Abstract
rom September 2008 to May 2009, 122 specimens were collected by sterile cotton swaps from patients suffering from eye infection at Ibn Al-Haitham Teaching Eye Hospital, 12 of them (9.83% isolation percentage) were diagnosed as Pseudomonas aeruginosa isolates by using biochemical tests, API 20 E system: they were named (P(1), P(2),……..P(12)). The agglutination sera for the grouping of P. aeruginosa test show that all the isolates belong to the P. aeruginosa strains (6, 9, 12, 16). The result show that P10 isolate have a greater ability to adhesion when all P. aeruginosa isolates were tested on Congo red agar medium and adherence to smooth surfaces. The chemical analysis of crude LPS that was extracted from P10 by using digestive enzyme and hot phenol water method showed that the percentage of carbohydrates was 7.3 and the percentage of protein was 1.3. No nucleic acids were found while the chemical analysis of partially purified LPS that was made by gel-filtration chromatography by using Sephacryl 200 S showed that the percentage of carbohydrates in the partial purified LPS was 19.5% and the percentage of binding proteins was 0.006%. No nucleic acids were found. When the molecular weight of LPS was measured by gel-filtration chromatography by using Sepharose C1- 6B- 200 it was found to be 630957 Dalton. The sera were prepared by using two wild type rabbits (weight 2-2.5 kg). They were injected with five doses over 70 days of different concentrations of partially purified LPS that have been extracted from P. aeruginosa P (10) isolate. One rabbit was used as a control that has been injected with PBS. The titer of antibodies in sera was determined by passive haemagglutination and it was in the immunized rabbit’s sera 160, and the best concentration of LPS was 10µg.
Highlights
Pseudomonas aeruginosa is the most commonly encountered gram-negative species that is not a member of the family Enterobacteriaceae [1]
This study aims to: Isolation of P. aeruginosa from patients suffering from corneal infection, extracting and partially purifying Lipopolysaccharide from the isolate of P. aeroginosa that has the greater ability of adhesion and antisera preparation against LPS and antibodies titer determination in rabbit serum using passive haemagglutination
Isolation and Identification of bacteria From September 2008 to May 2009, one hundred and twenty two specimens were collected from patients suffering from eye infection from Ibn Al-haithem Teaching Eye Hospital
Summary
Pseudomonas aeruginosa is the most commonly encountered gram-negative species that is not a member of the family Enterobacteriaceae [1]. 8. Adsorption of sera In order to get rid of natural heterophilic antibodies that may exist in rabbits, sera against sheep red blood cells (anti-sheep) which may lead to unspecific reactions, adsorption of sera must be done. 9. Determination of the best concentration of LPS for adsorption on the surfaces of SRBc In order to determine the best concentration of LPS of Pseudomonas aeruginosa for adsorption on the surface of sheep red blood cells, consecutive concentrations of LPS were prepared by using PBS: (1, 10, 25, 50, 100)μg/ml (these concentrations were depend on LPS contents of carbohydrates), 1 ml of each concentration was incubated in water bath at 100oC for 1 hr., left for cooling, 1 ml of red blood cells solution was added to each concentration in clean tubes, mixed gently. The red blood cells, that were covered with LPS antigens, were precipitated by centrifugation for 10 min at 1500 rpm, the cells were washed twice by PBS, suspended in PBS in a concentration of 1% in order to use them in titration reaction, within 24 hr [16]
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