The special dyeing technique of bacteria - spore staining film dyeing whiplash dyeing

one. Objective: To learn
the principles and methods of spores, film and whiplash dyeing.
two. Principle:
spore staining method is based on the bacteria spores and bacteria on the dye affinity of different principles, with different dyes for dyeing, so that spores and bacteria are different colors and easy to distinguish.
Spore wall thick, low permeability, coloring, decoloration are more difficult, when the use of weak alkaline dye peacock green in the case of
heating
staining, this dye can enter the bacteria and spores to color, and the dye into spores is difficult to penetrate. If re-dyed (red liquid), the bacteria are red and spores are green.
to observe the diaphragm usually uses negative staining method, that is, after the bacteria chromosomal, and then make the background coloring, so as to support the membrane out.
observation of whiplash is the use of dyeing at the same time to accumulate dye on the whiplash to make it bold. Bacteria only have whiplash when an individual develops to a certain period of time, and are generally dyed in its exuberant growth stage after multiple species transfers.
wednesday. Experimental materials: 1. Bacteria: B.megaterium: Nutrition
agar
slope
culture
20hs E. coli (Enterobacter aerogenes): Gravy and other oblique culture 20hs common phospheric bacteria (Proteus vulgaris): nutritional agar slope culture 18hs.
2． Dyes:
(1) spore staining with 7.6% saturated peacock green liquid, 0.5% red liquid (or charcoal acid red liquid and Lu's blue liquid)
(2) membrane dyeing with Congolese red, gelatin water solution, Lu's blue liquid and so on.
(3) whiplash dyeing with silver nitrate dyeing fluid (A, B liquid); Or Leifson staining fluid (A, B, C)
3. Other:
microscope
, carrier, inoculation ring, alcohol lamp, balsamic asphalt, xylene, sterile water,
1% hydrochloric acid, electric furnace, ink, 20% CuSO4, 95% ethanol, mirror paper, etc.
Thursday. Experimental methods and steps:(i) spore staining: introduction of two commonly used methods
1. Peacock green dyeing method: take a clean piece according to the sterile method to take a small amount of Bacillus spores made of smears, air-dried fixed, in the coating of 7.6% of the peacock green saturated water solution, dyeing 10 minutes later,
rinsed with water, and then with 0.5% red serum dye color 1 minute, water wash, air-dried mirror inspection. Spores are dyed green and the nutrients are red.
2. Charcoal acid red staining method: in a small
test tube
(10 x 100mm), drop 3-4 drops of steaming water, with the inoculation ring to take huge Bacillus spores in the water, fully stirred, so that the bacteria dispersed, to make a stronger bacteria suspension. Then drop plus equal volume (3-4 drops) of charcoal acid redness shake well. Put the test tube in a boiling water bath and cook for 10-15 minutes to color the spores and bacteria.
Take this bacteria liquid 2-3 ring on a clean film to make smears, natural
dyering
through the flame fixed, in tap water slowly buffer wash, so that the bacteria decolor, and then with Lu's blue liquid re-dye 1-2 minutes. Wash the excess dye with water, gently use absorbent paper to absorb moisture, dry mirror inspection, the result can be seen spores are dyed red, the bacteria appear blue.
(ii) membrane staining method
1. Method (1)
A drop of Congolese red water solution and gelatin solution is dropped on a clean carrier, bacterial culture or suspension is mixed and dried naturally on the carrier with inoculation ring, 1% HCl is rinsed so that the smear is blue, HCl is removed, and the water is re-dyed with blue for 1 minute, and the natural drying mirror is checked.
: bacteria with cystic membrane, bacteria blue, membrane colorless, background blue-purple; Due to the contraction of the dry bacteria, there may also be a narrow noncolored ring around the bacteria, but this is not a film. The part of the film that is not colored is wide.
2. Method (2) Wet ink method
(1) bacteria solution: add a drop of ink to the clean wave flakes, and pick a small amount of bacteria and mix well with it.
(2) cover glass: put a cleaning cover slide on the mixture, and then put a filter paper on the cover glass, press down, absorb excess bacteria.
(3) mirror examination.
results: the background is gray, the bacteria are darker, and a bright transparent circle is presented around it, namely the membrane.
3. Method (3) TyLer method
(1) Smear: smear according to the conventional method, naturally dry in the air.
(2) dyeing: dye with crystalline amethyst acetate for 5-7 minutes.
(3) washed with 20% CuSO4 aqueous solution and dried with fur-edged paper.
(4) mirror examination and observation results: membrane blue-purple, cell dark blue.
(iii) whip hair dyeing method
1. Silver salt staining method
(1) cleaning slides: choose smooth and non-cracked slides, preferably with new ones. To avoid the slides overlapping back, insert the slides on a dedicated metal frame. Then place the slide in the washing powder filter liquid (the washing powder is boiled first, then filter
with the filter paper
to remove coarse particles), boiling 20 minutes.
a little cold and rinse with tap water to dry. Soak in a thick lotion for another 5-6 days. Remove the slides before use, wash off the residual acid with water, and wash with distilled water. Drain the water and dehydrated it in 95% ethanol. Remove the slides, burn the alcohol on the flame and use immediately.
preparation and smearing of a large (2) bacterial fluid: the strains used for dyeing should be pre-continuously transferred for 5 to 7 generations. the
flibrium
used to pick up bacteria before dyeing should be freshly prepared, the surface wet, and there should be a little condensate at the bottom of the slope.
Inoculated Bacillus deformation on the slope of the broth, cultured at a suitable temperature for 15 to 18 hours, with a receiving ring to pick the bevel bottom bacteria moss ring, gently moved into a sterile water with 1 ml of the same temperature as the bacteria, do not vibrate, so that the active bacteria swim into the water, mildly cloudy.
keep warm for 10 minutes at the most suitable temperature, allowing the old bacteria to sink, while the young bacteria in sterile water can release the whiplash. Then pick a number of cyclic fluids from the upper end of the test tube and place them at one end of the clean slide, slightly tilting the slides so that the fluid flows slowly to the other end. naturally dry in the air.
(3) staining:
(1) drip with A, dye for 4 to 6 minutes.
(2) Gently and adequately wash the A liquid with distilled water.
(3) Wash the residual water with B-liquid, add B-liquid to the slide, heat it over a low heat until steaming, about 0.5 to 1 minute (the evaporated dye should be replenished at any time when heated, so that the slides do not dry out).
(4) washed and dried with distilled water.
(5) mirror
results: bacteria and whiplash are dark brown to black.