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Agar Plate and Agar Media Troubleshooting and Forum Topics
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Soil microorganisms such as bacteria are vital for crops and agriculture. Their study is crucial in the study and improvement of crop growth and culture.
Apparatus Needed for Study
Sterile soil borer; sterile paper bag; sterile large spatula; sterile large piece of paper; sterile paper 15 cm. Square; 500 c.c. wide-mouthed flask containing 200 c. c. sterile water; 90 and 99 c.c. dilution flasks (sterile water); ordinary agar; sterile Petri dishes; soil (different types), manure, etc.
Each student should use one type of soil and one type of manure for this experiment. Assignments will be made by the instructor. The results obtained by each student are to be compared with those of others.
1. Remove the coarser surface debris from the soil. Sink the soil borer to the depth of 30 cm., remove the borer and place the soil in the bag.
2. Take six borings so that your composite sample will be representative of the entire plot under considera tion.
3. At the laboratory, carefully mix and pulverize the composite sample with the spatula on the large piece of paper.
4. Weigh out 20 gms. On sterile paper and transfer immediately to the flask containing 200 c.c. of sterile water. A large amount of soil is used to reduce the error as much as possible. The manure should be treated in a similar manner.
5. Shake thoroughly for one minute, allow the coarser particles to settle and transfer 10 c.c. (equivalent to 1 gm. Of soil) of the supernatant liquid to 90 c.c. of .sterile water. Each cubic centimeter of this dilution then contains 0.01 gm. Soil.
6. Make and plate from the following dilutions: 1-10, 1-100, 1-1000, 1-10,000, 1-100,000, 1-1,000,000.
7. Incubate at room temperature for four to eight days.
8. Count and record the results as number of bacteria per gram of soil or manure, in tabular form.
9. Record the number of the various types of micro organisms. Note the numbers of chromogenic bacteria and the streptothrix forms.
10. Examine some of the manure in the hanging drop. What forms are seen? Make drawings. Are all of these forms found on the plates? Give reasons for what does occur. Add sterile water to the manure in a sterile deep culture dish or flask and examine every three or four days during the course of the experiment. Record your obser vations.
11. When the peat sample is obtained, at the same time partially fill a small sterile flask with swamp or marsh water. Examine immediately in the hanging drop and draw the forms seen.
12. Place this swamp water in the sunlight (more or less direct) for two or three days and examine again in the hanging drop for any forms of life present.
13. Compare the flora of these two microscopical prep arations. Suggest why each type of organism is present.
14. Compare all types of soil examined both quanti tatively and qualitatively as to their microflora. Which soils are most alike in their flora? Suggest a reason why. Why do various soils vary in the number of bacteria found?
B)
Apparatus
Soil or manure of same type as used for plating; sterile water; sterile Chinese ink; platinum loop of known capacity; sterile watch glass; cover-glasses, absolutely clean] ocular micrometer; stage (object) microm eter.
Method
1. To 1 gm. of soil or excrement (manure) in a test tube add 4 c.c. of sterile water and shake vigorously for five minutes.
2. Place 0.5 c.c. in a clean, sterile watch glass. Add 0. 5 c.c. of Chinese ink.
3. Mix with a platinum loop of known capacity.
Note. To determine the capacity of the platinum loop, weigh two watch glasses. Into one put exactly 1 gm. (1 c.c.) of water. Transfer five loopfuls from the glass containing water to the empty watch glass.
Weigh each. Then determine the weight and also the volume of one loopful.
4. Transfer one loopful of the " ink manure " solution to a clean, sterile cover-glass and spread in an even film over the entire surface.
5. Let this dry in air and fix by passing three times through the flame. Mount at once in balsam.
6. Measure the surface area of the cover-glass. Also the diameter of one field of the oil immersion lens (using the stage micrometer) and from that the area of the field.
7. Count fifty fields and determine the average.
8. From the data which you now have, determine the number of organisms on the cover-glass, which is the number in one loopful.
9. Then from this calculate the number in 1 gm. Of soil or excrement.
10. Also calculate the weight of bacteria in 1 gm. (See p. 88, Marshall's Microbiology.)
11. Compare the count thus obtained with the count obtained by the plate method. What is shown? How do you explain this result?
12. Compare the manure and soil counts. Draw con clusions and explain.
13. What other methods are used for obtaining numbers, etc., of organisms in soil and like materials?
14. State your data and observations in full. Draw any conclusions warranted and point out any practical applications.
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