Here is a list of top three experiments on bacteria: 1. Inoculation and cultivation of soil microorganisms 2. To isolate and cultivate bacteria from leaf sample 3. To stain bacterial smear by using gram staining technique.
Experiment # 1. Inoculation and cultivation of soil microorganisms.
Requirements:
Soil sample, petridishes, potato dextrose agar medium, incubator, metal loop, double distilled water, sieve and pipette.
Soil dilution plate method:
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Proceed step-wise as described below:
(i) Take 5 gm. of air dried sieved soil, add 500 ml of sterilized distilled water and shake the container in a mechanical shaker for 30 minutes (dilution 1: 100).
(ii) With the help of a pipette take out 10 ml of soil water suspension, add 90 ml of sterilized water in it and shake thoroughly (dilution 1: 1000).
(iii) Take 20 ml from (ii) and add an equal volume of sterilized distilled water (dilution 1: 2000).
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(iv) From each of the above dilutions (i, ii and iii) spread 1 ml of clear supernatant of soil suspension in separate well sterilized petridishes.
(v) Add 20 ml of cooled potato dextrose agar medium in each petridish and then rotate the petridishes so that the sample gets dispersed uniformly in the medium (hereafter referred to as petriplates or plates only).
(vi) Incubate a few petriplates at 20 ± 2°C and rest of them at 37 ± 2°C for 5-6 days.
(vii) Observe these plates regularly. Microbial colonies will be observed. The plates at 20 ± 2°C will have predominantly fungal colonies, while those at 37 ± 2°C will have mainly bacterial colonies.
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Precautions:
1. Keep the plates inverted while incubating when medium gets solidified.
2. Keep covered the plates after soil suspension is taken in step (iv) to prevent contamination of any air borne microorganism.
Preparation of medium:
Take cork peeled and sliced potatoes in 50 ml of distilled water. After sometime filter the potato extract and store. Dissolve 20 gm. agar-agar in 500 ml of distilled water by heating. Add potato extract in agar-agar solution just prepared and make it to 1000 ml by adding distilled water. Then add 20 gm. dextrose sugar in the extract so prepared. Sterilize the medium, thus, prepared and now the medium is ready for use.
Experiment # 2. To isolate and cultivate bacteria from leaf sample.
Requirements:
GYCA medium, sterilized scissors, sterilized distilled water, 0.1% mercuric chloride solution, petriplates, infected leaves, incubator, metal loop.
Preparation of GYCA medium:
Take 1000 ml of double distilled sterilized water, add 5 cm of glucose, 5 gm. of yeast extract + 4 gm. calcium carbonate and 15 gm. agar. Sterilized the solution so formed and then GYCA medium becomes ready for use.
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Microbiological Techniques
Procedure:
Prepare petriplates with GYCa medium in the manner described earlier. Then collect bacterial blight infected leaves with tiny black spots and old brown lesions. Sterilize the surface of these leaves by dipping them in 0.1% mercuric chloride solution and then wash them thoroughly with sterilized distilled water 3-4 times.
Cut the leaves into small pieces with the help of sterilized scissors. Place these leaf pieces over GYCA plates. Keep these plates in an incubator at 37°C for 4-5 days. Observe, you will note watery, pale yellow coloured growths at the cut ends of the leaf pieces after the incubation.
Take fresh petriplates having GYCA medium and transfer a small part of the growth so formed on the petriplates with the help of metal loops. Keep these petriplates in an incubater at 37°C for 4-5 days again and observe. Cultivation of bacteria occurs.
Experiment # 3. To stain bacterial smear by using gram staining technique.
Requirements:
Crystal violet solution, Lugol’s iodine, 1% Basic fushsin, 95% alcohol, cover-slips, microscopic glass slides, sterilized distilled water, metallic loop and Bunsen burner.
Preparation of Reagent:
Crystal violet solution: Take 0.5 gm. crystal violet in 20 ml of absolute alcohol and add 80 ml of 1% ammonium oxalate.
Lugol’s iodine:
Take 1000 ml of distilled water add 1 gm. iodine and 2 gm. potassium iodide.
1% Basic fuchsion:
Dissolve 100 mg of basic fuchsin in 100 ml of boiling distilled water.
Preparation of bacterial smear:
Take microscopic glass slides and cover-slips. Wash them carefully and rinse them with chromic acid solution. Wash them in tap water several times and then in distilled water.
Make the cover-slips and slides sterilized and then prepare smear of any one of them as suggested below:
Sputum:
Take a drop of sterilized distilled water on the slide. Touch the sputum by the sterilized metallic loop and transfer it to the drop of water. Put the cover-slip over the water drop gently so that the water drop spreads uniformly under the cover slip. Dry the smear so formed by gently warming it over Bunsen’s burner and stain (process given below).
Milk:
Take a drop of milk on the microscopic glass slide cleaned as above. Spread the milk drop over the slide by a sterilized needle to form a thin film. Dry the smear so formed over a Bunsen’s burner by gently warming it and stain.
Staining of Smear:
Stain the smear in crystal violet solution for 1 minute. Rinse it in water, then put Lugol’s iodine solution over the smear. Rinse it again in water and pour 95% alcohol over it till smear shows clear colour. Then rinse it again in water. Stain the smear again in basic fuchsin solution for 30 minutes, wash in water. Let in dry and observe under microscope.
Result:
The gram positive bacteria is stained deep violet or blue and gram negative bacteria is stained pink or red.