The Ability Of Different Sources Of Campylobacter Jejuni To Enter The Viable But Nonculture State At4℃Starvation Conditions And Its Morphological Changes Over Time

Objective:1To Observe different sources of Campylobacter jejuni in ourlaboratory whether can enter a viable but non-culturable state when the cellswere suspended in phosphate-buffered saline(PBS)(pH7.2+-0.1) at4℃during35days.2To observe the morphology changes in its Gram stain ofdifferent sources of Campylobacter jejuni in our laboratory at4℃starvationstress conditions during35days.Methods:1Recovery and passage of Campylobacter jejuni:Select the C.jejuni strains which is stored at-80℃from the Second Hospital of HebeiMedical University Laboratory of Neurology.There are C. jejuni strains LCand lulei for both are isolated form patients with Guillain-Barre syndrome(Guillain-Barre syndrome, GBS),while chicken is isolated from chicken fecesand NCTC11168strains is non-GBS sources.Naturally dissolved at roomtemperature,then tile the7%sterile defibrinated sheep blood agar Columbiamedium,placed them in three gas incubator,42℃microaerophilic (10%CO2,5%O2,85%N2) conditions, and cultured for24hours to obtain the recoveryof the generation of strain P1corresponding strain colonies taken threegenerations dividing line P1inoculation inoculated to7%sterile defibrinatedsheep blood agar medium Colombian placed three gas incubator,42℃microaerophilic (10%CO2,5%O2,85%N2) conditions, for24hours get thestrains corresponding P2and P3-generation strain.2Identification of Campylobacter jejuni: Gram stainingshowed Gram-negative strains Bacillus, hippurate hydrolysis test showedpositive.3Collect bacteria: Scrape amount of four generations of Campylobacter jejuni bacteria with a sterile inoculating loop to a sterile liquidamoxicillin bottle containing3ml PBS (PH7.2+-0.1).Shake bacterialsuspension with oscillator plenty,every plant strains make four backup andwell marked, all the turbidity of the bacterial suspension quantitative at3*108cfu/ml.4Starvation:The incubated cells were suspended in phosphate-buffered saline (PBS)(pH7.2±0.1) at4℃without shaking.5At the bacterial suspension were placed in the refrigerator day0, day3, day6..... with CTC-DAPI double staining counting the total bacterianumber and the live bacteria number, tablet directly count (HPC) counting thenumber of culturable bacteria.Every backup of Campylobacter jejuni countthe total bacterial,live bacteria and culturable bacteria number at the same timerespectively.6Spread plate counts.The culturability of each strain wasassayed every3days by spread plate counts. The samples were serially dilutedin double-distilled water and0.1ml of each distilled sample was spread intriplicate on to7%lysed sheep-blood Columbia agar, respectively. After48hof incubation at42℃under a micro-aerobic atmosphere, the cfucorresponding to each dilution were counted and compared with the originalsample. Subsequently, less than300cfu/ml of culturable cells were inoculatedon ten petri dishes containing0.1ml of microcosm water and7%lysedsheep-blood Columbia agar.7CTC-DAPI staining. In accordance with the techniquedeveloped by Cappelier et al.(1997), with appropriate modifications, amixture of0.5ml of brain-heart infusion (Biokar, Beauvais, France) and0.5ml of microcosm water was added to the cell suspensions to stimulate thecellular respiration. Then, CTC (5-cyano-2,3-di-tolyl tetrazolium chloride;Sigma-Aldrich, Japan), amended with double-distilled water to obtain a finalconcentration of5mM, was added to the cell cultures and incubated for4h at42℃under a micro-aerobic atmosphere. The cells were subsequentlyharvested by filtration through an isopore poly-carbonate black membrane filte（r0.2-μm pore size,25-mm in diameter; Millipore, Ireland）and treated for5min with a5μg/ml of DAPI solution for counterstaining purposes. Finally,the stain was removed by filtration, and the filter was air-dried and mounted innon-fluorescent immersion oil and covered using a coverslip. Four filters wereprepared for each sample. By using Olympus BX53epifluorescencemicroscope with a tight-fitted fluorescence source,405-nm excitation filter,and455-nm dichroic mirror, the filters were observed by simultaneouslyvisualizing both the dyes. The cells were counted randomly on the basis of20microscopic fields per filter. Both respiring cell counts, showing CTCformazan crystals, and total cell counts, exhibiting DAPI staining（i.e. bothviable and non-viable cells）,were determined. The results were expressed asthe number of corresponding bacteria per ml of the original sample. All theexperiments were performed in triplicate.8Light microscopy. At corresponding time points, remove asingle colony from the plate was obtained for Gram staining. In accordancewith the technique developed by Lane (20), the slide mounted with the cellswas flooded with crystal violet stain and allowed to stand for at least1minute.Then, the slide was quickly rinsed with tap water, flooded again with Gram’siodine, and allowed to stand for2minutes, or twice the time allowed forcrystal violet staining. Subsequently, the slide was rinsed thoroughly with tapwater. While holding the slide in a vertical position, the decolorizer wasslowly dripped onto the slide until the runoff was relatively clear. Occasionalquick tap water rinses during the decolorization step. The slide was rinsedwith tap water and flooded with safranine for at least30s. The stained slideswas placed on a slide rack and air-dried. After drying, the slide was viewedunder a microscope equipped with a l000×oil immersion lens. Thismagnification was necessary for easier and more accurate bacterialdifferentiation.Results:1Production of VBNC cells was obtained with all the4C.jejunistrains.VBNC state was reached after20days of starvation in microcosmwater for strains lulei, and after25days for strain11168, lc, chicken. 2The tested strains of C. jejuni showed different types ofmorphological change in microcosm water. Cj-11168, chicken, lc strainsexperienced a rod shape and spherical shape into each other at25dayobservation period. Most of the cells of Cj-11168，which is gram-negativebacilli at0day, becomes gram-negative coccus at the11th day, then at the21stday began to grow back. A majority of cells of Cj-lc strains,which isgram-negative bacilli at0day, turn to gram-negative globular at the10th day,then at the14th day become shorter, bacillus, at the20th day start to growback. Cj-chicken strains of gram-negative bacilli at0day, on the15th day is agram-negative bacteria, bacillus20days. The cells of Cj-lulei strain at0day isGram-negative bacilli, in the first11days convert into Gram-positive cocci.Conclusion:1Production of VBNC cells was obtained with all the4C.jejuni strains.VBNC state was reached after20days of starvation inmicrocosm water for strains lulei, and after25days for strain11168, lc,chicken.2The tested strains of C. jejuni showed different types ofmorphological change in microcosm water. Cj-11168, chicken, lc strainsexperienced a rod shape and spherical shape into each other at25dayobservation period. Most of the cells of Cj-11168，which is gram-negativebacilli at0day, becomes gram-negative coccus at the11th day, then at the21stday began to grow back. A majority of cells of Cj-lc strains,which isgram-negative bacilli at0day, turn to gram-negative globular at the10th day,then at the14th day become shorter, bacillus, at the20th day start to growback. Cj-chicken strains of gram-negative bacilli at0day, on the15th day is agram-negative bacteria, bacillus20days. The cells of Cj-lulei strain at0day isGram-negative bacilli, in the first11days convert into Gram-positive cocci.