CN104561229A - Nanobacteria culture and morphological identification method - Google Patents
Nanobacteria culture and morphological identification method Download PDFInfo
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- CN104561229A CN104561229A CN201510023747.3A CN201510023747A CN104561229A CN 104561229 A CN104561229 A CN 104561229A CN 201510023747 A CN201510023747 A CN 201510023747A CN 104561229 A CN104561229 A CN 104561229A
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Abstract
The invention discloses a nanobacteria culture and morphological identification method. The method comprises the following steps: performing sterile operation and recording primary urine as VB1, recording middle-section urine as VB2, EPS, and recording primary urine as VB3 after massage; treating an EPS specimen; treating a calculus specimen; culturing the specimen; performing indirect immunofluorescence staining and fluorescence microscope observation; performing transmission electron microscope scanning; performing ultrathin section observation; performing scanning electron microscope observation; performing Dahl McGec-Russel Alizarin red staining, thus obtaining the result determination standard. The EPS specimen and the calculus specimen are treated according to the standard procedure so as to be subjected to bacteria culture and identification, pathogenic bacteria are collected for researching the possible pathogenesis relationship between nanobacteria and type III prostatitis and prostatic calculus, the method is well used for guiding the clinical work, the pain of the mind and body and economical burdens of patients can be alleviated, and more medical resources are saved.
Description
Technical field
The invention belongs to biomedical sector, particularly relate to a kind of cultivation and Morphological Identification method of nanometer bacteria.
Background technology
According to the prostatitis sorting technique that the scorching collaboration network of International Prostate proposes for 1998, prostatitis is divided into four types.I type: acute bacterial prostatitis, refers to prostate acute bacterial infection; II type: chronic bacterial prostatitis, refers to prostate chronic recurrent bacteriological infection; III type: chronic prostatitis/chronic pelvic pain syndrome (CP/CPPS), it was defined as medical history more than 3 months, pelvic region pain or discomfort, in various degree urinate or sexual intercourse time uncomfortable, this kind of is divided into again struvite and non-inflammation two kinds of hypotypes according to white blood cell count(WBC) in massage of prostate liquid, seminal fluid; IV type, without clinical inflammatory prostatitis, does not have symptom, just finds at pathologic finding or when checking other symptoms that there is leucocyte in massage of prostate liquid is just accidentally diagnosed.Wherein I type, II type patient populations are few, and IV type prostatitis, because of without any symptom, must not process, also without important clinical significance.Only have III type prostatitis patient to fail to find clear and definite pathogen because of routine inspection and cultivation, etiology unknown, pathogenesis is unclear, and can not carry out immunotherapy targeted autoantibody, clinical efficacy is poor, and patient populations accounts for more than 60% of clinical patients with chronic prostatitis.
Research finds that nanometer bacteria can secrete calcification lipopolysaccharides biological membrane, has stronger toxicity.The nanometer bacteria of people's In vivo infection, mainly excretes through urinary system.When urinating, urethra high pressure can cause urine to backflow to enter in prostate duct and glandular cell, in the process, nanometer bacteria in urine is easily by adjoint transfer, and enter in prostate, this may infect in prostate process at nanometer bacteria and play main and positive effect.In addition, prostate duct is elongated, bending, opening part bore is little, and at a right angle or diagonal upwards enters urethra with urethra, is also conducive to urinary tract pathogens and enters body of gland, and be unfavorable for that body of gland inflammatory exudate is got rid of and drainage, pathogen can colonize in wherein for a long time.Above factors is that nanometer bacteria infects and the cause a disease condition that provides and possibility in prostate.
About the aetiology relation that nanometer bacteria and III type prostatitis and prostatic concretions may exist, minority scholar is only had to explore at clinicing aspect at present both at home and abroad.The original research result displays such as Jones, in prostatitis patient serum, nanometer bacteria antigen is significantly higher than normal male and Patients with Prostatic Hyperplasia through elisa assay recall rate.Shoke etc. are by accompanying calculus patient with after the anti-nanometer bacteria treatment of tetracycline to intractable III type prostatitis, find that patient clinical symptom is significantly improved, calculus diminishes or disappears; Somebody is separated respectively, turns out nanometer bacteria bacterium from the EPS of III type prostatitis patient and prostatic concretions, and finds that wherein nanometer bacteria has higher infection rate, reaches 62.5%, 65% respectively.After immunotherapy targeted autoantibody, in EPS, nanometer bacteria positive rate obviously declines.Therefore infer nanometer bacteria and there is comparatively substantial connection between III type prostatitis and prostatic concretions, being thought of as its Important cause of disease.
Summary of the invention
The object of the embodiment of the present invention is the cultivation and the Morphological Identification method that provide a kind of nanometer bacteria, be intended to for research nanometer bacteria and the cause of disease relation that may exist between III type prostatitis and prostatic concretions collect pathogen, be used to guide clinical position better, alleviate body and mind misery and the financial burden of sufferer, save more medical resource.
The present invention is achieved in that a kind of cultivation of nanometer bacteria and Morphological Identification method comprise:
The collection of step one, EPS sample:
Sterilization orificium urethrae externum, sterile working is got the first section urine of 5ml and is designated as VB1; Get 5ml midstream urine again after discharging about 50 ~ 100ml urine and be designated as VB2; Rectal prostatic of passing through is massaged, and 1 ~ 2mlEPS is got in sterile working, and final residual is applied on slide in 1 EPS of orificium urethrae externum, the conventional microscopy of row; Again sterilize orificium urethrae externum, after getting 5ml massage, just section urine is designated as VB3;
The process of step 2, EPS sample:
Get VB1, VB2, VB3, EPS1ml respectively, with normal saline dilution 5 times, through 0.45 μm of frit, 4 DEG C of centrifugal 40min (20,000 × g), abandon supernatant, leave and take 1ml at the bottom of pipe and fully mix, 5 times are diluted again by stroke-physiological saline solution, through 0.22 μm of frit, 4 DEG C of centrifugal 40min (20,000 × g), abandon supernatant, leave and take 1ml at the bottom of pipe and fully mix;
The process of step 3, calculus sample:
In operation, the aseptic calculus sample taking patient, soaks 30min, to demineralize in 1N HCl, add 1M Tris to neutralize, pulverize calculus, be made into 5% concentration with physiological saline, with 0.22 μm of membrane filtration, 4 DEG C of centrifugal 40min (20000 × g);
The cultivation of step 4, sample:
State in filtrate at step 3 gained and add the RPMI1640 nutrient culture media of 3 ~ 4ml containing 10% γ-FBS, at 37 DEG C, pH7.4,5%CO
2cultivate with under 95% air conditions, observe weekly and record the growing state of nanometer bacteria;
Step 5, indirect IF staining, fluorescence microscope:
The centrifugal 30min (20 of step 3 gained nutrient solution 4 DEG C, 000 × g), abandon supernatant, leave and take bottom 0.5ml fully to mix, getting 1 drips on slide, naturally dries, and 4% paraformaldehyde fixes 15min, at 70 DEG C of roasting 10min after washing, drip primary antibodie, hatch 60min for 37 DEG C, the PBS of PH7.4 rinses 2min × 3 time, drip two to resist, hatch 40min for 37 DEG C, the PBS of PH7.4 rinses 2min × 3 time, 80% glycerine mounting, be placed in fluorescence microscopy Microscopic observation, substitute 8D10 antibody as negative control with PBS;
Step 6, transmission electron microscopy (negative staining):
Step 3 gained nutrient solution 4 DEG C (20,000 × g), supernatant is abandoned after centrifugal 30 minutes, the glutaraldehyde precipitated with 2.5% is fixed, and dilution of bacteria drops on the copper mesh of film, leaves standstill after several minutes, sop up with filter paper point, with 3% phosphotungstic acid dyeing 1 ~ 2min, observe under being placed in PHILPS TECNAI10 transmission electron microscope and take a picture, operating voltage 80kV;
Step 7, ultra-thin section are observed:
Step 3 gained nutrient solution 4 DEG C (20, 000 × g), supernatant is abandoned after centrifugal 30 minutes, precipitation fixes 1 hour with 2.5% glutaraldehyde, 0.1mol/L sodium phosphate buffer (PBS) 10 minutes, 1% osmium tetroxide 1 hour, 0.lmol/L PBS10 minute, graded ethanol dewaters, epoxy resin embedding, ultra-thin section is carried out with diamond cutter, section is placed in 200 orders to be had on film copper mesh, copper mesh is floated on 10% to confirm not drip containing nanometer bacteria γ-FBS through cultivating, distilled water washes 3 minutes × 3 times, 5% acetic acid uranium dyes 5 minutes, distilled water washes 3 minutes × 3 times, citric acid lead dyeing 5 minutes, distilled water fully washs, observe under PHILPS TECNAI10 transmission electron microscope after drying and take a picture, operating voltage 80kV,
Step 8, scanning electron microscopic observation:
Step 3 gained nutrient solution 4 DEG C (20,000 × g), abandons supernatant after centrifugal 30 minutes, sediment PBS washes 2 times, and each 3 minutes, sediment entered six well culture plates, sterile cover slips is inserted cultivation 72 hours, PBS washes 2 times, each 3 minutes, 2.5% glutaraldehyde fixes 1 hour, graded ethanol dewaters, freeze drying, metal coating, observe under being placed in KYKY-EM3200 scanning electron microscope, operating voltage 30kV;
Step 9, Dahl McGec-RusselShi Alizarin red staining:
Get cultivation sample smear to fix, distilled water flushing 3 times, often all over 2min; 2% sodium alizarinsulfonate 2S dye liquor dye 2 ~ 5min, distilled water speed is washed; 0.2% pale green aqueous solution redyes 20 ~ 30s, and 0.5% aqueous acetic acid speed is washed; 95% ethanol and 100% ethanol dehydration, with the sealing of dimethylbenzene transparent neutral natural gum after drying, oily sem observation;
Step 10, result criterion:
Be spherical or club shape by the visible nanometer bacteria of electron microscopic observation, size is 100 ~ 500nm about, has the material of one deck black to hold around thalline; The visible positive sample of monoclonal antibody indirect IF staining is greeny shaft-like or spherical particle distribution in high power field, and the most clustering of calcium dyeing nanometer bacteria, in Grain-negative.
Further, the cultivation of the sample described in step 3, uses the RPMI 1640 not adding sample, the RPMI 1640 containing 10% γ-FBS not adding sample, physiological saline to add RPMI 1640 and carry out negative control cultivation respectively.
Accompanying drawing explanation
Fig. 1 is cultivation and the Morphological Identification method flow diagram of the nanometer bacteria that the embodiment of the present invention provides.
Embodiment
In order to make object of the present invention, technical scheme and advantage clearly understand, below in conjunction with embodiment, the present invention is further elaborated.Should be appreciated that specific embodiment described herein only in order to explain the present invention, be not intended to limit the present invention.
Below in conjunction with drawings and the specific embodiments, application principle of the present invention is further described.
The Object Selection of the embodiment of the present invention:
1. select the III type prostatitis patient that in January ,-2009 in September, 2008 goes to a doctor at Third Military Medical University's southwest hospital outpatient, filter out 55 examples as experimental subjects (with reference to America NI H1998 prostatitis criteria for classification) according to the result of routine urine examination,urine for routine microbe growth, the conventional microscopy of EPS before and after medical history, massage of prostate.26 ~ 48 years old age, average 36 years old.The course of disease 6 ~ 48 months.NAM 40 example is as normal control.21 ~ 43 years old age, 30 years old mean age.Without prostatitis or urinary tract infections history, before and after massage of prostate, urine bacteriology is cultivated and is feminine gender, WBC < 10/HP in EPS, lecithin >=+++/HP.
2. select the hyperplasia of prostate companion prostatic concretions patient that in September, 2008 ~ 2009 year January is in hospital at southwest hospital of Third Military Medical University Urology Surgery, filter out 30 examples, 40 ~ 62 years old age, average 51 years old according to medical history, x-ray, ultrasound diagnosis result.The calculus sample of aseptic collection prostatic concretions patient in operation.
As shown in Figure 1, a kind of cultivation of nanometer bacteria and Morphological Identification method comprise:
S101: sterile working gets just that section urine is designated as VB1, midstream urine is designated as VB2, EPS, massage afterwards just section urine be designated as VB3;
The process of S102:EPS sample;
S103: the process of calculus sample;
S104: the cultivation of sample;
S105: indirect IF staining, fluorescence microscope;
S106: transmission electron microscopy;
S107: ultra-thin section is observed;
S108: scanning electron microscopic observation;
S109:Dahl McGec-RusselShi Alizarin red staining;
S110: result criterion.
Concrete steps are as follows:
The collection of step one, EPS sample:
To sterilize orificium urethrae externum with bromogeramine, sterile working get 5ml just section urine be designated as VB1; Get 5ml midstream urine again after discharging about 50 ~ 100ml urine and be designated as VB2; Rectal prostatic of passing through is massaged, and 1 ~ 2ml EPS is got in sterile working, and final residual is applied on slide in 1 EPS of orificium urethrae externum, the conventional microscopy of row; Again to sterilize orificium urethrae externum with bromogeramine, after getting 5ml massage, just section urine is designated as VB3;
The process of step 2, EPS sample:
Get VB1, VB2, VB3, EPS1ml respectively, with normal saline dilution 5 times, through 0.45 μm of frit, 4 DEG C of centrifugal 40min (20,000 × g), abandon supernatant, leave and take 1ml at the bottom of pipe and fully mix, 5 times are diluted again by stroke-physiological saline solution, through 0.22 μm of frit, 4 DEG C of centrifugal 40min (20,000 × g), abandon supernatant, leave and take 1ml at the bottom of pipe and fully mix;
The process of step 3, calculus sample:
In operation, the aseptic calculus sample taking patient, soaks 30min, to demineralize in 1N HCl, add 1M Tris to neutralize, pulverize calculus, be made into 5% concentration with physiological saline, with 0.22 μm of membrane filtration, 4 DEG C of centrifugal 40min (20000 × g);
The cultivation of step 4, sample:
State in filtrate at step 3 gained and add the RPMI1640 nutrient culture media of 3 ~ 4ml containing 10% γ-FBS (irradiating process in 16 hours through 30kGy gamma-radiation in radiotechnology research institute of Third Military Medical University), at 37 DEG C, pH7.4,5%CO
2cultivate with under 95% air conditions, use RPMI 1640 (not adding sample) respectively, add RPMI 1640 carry out negative control cultivation containing the RPMI 1640 (not adding sample) of 10% γ-FBS, physiological saline, observe weekly and record the growing state of nanometer bacteria;
Step 5, indirect IF staining, fluorescence microscope:
The centrifugal 30min (20 of step 3 gained nutrient solution 4 DEG C, 000 × g), abandon supernatant, leave and take bottom 0.5ml fully to mix, getting 1 drips on slide, naturally dry, 4% paraformaldehyde fixes 15min, at 70 DEG C of roasting 10min after washing, drip primary antibodie (Mouse monoclonal antibody 8D10againstNanobacteria, 0.1mg/ml, NANOBAC OY), hatch 60min for 37 DEG C, the PBS of PH7.4 rinses 2min × 3 time, dropping two is anti-, and (FITC marks goat anti-mouse IgG, 0.03mg/ml, Beijing Ding Guo biotech firm), hatch 40min for 37 DEG C, the PBS of PH7.4 rinses 2min × 3 time, 80% glycerine mounting, be placed in fluorescence microscopy Microscopic observation, 8D10 antibody is substituted as negative control with PBS,
Step 6, transmission electron microscopy (negative staining):
Step 3 gained nutrient solution 4 DEG C (20,000 × g), supernatant is abandoned after centrifugal 30 minutes, the glutaraldehyde precipitated with 2.5% is fixed, and dilution of bacteria drops on the copper mesh of film, leaves standstill after several minutes, sop up with filter paper point, with 3% phosphotungstic acid dyeing 1 ~ 2min, observe under being placed in PHILPS TECNAI10 transmission electron microscope and take a picture, operating voltage 80kV;
Step 7, ultra-thin section are observed:
Step 3 gained nutrient solution 4 DEG C (20, 000 × g), supernatant is abandoned after centrifugal 30 minutes, precipitation fixes 1 hour with 2.5% glutaraldehyde, 0.1mol/L sodium phosphate buffer (PBS) 10 minutes, 1% osmium tetroxide 1 hour, 0.lmol/L PBS10 minute, graded ethanol dewaters, epoxy resin embedding, ultra-thin section is carried out with diamond cutter, section is placed in 200 orders to be had on film copper mesh, copper mesh is floated on 10% to confirm not drip containing nanometer bacteria γ-FBS through cultivating, distilled water washes 3 minutes × 3 times, 5% acetic acid uranium dyes 5 minutes, distilled water washes 3 minutes × 3 times, citric acid lead dyeing 5 minutes, distilled water fully washs, observe under PHILPS TECNAI10 transmission electron microscope after drying and take a picture, operating voltage 80kV,
Step 8, scanning electron microscopic observation:
Step 3 gained nutrient solution 4 DEG C (20,000 × g), abandons supernatant after centrifugal 30 minutes, sediment PBS washes 2 times, and each 3 minutes, sediment entered six well culture plates, sterile cover slips is inserted cultivation 72 hours, PBS washes 2 times, each 3 minutes, 2.5% glutaraldehyde fixes 1 hour, graded ethanol dewaters, freeze drying, metal coating, observe under being placed in KYKY-EM3200 scanning electron microscope, operating voltage 30kV;
Step 9, Dahl McGec-RusselShi sodium alizarinsulfonate (calcium dyeing) dye:
Get cultivation sample smear to fix, distilled water flushing 3 times, often all over 2min; 2% sodium alizarinsulfonate 2S dye liquor dye 2 ~ 5min, distilled water speed is washed; 0.2% pale green aqueous solution redyes 20 ~ 30s, and 0.5% aqueous acetic acid speed is washed; 95% ethanol and 100% ethanol dehydration, with the sealing of dimethylbenzene transparent neutral natural gum after drying, oily sem observation;
Step 10, result criterion:
Be spherical or club shape by the visible nanometer bacteria of electron microscopic observation, size is 100 ~ 500nm about, has the material of one deck black to hold around thalline; The visible positive sample of monoclonal antibody indirect IF staining is greeny shaft-like or spherical particle distribution in high power field, and the most clustering of calcium dyeing nanometer bacteria, in Grain-negative.
Nanometer bacteria sample cultivation results:
VB1, VB2, VB3 and EPS of III type prostatitis, normal control two groups observe discovery with prostatic concretions sample after being separated, cultivating 3-4 week, and major part occurs without growth-gen, occur the white precipitate being close to tube wall growth bottom part culture tube.The sedimentary test tube of adularescent is after concussion, and what have is cotton-shaped floating completely, and what have adheres at the bottom of pipe completely.Adhere to the situation at the bottom of pipe according at the bottom of pipe with or without culture tube white precipitate after white precipitate appearance and concussion, the result that sample is cultivated is divided into four groups.Concrete growing state is in table 1.
Growing state after the cultivation of table 1-1 sample
By centrifugal for whole sample culture material rear row monoclonal antibody indirect IF staining, identify further, find that white precipitate adheres to person bottom culture tube wholly or in part, nanometer bacteria positive rate reaches 73%, and precipitation is completely in cotton-shaped or without precipitation person, positive rate is only 11%, illustrate that having the sediment be closely adhered at the bottom of pipe is the important Microbiological Characteristics of in nanometer bacteria incubation one, as with cotton-shaped floating thing, then may be associated with other bacterial contaminations.In the urine specimen of III type prostatitis, normal control two groups, nanometer bacteria positive rate is respectively 6% and 3%, illustrates in urine that the probability finding nanometer bacteria is very little, also eliminates in EPS and finds that nanometer bacteria is possible of urine pollution.RPMI 1640 (not adding sample), to add containing the RPMI 1640 (not adding sample) of 10% hot deactivation γ-FBS, physiological saline and be showed no white depositions after RPMI 1640 cultivates and occur.
Nanometer bacteria Morphological Identification result:
After monoclonal antibody indirect IF staining, can there is specific binding in the nanometer bacteria cultivated and the anti-nanometer bacteria monoclonal antibody 8D10 of mouse, visible positive sample greeny shaft-like or spherical particle distribution in high power field under fluorescent microscope; Green fluorescence particle is not observed with the negative control that PBS replaces antibody 8D10 to do.Adopt monoclonal antibody indirect IF staining to identify nanometer bacteria, found that in the routine EPS sample of III type prostatitis patient group 55 and there is nanometer bacteria in 26 examples, positive rate is 48.3%; And in the routine EPS sample of Normal group 40, only having 2 examples to there is nanometer bacteria, positive rate is 5%.The two difference has statistical significance (P<0.01).In 30 routine calculus samples, find that there is 17 examples and there is nanometer bacteria, positive rate is 56.7%.RPMI 1640 (not adding sample), add the control groups such as RPMI 1640 all do not observe green fluorescence particle containing the RPMI 1640 (not adding sample) of 10% hot deactivation γ-FBS, physiological saline.
Observe under transmission electron microscope, nanometer bacteria is spherical or bat shape, and size is 50 ~ 200nm about, most clustering, has the material of one deck black to hold, and have hydroxyapatite to distribute around thalline; Alizarin red staining observations: the most clustering of bacterium, individuality is extremely small.Nanometer bacteria Grain stain is black particle shape.Result and criterion basically identical.
The present invention carries out microbe growth and qualification to EPS sample and calculus sample according to after standard program process, for research nanometer bacteria and the cause of disease relation that may exist between III type prostatitis and prostatic concretions collect pathogen, be used to guide clinical position better, alleviate body and mind misery and the financial burden of sufferer, save more medical resource.
The foregoing is only preferred embodiment of the present invention, not in order to limit the present invention, all any amendments done within the spirit and principles in the present invention, equivalent replacement and improvement etc., all should be included within protection scope of the present invention.
Claims (2)
1. the cultivation of nanometer bacteria and a Morphological Identification method, it is characterized in that, cultivation and the Morphological Identification method of described nanometer bacteria comprise:
The collection of step one, EPS sample:
Sterilization orificium urethrae externum, sterile working is got the first section urine of 5ml and is designated as VB1; Get 5ml midstream urine again after discharging about 50 ~ 100ml urine and be designated as VB2; Rectal prostatic of passing through is massaged, and 1 ~ 2mlEPS is got in sterile working, and final residual is applied on slide in 1 EPS of orificium urethrae externum, the conventional microscopy of row; Again sterilize orificium urethrae externum, after getting 5ml massage, just section urine is designated as VB3;
The process of step 2, EPS sample:
Get VB1, VB2, VB3, EPS1ml respectively, with normal saline dilution 5 times, through 0.45 μm of frit, 4 DEG C of centrifugal 40min (20,000 × g), abandon supernatant, leave and take 1ml mixing at the bottom of pipe, 5 times are diluted again by stroke-physiological saline solution, through 0.22 μm of frit, 4 DEG C of centrifugal 40min (20,000 × g), abandon supernatant, leave and take 1ml at the bottom of pipe and fully mix;
The process of step 3, calculus sample:
In operation, the aseptic calculus sample taking patient, soaks 30min, to demineralize in 1N HCl, add 1M Tris to neutralize, pulverize calculus, be made into 5% concentration with physiological saline, with 0.22 μm of membrane filtration, 4 DEG C of centrifugal 40min (20000 × g);
The cultivation of step 4, sample:
State in filtrate at step 3 gained and add the RPMI1640 nutrient culture media of 3 ~ 4ml containing 10% γ-FBS, at 37 DEG C, pH7.4,5%CO
2cultivate with under 95% air conditions, observe weekly and record the growing state of nanometer bacteria;
Step 5, indirect IF staining, fluorescence microscope:
Step 3 gained nutrient solution 4 DEG C of centrifugal 30min (20,000 × g), abandon supernatant, leave and take bottom 0.5ml mixing, get 1 and drip on slide, naturally dry, 4% paraformaldehyde fixes 15min, at 70 DEG C of roasting 10min after washing, drips primary antibodie, hatch 60min for 37 DEG C, the PBS of PH7.4 rinses 2min × 3 time, drips two and resists, hatch 40min for 37 DEG C, the PBS of PH7.4 rinses 2min × 3 time, 80% glycerine mounting, be placed in fluorescence microscopy Microscopic observation, substitute 8D10 antibody as negative control with PBS;
Step 6, transmission electron microscopy:
Step 3 gained nutrient solution 4 DEG C (20,000 × g), supernatant is abandoned after centrifugal 30 minutes, the glutaraldehyde precipitated with 2.5% is fixed, and dilution of bacteria drops on the copper mesh of film, leaves standstill after several minutes, sop up with filter paper point, with 3% phosphotungstic acid dyeing 1 ~ 2min, observe under being placed in PHILPS TECNAI10 transmission electron microscope and take a picture, operating voltage 80kV;
Step 7, ultra-thin section are observed:
Step 3 gained nutrient solution 4 DEG C (20, 000 × g), supernatant is abandoned after centrifugal 30 minutes, precipitation fixes 1 hour with 2.5% glutaraldehyde, 0.1mol/L sodium phosphate buffer (PBS) 10 minutes, 1% osmium tetroxide 1 hour, 0.lmol/L PBS10 minute, graded ethanol dewaters, epoxy resin embedding, ultra-thin section is carried out with diamond cutter, section is placed in 200 orders to be had on film copper mesh, copper mesh is floated on 10% to confirm not drip containing nanometer bacteria γ-FBS through cultivating, distilled water washes 3 minutes × 3 times, 5% acetic acid uranium dyes 5 minutes, distilled water washes 3 minutes × 3 times, citric acid lead dyeing 5 minutes, distilled water fully washs, observe under PHILPS TECNAI10 transmission electron microscope after drying and take a picture, operating voltage 80kV,
Step 8, scanning electron microscopic observation:
Step 3 gained nutrient solution 4 DEG C (20,000 × g), abandons supernatant after centrifugal 30 minutes, sediment PBS washes 2 times, and each 3 minutes, sediment entered six well culture plates, sterile cover slips is inserted cultivation 72 hours, PBS washes 2 times, each 3 minutes, 2.5% glutaraldehyde fixes 1 hour, graded ethanol dewaters, freeze drying, metal coating, observe under being placed in KYKY-EM3200 scanning electron microscope, operating voltage 30kV;
Step 9, Dahl McGec-RusselShi Alizarin red staining:
Get cultivation sample smear to fix, distilled water flushing 3 times, often all over 2min; 2% sodium alizarinsulfonate 2S dye liquor dye 2 ~ 5min, distilled water speed is washed; 0.2% pale green aqueous solution redyes 20 ~ 30s, and 0.5% aqueous acetic acid speed is washed; 95% ethanol and 100% ethanol dehydration, with the sealing of dimethylbenzene transparent neutral natural gum after drying, oily sem observation;
Step 10, result criterion:
Be spherical or club shape by the visible nanometer bacteria of electron microscopic observation, size is 100 ~ 500nm about, has the material of one deck black to hold around thalline; The visible positive sample of monoclonal antibody indirect IF staining is greeny shaft-like or spherical particle distribution in high power field, and the most clustering of calcium dyeing nanometer bacteria, in Grain-negative.
2. the cultivation of nanometer bacteria and Morphological Identification method as claimed in claim 1, it is characterized in that, the cultivation of the sample described in step 3, uses the RPMI 1640 not adding sample, the RPMI 1640 containing 10% γ-FBS not adding sample, physiological saline to add RPMI 1640 and carry out negative control cultivation respectively.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106367330A (en) * | 2016-11-19 | 2017-02-01 | 厦门大学 | Shooting identification device and method for morphology of microorganisms |
| CN110062880A (en) * | 2016-10-17 | 2019-07-26 | 巴塞尔大学 | Lossless freezing support grid is prepared by controlled sample evaporation |
-
2015
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Non-Patent Citations (3)
| Title |
|---|
| 明爱民: "纳米细菌在Ⅲ型前列腺炎和前列腺结石发生中的作用研究", 《中国博士学位论文全文数据库 医药卫生科技辑》 * |
| 沈学成: "纳米细菌与Ⅲ型前列腺炎以及前列腺结石发病关系的实验研究", 《中国优秀硕士学位论文全文数据库 医药卫生科技辑》 * |
| 沈学成等: "前列腺结石患者结石中纳米细菌的培养和形态学鉴定", 《第三军医大学学报》 * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110062880A (en) * | 2016-10-17 | 2019-07-26 | 巴塞尔大学 | Lossless freezing support grid is prepared by controlled sample evaporation |
| CN110062880B (en) * | 2016-10-17 | 2022-09-20 | 巴塞尔大学 | Preparation of non-destructive freezing grid by controlled sample evaporation |
| US11860071B2 (en) | 2016-10-17 | 2024-01-02 | Universitat Basel | Lossless cryo-grid preparation by controlled sample evaporation |
| CN106367330A (en) * | 2016-11-19 | 2017-02-01 | 厦门大学 | Shooting identification device and method for morphology of microorganisms |
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