Bacterial whiplash staining

(i) Experimental purpose:
to learn the bacterial whiplash dyeing method
(ii) experimental principle
the bacteria whip hair is very fine, the diameter is generally 10-20nm, only with electron
scope
can be observed. However, if special staining methods are used, they can also be seen under ordinary optical microscopes. Whiplash dyeing methods are many, but its basic principle is the same, that is, before dyeing with media dye treatment, let it deposited on the whiplash, so that the diameter of whiplash thickened, and then dyed. Commonly used media dyes are made from daninic acid and high-speed chloride or potassium alum. The following two dyeing methods are recommended.
(iii) the test equipment
1. Living
:
cultured
12-16h of Xanthomonas oryzae, adhesive Serratia marcescens, or pseudomonas sp.) sloping bacteria.
2. Staining fluid and
reagents
: silver nitrate staining fluid (see annex II, (i), 8), Leifson staining fluid (see annex II, (i-), 9), balsamic asphalt, xylene.
3. Equipment: slides, mirror paper, absorbent paper, marker pen, slide shelf, tweezers, inoculation ring, microscope.
(iv) The experimental method
1. Silver-plated dyeing
(1) cleaning slides Select smooth, crack-free slides, preferably new ones. In order to avoid the slides overlapping each other, the slides should be inserted on a special metal frame, and then put the slides in the washing powder
filtering
liquid (washing powder boiled with filter paper filter to remove coarse particles), boiling 20min. Remove slightly cold and rinse with tap water, dry, and then soak in a thick lotion for 5-6 days, remove the glass before use, wash the residual acid with tap water, and then wash with distilled water. Drain the water and dehydrated it in 95% ethanol.
(2) bacterial fluid preparation and production: bacteria older bacteria easy to lose whiplash, so before dyeing should be the newly prepared bacteria in the newly prepared beef paste protein
fase
slope (the base surface moist, the slope base contains condensate) continuous transfer 3-5 generations, to enhance the movement of bacteria. The last generation of bacteria put
rout temperature
box culture 12-16h. Then, with the inoculation ring to pick the bevel and condensate junction of the number of bacteria, moved to a test tube with 1-2mL sterile water, so that the bacteria liquid is mildly cloudy. Place the test tube in a temperature box at 37 degrees C and set aside 10min (the placement time should not be too long, otherwise the whiplash will fall off), so that the young bacteria whiplash loose unfold. Then, absorb a small amount of bacteria droplets at one end of the clean slide, immediately tilt the slide, so that the bacteria slowly flow to the other end, with absorbent paper to absorb excess bacteria. Smears are naturally
air
.
bacteria used for whiplash staining can also be cultured with semi-solid media. The method is to melt 0.3-0.4% of the
agar
paste medium into a sterile flat dish, after solidification in the center point of the plate to liven up 3-4 generations of bacteria, after constant temperature culture 12-16h, take the edge of the diffused bacteria to make smears.
(3) staining
(1) drops with A, dyeing 4-6min.
(2) wash the A liquid fully with distilled water.
(3) wash away the residual water with B liquid, add B liquid to the slide, heat on the flame of the alcohol lamp to the air, about 0.5-1min (when heating should be replenished at any time evaporated dye, do not make the slide dry area).
(4) is washed with distilled water and dried naturally.
(4) mirror inspection: first low times, then high times, and finally with oil mirror inspection.
results: the bacteria are dark brown, whiplash is light brown.
2. Improved Leifson staining method
(1) cleaning slide method is the same as 1.
(2) the preparation of dyes see Appendix II (i) 9. Dyes after the preparation to filter 15-20 times after dyeing effect is good.
(3) the preparation and smearing of the