| Part I Study on the bacterial infectious situation in 521 patients with tonsillitis through electron microscopeObjective:Recently,we find such a phenomenon:this is the patients with tonsillitis,who have been treated by the antibacterial experientially,without obvious clinical syndromes,still exist a large number of bacteria in tonsils as evidenced by electron microscope.Through checking bacteria in four different positions,i.e.,the irrigation solution of crypt,the epithelial surface of crypt,subepithelium,and the surface of tonsil,we attempted to seek an available and reliable technology for the detection of bacterial status of tonsillitis.Meanwhile,we aimed to guide the antibacterial treatments for patients with tonsillitis,through the evaluation of biofilm and the positions of bacteria.Materials and methods:Inclusion criteria:The tonsil was larger than 11° and AHI>5,or the tonsillitis occurred many times in a year.After the standard antibacterial treatment,the clinical syndrome disappeared,which lasted more than one week.Before operation,the hemanalysis was normal.Exclusion criteria:The age was either below 3 or above 60 years old and the pathological results confirmed a carcinoma.The patients were feeling pharyngeal hurt and discomfort.The tissues around the tonsil were infected.The patients had the autoimmune deficiency.We performed a normal plasma sterile tonsillectomy.Afterwards,the tonsil was put into the tube with 50 ml sterile saline to wash the surface,and then,the saline was collected.We washed the deep crypt immediately and collected the irrigation solution up to 50 ml.After flushing the deep crypt of tonsil,we immediately resected 2 pieces of crypt epithelial tissues(each was about 3 mm2),which should be prepared and observed under a sigma-300 scan electron microscope.Another tissue was prepared and observed through a JEOL-1200EX transmission electron microscope and recorded by an MORADA-G2.What we needed to record were the gender,age,the situation of bacteria in each position and whether there was a biofilm.The data were evaluated by the spssl9.0 software.According to the observation positions,the data were divided into 4 groups,which was analyzed to confirm whether different genders and ages could affect the infection in each position.It should be noticed that there was no distinction of bacteria,we only needed to record whether the bacteria were in the existence.The positive probability was recorded and analyzed.In this process,what we defined the golden standard was the non-bacterial infection in whole position.According to this golden standard,we compared each two of four positions about sensitivity,false positive rate,consistency analysis and so on.Then the permutation of four groups was analyzed.The enumeration data were processed by the Chi-squared test and consistency analysis.Results:Morphological results:we observed that numerous bacteria existed in the crypt and the formation of biofilm under the transmission electron microscope.The bacteria infected the epithelia cells and cells under them.At the same time,we observed that much bacteria gazed on the surface of crypt dispersively and rooted in the epithelia under the scan electron microscope.The epithelial formation could be observed clearly.It could be noticed that much bacterial aggregation existed in the deep irrigation solution with clear formation of bacteria.As well as exfoliative cells dispersively imbedded,the same situation happened on the surface irrigation solution,while the quantity of bacteria was much less than that of deep irrigation solution.We could also gain a further observation that many shedding cells,secretions and necrosis were around the bacteria.We noticed that the number of bacteria were the largest in the crypt,but in deeper tissues.The bacteria infected the crypt and cells underepithelia,and the number of bacteria gradually decreased from the surface of the crypt to the deep tissues.We noticed that there were the biofilms which were around the bacteria,and as the basement of bacteria existed in crypt 1.But when less bacteria appeared in the crypt,the biofilm could not be observed and there were more macrophages in crypt.Under the scan electron microscope,the epithelial cells were arranged densely.Meanwhile,the bacteria were dispersively scattered on and among them.The permutations of bacterial situation in four positions were analyzed to indicate the interdependence.The number of negative situation in all positions was 36 and the infection rate was 93.09%.The bacterial positive rate of the transmission electron microscope was 70.63%in the epithelial tissues,meanwhile,the positive rate of the scan electron microscope was 61.04%on the epithelial tissues.The bacterial positive rate of irrigation solution in crypt was 87.52%and the bacterial positive rate of irrigation solution on surface was 58.73%.After compared the data with each other,we drew a condition that the highest positive rate of bacteria was irrigation solution from crypt.Because the size and function were different in gender and age groups,we analyzed different gender and age groups to discover that the result was P>0.05 with no statistical significance.It means that neither the gender nor the age affected the rate of bacterial infection.Afterwards,we analyzed the gender and age whether affected the positive rate of bacteria in each position.The result showed that the minor group and the adult group were similar,p>0.05,without statistical significance.The male group and the female group were similar,p>0.05,no statistical significance.Then,we analyzed whether the gender and the age affected the positive rate in each position.But we did not discover the significant difference,p>0.05.On the other hand,we made the ROC curve which showed that the irrigation solution of deep crypt was the best for reflecting the situation of bacterial infection.We compared the data of each position about the sensitivity,the specificity,the false positive rate and the false negative rate.The results showed that the sensitivity decrease was as followed:the irrigation solution of deep crypt group>the epithelia under the TEM group>the epithelial under the SEM group>the irrigation solution of the surface group.The sensitivity of the crypt irrigation solution was 94.00%and the surface irrigation solution was 63.10%,and the specificity were all 100%.Meanwhile,we analyzed the four groups' consistency.The kappa values of each two groups were less than 0.4 except for the result of the epithelial under the TEM group and the irrigation solution of the surface group.There were 8 kinds of possibility with any samples.They were the only positive rate in the irrigation solution of surface group,the only positive rate in epithelial group and surface group but crypt group,the only positive rate in epithelial group,the only negative rate in crypt group,the only negative rate in epithelial group and the only positive rate in surface group and crypt surface group.There was an inevitable connection.When the positivity was in epithelial group,the irrigation solution in crypt group must be positive.When positivity was in the irrigation solution of surface group,the irrigation solution of crypt was positivity.However,when the irrigation solution in crypt was positivity,the positivity of irrigation solution of surface was not necessarily.When the irrigation solution of crypt was negativity,only the group of epithelia under the TEM could be positive.Conclusion:Although the clinical syndrome disappeared after antibacterial treatment,too many bacterial hidden in the tonsil.The characters of TEM or SEM in each position group and the crypt irrigation solution affected the situation of bacteria in the tonsil,which was helpful for antibiotic treatment.The biofilm was connective with number of bacteria in the tonsil.The bacteria in tonsil came from the outside,and this progress was from outside to inside of the tonsil.Part ? Study on reliability and availability for the 60 nm immune colloidal gold technique to detect Staphylococcus aureus and Group A hemolytic streptococcusObjective:The crypt irrigation solution reflected the situation of the bacterial infection in the tonsil,which was mentioned above.We need a convenient and reliable method for detection of bacterial species.Though the 18nm immune colloidal gold technology was mature,it was difficult to observe and wasted long time.Then,we aimed to check the availability and reliability of 60nm immunologic colloidal gold signature.Materials and methods:Because the SA is the most common pathogenic bacteria and the GAS is the one of the most dangerous bacteria in tonsil,we cultured standard strain of BNCC186335,staphylococcus aureus,and BNCC102663,haemolytic streptococcus in Patri dish.During the logarithmic phase,we took the 15 samples of staphylococcus aureus or haemolytic streptococcus by inoculating loop to immerse into 1%glutaraldehyde solution as soon as possible for 1h.Then each sample was divided to 6 parts and standby.To prepare the 60 nm colloidal gold antibody solution,we took the 60nm colloidal gold solution,1ml,40 ?g/m,into the standard bacterial solution,0.1 mg/ml.)and as the first antibody.We put the solution into the 4? constant temperature and shaken for 12 h.After wash,we took the standard bacterial solutions in cube with BSA separately for 1h.Then,they were centrifuged 5000 r/m,5 min and removed the supernatant three times.The sediment was divided into two groups and numbered group one and group two randomly.The group one was SA group,and the group two was GAS group.Each group had three samples,which were numbered 1,2 and 3.The NO.1 standard bacteria were put into 60 nm colloidal gold antibody solution,and the NO.2 standard bacteria were put into normal antibody solution and cultured at 37?for 1 h.In the meantime,the NO.3 samples were put into the 60 nm colloidal gold solution which did not combine with antibody.Then the standard bacterial solution was centrifuged 5000r/m,5min and removed the supernatant three times.The NO.1 and NO.3 standard bacterial solution was prepared for SEM.The NO.2,meanwhile,was put into the BSA in 37? for 1 h.Then the standard bacterial solutions were centrifuged 5000r/m,5min and removed the supernatant three times.Then the NO.2 samples were put into the 18 nm colloidal gold antibody(second antibody)solution in 37? for 1h.Then the standard bacterial solutions were centrifuged 5000r/m,5min and removed the supernatant three times,and prepared for SEM.These 15 groups,which contained 6 standard bacterial solutions,were prepared for SEM,and were checked under the SEM.Then we analyzed the photos of SEM.Results:The standard bacterial samples of 15 groups were all observed the 18 nm and 60 nm colloidal gold on the SA and GAS.And the NO.3 samples were not observed specific colloidal gold.The mean time of two kinds of colloidal gold signature was 6.25±0.15h and 8.23±0.19h,respectively.The photographs of 18nm groups were observed colloidal gold on the SA and GAS dispersedly,but the light points in the photograph were observed clearly in 100000x and 10kv.The same clear light points of 60nm groups' photograph were in 50000x and 3kv.conclusion:1.The Immunol colloidal gold signature technology could mark the SA and GAS in bacterial solution.2.The 60 nm colloidal gold signature technology marked faster and clearer than the 18nm technology.3.The 60nm colloidal gold signature technology was available and reliable for identification of bacteria. |
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