Influence Of Solid And Liquid Media On Bitrichous Flagella In Pseudomonas Aeruginosa

1.To Develop a new technique for bacterial flagella staining. Methods ReagentA was acidized ferric chloride solution and B was tannic acid Containing formalin.The mixture of A and B was heated slightly, the Smears were covered with the cooling mixture for 50 sec. Washed gently with distilled water,the Smears were stained with silver solution.228 Strains of 19 genera 34 species were demonstrated for flagella.Each Culture was incubated into a tube of flagella broth medium and onto a sheep blood agar(SBA)plate.All stained Smears were rated by WEST′smethod. Results The flagella and their position on the bacteria were easily dis-Cerned under the microscope, 228 strains of organism growing on SBA plates and in broth medium had the highly Ratings with the mean of 4.7 and 4.6, each rating of 100 cultures of nonfermentative rods grown on SBA was highly scored 5 different from that of 104 cultures of enterobacteria grown in flagella broth medium with rating score above 4. As to some strains of Vibrioraceae,flagellar arrangement may differ with the two kinds of incubation media.Sin- glelateral flagellum and subterminal flagellum were demonstrated in 1 strain of V.cholerae non-O1.Conclusions This simple and fast method with the stable mordant was good in reliability. This technique overcomed almost all the difficulties in flagella staining And so can be used as a routine method. Nonfermentative bacilli growing on solid Mediumand enterobacteria growing in flagella broth were more suitable for flagella-staining.2.To devise a membrane filter method for reliably demonstrating bacterial flagella in broth cultures. Methods The composition of flagella broth was modified .The liquid medium was solidified with DIFCO agar for making plates. Each culture was incubated into two tubes of flagella broth and onto a agar plate at 28℃for 5～18 hours . one of broth cultures was filtered by pore size 0.1μm sterilization membranes. After filtration, the membrane was taken off from the fitration holder, then was put into sterile distilled water. Three smears were prepared for each culture. All smears were stained by Gu's method for flagella. One handred independent cells were examined on each stained smear under microscope, and the cells carrying flagella were numbered simultaneously stained smears were scored by stain rating for the cells carrying flagella. Results A total of 411 strains of 39 species were prepared for smears by three different methods, The smears stained with bacterial suspensions from plate cultures had a mean ratio of 71%. The smears stained with centrifuged broth cultures had a mean radio of 69%. The smears stained with filtered broth cultures had a mean ratio of 78%. Conclusions The liquid medium is the most appropriate for flagella development. The smears for staining flagella may be prepared quickly to be used for the flagella broth cultures. The best results can be obtained by using the membrane filter method for demonstrating bacterial flagella in broth cultures. In contrast to the centrifuged method, the membrane filter method is rather rapid and reliable.3.To observe flagellation of P.aeruginosa by Gu's method for staining flagella and electron microscopy. Methods 84 strains of P.aeruginosa were isolated from clinical specimens and two type strains of P.aeruginosa ATCC27853,ATCC43088 were used in this study.Each culture was incubated onto a sheep blood agar at 35℃, and then was stained by three smears prepared .100 independent cells with a single flagellum were observed on each stained smear.The number of one cell carrying two polar flagella was counted among the 100 polar monotrichous cells.The flagellation of each strain was examined by electron microscopy. Results Two polar bitrichous cells were seen on three smears of each culture. Eight types of flagellar status were found on all smears. The ratios of 86 cultures between the number of bitrichous cells and monotrichous cells were given from 0.67 to 15%. A cell carrying two polar flagella was seen by the method for staining flagella and electron microscopy. Conclusions Pseudomonas aeruginosa possesses one and two polar flagella, that is bitrichous flagella instead of polar monotrichous flagellum.4.Observe flagellation of P.aeruginosa by Gu's method for staining flagella and electron microscopy. Methods :88 strains of P.aeruginosa were isolated from clinical specimens and two type strains of P.aeruginosa ATCC27853,ATCC43088 were used in this study.The composition of flagella broth was modified . The liquid medium was solidified with DIFCO agar for making plates .Each culture was incubated into two tubes of flagella broth and onto a agar plate at 35℃for 18 hours.The smears of broth cultures were made by the membrane filter..100 independent cells with a single flagellum were observed on each stained smear.The number of one cell carrying two polar flagella was counted among the 100 polar monotrichous cells.The flagellation of each strain was examined by electron microscopy. Results Two polar bitrichous cells were seen on three smears of each culture. Eight types of flagellar status were found on all smears. The ratios of 90 plate cultures between the number of bitrichous cells and monotrichous cells were given from 0.33 to 15%.The ratios of 90 broth cultures were given from 1.7% to 26.3%. A cell carrying two polar flagella was seen by the method for staining flagella and electron microscopy. Conclusions Pseudomonas aeruginosa possesses one and two polar flagella, that is bitrichous flagella instead of polar monotrichous flagellum.The cells carrying two polar flagella must be easily seen in the broth cultures than onto plate cultures.