Procedure for Isolation Identification and Gram Staining of Microorganisms


To lay down the Procedure for Isolation,Identification and Gram Staining of Microorganisms

1.0       Purpose          : 

To lay down the Procedure for Isolation,Identification and Gram Staining of Microorganisms.

2.0       Scope              : 

This procedure applies to isolation, identification and gram staining of microorganisms during the course of regular microbiological analysis.

3.0       Responsibility: 

3.1       The responsibility of isolation, identification and gram staining of microorganisms lies with the Microbiologist/Analyst.

4.0       Definitions/Abbreviations:

4.1       Gram Staining: A differential staining procedure that divides bacteria into Gram positive and Gram negative groups based on their ability to retain crystal violet when decolorized with an organic solvent such as ethanol or acetone.

5.0       Procedure      : 

5.1       Isolation of Microorganisms:

5.1.1    At the end of incubation of the bacterial culture, check for the growth in broth / solid media for turbidity / colony forming units.

5.1.2    If turbidity / colony forming units observed, the same shall be isolated as described here under.

5.1.3    Streak the colony or culture on any non-selective media.

5.1.4Incubate for 24 hours.

5.1.5Select an isolated colony and proceeds for identification.

5.2       Identification of Microorganisms:

5.2.1    Initiate the identification of isolated microorganisms as per the procedure given below.       Morphological characteristics like shape, arrangement, motility, capsular nature, elevation, margins, surface, edges, structure and consistency shall be checked.       Shape: The shape may be spherical, rod shaped, filamentous, comma shaped or spiral.       Arrangement: For cocci:  Arranged in singly, in pairs, in tetrads, in chains, short or long. For bacilli: Arranged in random, in short or long chains, in Chinese letter pattern, in bundles or as palisades. For vibrios: Arranged in single or spiral forms.       Motility: May be motile or non- motile.       Spores:  May be in oval, equatorial, sub terminal, or terminal.       Capsules: May or may not be present.       Elevation: Effuse, elevated, convex, concave, umbonate or umbilicate.       Margins: Beveled.       Surface: Smooth, wavy, rough, granular, papillae or glistening.     Edges: Entire, undulate, crenated, fimbriate or curled.     Structure: Opaque, translucent or transparent.     Consistency: Membranous, friable and viscid.

5.2.2    Observe the growth in stock culture and fluid medium. In stock culture: Degree of growth- scanty, moderate or profuse. Nature – discrete or confluent, filiform rhizoid. Their elevation, surface, edges, color, structure, odour, consistency, and changes in the medium.  In a fluid medium: The degree of growth, presence of turbidity and its nature, presence of  deposit and its character, nature of surface growth such as pellicle and its quality and ease of disintegration, and odour shall be noted.

5.3       Staining Techniques: Staining reactions mainly by differential staining.

5.3.1Gram staining:  Bacteria may be examined under the compound microscope, either in the Living state or after fixation with Gram stain.

5.3.2Take a clean slide and mark a field with glass marking pencil. Turn over the slide and make a    smear of the bacterial culture suspension in the marked area with the help of sterile loop and if it is a colony take a drop of water on the slide then add loopful of colony and smear it.

5.3.3Heat the smear over blue flame of a burner for 30-60 seconds by wavy motion.

5.3.4Stain the smear with basic dye- crystal violet, the primary stain and kept for 30 seconds.

5.3.5Drain off and rinse the slide gently with purified water to remove excess crystal violet.

5.3.6Followed by treatment with an Iodine solution functioning as a Mordant i.e. the iodine    increases the interaction between the cell and the dye, so that the cell is stained more strongly    and allow it for one minute.

5.3.7Decolorise the smear by washing with 95 % acetone or ethanol and keep for 30 seconds. This    step generates the differential aspects of Gram stain; Gram-positive bacteria retain the crystal    violet, where as Gram-negative loss their crystal violet and become colorless.

5.3.8Finally counter stain the smear with a basic dye Safranin, which is different in color from crystal violet and allow it for one minute.

5.3.9Wash the smear with purified water and allow smear to air dry.

5.3.10Put the drop of cedar wood oil on the center of the stained side and examine under oil immersion lens of microscope.

5.4Gram-positive bacteria will take crystal violet stain and appears in purple violet color. Where    as Gram-negative bacteria, appear in pink or red in color.


5.5.1Hanging Drop Method: This method is useful to check the motility of microorganisms. For this purpose the organisms under test is subjected to grow in the liquid media. Place a loopful of organism in the form of a droplet over the surface of a clean cover slip. Now apply petroleum jelly or Vaseline on the corners of the cover slip and close the cover slip with cleanslide. Now mount this slide over the stand of a microscope and observe the drop under 45X and observe the motility.

5.5       Metabolism: The requirements of oxygen, the need for carbon dioxide, the capacity to form pigments and the production of haemolysis helps in classification.

5.6       Biochemical Properties:

5.6.1Sugar Fermentation: This test is carried out in presence of sugar media. In this observe the production of acid and gas.

5.6.2Indole Test: This test is demonstrating the presence of E.coli. Inoculate the test organism in the peptone water and incubates at 30- 35 °C for 24 hours. After the 24 hours incubation completed, add 0.5 ml of Kovac’s reagent and shake gently. A red color indicates the presence of E.coli. Kovac’s reagent consists of p-dimethyl aminobenzaldehyde (10g), amyl or isoamyl alcohol (150ml) and concentrated HCL (50ml).

5.6.3Methyl-red Test: This test is employed to detect the production of acid during the fermentation of glucose and the maintenance of pH below 4.5. This is qualitative, a bright red color is a positive and yellow or orange is a negative.

5.6.4Voges-Proskauer Test: This test is depends on the production of acetyl methylcarbinol from pyruvic acid. In the presence of alkali and atmospheric oxygen, the small amount of acetyl methyl carbinol present in the medium is oxidized to diacetyl, which reacts with the peptone of the broth to give a red color.

The test is performed by adding 0.6ml of 5%solution of α-napthol and 0.2ml of 40% KOH in glucose phosphate medium and incubate at 37°C for 48hours. In a positive reaction, pink   color appears in 2-5minutes. In a negative reaction, pink coloration should be ignored.

5.6.5Citrate Utilization Test: Kovac’s citrate medium has citrate as the sole source of carbon. Ability to use this substance is indicated by the production of turbidity in the medium.  Indole, Methyl red, Voges-proskauer and citrate utilization tests are very useful in the identification of Enteric Gram negative bacteria.

5.6.6Catalase Test: Place a loopful H2O2 on colonies on nutrient agar. Prompt effervescence indicates catalase production.

5.6.7Oxidase Test: This reaction is due to a cytochrome oxidase which catalyses oxidation of cytochrome by oxygen. 1 to 1.5% solution of tetra methyl para-phenylene di-amine hydrochloride is poured over the colonies. Oxidase positive colonies become purple, maroon, and black in 10 to 30 minutes.

5.7Commercially available identification kits for specific organisms shall also be employed.

6.0       Related Documents: Nil.



Microbiology, Isolation, Identification, Microorganisms


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