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In this article we will discuss about the Isolation of Various Microorganisms:- 1. Isolation of Algae, Fungi, Bacteria and Actinomycetes from Soil 2. Isolation of Bacteria from Milk Samples by Agar Plate Method 3. Isolation of Microorganisms from Air by Exposure Plate Technique 4. Isolation of Microorganisms from Water or Waste Water Samples 5. Isolation of Root Nodule Bacteria.
Contents:
- Isolation of Algae, Fungi, Bacteria and Actinomycetes from Soil
- Isolation of Bacteria from Milk Samples by Agar Plate Method
- Isolation of Microorganisms from Air by Exposure Plate Technique
- Isolation of Microorganisms from Water or Waste Water Samples
- Isolation of Root Nodule Bacteria
1. Isolation of Algae, Fungi, Bacteria and Actinomycetes from Soil:
Principle:
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Few environments on earth provide as greats variety of microorganisms as fertile soil. Bacteria, fungi, algae, protozoa and viruses make up this microscopic menagerie, which may reach a total of billions of organisms per gram of soil.
Various methods are available for counting the organisms in soil as well as for isolating various species in pure culture. This is generally done by plating method following dilution. Different kinds of organisms are isolated in specific media. The developing colonies are then counted to get an idea of the microbial population of the soil.
Material:
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Soil sample – 10 gms.
Requirements:
(i) Sterilised distilled water.
(ii) Sterilised petridishes.
(iii) Sterilised pipettes, culture tubes.
(iv) Ingredients of media as stated below.
(v) Inoculating media, flasks.
(vi) Burner, Balance, Weight box etc.
Details of Soil Sample:
Samples of soil arc collected from different places. The colour of the soil is noted and its water content is determined.
A. Preparation of Media for Isolation of Bacteria:
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Media Composition:
K2 – 0.4 gm.
NH4HPO4 – 0.5 gm.
MgSO47H2O – 0.05 gm.
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MgCl2 – 0.1 gm.
CaCl2 – 0.1 gm.
FeCl3 – 0.01 gm.
Peptone – 1.0 gm.
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Yeast extract – 1.0 gm.
Agar agar – 20 gm.
Distilled Water – 750 ml.
pH – 7.4
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20 gms. of agar is weighed, taken in a flask containing 500 ml distilled water and heated in a water-bath until the agar melts. In another flask all the other ingredients are taken along with 250ml. of water. This solution is added to the melted agar solution and the pH of the medium is adjusted to 7.4.
The medium is then dispensed in tubes, each containing about 18 c.c. of the medium. The tubes are then plugged and autoclaved at 15 lbs. pressure for 20 min.
B. Isolation of Actinomyces:
(Dextrose-casein-agar Medium)
Composition:
Dextrose – 2.0 gm.
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Caesin – 0.2 gm.
(dissolved in 10 ml. of 0.1(N) NaOH soln.)
K2HPO4 – 0.5 gm.
MgSO4, 7H2O – 0.2 gm.
FeCl3, 6H2O – trace.
Agar agar – 20 gm.
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Distilled water – 1000 ml.
pH – 6.5 to 6.6.
20 gms of agar is weighed and added to 500 ml. of distilled water in a flask and heated in a water-bath until the agar melts. All the other ingredients except casein are weighed and added to 490 ml. water in a flask. Caesin is dissolved in 10 ml. of 0.1 (N) NaOH solutions and then added to the solution containing all the ingredients.
The agar solution is mixed thoroughly with the other solution. Now the pH is adjusted to 6.5 – 6.6. The medium is then dispensed in tubes and each tube is plugged and autoclaved at 15 lbs. pressure for 20 minutes.
C. Isolation of Fungi:
(Streptomycin-peptone-dextrose Medium of Johnson 1957)
Composition:
KH2PO4 – 1.0 gm.
MgSO4, 7H2O – 0.5 gm.
Peptone – 5.0 gm.
Dextrose – 10.0 gm.
Distilled Water – 1000 ml.
Rose Bengal – 10.0 ml. (1:30,000)
Streptomycin – 30 µg/ml.
or Aureomycin – 20 µg/ml.
20 gms. of agar is dissolved in 250 ml. of distilled water in a flask by heating on a water-bath. All the other ingredients except Rose-Bengal and antibiotics are dissolved in 750 ml. water in a flask and then this solution is poured in the agar solution.
The mixture is then heated and stirred continuously, till it boils. After removing from the heater 10 ml. of Rose-Bengal (1:30,000) dilution is added at the rate of 1 ml/100 ml. of medium.
The medium is then dispensed in 100 ml. conical flasks at the rate of 100 ml. per flask. The flasks are then plugged and autoclaved at 15 lbs. pressure for 20 minutes. The antibiotic solution (streptomycin) is added before plating the medium at nearly 45°C (streptomycin inhibits the growth of bacteria while Rose-Bengal is a growth retardant).
D. Isolation of Algae:
(Chou-10-Medium)
Composition:
K2HPO4 – 0.01 gm.
Ca(NO3)2, 4H2O – 0.04 gm.
MgSO4, 7H2O – 0.025 gm.
Na2SiO3, 9H2O – 0.025 gm.
FeCl3 – 0.008 gm.
Agar agar – 15.0 gm.
Distilled Water – 1000 ml.
15 gms. of agar is dissolved in 500 ml. distilled water. The other ingredients are dissolved in 500 ml. distilled water. The two solutions are mixed; the pH is adjusted and then dispensed in tubes. They are autoclaved at 15 lbs. pressure for 15 minutes after plugging.
Procedure:
(a) Preparation of Soil Suspension:
10 gms. of sieved soil sample is added to 90 ml. of sterilised distilled water. The flask is shaken vigorously to ensure thorough mixing. Serial dilutions are made by transferring 1 ml. of suspension to 9 ml. of sterilised water in a culture tube.
This is soil suspension of 10-1 dilution. After mixing this solution in a vortex mixer, 1 ml. of this suspension of 10-1 dilution is transferred to 9 ml. of sterilised water. This is suspension of 10-2 dilution. This process is repeated until 10-7 dilution is obtained. The whole process is carried out aseptically.
(b) Inoculation and Plating:
(i) Incorporation:
The stabs containing different isolation media are heated in a water-bath for uniform melting. These are cooled to nearly 45°C and 1 ml. of soil suspension of definite dilution is poured in each of the tubes by a pipette. Each tube is rolled in a vortex mixer for thorough mixing.
The medium with soil suspension of each tube is plated in sterilised petridishes aseptically and allowed to solidify. After solidification, the petridishes are incubated at 30°C in an incubator inversely except petridishes for algae isolation which are placed in an open air lighted environment keeping them straight (upper lid upward). Soil suspension of 10-6 and 10-7 dilution are used for isolating actinomycetes and bacteria; 10-4 and 10-5 for fungi and 10-2 and 10-3 for algae.
(ii) Spreading:
Some selected media are inoculated in this technique. In this method, the media contained in, the flasks are melted and antibiotic added after cooling to 45°C. After thorough mixing, these are plated in sterilised petridishes and allowed to solidify.
After solidification, 0.2 ml. of soil suspension of 10-4 and 10-5 dilution are added separately to the medium by a pipette and spread out by a glass spreader. The whole thing is done aseptically. Then they are incubated invertedly at 30°C or at 27°C.
(c) Plate Reading:
After 48 hrs. and 96 hrs. The plates are taken out from incubator, the number of colonies in each plate is counted by a colony counter and at the same time the nature of the colonies is observed with the naked eye (Fig.3.1).
(d) Determination of Water Content of Soil Sample:
10 gms. of soil sample is taken in a clean petridish and kept in a hot air oven at 110°C for 3 – 5 hrs. and then kept at 60°C overnight. On the next day the weight of the soil is taken and thus the dry weight is found. Loss of weight is due to evaporation of water from the soil.
Results:
2. Isolation of Bacteria from Milk Samples by Agar Plate Method:
Requirements:
1. Milk sample.
2. 9 ml. dilution blank.
3. Sterilised 1 ml. pipettes.
4. Sterilised petridishes.
5. Tryptone-glucose-yeast-agar medium.
Medium:
Tryptone – 5 gm., yeast extract – 2.5 gm.,
Glucose – 1 gm., Agar – 20 gm.,
Distilled Water – 100 ml., pH – 7.
Procedure:
1. Invert 3 sterilised petridishes and mark on them the following dilutions: 1: 10, 1: 100 and 1: 1000.
2. Shake the milk sample vigorously at least 25 times to obtain a homogeneous suspension of the organism.
3. Remove 1 ml. with a sterilised 1 ml. pipette and transfer to a 9 ml. dilution blank.
4. Mix thoroughly by drawing into the pipette and expelling: Repeat several times.
5. With the same pipette, transfer 1 ml. of 1: 10 dilution to the plate so marked and 1 ml. to a second 9 ml. dilution blank.
6. Mix thoroughly as before.
7. Transfer 1 ml. of 1: 100 dilutions to the plate so marked and 1 ml. to a third 9 ml. dilutions blank.
8. Again mix as before.
9. Transfer 1 ml. of the 1: 1000 dilutions to the remaining plate.
10. Melt 3 tubes of tryptone-glucose-yeast-agar medium in an Arnold sterilizer or in boiling water.
11. Allow the agar to cool to about 50°C.
12. Pour the agar medium into the petridishes.
13. Mix thoroughly by tilting and rotating the plates.
14. When the agar has hardened inverts the plates and incubates at 35°C for 48 hrs.
15. Only those plates showing from 30 to 300 colonies should be counted and recorded as per the following format.
Result:
3. Isolation of Microorganisms from Air by Exposure Plate Technique:
Principle:
‘AIR’ is considered to be one of the major microbial reservoirs. In air microbes are suspended alone or attached to the surfaces of fine particles. They include intact cells/colonies, spore bodies and/or mycellium or filament fragments. These components can be isolated by the air samples or by the exposure plate technique.
Requirements:
(i) Culture media,
(ii) Petridishes,
(iii) Stop watch,
(iv) Glass marker,
(v) Water-bath, incubator.
(vi) Colony counters, etc.
Procedure:
About 15-20 ml. of suitable melted culture medium (Potato-dextrose-agar medium for isolation of fungi, Nutrient agar medium or isolation of bacteria, Thornton medium for isolation of actinomycetes) is plated on a sterilised petridish and then allowed to settle for 30 min. on the laboratory desk. Then each petridish is marked for the specific kind of isolation and period of aerial exposure.
The petridishes are exposed in air separately for a definite period of 1, 2, 3, 4 and 5 mins. Respectively (as per their marking). During exposure of each plate the upper cover is fully removed for the desired period. Then each petridish is incubated for 2 – 5 days or more for the appearance of colonies. Then colonies are observed and counted by the colony counter (Fig. 3.2). For each reenactment there should be at least five replicas.
Result:
No. of colonies per exposure time are tabulated separately as follows:
Then the data are graphically plotted to determine the minimum time exposure needed for isolation of maximum numbers of microorganisms from air.
4. Isolation of Microorganisms from Water or Waste Water Samples:
Principle:
Water is one of the major habitats of the microbes. It contains different kinds of microbe’s viz. bacteria, fungi, algae, actinomycetes, viruses, etc. Bacteriological analysis of water is mostly done for water quality assessment.
As water is an important source through which diseases may be transmitted, it is important that water must be regularly analysed to detect whether it is potable or polluted by the sewage or intestinal bacteria. These sewage or intestinal bacteria are mostly of coliform type.
However, for routine bacteriological procedures there are two major steps:
(a) Plate count to determine the number of bacteria present and
(b) Test to reveal the presence of coliform bacteria.
The details of standard plate count technique are described below:
(a) Collection of water samples in sterilised bottles.
(b) Standard plate count technique.
Collection of Water Samples:
i. The sample must be collected in a sterilised bottle.
ii. The sample must be representative of the supply from which it is taken.
iii. Contamination of the sample must be avoided during and after sampling.
iv. The samples should be tested as promptly as possible after collection.
v. If there is a delay in examination of the sample, it should be stored at a temperature between 0 – 10°C.
Standard Plate Count:
Colony counts are performed after plating the water sample. The interpretation of the results of the standard plate count must take into account the fact that the presence of a few pathogenic microorganisms is more significant than water containing many saprophytic bacteria. However, water of good quality is expected to give a low count, less than 100 per milliliter.
Plate counts are useful in determining the efficiency of operations for removing or destroying organisms e.g. sedimentation, filtration and chlorination. A count can be made before and after specific treatment. The results indicate the extent to which the microbial population has been reduced.
Requirements:
(i) Sterilised water collection bottles.
(ii) Sterilised petridishes.
(iii) Culture media.
(iv) Water-bath, incubator, etc.
(v) Sterilised pipette.
(vi) Sterilised water tubes (9 ml.).
(vii) Vortex apparatus, etc.
Procedure:
Sample water must be collected in a sterilised bottle and aseptically transported to the laboratory for microbiological testing. One ml. of sample water is transferred to the sterilised water tube (9 ml.) and thoroughly shaken by vortex or by hand. Then serial dilutions are made from 10-1 to 10-5 dilutions.
0.1 ml. diluted sample of each dilution (10-1, 10-2, 10-3, 10-4 and 10-5) is transferred separately to sterilised petridishes aseptically. Then 10 – 20 ml. melted media is quickly poured on each petridish aseptically and the petridish is gently rotated (clockwise and anticlockwise) to mix the medium and water sample. Then the petridishes are allowed to cool and finally placed in the incubator (37°C) for 2 – 3 days.
Results:
The total number of bacterial colonies is counted from each petridish.
5. Isolation of Root Nodule Bacteria:
Principle:
Plants belonging to the family Fabaceae (Leguminosae) commonly enter into symbiotic relation with the soil bacteria of the genus Rhizobium. The bacteria can generally fix atmospheric nitrogen in such association.
Within the nodules, bacteria generally lose their normal appearance and assume convoluted form known as bacteroids. Nodules are distributed mainly on the basal portion of the branch roots. They are mostly globose to ellipsoidal in shape while some are irregular in outline.
Their diameter ranges from 1 to 2 mm. The internal portion of the nodule is reddish in colour. This flesh colour is due to the presence of leghaemoglobin. Only the effective nodules show this colour but the in-effectives do not. On isolation from the nodules the bacteria can be grown on artificial medium but generally do not fix nitrogen if it is not supplemented by Myoinositol and L-Arabinose.
Requirements:
(a) Freshly collected nodules,
(b) Carbol fuchsin stain,
(c) Sterilised glass slides,
(d) Sterilised petridishes,
(e) Inoculating needles,
(f) Rectified spirit,
(g) Spirit lamp,
(h) Yeast extract – mannitol – agar – media:
Mannitol – 10.0 gm.
K2HPO4 – 0.5 gm.
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MgSO4, 7H2O – 0.2 gm.
NaCl – 0.1 gm.
CaCO3 – 3.0 gm.
Yeast extract – 1.0 gm.
Agar – 20.0 gm.
Distilled water – 1.0 lit.
(i) 0.1% HgCl2 Soln.
(j) Distilled water.
Procedure:
At first the distribution of the nodules on the root system of the supplied plant, tap root and branch root are examined. Next, only healthy nodules are selected and cut to observe the internal structure of the nodules by a lenses, to see the bacteroid of the nodules. It is first washed with water to remove soil particles adhering to the surface.
Then it is crushed with a sterilised scalpel on a sterilised slide. The extracted milky sap is taken on another sterilised slide, spread uniformly, allowed to dry in air and then heat fixed. The slide is then covered with the stain carbol fuchsin for two minutes, washed, blotted dry and observed under oil immersion lens of microscope.
A few healthy nodules from the roots are removed and washed thoroughly in water. Then they are passed through 0.1% HgCl2 solution for 2 minutes followed by three changes in sterilised distilled water keeping one minute in each. A nodule is aseptically transferred on a sterilised tube containing 9 ml distilled water.
The nodule then crushed inside aseptically. This is original bacterial inoculum. 0.1 ml of such inoculum is then transferred to sterilised petridish in which latter 15 ml melted yeast extract media is poured. The petridish is then rotate for mixing inoculum and finally incubated for Rhizobium colony development at 37 ± 1°C for 48 hours.
A portion of the colony is inoculated on the surface of a yeast extract-mannitol-agar slant with inoculating needle. The slant is incubated at 37°C for 3-4 days. A slide is prepared with the bacteria from the slant and stained with carbol fuchsin and examined under oil immersion lens of microscope.
Observation:
The bacteroids are small in size with convoluted margins. Their shape varies from simple rod to V, L, T, Y. etc. shapes. The degree of convolution of the margin and the shape of the bacteriod is found to be dependent on host species.
Bacteria from culture is found to be small, straight rods with smooth margin. Both the bacteroids and bacteria contain conspicuous granules of poly β hydroxy butyric acid as characteristic reserve food material.
