CN106018404A - Staining microscopic examination method for oral candida hyphae - Google Patents
Staining microscopic examination method for oral candida hyphae Download PDFInfo
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- CN106018404A CN106018404A CN201610326401.5A CN201610326401A CN106018404A CN 106018404 A CN106018404 A CN 106018404A CN 201610326401 A CN201610326401 A CN 201610326401A CN 106018404 A CN106018404 A CN 106018404A
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- 208000007027 Oral Candidiasis Diseases 0.000 title claims abstract description 45
- 238000000034 method Methods 0.000 title claims abstract description 43
- 238000010186 staining Methods 0.000 title claims abstract description 26
- IQFVPQOLBLOTPF-HKXUKFGYSA-L congo red Chemical compound [Na+].[Na+].C1=CC=CC2=C(N)C(/N=N/C3=CC=C(C=C3)C3=CC=C(C=C3)/N=N/C3=C(C4=CC=CC=C4C(=C3)S([O-])(=O)=O)N)=CC(S([O-])(=O)=O)=C21 IQFVPQOLBLOTPF-HKXUKFGYSA-L 0.000 claims abstract description 23
- 238000001514 detection method Methods 0.000 claims abstract description 23
- 239000000243 solution Substances 0.000 claims abstract description 22
- 210000003296 saliva Anatomy 0.000 claims abstract description 19
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 claims abstract description 18
- 239000006228 supernatant Substances 0.000 claims abstract description 13
- 239000000725 suspension Substances 0.000 claims abstract description 13
- 238000001035 drying Methods 0.000 claims abstract description 6
- 238000000926 separation method Methods 0.000 claims abstract description 4
- 239000000428 dust Substances 0.000 claims description 63
- 238000004043 dyeing Methods 0.000 claims description 36
- 230000006978 adaptation Effects 0.000 claims description 32
- 210000000214 mouth Anatomy 0.000 claims description 30
- 238000013517 stratification Methods 0.000 claims description 24
- 239000000178 monomer Substances 0.000 claims description 22
- 206010007134 Candida infections Diseases 0.000 claims description 20
- 238000000386 microscopy Methods 0.000 claims description 19
- 238000005119 centrifugation Methods 0.000 claims description 13
- 238000004140 cleaning Methods 0.000 claims description 13
- 238000001556 precipitation Methods 0.000 claims description 11
- 238000000879 optical micrograph Methods 0.000 claims description 9
- 238000012545 processing Methods 0.000 claims description 8
- 239000002504 physiological saline solution Substances 0.000 claims description 4
- 239000002470 thermal conductor Substances 0.000 claims description 4
- 230000005611 electricity Effects 0.000 claims description 3
- 239000000203 mixture Substances 0.000 claims description 3
- ZZUFCTLCJUWOSV-UHFFFAOYSA-N furosemide Chemical compound C1=C(Cl)C(S(=O)(=O)N)=CC(C(O)=O)=C1NCC1=CC=CO1 ZZUFCTLCJUWOSV-UHFFFAOYSA-N 0.000 claims description 2
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 claims description 2
- 239000010931 gold Substances 0.000 claims description 2
- 229910052737 gold Inorganic materials 0.000 claims description 2
- 230000011218 segmentation Effects 0.000 claims description 2
- 230000035945 sensitivity Effects 0.000 abstract description 12
- 239000007788 liquid Substances 0.000 abstract description 9
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 abstract description 4
- 238000003958 fumigation Methods 0.000 abstract 1
- 239000002324 mouth wash Substances 0.000 abstract 1
- 230000003287 optical effect Effects 0.000 abstract 1
- 239000002244 precipitate Substances 0.000 abstract 1
- 230000000052 comparative effect Effects 0.000 description 15
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 9
- 239000000975 dye Substances 0.000 description 8
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 6
- 210000004877 mucosa Anatomy 0.000 description 6
- 239000008399 tap water Substances 0.000 description 6
- 235000020679 tap water Nutrition 0.000 description 6
- 201000010099 disease Diseases 0.000 description 5
- 238000009825 accumulation Methods 0.000 description 4
- 210000002200 mouth mucosa Anatomy 0.000 description 4
- 238000000399 optical microscopy Methods 0.000 description 4
- 241000222122 Candida albicans Species 0.000 description 3
- 201000003984 candidiasis Diseases 0.000 description 3
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- ZCYVEMRRCGMTRW-UHFFFAOYSA-N 7553-56-2 Chemical compound [I] ZCYVEMRRCGMTRW-UHFFFAOYSA-N 0.000 description 2
- 229920000832 Cutin Polymers 0.000 description 2
- 229910000896 Manganin Inorganic materials 0.000 description 2
- 230000002745 absorbent Effects 0.000 description 2
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- 230000002378 acidificating effect Effects 0.000 description 2
- 230000003044 adaptive effect Effects 0.000 description 2
- VBIXEXWLHSRNKB-UHFFFAOYSA-N ammonium oxalate Chemical compound [NH4+].[NH4+].[O-]C(=O)C([O-])=O VBIXEXWLHSRNKB-UHFFFAOYSA-N 0.000 description 2
- 238000009835 boiling Methods 0.000 description 2
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- 238000007790 scraping Methods 0.000 description 2
- 230000003068 static effect Effects 0.000 description 2
- NMWSKOLWZZWHPL-UHFFFAOYSA-N 3-chlorobiphenyl Chemical compound ClC1=CC=CC(C=2C=CC=CC=2)=C1 NMWSKOLWZZWHPL-UHFFFAOYSA-N 0.000 description 1
- 241000894006 Bacteria Species 0.000 description 1
- 241000222120 Candida <Saccharomycetales> Species 0.000 description 1
- 238000003794 Gram staining Methods 0.000 description 1
- 229910000792 Monel Inorganic materials 0.000 description 1
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 1
- 101001082832 Saccharomyces cerevisiae (strain ATCC 204508 / S288c) Pyruvate carboxylase 2 Proteins 0.000 description 1
- 208000005946 Xerostomia Diseases 0.000 description 1
- 102000011759 adducin Human genes 0.000 description 1
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Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/84—Systems specially adapted for particular applications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N1/2813—Producing thin layers of samples on a substrate, e.g. smearing, spinning-on
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N1/30—Staining; Impregnating ; Fixation; Dehydration; Multistep processes for preparing samples of tissue, cell or nucleic acid material and the like for analysis
Abstract
The invention relates to a staining microscopic examination method for oral candida hyphae. The method comprises steps as follows: saliva of a patient infected with oral candida or a gargle obtained after gargling with sterile normal saline by a patient infected with the oral candida is collected; the saliva or the mouth rinse is subjected to centrifugal separation; and a suspension liquid is prepared from precipitate and a supernatant; the suspension liquid is smeared to form a thin layer and dried; a Congo red solution is dropwise added to the dried thin layer, the thin layer is completely covered with the Congo red solution, and then the thin layer is subjected to secondary drying; the thin layer after secondary drying is subjected to fumigation and negative staining with volatile gas of concentrated hydrochloric acid to become blue; the thin layer after negative staining is placed under an optical microscope for observation, and white oral candida hyphae under the blue background can be seen. According to the staining microscopic examination method for the oral candida hyphae, the operation is simple and convenient, a result is clear, the sensitivity is high, and the detection efficiency of the oral candida hyphae is improved.
Description
Technical field
The present invention relates to biological field, be specifically related to a kind of dyeing microscopic examination method of fungal mycelia.
Background technology
Candidiasis (Candida) belongs to conditioned pathogen, is in commensalism with body under normal circumstances,
Do not cause disease, but when poised state is destroyed, and candidiasis is changed into bacterium by circular or ovate Yeast Phase
Silk phase, breeds at local raised growth, causes clinical visible infection disease damage.
At present, conventional oral Candida mycelia detection method includes oral mucosa exfoliative cyte 10%KOH
Smear for microscopic examination and Gram's staining smear for microscopic examination.
10%KOH smear for microscopic examination operating process is as follows: use Dispoable medical spatula in disease damage district repeatedly
Painting is scraped 5～8 times, scrapes a little mucosa exfoliative cyte, is uniformly applied to microscope slide and becomes a thin layer；On microscope slide
Dripping a droplet 10%KOH solution, covered is also gently pressed, and expulsion bubble is the most ironed by specimen；Light
Alcohol burner, hold microscope slide on flame quickly through 2～3 times to dissolve cutin, fixed preparation, note avoiding
Heat time heating time is long causes specimen boiling, crystallization；Microscope slide is placed in optical microphotograph Microscopic observation, and background is colourless,
Mycelia is the transparent filament that refractivity is strong.
Gram's staining smear for microscopic examination operating process is as follows: routine smear is dried, and microscope slide is successively through ammonium oxalate
At the beginning of crystal violet dye liquor, dye 1 minute, tap water flushing 1 minute, iodine liquid mordant dyeing 1 minute, tap water rinse 1
Minute, 95% ethanol decolouring 20s, luxuriant red colouring liquid redye 1 minute, tap water rinse 1 minute, natural
After drying, microscope slide is placed in optical microphotograph Microscopic observation, and mycelia is Grain-positive and dyes uneven thread
Thing.
There are respective pluses and minuses in existing two kinds of microscopy methods.10%KOH smear for microscopic examination is simple and easy to do, behaviour
Making low cost, specificity is high；But because its object of observation is little with background reflectance, so sensitivity is low, report at present
The mycelia positive rate in road is 9.67%～47.27%, easily causes false negative and fails to pinpoint a disease in diagnosis.Gram's staining smear mirror
Inspection sensitivity is high；But complex operation, need to repeatedly dye, rinse, and background component is many and complicated, disturb because of
Element is many.Two kinds of methods are the most easily affected by many factors such as easy examined person's experience, smear fabricating quality.
Summary of the invention
It is contemplated that one of technical problem solved the most to a certain extent in correlation technique.
To this end, it is an object of the present invention to propose a kind of easy and simple to handle, result is clear, sensitivity is high
The dyeing microscopic examination method of oral Candida mycelia, to improve the detection efficiency of oral Candida mycelia.
The dyeing microscopic examination method of a kind of oral Candida mycelia according to embodiments of the present invention, including walking as follows
Rapid: collection of specimens: to gather the saliva of oral cavity monilial infection patient or gather oral cavity monilial infection patient use
The gargarism that physiological saline solution rinsing the mouth is crossed；Sample disposal: by saliva or gargarism centrifugation, take precipitation
It is prepared as suspension with supernatant；Smear: suspension is coated with straticulation and is dried；Dyeing: after the drying
Drip Congo red solution on thin layer, make Congo red solution that thin layer is completely covered, then redrying；Negative staining:
By the thin layer after redrying, through the volatilization gas of concentrated hydrochloric acid, stifling to make it be blueness；Microscopy: after negative staining
Thin layer is placed in optical microphotograph Microscopic observation, i.e. it can be seen that oral Candida mycelia white under blue background.
The dyeing microscopic examination method of a kind of oral Candida mycelia according to embodiments of the present invention, it is only necessary to gather oral cavity
The rinsing the mouth that the saliva of monilial infection patient or collection oral cavity monilial infection patient cross with normal saline rinsing the mouth
Liquid, easy and simple to handle, improve the efficiency of microscopy, additionally use Congo red solution to carry out microscopy, at highly acid
Under the conditions of, Congo red solution becomes blue, and the mycelia of white is the most high-visible, so that mirror
The sensitivity of inspection improves.
It addition, the dyeing microscopic examination method of a kind of oral Candida mycelia according to the above embodiment of the present invention, also
Can have a following additional technical characteristic:
Further, in sample processing steps, take precipitation 40 μ L～60 μ L, take supernatant 40 μ L～60 μ L,
In smear step, a diameter of 1.3cm～1.8cm of thin layer.
Further, in sample processing steps, take precipitation 50 μ L, take supernatant 50 μ L, walk at smear
In Zhou, a diameter of 1.5cm of thin layer.
Further, in staining procedure, the mass concentration of Congo red solution is 1%～3%.
Further, in negative staining step, the mass fraction of concentrated hydrochloric acid is 35%～40%.
Further, in sample processing steps, the rotating speed of centrifugation is 10000r/min～15000r/min,
The time of centrifugation is 5min～15min.
The dyeing microscopic examination method of a kind of oral Candida mycelia according to the above embodiment of the present invention, inventor exists
In centrifugal separation processes, find that too many dust the most usually accumulated by centrifuge, affect microscopy
Efficiency.Based on this, centrifuge is correspondingly improved by inventor.
The dyeing microscopic examination method of a kind of oral Candida mycelia according to the present invention, in sample processing steps
In centrifugal separation processes, using centrifuge to be centrifuged separating, centrifuge includes shell and PCB,
Component it is provided with on pcb board.
Be additionally provided with dust cleaning system in housing, dust cleaning system include the first dust stratification detection device, the
Two dust stratification detection devices, exhaust outlet and dust blow device, dust blow device includes being swept into device of air,
It is swept at the air inlet of device of air and defecator is set, the circuit being swept into device of air with being arranged at pcb board
The cleaning pipeline connection of element installed surface side, cleans pipeline and is positioned at above component, and generate heat in correspondence
The position of element is provided with self adaptation gas outlet, arranges multi-disc sector bimetallic at each self adaptation gas outlet
Sheet, time below deformation temperature, the mutually adjacent composition of multi-disc sector bimetal leaf is discoid, thus by adaptive
Answer gas outlet to close, bend to self adaptation gas outlet lateral direction higher than sector bimetal leaf during deformation temperature,
Self adaptation gas outlet is opened, and when the temperature of fan-shaped bimetal leaf is the highest, self adaptation gas outlet is opened
Amplitude the biggest.
First dust stratification detection device include for measuring cell fin capacitance the first capacitometer and
Comparator, element radiating sheet is made up of multiple fin monomers, electric insulation between multiple fin monomers, the
Electric capacity between fin monomer is detected by one capacitometer, when comparator is judged to measure capacitance
During less than threshold value electric capacity, start and be swept into device of air, adaptive via clean on pipeline through filtered air
Answer gas outlet to blow out, component is cleaned.
Second dust stratification detection device includes light-beam generator and at least one reflecting mirror being arranged on pcb board
Face, this light-beam generator is arranged in actuator housing internal side wall, and light-beam generator will with fixing angle
Rectilinear light beam is transmitted into mirror surface, and after being reflected direct reflection, light beam is projected onto and is positioned at actuator housing
Photosensitive switch in internal side wall, photosensitive switch remains on after receiving light beam；When on mirror surface
Dust stratification when reaching predetermined thickness, photosensitive switch sensing less than reflection light, then photosensitive switch Guan Bi, automatically
Start and be swept into device of air.
Any one in this first dust stratification detection device and the second dust stratification detection device starts cleaning air inlet dress
When putting, this is swept into device of air and starts, and after the scheduled time, this is swept into device of air and is automatically switched off.
Further, light-beam generator is laser generator.
Further, light-beam generator includes LED and concave mirror, and light is projected concave surface by LED
On mirror, and by sending directional light after concave mirror.
Further, fin monomer is arranged on an insulating thermal conductor, edge between each fin monomer
The bearing of trend segmentation of diffusion sheet, or, along circular dividing between each fin monomer.
The additional aspect of the present invention and advantage will part be given in the following description, and part will be retouched from following
Become obvious in stating, or recognized by the practice of the present invention.
Accompanying drawing explanation
Fig. 1 is the dyeing microscopic examination method flow diagram of a kind of oral Candida mycelia of the embodiment of the present invention；
Fig. 2 is the optical microscopy map of the dyeing microscopic examination method of the oral Candida mycelia of the embodiment of the present invention 1；
Fig. 3 is the optical microscopy map of the dyeing microscopic examination method of the oral Candida mycelia of comparative example 1 of the present invention；
Fig. 4 is the optical microscopy map of the dyeing microscopic examination method of the oral Candida mycelia of the embodiment of the present invention 2；
Fig. 5 is the optical microscopy map of the dyeing microscopic examination method of the oral Candida mycelia of comparative example 2 of the present invention；
Fig. 6 is the structural representation of the control system of the embodiment of the present invention 3；
Fig. 7 is the circuit part schematic diagram in the embodiment of the present invention 3；
Fig. 8 is the partial view to self adaptation gas outlet in the embodiment of the present invention 3；
Fig. 9 is the partial view of the element radiating sheet in the embodiment of the present invention 3.
Detailed description of the invention
Embodiments of the invention are described below in detail, and the embodiment described below with reference to accompanying drawing is exemplary
, it is intended to it is used for explaining the present invention, and is not considered as limiting the invention.
As it is shown in figure 1, the dyeing microscopic examination method of a kind of oral Candida mycelia according to the present invention, including such as
Lower step:
S101: gather the saliva of oral cavity monilial infection patient or gather oral cavity monilial infection patient with aseptic
The gargarism that normal saline rinsing the mouth is crossed.Because the patient oral cavity of a lot of xerostomias has monilial infection, but he
Cannot naturally flow out saliva, for this type of patient, taking gargarism is than better suited sample.
S102: by saliva or gargarism centrifugation, takes precipitation and supernatant is prepared as suspension.Specifically,
The rotating speed of centrifugation is about 10000r/min～15000r/min, and the time of centrifugation is at 5min～15min
Left and right.
S103: suspension is coated with straticulation and is dried.In specific operation process, it is heavy to extract in step S102
When forming sediment with supernatant, take precipitation 40 μ L～60 μ about L, take supernatant 40 μ L～60 μ about L, thin layer
Diameter is about 1.3cm～1.8cm, suspension substantially can be made to be launched into relatively thin one layer of ratio, and shape is regular,
Can avoid missing the when of observation the thin layer that part is to be seen with microscope.Preferably, precipitation can be taken
50 μ L, take supernatant 50 μ L, in smear step, and a diameter of 1.5cm of thin layer so that the thickness of thin layer
Degree and shape are more conducive to observe.
S104: drip Congo red solution on thin layer after the drying, make Congo red solution that thin layer is completely covered,
Then redrying.Specifically, the mass concentration of Congo red solution is about 1%～3%.
S105: stifling to make it be blueness through the volatilization gas of concentrated hydrochloric acid by the thin layer after redrying, Congo red
Can become blue under strongly acidic conditions, in this context, the mycelia of white is the most high-visible.Wherein, dense
The mass fraction of hydrochloric acid is 35%～40%, provides strong acidic condition for Congo red variable color.
S106: the thin layer after negative staining is placed in optical microphotograph Microscopic observation, i.e. it can be seen that in vain under blue background
The oral Candida mycelia of color.
A kind of oral Candida mycelia of the present invention is described in detail below in conjunction with specific embodiment and comparative example
Dyeing microscopic examination method.
Embodiment 1
Embodiment 1 is to utilize Congo red solution to enter the saliva or gargarism of infecting oral cavity monilial infection person
The microscopy method of row negative staining.
Collection of specimens: choose 20 patients and use 50ml sterile centrifugation tube to leave and take static non-stimulated saliva 1ml,
10 patients tell back 50ml sterile centrifugation tube after using 5ml physiological saline solution rinsing the mouth 1min, leave and take and contain
Gargarism, altogether 30 parts of salivas or gargarism specimen.
Sample disposal: 30 parts of specimen are all drawn 1ml and are placed in aseptic EP pipe, and 12000r/min is centrifuged 10min,
Precipitation and supernatant are respectively drawn 50 μ L and are placed in another aseptic EP pipe, and piping and druming is mixed into suspension.
Smear: take a clean slide, draws whole suspension, is uniformly applied on the right side of microscope slide 1/2, shape
Become a diameter to be about 1.5cm big small circular thin layer, be then dried, microscope slide can be placed in air and dry,
Can also suitably heat auxiliary with alcohol burner to be dried.
Dyeing: the thin layer surface at microscope slide drips 2% Congo red solution 1～2 so that it is be completely covered thin
Layer, is then placed in microscope slide in air and naturally dries.
Negative staining: be inverted in dried microscope slide to include and fumigate on the wide mouthed bottle bottleneck of 37.5% concentrated hydrochloric acid,
Congo red is become blue from redness immediately.
Microscopy: the microscope slide after negative staining is placed in optical microphotograph Microscopic observation, as in figure 2 it is shown, arrow indication
Being oral Candida mycelia, under blue background, the oral Candida mycelia of white is high-visible.
Comparative example 1
Comparative example 1 is that the oral mucosa infecting oral cavity monilial infection person is taken off by the KOH solution utilizing 10%
The cell that falls carries out microscopy.
Collection of specimens: use Dispoable medical spatula anti-in disease damage district 30 patients in embodiment 1
Overcoating is scraped 5～8 times, scrapes a little mucosa exfoliative cyte.
Scraping mucosa exfoliative cyte are uniformly applied on the right side of microscope slide 1/2 by smear: take a clean slide,
Form a diameter and be about 1.5cm big small circular thin layer.
Film-making: drip the KOH solution of a droplet 10% on microscope slide thin layer, covered is also gently pressed,
Expulsion bubble is the most ironed by specimen.
Fixing: to light alcohol burner, hold microscope slide on flame quickly through 2～3 times to dissolve cutin, fixing
Specimen, notes avoiding long cause specimen boiling, crystallization heat time heating time.
Microscopy: microscope slide is placed in optical microphotograph Microscopic observation, as it is shown on figure 3, arrow indication is oral cavity
Candidiasis mycelia, background is colourless, and mycelia is the transparent filament that refractivity is strong.
The operating time of embodiment 1 is 9.73 ± 0.68min, and the operating time of comparative example 1 is 4.39 ± 0.95
Min, embodiment 1 differs only by 5min with the operating time of comparative example 1, but, the sensitivity of embodiment 1
Property (i.e. mycelia positive rate) is 82.3%, and the sensitivity of comparative example 1 only has 51.6%.Real by contrast
Execute example 1 and comparative example 1 finds, the microscopy method of a kind of oral Candida mycelia according to embodiments of the present invention,
Saliva or gargarism to infecting oral cavity monilial infection person is only needed to carry out negative staining, it is not necessary to extract the mouth of patient
Chamber mucosa exfoliative cyte, easy and simple to handle and sensitivity is high.
Embodiment 2
Embodiment 2 is to utilize Congo red solution to enter the saliva or gargarism of infecting oral cavity monilial infection person
The microscopy method of row negative staining.
Collection of specimens: 20 patients use 50ml sterile centrifugation tube to leave and take static non-stimulated saliva 1ml, 10
Name patient tells back 50ml sterile centrifugation tube after using 5ml physiological saline solution rinsing the mouth 1min, leaves and takes rinsing the mouth
Liquid, altogether 30 parts of saliva (or gargarism) specimen.
Sample disposal: 30 parts of specimen are all drawn 1ml and are placed in aseptic EP pipe, and 12000r/min is centrifuged 10min,
Precipitation and supernatant are respectively drawn 50 μ L and are placed in another aseptic EP pipe, and piping and druming is mixed into suspension.
Smear: take a clean slide, draws whole suspension, is uniformly applied on the right side of microscope slide 1/2, shape
A diameter is become to be about 1.5cm big small circular thin layer.Then microscope slide is placed in air and dries, it is also possible to use
Alcohol burner suitably heats auxiliary and is dried.
Dyeing: the thin layer surface at microscope slide drips 2% Congo red solution 1～2 so that it is be completely covered thin
After Ceng, microscope slide is placed in air and naturally dries.
Negative staining: be inverted in dried microscope slide to include and fumigate on the wide mouthed bottle bottleneck of 37.5% concentrated hydrochloric acid,
Congo red is become blue from redness immediately.
Microscopy: the microscope slide after negative staining is placed in optical microphotograph Microscopic observation, as shown in Figure 4, arrow indication
Being oral Candida mycelia, background is blue, and oral Candida mycelia is white clearly.
Comparative example 2
Comparative example 2 utilizes the Gram staining oral mucosa exfoliative cyte to infecting oral cavity monilial infection person
Carry out microscopy.
Collection of specimens: use Dispoable medical spatula anti-in disease damage district 30 patients in embodiment 2
Overcoating is scraped 5～8 times, scrapes a little mucosa exfoliative cyte.
Scraping mucosa exfoliative cyte are uniformly applied on the right side of microscope slide 1/2 by smear: take a clean slide,
Form a diameter and be about 1.5cm big small circular thin layer.Then microscope slide is placed in air and dries, it is also possible to
Suitably heat auxiliary to be dried with alcohol burner.
Just dye: add ammonium oxalate crystal violet dye liquor few drops in slide surface and exfoliative cyte thin layer is completely covered, dye
Color rinses 1 minute after 1 minute under tap water.
Mordant dyeing: add iodine liquid few drops and exfoliative cyte thin layer is completely covered, rinses after dyeing 1 minute under tap water
1 minute, suck moisture with absorbent paper.
Decolouring: add 95% ethanol few drops, and be shaken gently for decolouring, washes after 20 seconds, uses absorbent paper
Suck moisture.
Redying: add luxuriant red colouring liquid few drops, after dyeing 1 minute, tap water rinses 1 minute.Then glass will be carried
Sheet is placed in air and naturally dries.
Microscopy: microscope slide is placed in optical microphotograph Microscopic observation, as it is shown in figure 5, arrow points to is mycelia,
Visible mycelia is Grain-positive and uneven filament of dyeing.
The operating time of embodiment 2 is 9.73 ± 0.68min, and the operating time of comparative example 2 is 16.66 ± 0.85
Min, embodiment 2 saves nearly 7min than comparative example 2, and, sensitivity (the i.e. mycelia of embodiment 2
Positive rate) it is 82.3%, and the sensitivity of comparative example 2 only has 71.0%.By comparative example 2 and right
Ratio 2 finds, the microscopy method of a kind of oral Candida mycelia according to embodiments of the present invention, it is only necessary to sense
Saliva or the gargarism of dye oral cavity monilial infection person carry out negative staining, it is not necessary to the oral mucosa extracting patient takes off
Fall cell, easy and simple to handle and sensitivity is high, improves the microscopy efficiency of oral Candida mycelia.
Embodiment 3
Embodiment 3 is according to centrifuge in the dyeing microscopic examination method of a kind of oral Candida mycelia of the present invention
Detailed description of the invention.
As shown in Fig. 6～Fig. 7, centrifuge includes shell 1 and PCB 2, and pcb board 2 is arranged
There is component 19.Being additionally provided with dust cleaning system in housing 1, dust cleaning system includes that first amasss
Ash detection device, the second dust stratification detection device, exhaust outlet and dust blow device, dust blow device includes
Being swept into device of air 11, in the present embodiment, being swept into device of air can be supply fan or inlet air pressure
Contracting machine, is swept at the air inlet of device of air 11 and arranges defecator 12, be swept into device of air 11 and set
The cleaning pipeline 13 of the component 19 installed surface side being placed in pcb board 2 connects, and cleans on pipeline 13
Also in multiple self adaptation gas outlet 14 that is arranged over of multiple components 19, each self adaptation gas outlet 14
Place arranges multi-disc sector bimetal leaf 15, and the fan-shaped bimetal leaf 15 at each self adaptation gas outlet 14 exists
Self adaptation gas outlet 14 is closed when being in relatively cold state by fan-shaped bimetal leaf 15, at fan-shaped bimetal leaf
15 bend to self adaptation gas outlet 14 lateral direction, by 14 dozens, self adaptation gas outlet when being in relatively Warm status
Open, and when the temperature of fan-shaped bimetal leaf 15 is the highest when, the amplitude that self adaptation gas outlet 14 is opened
The biggest；
First dust stratification detection device includes the first capacitometer for measuring cell fin and comparator,
Element radiating sheet is made up of multiple fin monomers 16, electric insulation between multiple fin monomers 16, the first electricity
Holding measuring device be connected with comparator and survey capacitance to comparator feedback, comparator is also connected with memorizer,
Memory Reference capacitance in memorizer, reference capacitance value is multiple heat radiations when element radiating sheet non-accumulation dust
Capacitance between sheet monomer 16, when actual measurement capacitance is beyond the threshold value set so that reference capacitance value is as the criterion
Time, it is swept into device of air 11 and starts, through filtered air via the self adaptation gas outlet cleaned on pipeline 13
14 blowouts, clean component 19, dirty air through exhaust outlet 20 from actuator housing 11
Interior discharge.
Second dust stratification detection device light-beam generator and at least one component 19 being arranged on pcb board 2
Between the mirror surface 17 of position, it is internal that light-beam generator is arranged on actuator housing 1, light-beam generator with
Rectilinear light beam is transmitted into mirror surface 17 by fixing angle, and after being reflected minute surface 17 reflection, light beam is projected
On the photosensitive switch 18 being provided with within actuator housing 1, when photosensitive switch 18 senses less than reflection light
Time, automatically start and be swept into device of air 11.
Owing to self adaptation gas outlet 14 being closed when fan-shaped bimetal leaf 15 is in relatively cold state, fan-shaped double
Sheet metal 15 bends to self adaptation gas outlet 14 lateral direction, by self adaptation gas outlet 14 when being in relatively Warm status
Open, and when the temperature of fan-shaped bimetal leaf 15 is the highest when, the amplitude that self adaptation gas outlet 14 is opened
The biggest；So that the area of each self adaptation gas outlet 14 is along with the unit below self adaptation gas outlet 14
The temperature of part is different and different.Because for the element that temperature is the highest, its area of dissipation can be designed to the biggest,
Or the surface of radiator portion is more conducive to the exchange of heat, but be exactly more conducive to the structure of heat exchange
Also being to cause air flowing change more acutely or to make air pass through thinner gap, this structure is the most more
Being conducive to generation and the increase of dust stratification, the dust accumulated after the identical time is the most, cleaning when just
Clear up by more air mass flow.
When fin accumulation dust is more when, the gap between fin can reduce, and fin is as one
The electric capacity of individual entirety can diminish, so when electric capacity is varied down to certain value when, may mean that dust accumulates
Arrive certain stage, so needing to clear up dust.
When pcb board 2 and component 19 dust stratification are more when, the dust stratification on mirror surface 17 is the most more,
When dust stratification is more when, dust can produce obvious diffuse-reflectance effect, weakens mirror surface 17 greatly
Direct reflection effect, photosensitive switch 18 can be caused cannot to receive the light intensity of sufficient intensity to be triggered.Logical
Cross another mode and also achieve the automatic monitoring for element surface dust stratification degree, thus significantly improve
Element surface dust stratification is more and causes short circuit or heat radiation is smooth and the element burnout problems that causes.
By arranging capacitance detecting and light detection two ways, can backup each other, to guarantee when dust accumulates
To dust cleaning system when of certain stage can system in time, remove the dust of element surface covering.
Particularly, light-beam generator is laser generator 24.
Laser generator 24 directionality is good, and the light after direct reflection will not occur significantly to dissipate,
But when some surface attachment after dust, after particularly dust accumulation is more, it will occur significantly
Diffuse-reflectance.Thus when having improve dust and free from dust or the when that dust being many and dust is few, photosensitive switch
The discrimination of 18 light intensity being able to receive that, improves the reliability for light-intensity test, reduces by mistake
The probability started.
Particularly, fin monomer 16 is arranged on an insulating thermal conductor 23, each fin monomer
Between 16, the bearing of trend along diffusion sheet is split.
Insulating thermal conductor 23 can be attachment insulating barrier on metal material, is significantly reducing heat conductivility
In the case of so that each fin monomer 16 mutually insulated, so that structure between each monomer of fin
Become electric capacity.By making each fin split along bearing of trend, permissible between each fin monomer 16
Independent composition electric capacity, it is simple to wiring.
Particularly, the quantity of the fan-shaped bimetal leaf 15 at each self adaptation gas outlet 14 is 6.
Fan-shaped bimetal leaf 15 includes active layers and passive layer, and active layers is manganin manganin alloy, the material of passive layer
Mainly dilval.
When the quantity of fan-shaped bimetal leaf 15 is 6 when, lateral in each fan-shaped bimetal leaf 15
Restriction tension force is the most less, so that fan-shaped bimetal leaf 15 forms bigger flexural deformation, fan-shaped gold
The quantity belonging to sheet is the most abundant, it is not necessary to be further added by the quantity of fan-shaped sheet metal to reduce the reliability of system.
Fan-shaped bimetal leaf 15 uses monel as active layers, and its deformation quantity is relatively big, thus obtains ratio
Significantly opening effect.
Particularly, on the basis of the lower limit of threshold value 0.9 times of capacitance.
On the basis of capacitance when of 0.9 times of capacitance, the dust on electric elements is the most more, as
Fruit is cleared up not in time, it will causes element radiating not smooth, thus reduces the serviceability of detection device.
It is as follows that the dust of the present embodiment cleans operating principle:
The when that between component or fin, upper dust stratification being more, the reality between fin monomer away from
Defection declines, the air of the non-conductor as electricity between metal fin, also has shared by partial air
Space replaced by dust, so the electric capacity between fin monomer can along with the accumulation of dust step by step
Decline.When the low lower limit to threshold value of capacitance, start and be swept into device of air.
In addition to this detection mode, it is also provided with the mode of light detection, when dust amasss on mirror surface
Tired more when, the reflectance of direct reflection can be reduced, reduce the light that photosensitive switch can receive
According to intensity, photosensitive switch sensing is less than reflection light, then photosensitive switch Guan Bi, startup cleans air inlet dress automatically
Put.
It is swept into after device of air starts and absorbs the most filtered cleaned air from the external world, send into and clean pipeline
In.Shape is altered in steps owing to the fan-shaped bimetal leaf at self adaptation gas outlet is as variations in temperature,
So self adaptation gas outlet now has been opened, and, the hotter self adaptation above element is given vent to anger
Mouth is opened bigger, and the self adaptation gas outlet above the most colder element is opened less, so that dry
The flow of net air can be different according to the different temperatures of element distribution, to improve the utilization of cleaned air
Efficiency.Can allow be swept into after device of air first blows the very first time and be automatically switched off, the such as very first time can be half
Minute.Then second time that waited allows and is suspended in the ash fall of actuator housing inner space, because while
The dust of component surface is blown afloat by giving vent to anger of self adaptation gas outlet, and constantly inside actuator housing
Space make-up air, says from main trend, and the air being mixed with dust can be discharged from exhaust outlet, but can not protect
Card after some time, will all discharge from actuator housing inner space by the air being mixed with dust.Institute
After blowing over a period of time, the air in space in drive shell body, in fact still for be mixed with a small amount of dust
Air, so needing to wait for a period of time, allows dust in air settle.Such as second time can be 3
Minute or 2 minutes.After ash fall in drive shell body, then detecting electric capacity, being less than if remained
Threshold value, then continue to clean, the most repeatedly, so that on element and circuit board
Dust is cleaned up.
The dyeing microscopic examination method of a kind of oral Candida mycelia according to embodiments of the present invention, it is only necessary to gather oral cavity
The rinsing the mouth that the saliva of monilial infection patient or collection oral cavity monilial infection patient cross with normal saline rinsing the mouth
Liquid, easy and simple to handle, improve the efficiency of microscopy, additionally use Congo red solution to carry out microscopy, at highly acid
Under the conditions of, Congo red solution becomes blue, and the mycelia of white is the most high-visible, so that mirror
The sensitivity of inspection improves.
In describing the invention, it is to be understood that term " " center ", " longitudinally ", " laterally ", " length ",
" width ", " thickness ", " on ", D score, "front", "rear", "left", "right", " vertically ", " level ", " top ",
The orientation of the instruction such as " end " " interior ", " outward ", " clockwise ", " counterclockwise ", " axially ", " radially ", " circumferential "
Or position relationship is based on orientation shown in the drawings or position relationship, it is for only for ease of the description present invention and Jian
Change and describe rather than indicate or imply that the device of indication or element must have specific orientation, with specifically
Azimuth configuration and operation, be therefore not considered as limiting the invention.
Additionally, term " first ", " second " are only used for describing purpose, and it is not intended that instruction or hint phase
To importance or the implicit quantity indicating indicated technical characteristic.Thus, define " first ", "
Two " feature can express or implicitly include one or more this feature.In description of the invention
In, " multiple " are meant that two or more, unless otherwise expressly limited specifically.
In the present invention, unless otherwise clearly defined and limited, term " install ", " being connected ", " connection ",
The term such as " fix " should be interpreted broadly, connect for example, it may be fixing, it is also possible to be to removably connect,
Or it is integral；Can be to be mechanically connected, it is also possible to be electrical connection；Can be to be joined directly together, it is also possible to pass through
Intermediary is indirectly connected to, and can be connection or the interaction relationship of two elements of two element internals.
For the ordinary skill in the art, above-mentioned term can be understood as the case may be in the present invention
Concrete meaning.
In the present invention, unless otherwise clearly defined and limited, fisrt feature second feature " on " or
D score can be that the first and second features directly contact, or the first and second features are connect indirectly by intermediary
Touch.And, fisrt feature second feature " on ", " top " and " above " but fisrt feature second
Directly over feature or oblique upper, or it is merely representative of fisrt feature level height higher than second feature.Fisrt feature
Second feature " under ", " lower section " and " below " can be fisrt feature immediately below second feature or tiltedly under
Side, or it is merely representative of fisrt feature level height less than second feature.
In the description of this specification, reference term " embodiment ", " some embodiments ", " example ",
The description of " concrete example " or " some examples " etc. means to combine the concrete spy of this embodiment or example description
Levy, structure, material or feature are contained at least one embodiment or the example of the present invention.In this explanation
In book, the schematic representation of above-mentioned term is necessarily directed to identical embodiment or example.And,
Describe specific features, structure, material or feature can with in one or more embodiments in office or example with
Suitably mode combines.Additionally, in the case of the most conflicting, those skilled in the art can be by this
The feature of different embodiments described in description or example and different embodiment or example be combined and
Combination.
Although above it has been shown and described that embodiments of the invention, it is to be understood that above-described embodiment
It is exemplary, it is impossible to being interpreted as limitation of the present invention, those of ordinary skill in the art is the present invention's
In the range of above-described embodiment can be changed, revise, replace and modification.
Claims (10)
1. the dyeing microscopic examination method of an oral Candida mycelia, it is characterised in that comprise the steps:
Collection of specimens: gather the saliva of oral cavity monilial infection patient or gather oral cavity monilial infection patient use
The gargarism that physiological saline solution rinsing the mouth is crossed；
Sample disposal: by described saliva or gargarism centrifugation, takes precipitation and supernatant is prepared as suspension；
Smear: suspension is coated with straticulation and is dried；
Dyeing: drip Congo red solution on thin layer after the drying, make Congo red solution be completely covered described thin
Layer, then redrying；
Negative staining: the thin layer after redrying is fumigated negative staining through the volatilization gas of concentrated hydrochloric acid and makes it be blueness；
Microscopy: the thin layer after negative staining is placed in optical microphotograph Microscopic observation, i.e. it can be seen that in vain under blue background
The oral Candida mycelia of color.
The dyeing microscopic examination method of a kind of oral Candida mycelia the most according to claim 1, its feature exists
In, in sample processing steps, take precipitation 40 μ L～60 μ L, take supernatant 40 μ L～60 μ L, walk at smear
In Zhou, a diameter of 1.3cm～1.8cm of thin layer.
The dyeing microscopic examination method of a kind of oral Candida mycelia the most according to claim 2, its feature exists
In, in sample processing steps, take precipitation 50 μ L, take supernatant 50 μ L, in smear step, thin layer
A diameter of 1.5cm.
The dyeing microscopic examination method of a kind of oral Candida mycelia the most according to claim 1, its feature exists
In, in staining procedure, the mass concentration of Congo red solution is 1%～3%.
A kind of dyeing microscopic examination method of oral Candida mycelia, it is characterised in that
In negative staining step, the mass fraction of concentrated hydrochloric acid is 35%～40%.
The dyeing microscopic examination method of a kind of oral Candida mycelia the most according to claim 1, its feature exists
In, in sample processing steps, the rotating speed of centrifugation is 10000r/min～15000r/min, centrifugation
Time be 5min～15min.
7. according to the dyeing microscopic examination method of a kind of oral Candida mycelia described in any one of claim 1 or 6,
It is characterized in that, in the centrifugal separation processes in sample processing steps, use centrifuge to be centrifuged separating,
Described centrifuge includes shell and PCB, and pcb board is provided with component；
Being additionally provided with dust cleaning system in described housing, described dust cleaning system includes the first dust stratification detection
Device, the second dust stratification detection device, exhaust outlet and dust blow device, described dust blow device includes clearly
Sweep into device of air, described in be swept at the air inlet of device of air defecator be set, described in be swept into device of air
With the cleaning pipeline connection of the component installed surface side being arranged at pcb board, described cleaning pipeline is positioned at institute
State above component, and be provided with self adaptation gas outlet in the position of corresponding heater element, each described from
Adapt to arrange multi-disc sector bimetal leaf at gas outlet, time below deformation temperature, the fan-shaped double gold of described multi-disc
Belong to the mutually adjacent composition of sheet discoid, thus described self adaptation gas outlet is closed, higher than deformation temperature time institute
State fan-shaped bimetal leaf to bend to described self adaptation gas outlet lateral direction, described self adaptation gas outlet opened,
And when the temperature of described fan-shaped bimetal leaf is the highest, the amplitude that described self adaptation gas outlet is opened is the biggest；
Described first dust stratification detection device includes the first capacitometer for measuring cell fin capacitance
And comparator, described element radiating sheet is made up of multiple fin monomers, and between multiple fin monomers, electricity is absolutely
Edge, the electric capacity between fin monomer is detected by described first capacitometer, when comparator is judged
Measure capacitance less than threshold value electric capacity time, start be swept into device of air, through filtered air via described clearly
Sweep the self adaptation gas outlet blowout on pipeline, described component is cleaned；
Described second dust stratification detection device includes light-beam generator and at least one reflection being arranged on pcb board
Minute surface, this light-beam generator is arranged in described actuator housing internal side wall, and light-beam generator is with fixing
Rectilinear light beam is transmitted into described mirror surface by angle, and after described mirror surface reflects, light beam is projected onto
Being positioned at the photosensitive switch in actuator housing internal side wall, photosensitive switch remains on after receiving light beam；
When the dust stratification on mirror surface reaches predetermined thickness, photosensitive switch sensing, less than reflection light, the most photosensitive is left
Close Guan Bi, automatically start and be swept into device of air；
Any one startup in this first dust stratification detection device and the second dust stratification detection device is swept into device of air
Time, this be swept into device of air start, after the scheduled time, this is swept into device of air and is automatically switched off.
The dyeing microscopic examination method of a kind of oral Candida mycelia the most according to claim 7, its feature exists
In, described light-beam generator is laser generator.
A kind of dyeing microscopic examination method of oral Candida mycelia, it is characterised in that
Described light-beam generator includes LED and concave mirror, and light is projected described concave mirror by described LED
On, and by sending directional light after concave mirror.
A kind of dyeing microscopic examination method of oral Candida mycelia, its feature exists
In, described fin monomer is arranged on an insulating thermal conductor, along dissipating between each described fin monomer
Penetrate the bearing of trend segmentation of sheet, or, along circular dividing between each described fin monomer.
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