The preparation and counting of the culture base of Bacillus tuberculosis

TB bacteria
culture
base preparation
netizen ljksbs gave the TB bacteria
medium
preparation method:
TB culture using Ro-chin medium, in order to prevent the medium dry cracking, can add a moderate amount of glyceros, we are in 1600 ml of medium added to the glycemic 12 ml.
- Qin culture base ingredients:
hydrophosphate potassium 2.4g; a water magnesium sulfate 0.24g;magnesium acid 0.6g; Tianmen donglin 3.6g; 2% peacock green water solution 20ml; distilled water 600ml; potato starch 30g; glycerin 12ml; fresh egg liquid 1000ml.
specific preparation methods are as follows:
1,
heating
the above weight of dissolved phosphate, magnesium sulfate, magnesiumacid, Tianmen dongsin and glycerin in 600 ml distilled water.
2, stir and add potato starch, dissolve in boiling water for 1 hour, and place in a 56-degree water bath for 1 hour.
3, after washing the fresh eggs, in 75% ethanol disinfection for 30 minutes, remove after ignition and burn the ethanol on the egg shell, sterile break the eggshell, protein and egg yolks collected in the sterilization container, fully stirred, sterile gauze
filter
, in the collection of fresh egg liquid 1000ml;
4, add sterilization of 2% peacock green water solution, evenly mixed, divided into sterilized
tit tube
, plug closed, obliquely placed in the
serum
coagulator, 85 degrees 1 hour intermittent sterilization 2 times. Keep a spare in the fridge.
Synthic Bacillus count
netizen Benrong gave a counting scheme:
TB bacteria growth is slow and difficult to cultivate, not suitable for plate culture, one is easy to pollute the environment, the other is long training time, the tablet will soon dry out. Therefore, the cfu determination of tuberculosis bacteria cannot be made using flat-panel colony counting as E. coli. Generally few people do cfu count of TB bacteria, if necessary, can be diluted with Midlebrook 7H9 liquid culture, and then inoculated in Roche sloped culture base or Midlebrook 7H10, the count can be seen to the number of colonies on the culture slope of a single colony, calculate cfu.
general slope area of up to 4 to 5 square centimeters, as long as the number of live bacteria in the diluted bacteria solution is small enough, you can certainly get the number of single bacteria can be visually detected (n). Assuming that the volume of each dilution bacteria solution is v1 and the inoculation volume is V2, then cfu/ml × V1× dilution multiply/V2.
has been cultured TB colonies, can be used a glass test tube, plus a few glass beads, 1 ml of physiological saline, together with high-pressure steam sterilization. Then, pick the bacteria into this test tube, shake to obtain a uniform bacteria suspension, and then dilute to the desired wheat turbidity. Pure bacteria can also be inoculated to 7H9 broth culture, waiting for bacteria to grow and then diluted according to your needs to a certain degree of wheat turbidity of the bacteria. With the addition of OADC and Twain-80, Midlebrook 7H9 brooth is not only nutritious, but also bacteria can grow evenly.
netizen tb9981 think:
TB bacteria and general bacteria are not the same, easy to form bacterium groups, should be ground evenly before inoculation, preparation of 1mg /ml bacteria suspension with 10 times dilution, if inexperienced, Several more dilutions, each dilution of at least 3, each inoculation 0.1 ml, calculated the last full non-calculation, can count dilution colonies on it, generally qualified culture per milligram of Bacillus tuberculosis about 1-20 million live bacteria.
the first time, you can take the card seedling (0.5mg/branch) as a control (2-10m cfu/mg) to make sure your experimental method is correct.