Exercise.Ⅰ Agglutination
Slide Agglutination Test Identification of strains
Purpose：
Observe the bacterial agglutination phenomenon in which bacteria bind to their corresponding antibodies on the slide and understand the specificity of the antigen-antibody reaction.
Experimental principle：
The known bacterial antibody is mixed with the bacteria to be tested，and if the antigen corresponds to the antibody，the bacteria are caused to aggregate，and conversely，the agglutination is not performed，and the bacterial species can be judged according to the agglutination phenomenon.
Experiment equipment：
1.1∶20 Salmonella typhimurium immune serum，Salmonella typhimurium and Escherichia coli 24 hours agar slant culture.
2.saline，slides，markers，inoculation rings，alcohol lights，etc.
Experimental method：
1.Take a clean slide，add 1 drop of Salmonella typhimurium to the left and middle of the slide，and add 1 drop of normal saline on the right side；
2.Pick the smear culture of Salmonella typhimurium with a sterile inoculating loop，mix well with the normal saline and intermediate diagnostic serum on the right side，and also mix the Escherichia coli slant culture with the left diagnosis serum；operating.
3.Shake the slide gently and observe it after 1 to 2 min.
Salmonella typhimurium immune serum Salmonella typhimurium immune serum saline（ns）
Escherichia coli Salmonella typhimurium Salmonella typhi.Results observation：
Positive：The liquid became clear and there was a milky white agglomerate.
Negative：The liquid is still turbid and no agglomerates appear.
After recording the results，place the slide in the designated container containing the disinfectant.Do not place or rinse it.
Note：When taking the bacterial culture，it should not be too much.When mixing with the immune serum，the bacteria must be spread and evenly coated，but it is not appropriate to coat the area too much to avoid drying up quickly and affecting the observation.In the identification of bacteria，especially the Salmonella or Shigella of the intestinal bacteria，in principle，the multivalent diagnostic serum is used for detection.If it is positive，the monovalent diagnostic serum is used for grouping or stereotyping.When the blood type is measured，the room temperature needs to be kept at about 20℃.If it is lower than 10℃，the condensation phenomenon is likely to occur and the false positive diagnosis is caused.
Tube Agglutination Test
Principle
Dilute the examinee's serum consecutively.Mix it with antigen suspension of known concentration in test tube.Observe the agglutination phenomenon in certain reaction time.
In this experiment，typhoid bacterium of certain quantity acts as antigen.Whether serum contains corresponding antibody or not is up to the agglutination reaction to decide.Furthermore，we could decide the titer of antibody by the agglutination extent in different tubes.We could diagnose and tell patient condition and prognosis by this principle.
Materials
1.Inactivated typhoid bacteria（7×108/ml）
2.Animal serum to be examined（Immunized by typhoid bacteria‘H'antigen）(https://www.daowen.com)
3.Normal saline（NS）
Methods
1.Take 6 test tubes.Mark them and put into rack in turn.
2.Add NS 0.9ml into No.1 test tube.Add 0.5ml NS into other tubes.Then add typhoid immunized serum 0.1 ml to No.1 tube and mix it with NS evenly.Draw 0.5 ml mixed fluid from No.1 tube to No.2 tube.Mix the fluid evenly in No.2 tube and draw 0.5 ml to No.3 tube.Also in such way the serum is diluted in other tubes.When it turns to No.5 tube，after the mixation of fluid，draw 0.5 ml and discard it.So the No.6 tube contains no typhoid immunized serum and acts as the negative control tube in this experiment.At last，add 0.5 ml typhoid bacteria fluid in each tube.The table below indicates the whole process.
3.shake rack gently to mix the fluid in test tubes.Put rack in 56℃water bathe for 1 to 2 hour.（Table 1）
Table 1 Adding sample table of tube agglutination test
Results
1.Control tube（No.6 tube）：Supernatant is turbid.Precipitated bacteria lie in the tube bottom.Shake gently and bacteria disperse into evenly turbid fluid.
2.Experiment tube：Observe result in turn from tube 1 to tube 5.Positive result tube bottom has irregular and loose agglutination clot.Negative result tube has same result with control tube.
3.Classification of agglutination extent.
++++：Fluid is clear and transparent.Bacteria agglutinate completely to the tube bottom.Shake gently and large agglutination clot could be seen.
+++：Fluid is slightly turbid.Bacteria agglutinate mostly to the tube bottom.Shake gently and comparatively smaller agglutination clot could be seen.
++：Fluid is obviously turbid.Shake gently and agglutination clot could be seen obviously.Agglutination clot is smaller.
+：Fluid is turbid.Observed carefully，small agglutination particle could be seen.
-：Same as control tube.
4.Judgement of agglutination titer.
The titer is customarily reported as the reciprocal of the highest dilution that causes an obvious agglutination（++）.
Attentions
1.Mark the tubes in turn.Pipette of different reagent should not be mixed up.
2.Be sure to mix evenly when dilute the antiserum and then add to next tube.
3.Add reagent accurately.Avoid air bubble.
4.Shake gently after all reagents are added and then have water bathe.
5.During water bathe，keep the tubes from hot water dropping into them.
6.After experiment，take out the tubes gently，in case agglutination would disperse and result is hard to observe.
7.Observe the control tube to decide the experiment credibility.Then observe the experiment tubes.