Exercise.Ⅳ Bacteriolysis of Lysozyme
Experimental purpose
Conform the existence of lysozyme in body fluids and its solubility to grampositive bacteria.
Basic principles
The bactericidal mechanism of lysozyme is that it ACTS on the mucous peptide layer of bacterial cell wall，which is the main component of bacterial cell wall.Lysozyme can cut off the-1，4 glycosidic bond between n-acetylglucosamine and n-acetylated cytosolic acid in the structure of viscopeptides，thus damaging the viscopeptide scaffold and cell wall.Because one of the important functions of the bacterial cell wall is to protect the bacteria，that is，to resist low permeability，the bacteria can be dissolved in the low permeability environment after losing the protection of the cell wall.The main target of lysozyme is gram-positive bacteria.There are lipopolysaccharides，outer membrane and lipoproteins in the cell wall mucopeptide layer of gram-negative bacteria，therefore，lysozyme is not easy to play a direct role under normal circumstances.
Experimental materials
1.Staphylococcus：a gram-positive bacterium，which grows well in ordinary AGAR medium.
2.Standard lysozyme：standard pure lysozyme was weighed and prepared into 1000μg/ml original solution with 1/15mol.L-1 PBS and diluted into 100，50，10 μg/ml standard liquid，stored in the refrigerator before use.Used for positive control and preparation of standard curve.
3.Saliva：collect saliva in a sterile plate，which can be collected among students.
4.Others：aseptic punch（hole diameter：2mm），aseptic capillary straw，mm ruler，etc.(https://www.daowen.com)
Experimental methods
1.Perforate the staphylococcus AGAR plate with a sterile perforator，and pick out the AGARin the hole with a needle，2mm in diameter and 5 to 20 mm in spacing.
2.Fresh collected saliva was taken from the capillary straw and added into the AGAR hole，one drop per hole，about 0.025ml per drop，and standard lysozyme was added as positive control.
3.Observe the result after cultivation for 12 to 18 h under 24℃to 28℃.
Experimental results
（1）The diameter of the lysozyme ring around the hole was measured with mm ruler or triangle plate and recorded，which could be compared with the standard lysozyme positive control.
（2）If the content of lysozyme in the test sample（saliva）needs to be quantitatively determined，the standard solution of lysozyme with the above-mentioned dilutions can be added into the holes respectively，and the diameter of the lysozyme ring can be determined by the same method.The standard curve is made of semi-logarithmic paper，and the content of lysozyme in the test sample can be detected from the standard curve（Figure 1）.
Figure 1 Bacteriolysis of lysozyme NO.9