Characterization Of Bacteria Isolated From Soil Sample

Microorganisms are extremely minute lifeforms which are omnipresent. These lifeforms exist in the most varied habitats of the world. Soil, water, excreta, etc are some of the richest sources of microorganisms especially of a wide variety of bacteria.

A number of microbiological techniques are employed for the isolation and characterization of bacteria. Bacteria can be allowed to grow and multiply in the laboratory in a controlled environment. A pure culture of such colonies can be obtained using techniques such as serial dilution, spread plate culture and streaking. Such isolated bacteria can be characterized on the basis of various properties using a wide range of tests some of them being popular techniques like Gram's staining and also tests such as MR and indole production tests.

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This study involved the isolation of bacteria from soil sample and their characterization based on Gram's technique, mixed acid production (using MR test) and indole production (using indole test) and the results were duly noted and analysed.

COLLECTION OF SOIL SAMPLE AND SERIAL DILUTION

Principle

Bacteria can subsist in almost any type of environment and habitat. Soil found near and around the roots of trees and small plants is significantly rich in a humongous diversity of bacteria. These bacteria can be isolated from soil by an important microbiological technique known as serial dilution.

A solution containing a certain amount of a substance or solute can be diluted in a stepwise fashion to obtain solutions with much lower concentrations. Generally, the dilution factor used at every step is constant. This method is termed as serial dilution. Even a very minimal amount of soil may contain a huge number of bacteria which becomes difficult to work with experimentally. In microbiology, the purpose of serial dilution is reduction of a dense culture of cells to obtain a much lower concentration of cells such as those of bacteria.

Methodology Used

Soil was collected from the vicinity of the roots of trees found in the neighbourhood of the organization. 1g of the collected soil was weighed using a weighing balance and was used in the experiment. The weighed 1g of soil sample was dissolved in distilled water and the total volume of the solution was made up to 10 ml in an autoclaved test tube labelled as 1. 4 other autoclaved test tubes were taken and duly labelled as 10-1, 10-2, 10-3 and 10-4 respectively. 9ml of distilled water was added to each of the 4 test tubes. Carefully 1ml of solution was taken from test tube 1 using a micropipette and added to the test tube labelled as 10-1 and mixed thoroughly so that the total volume in the test becomes 10ml. Now 1ml solution was drawn from this test tube labelled 10-1 and added to the test tube labelled 10-2. The same procedure was repeated 2 more times to obtain the concentrations 10-3 and 10-4.

Result

Finally, solutions of 10-1, 10-2, 10-3 and 10-4 concentrations were obtained at the end of this process of serial dilution which were used further for isolation of bacteria.

CULTURE OF BACTERIA ON NUTRIENT AGAR MEDIUM BY SPREAD PLATE METHOD AND SUCCESSIVE STREAKING

Principle

Nutrient Agar is a commonly used nutrient medium which is employed for cultivation of microorganisms. It supports the growth of a broad range and variety of non-fastidious organisms. It owes its popularity to its ability to allow the growth a wide range of bacteria and fungi. It is able to provide a lot of nutrients required by bacteria for growth.

Nutrient Agar is composed of 0.3% beef extract (provides carbohydrates, vitamins, salts and organic nitrogen and helps bacterial growth), 0.5% Peptone (major source of organic nitrogen for bacteria), 0.5% NaCl (maintaining salt concentration which resembles the cytoplasm of microorganisms) and 1.5% agar (solidifying agent) in Distilled water.

Spread Plate method is a technique used for isolation of microorganisms from a mixed culture and to distribute it uniformly. It involves application of a little quantity of bacteria which is suspended in solution over a media plate by using a thoroughly sterilized glass spreader.

Streaking is a common microbiological technique utilized for obtaining a pure culture of microorganisms from a very mixed population. The principle behind the techniques is to thin out bacteria by streaking the inoculum over fresh agar medium. This allows separation of individual bacterial cells. During streaking, the dilution of the inoculum occurs to the stage where the deposition of single bacterial cell takes place at every few millimetres. Pure cultures can be secured by careful selection of nicely isolated colonies and re-streaking them on freshly prepared agar plates.

Methodology Used

Fresh Nutrient Agar Medium was prepared using Nutrient Broth containing appropriate quantities of peptone, beef extract, and sodium chloride along with Bacteriological Agar. 1.3 g of the Nutrient Broth and 1.5 g of Agar were weighed and dissolved in 50ml of distilled water and the total volume was made up to 100 ml. The media was heated over a hot plate with continuous shaking to allow the proper dissolution of the ingredients. After proper dissolution the flask containing media was well covered by a cotton plug and autoclaved at 121 ̊C for 30 minutes.

The autoclaved media is allowed to cool but not to the point of solidification. It is then carefully used for pouring of 4 well sterilized petri dishes inside a laminar air flow. The media is allowed to solidify on the plates. Further the previously prepared dilutions of bacteria are used for inoculation. 200 l of inoculum is carefully drawn from tube 1 labelled 10-1 using a micropipette and placed over the first agar plate. The inoculum is spread over the medium using a glass spreader sterilized using ethanol. The plate is rotated during spreading to allow uniform distribution. Similarly, 3 more agar plates are inoculated using the 10-2, 10-3 and 10-4 concentration inoculums. The inoculated agar plates are incubated at 37 C overnight.

A bacterial lawn is obtained after incubation which shows isolated bacterial colonies. 2 fresh Nutrient Agar plates are poured and streaked by using the isolated bacterial colonies from previous step. 8 well isolated bacterial colonies are obtained and streaked onto agar plates by dividing each plate into 4 parts. The streaked plates are again incubated at 37 C overnight.

Result

8 purely cultured and isolated bacterial colonies are obtained on 2 Nutrient Agar Plates after the above procedure has been completed. Isolation of bacteria from soil sample is concluded at this step.

CHARACTERIZATION OF ISOLATED BACTERIA USING GRAM'S STAINING METHOD

Principle

Gram staining is a significant microbiological procedure which was introduced by Hans Christian Gram, a Danish physician, in 1884. It is considered a cornerstone and fundamental for bacterial taxonomic division and identification. This differential staining method categorizes bacteria into 2 groups on the basis of composition of cell wall: Gram-positive and Gram-negative bacteria.

Gram-positive bacteria possess a thick layer of peptidoglycan together with plenty of teichoic acid cross-linking that resist decolorization. Gram staining technique makes use of crystal violet and iodine. Crystal violet dissociates in aqueous solution and penetrates the membrane of both types of bacteria. It interacts with the elements of bacterial cells consequently staining them purple. Iodine on addition forms crystal violet-iodine complex and acts as a mordant. Ethanol acts as a decolorizing agent and interacts with membrane lipids. Gram-negative cells lose their outer lipopolysaccharide membrane layer and the peptidoglycan layer is left exposed. As a result, the Gram-negative cells turn leaky and permit the crystal violet-iodine complex to be washed out and in turn takes up the pink colour of the positively charged counter stain safranin while the multi-layered and cross-linked peptidoglycan layer of Gram-positive retains the complex and remains purple, hence the basis of distinction.

Methodology Used

The previously isolated bacterial colonies were identified using the following procedure:

• i. Carefully pick up a very small amount of bacterial colony and prepare a bacterial smear onto a thoroughly washed glass slide.
• ii. Gently heat fix it onto the slide over a flame.
• iii. Place few drops of crystal violet onto the slide and wait for a minute before washing off with tap water.
• iv. Add a few drops of Gram's iodine and halt for 1 minute and then wash with tap water.
• v. Put a few drops of 95% ethanol dropwise to allow decolorization and rinse it.
• vi. Counter stain with safranin for 45 seconds and wash off with tap water.
• vii. Observe slide under a light microscope.

Results

The above procedure is repeated 8 times to observe and analyse all 8 isolated bacterial colonies. The bacteria are classified as Gram-positive or negative and the shape and morphology of the bacteria is observed and the results are tabulated in Table 1.

Colony No. Colour retained after Gram's staining procedure Gram's characterization (Gram positive/ Gram negative) Shape

1. Pink Negative Round
2. Pink Negative Rod
3. Purple Positive Rod
4. Pink Negative Round
5. Purple Positive Rod
6. Purple Positive Rod
7. Pink Negative Rod
8. Pink Negative Round

5 of the obtained 8 colonies were found to be Gram negative out of which 3 were round while 2 were rod shaped. Remaining 3 out of 8 colonies were found to be Gram positive and all of them being rod shaped.

METHYL RED (MR) TEST

Principle

Methyl Red (MR) test is a test used to ascertain whether on supplying glucose a particular microbe performs mixed acid fermentation or not. Proportion and types of fermentation products generated by anaerobic glucose fermentation is one of the significant taxonomic characteristics which assist the differentiation of several genera of enteric bacteria.

Three acids namely lactic, succinic and acetic acid are produced in notable amounts in mixed acid fermentation. 1 mol of ethanol (neutral product of fermentation), 1 mol H2, 1 mol of CO2 and 4 mol of acidic products (especially acetic and lactic acid) are given by the mixed acid pathway per mol of glucose fermented. Such huge quantities of acid cause the pH of the medium to decrease significantly and fall to 4.4 or below. This fall of pH is indicated by methyl red, a pH indicator, that is red at any pH below 4.4 and is yellow above 5.1.

On addition of methyl red if the culture medium turns red then the bacteria is said to be MR positive due to which it can decrease the pH below 4.4 by fermentation of carbohydrate substrate. On the other hand, if the culture medium remains yellow due to low amounts of acid production the bacteria is said to be MR negative.

Methodology Used

The Nutrient Broth medium for the MR test is prepared by weighing 0.7g of Peptone, 0.5g of Glucose, 0.5g potassium phosphate and dissolving it in 50ml of distilled water and making the volume up to a total of 100ml. The media is gently heated for proper dissolution and autoclaved for 30 minutes. Media is allowed to cool and then poured into 8 culture tubes, one for each bacterial colony. The media is inoculated with the respective bacteria and incubated at 37 C for 48 hours. After incubation 5 drops of methyl red indicator is added to each tube and the colour change is observed and noted. Red colour indicates a positive test whereas yellow colour indicates a negative test.

Results

The results obtained after following above procedure are tabulated in Table 2.

Colony No. Colour observed on addition of Methyl Red Characterization (MR Positive/ MR Negative) Mixed Acid Fermentation

1. Red Positive Present
2. Red Positive Present
3. Red Positive Present
4. Red Positive Present
5. Red Positive Present
6. Red Positive Present
7. Red Positive Present
8. Red Positive Present

All 8 test bacterial samples are found to give red colour that is positive MR test indicating presence of mixed acid fermentation causing lowering of pH below 4.4. Fig.2. shows the results obtained.

INDOLE PRODUCTION TEST

Principle

Indole test is employed to ascertain if a microorganism is able to produce indole by splitting amino acid tryptophan or not. Hydrolysis of tryptophan is facilitated by the enzyme trytophanase which results in formation of 3 products one of them being indole. The production of indole can be detected by using Kovac's reagent which can react with indole to form a red coloured compound. Kovac's reagent is combination of p-dimethylaminobenzaldehyde, isoamyl alcohol and HCl.

Methodology Used

Nutrient broth for the indole test is prepared by dissolving 1g Tryptone in 100ml of distilled water. The media is autoclaved for 30 min, allowed to cool and poured into 8 culture tubes. Each is inoculated with respective bacteria and incubated at 37 C for 48 hours. After incubation, 2-3 drops of Kovac's reagent is added to each tube and colour change is noted after 2 minutes. Appearance of a cherry red band indicates a positive indole test while no change in colour implies a negative test. Also, in case of positive test a paper strip impregnated with oxalic acid turns pink due to diffusion of indole above the medium.

Result

The results observed are listed in Table 3 for all 8 test samples.

Colony No. Colour Change Indole Characterization (Positive/Negative) Indole Production

1. No Colour Change Negative Absent
2. No Colour Change Negative Absent
3. No Colour Change Negative Absent
4. No Colour Change Negative Absent
5. No Colour Change Negative Absent
6. No Colour Change Negative Absent
7. No Colour Change Negative Absent
8. No Colour Change Negative Absent

All 8 test samples are found to give a negative indole test indicating a lack of indole production in all samples.

Summary of Results

In this study, soil was used to isolate bacteria and characterize them using various microbiological techniques namely Gram's staining, MR test and Indole production test. Such techniques are a key part of microbiology and widely used for the categorization of bacteria. Microbiological techniques such as serial dilution, spread plate culture, streaking and sterilization are also indispensable procedures popularly utilized by researches and scientists used worldwide. Knowledge and understanding of such fundamental techniques is the foremost step towards greater and extensive research in the field of microbiology.