Experiments on the preparation of heterogeneous dyeing particles and dyeing methods

Summary

Experiments with heterochromatic particle staining preparations and staining methods can be used to (1) detect the presence of Corynebacterium diphtheriae and (2) provide good contrast between heterochromatic particles and the organism.

Operation method

Heterogeneous particle stain preparation and Gram staining

Principle

For diphtheria bacillus staining, heterochromatic particles can be clearly shown, and specimens can be stained with modified Abbott's method to visualize heterochromatic particles in both direct smears or bacterial smears.

Materials and Instruments

Corynebacterium diphtheriae
Toluidine blue Malachite green Glacial acetic acid Ethanol
Culture medium Slide

Move

Experimental reagents:

Liquid A: Toluidine blue: 0.15g Malachite green: 0.2g

Glacial acetic acid: lml 95% ethanol: 2ml

Distilled water: 100ml

Dissolve the dyes in ethanol, and then add them into the mixture of water and glacial acetic acid, and mix thoroughly. Let it stand for 24h and then filter it.

B solution: iodine: 2g Potassium iodide: 3g

Distilled water: 300ml

Add potassium iodide with a little distilled water (about 2ml) and shake it thoroughly, wait until it is completely dissolved, then add iodine to make it completely dissolved, and then add distilled water up to 300ml.

Staining method:

1. Smear is fixed by flame, and then added with A staining solution to stain it for 3-5min, and then rinsed with water.

2. Add B solution to it, and then stain it for 1min, and then rinsed with water.

3. After drying and microscopic examination, the bacterium is green and the foreign stained particles are blue-black.

Caveat

After the bacteria are repeatedly transferred to the egg slant, the bacterial body is elongated and more consistent, and the staining effect is good. Generally, 3 or 4 times can be transferred.

Common Problems

The effect of A and B liquid overflowing out of the circle on the staining result: if A liquid or B liquid overflows out of the circle after addition, the bacterium will be purple after staining, and there will be several heterogeneous staining particles in the form of beads on it, and the contrast will not be clear.

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