Two whiplash staining methods for bacteria

(i) Experimental purpose: to learn the bacterial whiplash dyeing method
(ii) experimental principle: bacteria whiplash is very fine, 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 materials:
culture
12-16h of Xanthomonas oryzae, viscous Serratia marcescens, or pseudomonas sp.) oblique strains.
2. Dyeing fluid and
reagents
: silver nitrate dye, Leifson dye, balsamic tar, xylene.
3. Equipment: slide, 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 and non-cracked slides, preferably new ones. To avoid the slides overlapping with each other, insert the slides on a special metal frame, then place the slides in a washing powder
filtering
solution (the washing powder is boiled and filtered with filter paper to remove coarse particles) and boil for 20min. After taking out a little cold with tap water rinse, dry, and then put in a thick lotion soaked for 5-6 days, before use to remove the slide, with tap water to flush off residual acid, and then washed 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
cultured
12-16h in a thermostat.
then, the number of bacterial fluid rings at the junction of the bevel and condensate is selected with the inoculation ring and moved to a test tube containing 1-2mL sterile water, so that the bacterial fluid is mildly cloudy.
put 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 whiplash of the young bacteria to expand. 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) drop plus A liquid, 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 up 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 appear 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 1.
(2) the dye dye after the dye is well-matched to filter 15-20 times after dyeing effect is good.
(3) the preparation of the bacteria liquid and the preparation of
(1) bacteria liquid with 1.
(2) divide 3-4 equal areas on clean slides with a marker pen.
(3) put 1 drop of bacteria at one end of the first sub-region, tilt the slide, let the bacteria flow to the other end, and use filter paper to absorb excess bacteria.
(4) dry naturally in the air.
(4) staining
(1) with dyeing fluid in the first region, so that the dye covers the smear. The dye is added to the second zone after a few minutes, and so on (the time is at your discretion), with the aim of determining the most appropriate dyeing time and saving material.
(2) washing: without dumping the dye, gently flush the dye with distilled water, otherwise it will increase the precipitation of the background.
(3) dry: naturally dry.
(5) mirror inspection first low-fold observation, then high-fold observation, and finally with oil mirror observation, observation to find more vision, do not attempt to see bacteria in 1-2 field of view whiplash.
result: the bacteria and whiplash are dyed red.
(v) Experimental operation: give the form map of whiplash bacteria
(vi) Note
1. Silver plating dyeing is relatively easy to master, but the dyeing liquid must be used now every time, can not be stored, more troublesome.
2. Leifson dyeing method is affected by factors such as bacteria, sterile age and room temperature, and the dyeing fluid must be filtered 15-20 times, in order to master the dyeing conditions must go through some exploration.
3. Bacterial whiplash is extremely fine and easy to fall off, and care must be taken throughout the operation to prevent whiplash from falling off.
4. Staining with slides clean and oil-free is a prerequisite for the success of whiplash dyeing.
: The motion observation of bacteria
(i) experimental principle Whether bacteria have whiplash is one of the important characteristics of bacterial classification and identification. Although the pattern, living position and number of whiplash can be observed by whiplash dyeing, it is time-time-time and troublesome. If you only need to know if a bacteria has whiplash, you can use suspension or water sealing method (i.e., pressure drop method) directly under the optical microscope to check whether the living bacteria have the ability to move, to determine whether bacteria have whiplash. This method is faster and easier.
the suspension drip method is to add the droplets to the center of a clean cover glass, coat Vancelin around it, and then cover it upside down in the center of the slide with grooves, which can be observed under a normal optical microscope. The water seal method is to drop the bacteria on the ordinary slide, and then cover the slide, under a microscope to observe.
most of the bacteria do not have whiplash, some of the bacillus has whiplash and some have no whiplash, Vibrio and screw bacteria almost all have whiplash. Bacteria with whiplash have strong motor force at an early age, and aging cell whiplashes are easy to fall off, so it is advisable to choose young bacteria when observing.
(ii) The experimental equipment
1. Living materials: Culture 12-16h hours of Bacillus subtilis, Staphylococcus aureus, pseudomonas sp.
2. Reagents: asphalt, xylene, Vladlin, etc.
3. Equipment: concave slides, cover slides, tweezers, inoculation rings, droppers, wipe paper, microscopes.
(iii) Experimental method:
1. Preparation of bacteria liquid: on the slope of young bacteria, drops plus 3-4mL sterile water, to make mildly cloudy bacteria suspension.
. Tufanslin: Take 1 piece of clean, oil-free cover slide and apply a small amount of Vieslin around it.
3. Drop-added liquid: add 1 drop of bacteria in the center of the cover glass, and use a marker pen at the edge of the bacteria liquid to make a mark, so that when viewed under the microscope, it is easy to find the location of the bacteria.
4. The cover concave slide Aligns the groove of the concave glass at the liquid in the center of the cover glass and gently covers the lid glass so that the two stick together, then flips the concave slide so that the liquid hangs right in the middle of the groove, and then gently press the glass with a pencil or matchstick to close the edges around the slide to prevent the liquid from drying out.
if a water seal is made, a drop of fungus is added to the slide and the cover glass can be viewed under a microscope.
5. The mirror first uses a low-fold mirror to find the marker, then slightly moves the concave slide to find the edge of the droplet, and then moves the liquid to the center of the field of view for a high-fold mirror observation. Since the bacterial body is transparent, the aperture can be appropriately reduced or the spotlight can be lowered to increase the contrast for easy observation. During the mirror examination, carefully identify whether it is the movement of bacteria or the movement of molecules (i.e. brown motion), the former can be seen in the field of view bacteria swimming from one place to another, while the latter only swings left and right in place. The speed of movement of bacteria depends on the type of bacteria, should be carefully observed.
results: Staphylococcus aureus with whiplash and fake monocytobacteria can be seen to be active, while staphylococcus aureus without whiplash does not.
(iv) experimental operation: draw a morphological map of the bacteria you see and use arrows to indicate the direction of their movement.
(v) Precautions are
1. Check the movement of bacteria slides and cover slides should be clean and oil-free, otherwise it will affect the movement of bacteria.
2. When making water seals, the bacteria can not be added too much, too much bacteria will flow under the cover glass, so in the field of vision only see a large number of bacteria moving in one direction, thus affecting the normal movement of bacteria observation.
. If observed using an oil mirror, add a drop of balsamic tar to the cover glass.
editor's note: Because xylene is harmful to the body, can be used waterless ethanol: waterless ether 2:8 or 3:7 instead, ether by anesthesia, also harmful to the body, but relatively better, and can better protect the lens.