Reagents and instruments
Bright green lactose bile salt broth (BGLB broth)
Crystal violet neutral red bile salt agar
Constant temperature incubator
Automatic diluter
homogenizer
converter
Two-sample processing
Solid samples should be fully crushed under aseptic conditions. This demonstration takes milk powder as an example, and samples with good sealing condition are taken for later detection.
Three. Detection process
Preparation before experiment
Before entering the sterile operating room, you should change your sterile lab clothes, wear a hat, a mask and a sterile gloves. After entering the aseptic operation room, first take a proper amount of alcohol cotton with tweezers and wipe your hands thoroughly, and then carry out the experimental operation.
Alcohol is flammable, so you should keep a certain distance from the flame of alcohol lamp in the process of wiping your hands to prevent danger, and do the experiment after the alcohol on your hands is completely volatilized.
Sample dilution
Put a clean and sterile homogeneous bag on the automatic diluter, wipe the sample bag with alcohol cotton, put the scissors on the external flame of alcohol lamp for full combustion, and carefully cut off the sample bag. Burn the weighing spoon in the external flame of the alcohol lamp for a moment, and accurately weigh 25g of sample in the aseptic homogenization bag. Burn the water injection port with the external flame of alcohol lamp for a moment, and inject 225mL sterile physiological saline or phosphate buffer into the homogeneous bag containing the sample. Take off the homogenate bag, put it into a vibrating homogenizer, and beat 1 min ~ 2 min with the homogenizer to make a sample homogenate of 1: 10.
Note: The pH value of the sample homogenate should be between 6.5 and 7.5, and it can be adjusted by 1 mol/L sterile sodium hydroxide or 1mol/L sterile hydrochloric acid solution if necessary.
After homogenization, mark it, and put the homogenization bag into the sample rack.
Mark the sterile test tube containing 9mL of normal saline, suck the 1: 10 sample with a 1mL sterile pipette, and slowly inject 1mL into the sterile test tube containing 9mL of normal saline along the tube wall. Before and after dilution, the test tube mouth should be burned on an alcohol lamp for a while. After adding the stopper, mix it evenly on the oscillator to make a sample homogenate of 1: 100. Similarly, according to the above operation, a series of diluted sample homogenates were made in ten-fold increments in turn. When diluting the sample, be careful that the tip of the straw or suction head should not touch the surface of the diluent; For every increase in dilution 1 fold, replace it with 1 sterile pipette or 1 ml suction head.
Inoculation and culture
Mark the bottom of the sterilized Petri dish in advance. According to the estimation of the pollution status of the sample, 2 ~ 3 suitable continuous dilutions are selected. In this demonstration, only samples of 1: 10 and 1: 100 are diluted.
Use a sterile pipette to suck 1: 10 sample diluent 1mL, and add it to a Petri dish for later use. Similarly, add 1: 100 diluent and blank solution. Note that two sterile Petri dishes should be inoculated for each dilution, and the blank solution is 1mL saline instead of 1mL sample diluent.
After the sample gradient diluent is added, the culture medium can be poured. Before dumping, the mouth of the conical bottle should be fully burned on the alcohol lamp. After burning, timely pour 15 ml ~ 20 ml crystallized purple neutral red bile salt agar into each Petri dish, and carefully rotate the Petri dish to fully mix the culture medium with the sample solution. When pouring the culture medium, the bottle mouth of the conical flask should extend into the culture dish as far as possible to prevent the culture medium from hanging on the inner wall or outer wall of the culture dish when pouring, and the pouring process should be decisive to prevent the culture medium from flowing out and dripping along the outer wall of the conical flask. The temperature of the culture medium should be 46℃, neither too high nor too low. Too high temperature is not conducive to operation and harmful to bacteria. Too low temperature makes the culture medium solidify rapidly, and the bacteria can not be evenly distributed in the Petri dish, which makes it difficult to count the colonies. When rotating the culture medium, the force should be even to prevent the culture medium from touching the top of the culture dish or overflowing.
After pouring the culture medium, let the culture dish stand and wait for the culture medium to cool and solidify.
After the culture medium is solidified, add 3 ml ~ 4 ml of crystal purple neutral red bile salt agar to cover the surface of the plate. The main purpose of adding culture medium twice is to make bacteria grow in the culture medium in order to observe the typical colony morphology. Wait for the culture medium to cool and solidify. After the culture medium was solidified, the plate was turned over and placed in an incubator at 36℃ 65438 0℃ for 65438±08h ~ 24h.
Plate colony count
Select the plate with the colony number between 15 cfu ~ 150 cfu, and count the typical and suspicious coliforms on the plate respectively, in which the typical colony is purplish red, surrounded by red bile salt precipitation ring, and the colony diameter is more than 0.5 mm.
Hypothesis verification experiment
Mark the bright green lactose bile salt broth tube first. Sterilize the inoculation ring in an infrared sterilizer, then select 10 different types of typical and suspicious colonies from the crystal violet neutral red bile salt agar plate with inoculation ring, transplant them into a bright green lactose bile salt broth tube, and shake well. After each inoculation, the inoculation ring should be sterilized in a sterilizer in time. The inoculated bright green lactose bile salt broth tube was placed in an incubator and cultured at 36℃ 65438 0℃ for 24 ~ 48. After culture, the gas production in the bright green lactose bile salt broth tube was observed. Anyone who has bubbles in an inverted test tube can be reported as positive for Escherichia coli.