zoological specimens Prevention of Mould Development field, especially a kind of mold-proof method of marine organism specimen；With the region for being stained with spirituous cotton swab and being marked on sample several same homalographics, with alcohol by each region wiped clean, after the vaporized alcohol in each region is complete, carbendazim solution is coated respectively；Timing observes and records；The concentration of carbendazim mould inhibitor used in the present invention is safe and non-toxic, and to no skin irritation, no sensitization；The zoological specimens mold-proof method of the present invention, minimum inhibitory concentration is low, and anti-mold effect is good, and using the carbendazim solution of 0.02ppm, zoological specimens are up to mould not long in the time of 30 days.
Description
Technical field
The present invention relates to zoological specimens Prevention of Mould Development field, especially a kind of mold-proof method of marine organism specimen.
Background technology
The type of animal is various, and the method for its preparation of specimen of different types of animal and preservation also differs, by making side
Zoological specimens can be divided into skinned-specimen, humid preparation, skeleton specimen, dry preserved specimen and specimen embedding etc., manufactured animal by method
It can be with persistence on branch and root.However, being limited to various subjective and objective conditions, zoological specimens can meet with often during preserving
The damage of various hazard factor is met, sample is caused to be shortened dramatically in service life.
It is the most serious with the harm of mould in the factors of damage zoological specimens.In a humidity environment, mould is non-
Chang Rongyi is largely grown on sample, and sample is caused to go mouldy.Since the spore naked eyes difficulty of mould nascent is distinguished, so holding very much
Easily ignored by people, but wait and grow bacterium colony, for quilt it is seen that when forming mildew, zoological specimens have received serious damage.It is mould
Damage of the bacterium to zoological specimens, gently then cause fur fade, come off influence sample surface aesthetic, it is heavy then cause sample expansion change
Shape becomes broken, rotten, causes destructive destruction.More importantly mould causes serious harm to the health of visitor.
Bacterium spirit (also known as carbendazol, carbendazol).Carbendazim is a kind of broad-spectrum germicide, to various crop by true
Disease caused by bacterium (such as Fungi Imperfecti, more sac fungus) has control effect.
People is by carbendazim on sample is mould proof not yet both at home and abroad at present, while there is presently no about to sea both at home and abroad
The research of prevention this aspect that foreign biological sample goes mouldy.
Invention content
The technical problem to be solved by the present invention is to：It provides a kind of safe and environment-friendly and sample will not be generated dysgenic
The mold-proof method of marine organism specimen.
In order to solve the above technical problems, the technical solution adopted by the present invention is as follows：
A kind of mold-proof method of marine organism specimen, the mold-proof method include the following steps：
(1) go mouldy bacterial strain acquisition and isolate and purify：With on sterilized sampling pipe and cotton swab acquisition marine organism specimen
The bacterial strain that goes mouldy, by the collected sample inoculation that goes mouldy of sterile working in PDA plate, in 28 DEG C of constant incubators be inverted training
After supporting 3-5d, with the single bacterium colony on oese picking tablet, streak inoculation continues to cultivate on blank PDA plate；Treat that bacterium colony exists
When plated growth enters animated period, make plate streaking with a small amount of spore of aseptic inoculation ring picking, repeat 2-3 plate streaking up to
Obtain pure culture bacterial strain；
(2) Morphological Identification of mould：Pure culture bacterial strain in step (1) is again it is observed that colonial morphology and the micro- shape of bacterial strain
State, removal saccharomycete and repetition bacterial strain, obtain the different mould of kind；
(3) rDNA-ITS sequencings identification：DNA is extracted with fungal DNA extraction kits, using primer amplification rDNA-ITS,
Then it is sequenced；It is multiple right that the reference sequences obtained to measured ITS sequence and from GenBank by Blast retrievals carry out
Position parallelism, and phylogenetic tree construction, the significance,statistical analysis of each branch of genealogical tree are examined in Bootstrap methods
It tests；
(4) the inhibition experiment of the mould obtained in step (3)：The inhibitor of mould uses carbendazim, and experiment is inhibited to use
Filter paper enzyme, so as to obtain the minimum inhibitory concentration of the carbendazim of various moulds and optimum inhibition concentration；
(5) the mould proof processing of biological sample：With the region for being stained with spirituous cotton swab and being marked on sample several same homalographics, use
Each region wiped clean after the vaporized alcohol in each region is complete, is coated carbendazim solution, then room temperature is put by alcohol respectively
It puts.
Preferably, the step (1) is further included pure culture inoculation in 20% glycerine in -20 DEG C of Cord bloods.
Preferably, the step (2) further includes is made -20 DEG C of low temperature conservations of 20% glycerine bacterium suspension by mould.
Preferably, the primer is ITS1：5'-TCCGTAGGTGAACCTGCGG-3', ITS4：5'-
TCCTCCGCTTATTGATATGC-3'PCR。
Preferably, the filter paper enzyme in the step (4) is specific as follows：The series that carbendazim is made to various concentration is molten
Liquid is placed on circular filter paper on piece with liquid-transfering gun absorption respectively, the mould inhibitor filter paper of different content is made；What is made in advance
A filter paper for containing different mould inhibitor doses is put on the tablet of each strain, 3 parallel tests, solvent is blank control；
The 28 DEG C of constant temperature of tablet for posting filter paper are inverted culture；Observation daily and with vernier caliper measurement antibacterial circle diameter.
Preferably, a concentration of 200ppm-900ppm of the carbendazim.
Preferably, the minimum inhibitory concentration of step (4) carbendazim is 0.2ppm-1.6ppm.
Preferably, a concentration of 0.2ppm-300ppm of the carbendazim solution in the step (5).
The advantageous effect of technical solution using the present invention is：
1st, the concentration of carbendazim mould inhibitor used in the present invention is safe and non-toxic, and to no skin irritation, no cause
Quick property；40% carbendazim sulphur suspending agent is to female male SD rat orals LD50Respectively 4300mg/kg and 2710mg/kg；It is right
The percutaneous LD of female male SD rat acutes50>2000mg/kg (experiment maximum concentration 300ppm is well below the value)；Acute inhalation
LD50>2000mg/m3；Skin wound repair integral mean value is 1.0；Without acute Eye irritation；Skin sensitization rate is 0, so carbendazim
There will not be undesirable influence on sample；
2nd, mold-proof method of the invention, minimum inhibitory concentration is low, and anti-mold effect is good, using the carbendazim solution of 0.02ppm,
Zoological specimens are up to mould not long in the time of 30 days.
Description of the drawings
The picture of control group when Fig. 1 is the 3rd day in the present invention.
The picture of control group when Fig. 2 is the 15th day in the present invention.
The picture of control group when Fig. 3 is the 30th day in the present invention.
0.2ppm experimental group pictures when Fig. 4 is the 3rd day in the present invention.
0.2ppm experimental group pictures when Fig. 5 is the 15th day in the present invention.
0.2ppm experimental group pictures when Fig. 6 is the 30th day in the present invention.
Specific embodiment
In order to make the foregoing objectives, features and advantages of the present invention clearer and more comprehensible, With reference to embodiment
The present invention is described in further detail.
1st, go mouldy bacterial strain acquisition and isolate and purify
It is with the bacterial strain that goes mouldy on sterilized sampling pipe and cotton swab acquisition marine organism specimen, sterile working is collected
Sample inoculation go mouldy in PDA plate, after culture 3-5d is inverted in 28 DEG C of constant incubators, on oese picking tablet
Single bacterium colony, streak inoculation continue to cultivate on blank PDA plate.When bacterium colony when plated growth enters animated period, connect with sterile
The kind a small amount of spore of ring picking makees plate streaking, repeats 2-3 plate streaking until obtaining pure culture bacterial strain.Take strain after purification
It is inoculated in 20% glycerine in -20 DEG C of Cord bloods.
2nd, the Morphological Identification of mould
After isolating and purifying, 8 plants of bacterial strains are obtained altogether.Again it is observed that colonial morphology and bacterial strain microscopic morphology, remove saccharomycete
With repeat bacterial strain, finally obtain 6 plants of moulds, respectively number be D1, D2, D3, D4, D5 and D6, be made 20% glycerine bacterium suspension-
20 DEG C of low temperature conservations.
3rd, rDNA-ITS sequencings identification
DNA is extracted with fungal DNA extraction kits, using primer I TS1：5'-TCCGTAGGTGAACCTGCGG-3',
ITS4：5'-TCCTCCGCTTATTGATATGC-3'PCR expands rDNA-ITS, and company is sent to be sequenced.With MEGA5.1 softwares to being surveyed
The ITS sequence obtained and the reference sequences obtained from GenBank by Blast retrievals carry out multiple contraposition parallelism, and build and be
System development tree, the significance,statistical analysis of each branch of genealogical tree are tested in Bootstrap methods, and number of repetition is
1000 times.
6 plants of moulds are initially identified as aspergillus sydowii Aspergillus sydowii D1, aspergillus japonicus A.japonicas
D2, microorganism Aspergillus aculeatus A.aculeatus D3, penicillium chrysogenum Penicillium chrysogenum D4, aspergillus flavus A.flavus
D5 and Penicillium notatum P.hispanicum D6
4th, carbendazim tests (filter paper enzyme) to the inhibition of 6 plants of moulds
By carbendazim be made various concentration serial solution (concentration of test have 200ppm, 300ppm, 400ppm,
500ppm, 550ppm, 600ppm, 650ppm, 700ppm, 800ppm and 900ppm), 2.5 μ L are drawn with liquid-transfering gun be placed in respectively
In circular filter paper piece (Φ=6mm), the mould inhibitor filter paper of different content is made.It is put on the tablet of each strain made in advance
Enter a filter paper for containing different mould inhibitor doses, 3 parallel tests, solvent is blank control.The flat of filter paper will be posted
28 DEG C of constant temperature of plate are inverted culture.Observation daily and with vernier caliper measurement antibacterial circle diameter.Experimental data is shown in Table 1.
Mould inhibitor carbendazim has significant difference in the range of experimental concentration to 6 plants of mould inhibiting effect.Wherein to D4 bacterium
The inhibiting effect of strain is most strong, a concentration of 550ppm of optimal inhibition, and antibacterial circle diameter is 45.5 ± 1.7mm；Inhibition to No. 5 bacterial strains
Effect is most weak, a concentration of 400ppm of optimal inhibition, and antibacterial circle diameter is 10.2 ± 1.2mm.In 550ppm, the D4 of Penicillium and
D6 antibacterial circle diameters are significantly higher than aspergillus bacterial strain (P<0.05), and D4, D6 antibacterial circle diameter are without significant difference (P>0.05) it is, bent
D1, D2 and D3 antibacterial circle diameter of mould category are significantly higher than D5 (P<0.05), and D1, D2, D3 antibacterial circle diameter are without significant difference (P>
0.05).In 200ppm, D6 antibacterial circle diameters are significantly higher than other bacterial strains (P<0.05), D3, D4 antibacterial circle diameter are significantly higher than
D2 and D5 (P<0.05), D2, D3 antibacterial circle diameter are without significant difference (P>0.05).
Table 1
5th, carbendazim minimum inhibitory concentration
Carbendazim solution test concentrations are configured to 0.2ppm-1.6ppm totally 7 gradients, each concentration of every plant of bacterium parallel 3
Group.Every group of proportioning is：4mL Cha Shi fluid nutrient medium+0.5mL spore suspension+0.5mL carbendazim solutions.If blank control
For pure culture base and the culture medium for adding spore suspension.Above-mentioned test group and control group are finally put into shaking table 140r/min, 28
DEG C constant temperature incubation, observation whether there is mould generation.
6th, carbendazim is in the experiment on sample of going mouldy
(1) square that the length of side is 4cm (drawing 18) is marked on sample going mouldy with being stained with spirituous cotton swab.
(2) with alcohol by the mould wiped clean in each figure.
(3) after the vaporized alcohol in square area is complete, coat respectively a concentration of 0.2ppm, 20ppm, 100ppm,
The carbendazim solution of 200ppm, 300ppm (each concentration is parallel to apply 3 squares, and 3 control groups apply distilled water).
(4) it periodically observes and records.
Experimental result
It was found that (each concentration is parallel to apply 3 just to the carbendazim solution of 0.2ppm, 20ppm, 100ppm, 200ppm, 300ppm
Rectangular, 3 control groups apply distilled water) it is all fairly obvious to the inhibition of mould, we observe 3 days, 15 days, 30 days respectively
When carbendazim inhibition, find experimental group all concentration square area in not long mould, control group is long
Mould.
The result is shown in Figure 1-Fig. 6.
The picture of control group when Fig. 1 is the 3rd day in the present invention, does not grow mould also in square area.
The picture of control group, can significantly observe and mould has been covered in square area when Fig. 2 is the 15th day in the present invention.
The picture of control group, can significantly observe and mould has been covered in square area when Fig. 3 is the 30th day in the present invention.
0.2ppm experimental group pictures when Fig. 4 is the 3rd day in the present invention, not long mould in 3 regions.
0.2ppm experimental group pictures when Fig. 5 is the 15th day in the present invention, not long mould in 3 regions.
0.2ppm experimental group pictures when Fig. 6 is the 30th day in the present invention, not long mould in 3 regions.
The oxicity analysis of carbendazim
All carbendazim solution concentration prepared in experiment are well below the standard.
1st, carbendazim solution is to the acute oral LD of big white mouse50For 5000mg/kg.
2nd, 40% carbendazim sulphur suspending agent is low toxicity, without Eye irritation, mild skin stimulation, weak sensitization pesticide：
40% carbendazim sulphur suspending agent is to female male SD rat orals LD50Respectively 4300mg/kg and 2710mg/kg；To male and female
The percutaneous LD of property SD rat acutes50>2000mg/kg (experiment maximum concentration 300ppm is well below the value)；Acute inhalation LD50>
2000mg/m3；Skin wound repair integral mean value is 1.0；Without acute Eye irritation；Skin sensitization rate is 0, so carbendazim will not
There is undesirable influence on sample.
Using above-mentioned desirable embodiment according to the present invention as enlightenment, by above-mentioned description, relevant staff is complete
Various changes and amendments can be carried out without departing from the scope of the technological thought of the present invention' entirely.The technology of this invention
Property range is not limited to the content on specification, it is necessary to determine its technical scope according to right.
Claims (8)
1. a kind of mold-proof method of marine organism specimen, which is characterized in that the mold-proof method includes the following steps：
(1) go mouldy bacterial strain acquisition and isolate and purify：It is acquired with sterilized sampling pipe and cotton swab mould on marine organism specimen
Become bacterial strain, by the collected sample inoculation that goes mouldy of sterile working in PDA plate, culture 3- is inverted in 28 DEG C of constant incubators
After 5d, with the single bacterium colony on oese picking tablet, streak inoculation continues to cultivate on blank PDA plate；Treat bacterium colony in tablet
When growing into animated period, make plate streaking with a small amount of spore of aseptic inoculation ring picking, repeat 2-3 plate streaking until obtaining
Pure culture bacterial strain；
(2) Morphological Identification of mould：Pure culture bacterial strain in step (1) again it is observed that colonial morphology and bacterial strain microscopic morphology,
It removes saccharomycete and repeats bacterial strain, obtain the different mould of kind；
(3) rDNA-ITS sequencings identification：DNA is extracted with fungal DNA extraction kits, using primer amplification rDNA-ITS, then
Sequencing；The reference sequences obtained to measured ITS sequence and from GenBank by Blast retrievals carry out multiple contraposition and arrange
Than, and phylogenetic tree construction, the significance,statistical analysis of each branch of genealogical tree are tested in Bootstrap methods；
(4) the inhibition experiment of the mould obtained in step (3)：The inhibitor of mould uses carbendazim, inhibits experiment using filter paper
Piece method, so as to obtain the minimum inhibitory concentration of the carbendazim of various moulds and optimum inhibition concentration；
(5) the mould proof processing of biological sample：With the region for being stained with spirituous cotton swab and being marked on sample several same homalographics, alcohol is used
By each region wiped clean, after the vaporized alcohol in each region is complete, carbendazim solution is coated respectively, is then placed at room temperature for.
2. a kind of mold-proof method of marine organism specimen according to claim 1, it is characterised in that：The step (1) is also
Including by pure culture inoculation in 20% glycerine in -20 DEG C of Cord bloods.
3. a kind of mold-proof method of marine organism specimen according to claim 1, it is characterised in that：The step (2) is also
Including mould is made -20 DEG C of low temperature conservations of 20% glycerine bacterium suspension.
4. a kind of mold-proof method of marine organism specimen according to claim 1, it is characterised in that：The primer is
ITS1：5'-TCCGTAGGTGAACCTGCGG-3', ITS4：5'-TCCTCCGCTTATTGATATGC-3'PCR.
5. the mold-proof method of a kind of marine organism specimen according to claim 1, which is characterized in that in the step (4)
Filter paper enzyme it is specific as follows：Carbendazim is made to the serial solution of various concentration, is placed on circle with liquid-transfering gun absorption respectively
On filter paper, the mould inhibitor filter paper of different content is made；One is put on the tablet of each strain made in advance containing not
With the filter paper of mould inhibitor dose, 3 parallel tests, solvent is blank control；The 28 DEG C of constant temperature of tablet for posting filter paper are fallen
Put culture；Observation daily and with vernier caliper measurement antibacterial circle diameter.
6. a kind of mold-proof method of marine organism specimen according to claim 5, it is characterised in that：The carbendazim it is dense
It spends for 200ppm-900ppm.
7. a kind of mold-proof method of marine organism specimen according to claim 1, it is characterised in that：The step (4) is more
The minimum inhibitory concentration of bacterium spirit is 0.2ppm-1.6ppm.
8. a kind of mold-proof method of marine organism specimen according to claim 1, it is characterised in that：In the step (5)
Carbendazim solution a concentration of 0.2ppm-300ppm.
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| CN201810013825.5A CN108130279B (en) | 2018-01-08 | 2018-01-08 | Mildew-proof method for marine organism specimen |
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101653113A (en) * | 2009-09-10 | 2010-02-24 | 边望之 | Method for manufacturing nontoxic specimen of marine organism |
| CN104365581A (en) * | 2014-11-05 | 2015-02-25 | 中山大学 | Novel specimen conservation solution |
-
2018
- 2018-01-08 CN CN201810013825.5A patent/CN108130279B/en not_active Expired - Fee Related
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101653113A (en) * | 2009-09-10 | 2010-02-24 | 边望之 | Method for manufacturing nontoxic specimen of marine organism |
| CN104365581A (en) * | 2014-11-05 | 2015-02-25 | 中山大学 | Novel specimen conservation solution |
Non-Patent Citations (5)
| Title |
|---|
| 吴光金，等: "竹材霉菌的鉴定及防霉剂的筛选", 《经济林研究》 * |
| 杨登嵩，等: "8712腐剂对标本防腐作用的研究", 《科技通报》 * |
| 王康，等: "生物标本霉菌防治研究现状及发展前景", 《中国环境科学学会科学与技术年会论文集》 * |
| 金晶，等: "皮革中多菌灵等三种防霉剂的检测方法研究", 《皮革与化工》 * |
| 魏中华，等: "国内多菌灵的研究进展", 《今日农药》 * |
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