第二次微生物學實驗

1、Continuous streak / Quadrant streak methodPrincipleIsolation cultivationStreak a bacterial culture on a nutrient agar plate to separate the individual bacterial cells, each isolated cell will develop into a pure colony after incubation. There are two methods, including continuous streak method and q

2、uadrant streak dilution method.MaterialE.Coli Agar plate spirit-lamp inoculation oeseProcedure1)Pick up bacteria: choose smooth inoculation rings and pick up a small amount of bacteria samples according to aseptic manipulation. 2) Divide the first area: the flat plate is inverted to the flame of the

3、 alcohol lamp, and the plate bottom is taken out with the left hand to make the flat surface roughly perpendicular to the desktop and let the flat plate face the flame. The right hand holds the inoculation ring containing bacteria. First, the 34 parallel lines are lightly delimit in the first area a

4、s the initial dilution bacteria source. Burn out the residual bacteria on the inoculation ring.In the rest area, the inoculation ring was cooled on the edge of the plate culture base, and the second area was transferred to the line position. The inoculation ring was moved to the second area through

5、the first region (the source area), and then the 67 line was lightly divided in the second area.With the same operation, there are more parallel lines in the third area and the last zoning, and the lines in the last area are parallel to the first area (but not in line with the lines in the first are

6、a or second area! Finally, put the left-handed container bottom into the lid. Burn out the residual bacteria on the ring. 5, incubator of constant temperature3)The flat plate will be placed at 37 C for 23 days.ResultAnalysisIt can be seen clearly that individual bacterial cell was separated in the t

7、hird region. The separation is successful. The separated bacterial cell is round, not transparent, white, big, flat and its surface is smooth, its margin is neat.Ultraviolet Ray SterilizationPrincipleUV effects DNA replication due to inducing the formation of dT dT dimers, therefore leads bacteria t

8、o die. But UV cant go through even a piece of paper. Thus, the exposed bacteria will die while bacteria covered under the paper will survive.UV dose (J/m2) = irradiation time (s) * UVC intensity (W/m2)MaterialCulture plateAlcohol lampE.coliMicroorganism operating tableInoculation ringUV collimated b

9、eam apparatusProcedure1) Use aseptic technique to pick up a small amount of bacteria sample and inoculate in the culture plate.2) Apply the quadrant streak method to separate the individual bacterial cell.3) Cover the agar plate with a piece of flower-shaped paper.4) Use the UV light to sterilize ba

10、cteria.5) Invert cultivation under 37 degree Celsius for 24 hours.ResultAnalysisThe flower-shaped bacteria can be seen obviously, so the experiment is successful. The UV light did sterilize exposed bacteria, and the bacteria hided under the paper survive and multiple.Morphology and Culture of M. tub

11、erculosisMorphologySlender,slightly curved bacilliacid-fast stain positivePrinciple1.Cell wall of M. tuberculosis is impermeability to stains and dyes. But M. tuberculosis can be stained by acid-fast stain with long time heating. Bacteria except M. tuberculosis can be decolorized by 3% acid alcohol

12、. 2.So the color of M. tuberculosis is red and that of other mycolic acid negtive bacteria is blue after counterstained with methylene blue . Procedure1. Make a smear of the sputum, dry and fix it.2. Ziehl-Neelsen acid-fast stain:(1) Flood the slide with carbolfuchsin. Heat the slide to steaming for

13、 5mins. Do not boil or allow the smear to dry. As stain evaporated from the slide, replenish with additional carbolfuchsin. Allow the slide to cool and rinse it thoroughly with water.(2) Decolorize the slide with acid alcohol until the red color no longer comes off in the decolorizer. It takes about

14、 30secs. Rinse the slide with water.(3) Counterstain it with methylene blue, allow the stain to react for 1min, rinse as above.(4) Dry the slide carefully. Examine under microscope.ResultAnalysisI didnt obtained the acid-fast stained M.tuberculosis successfully.The reasons why the experiment was failed may be as follows:1) The procedure wasnt correct, so the stain wasnt successful2) The