How to distangushing Gram positive and Gram negative bacteria if you don’t have dyes and microscope? Describe this method and explain it.
Methodical instructions
In one glass of skim make strokes of different microorganisms in the center - a smear of cells studied culture, left and right - the control of microorganisms. The cells of one of the test organisms (eg, Staphylococcus aureus) Gram stain, and the cells of another (for example, Escherichia coli) - not colored. Smears must be thin so that the cells are uniformly distributed over the surface of the glass and did not form clusters, since the thickness of the stroke depends on the results of staining. Smears air-dried and fixed over the burner flame.
Rapid method for determining membership gram-positive bacteria or gram-negative species
The culture of the bacteria studied by means of a loop is transferred to the dense medium on a glass slide in a drop of 3% KOH solution and mix thoroughly. After 10 sec loop drops sharply raised above. Under these conditions, Gram-negative bacteria is characterized by mucus, which stretches for a loop at 0.5-1.0 cm mucus occurs as a result of the destruction of the cell walls of gram-negative bacteria and leaving the nucleic acids. If the mucus is not produced, the bacterium belongs to the gram-positive.
Characterize the methods of revealing reserve nutrients in microorganisms. What kind of methods do we use to reveal glycogen, starch, granulosis, volutin and lipid substances in microorganisms? Describe it.
To identify volutin in yeast usually used the following method.
PROCEDURE:
Fixed smear stained with methylene blue (Loeffler's methylene blue staining) for 3 min.
The dye is poured, the drug is washed with water and without drying, applied to smear a small drop of 1% solution of sulfuric acid.
A smear covered with a coverslip.
Volutin appears the form of drops of blue-purple color on the little-blue background of the cytoplasm.
Detection and visualization of Polyphosphate (volutin granules) in Bacteria.
Volutin detected by the method of coloring Omelyansky. Coloring is based on the metachromatic granules of low solubility in acid solutions.
PROCEDURE:
To skim the slide is prepared thin smear of bacteria, it is dried in air and fixed over the burner flame.
On the fixed smear Ziehl's solution is poured and stained the cells for 0.5 min without heating.
The dye is poured, the drug was washed with water and additionally stained with methylene blue (1:40) for 20-30 sec.
The drug is again washed with water and dried.
When properly stained grains volutin are red and clearly visible agains the background of blue cytoplasm.
Granules of polyglucose. (Glycogen, starch, granulosis).
Glycogen inclusions in cells of the well to investigate in Saccharomyces cerevisiae and Bacillus mycoides one-two-day age. To detect an object in granulosis cane use enrichment culture of Clostridium. These substances are detected microchemical processing cells Lugol's iodine solution.
3 a. Glycogen, starch (in Saccharomyces cerevisiae).
PROCEDURE:
A drop of cell suspension test organisms on a slide add a drop of Lugol's solution.
The drug is covered with a coverslip.
Granules of starch substances stained blue, and the pellets glycogen - a russet.
3 b. Granulosis (in Clostridium butyricum, Cl. butylicum, Cl. Pasteurianum).
PROCEDURE:
1. Apply a drop of microbial culture on a glass slide.
2. Add a drop of enrichment cultures Lugol's solution .
3. Covered with a coverslip, on which is placed a drop of immersion oil.
In places the cells, which contain granulosis, there is a blue color.
How to discover the mobility of bacteria? How to do it without microscope?
The motility of bacteria can be determined by several methods:
It can be determined microscopically by observing cells in a wet mount. This method is called the hanging drop technique. In this procedure a drop of cells is placed on a cover slip which is then placed on a special slide with a concave depression in its center. The coverslip is held in place with petroleum jelly. This creates an enclosed glass chamber that prevents drying. It is important to distinguish between cells that are moving due to the vibrations of the table and microscope and cells that are actually motile.
Fig.1 - Hanging drop technique
An alternative method, one that is safer when working with potential pathogens, is the motility stab. In order to determine if an organism is motile in semi-soft medium (SSM), examine the stab line where the tube was inoculated. If an organism is non-motile, it will be concentrated along the stab line. If the organism is motile, uniform turbidity is observed throughout the media. This distribution of cells within the agar is an indication the organism possessed flagella which allowed movement into the media.
Fig.2 - Reaction of motile and non-motile microbes in SSM
Methodical instructions
«Нanging drop» or a simple «wet mount».
PROCEDURE:
Using a toothpick applying a circular "ring" of Vaseline around the edge of depression on the slide (fig.3, step 1).
Take a cover glass and clean it thoroughly. It may be dipped in alcohol and polished dry with tissue.
Using good aseptic technique, sterilize the wire loop, remove the cap of the tube, and take up a loopful of culture. Be certain the loop has cooled to room temperature. Close and return the tube to the rack.
Place the loopful of agar culture in drop of water in the cover glass as in figure 3, step 2 (do not spread it around). (Place 5-10 loopfuls of the mixed overnight incubated broth onto a coverslip. Sterilize the loop and put it down.
Hold the hollow-ground slide inverted with the well down over the cover glass (fig.3, step 3), then press it down gently so that the petroleum jelly adheres to the cover glass. Now turn the slide over. You should have a sealed wet mount, with the drop of culture hanging in the well (fig.3, step 4).
Place the slide on the microscope stage, cover glass up. Make your examination with the high-dry and oil-immersion objectives (be very careful not to break the cover glass with the latter). Reduce your Iris to very LOW LIGHT! Focus under oil immersion and look for "tumbling" behavior.
Fig. 3- Hanging drop setup for observing motility under the microscope
Note:
To make visualization easier you can be added to the bacterial suspension drop of a weak solution of methylene blue.
Vibrating Microbes are NOT motile. You MUST focus within 3 minutes of preparation of the slide as by 5 minutes all the microbes are NON-MOTILE as they are dead - killed from the heat produced by the light source.
The motility gel deep/STAB test
PROCEDURE: