ازا اردت زرع بعض انواع البكتيريا الاكثر انتشارا فهناك e.coli & pseudomonas aeruginosa
لكن الا يمكنك ان تطلبي عينة من المختبر؟
اما بالنسبة لطريقة عزلها وتلوينها فهزا اسهل موقع مع الصور
Single Colony Isolation of Bacteria
It is necessary to separate individual bacterial cells from one another on an agar surface in order to produce pure colonies. A colony of bacteria is made up of millions of identical bacterial cells, each originating from one single cell.
Students should streak gently across the agar surface to avoid tearing the agar. If the cells are embedded in the agar it will not be possible to spread them out.
You will need:
• A source plate with colonies of several different species of bacteria growing on it
• 1 sterile agar plate per student
• Sterile toothpicks
• Sharpie marker for labeling
• Incubator, and extra plates for streaking practice
What to do:
Label the bottom of the plate with the date, type of agar being used, source of bacteria, and your initials.
Begin by looking at the bacteria growing on the plate you have already swabbed. Look for a colony of bacteria that you believe you can touch with a toothpick without touching any other colonies.
Once a bacterial colony is chosen for study, making sure you have chosen one that is bright pink or red in color, smooth, and uniformly round in shape, you are ready to begin the streak plate method.
Remove a colony from the MacConkey agar plate using a sterile inoculation loop or toothpick.
Replace the lid.
Remove the lid of the sterile agar plate, and place it upside-down on the table top.
Then with a gentle wiping motion, a series of horizontal streaks spread the bacteria across the edge of the new LB agar plate. The streaks should appear as smudges on a mirror. If too much pressure is applied during streaking, the agar surface may be torn.
After the first set of streaks is applied, the plate is rotated slightly. Then, using a new sterile toothpick, make three parallel streaks coming away from the first streak. The last streak is applied by streaking through the end of the three small parallel streaks and then streaking back and forth over the remainder of the plate without crossing over.
The object is to spread the bacteria so thin that individual cells are isolated on the surface of the agar.
Each of the isolated cells will give rise to a colony of its descendants when the plate is incubated.
After incubating at 37o C for 24 hours, you should have a genetically pure colony.
www.okscienceproject.org/core/StudyingBacteriaPart2.pdfومن هزا الموقع ايضا طريقة عزلها
اما بالنسبة لتلوينها فبواسطة gram stain
A Gram Stain is usually performed on a smear preparation that has been heat fixed. One function of fixation is to secure (fix) the cells to the slide. In a biofilm, however, the cells are already fixed. Furthermore, a heat fixed slide is dry, but a biofilm is mostly water. Drying alters the biofilm virtually beyond recognition. Described here is a method for obtaining a Gram Stain on a minimally altered biofilm.
Instructions
Preparing the biofilm for staining.
Prepare a biofilm slide (by the micro-fishing technique for example).
Wipe the top and bottom 2 mm of the slide clean.
Thinly smear some petroleum jelly on a sheet of paper. Hold a cover slip at right angles to the petroleum jelly and scrape it in such a way as to build up a thin ridge along one edge of the cover slip.
Repeat the process with the opposite side of the cover slip.
Place the cover slip onto the biofilm slide preparation.
Preparing the Gram stain.
The Gram stain reagents are added at one open end of the slide and are drawn through the space under the slide by means of an absorbent paper towel. The reagent sequence is similar to that in the standard Gram Stain but the timing is somewhat longer to compensate for a possible dilution effect of the aqueous phase of the biofilm. It is important to be certain that sufficient reagent has been drawn under the coverslip so that dilution by the volume of water present or the previous reagent does not occur. The bacteria stain as in a traditional Gram Stain, but because of the thickness of the preparation and the presence of water the slides do look different from a typical Gram Stain.
Add the Crystal Violet dye to one edge of the coverslip. Draw the dye through using an absorbant paper towel applied to the opposite side of the cover slip. Draw the dye through until all the water has been replaced by dye. Stain for 30 seconds.
Wash the slide by drawing water under the coverslip with a paper towel until no further purple color is removed.
Add the Gram's Iodine solution by adding several drops of the reagent to the same edge of the coverslip and drawing it through with a paper towel. About 1.5 minutes.
Decolorize the slide by drawing 95%alcohol under the slide until no further purple due is removed.
Wash the slide as in step two.
In a similar manner, counter stain the slide with Safranin dye for .5 minute.
Wash the slide as in step two.
Observe the slide under high dry or oil immersion microscopy.
The Following is a Diagram of the above instructions
وطريقتها سهلة جدا من خلال هزه الرسمة
http://www.personal.psu.edu/faculty/j/e/jel5/biofilms/graminstr.jpgوهزه صورة staph aureus
http://www.life.umd.edu/classro....ain.jpgوهزه صورة للe.coli
http://biology.clc.uc.edu/fankhau....897.JPGانشاء الله يفيدك
