Identification of Environmental Flora

1.0 Equipment Required

Microscope
LAF

2.0 Material Required

Loop
Slides
Coverslip
Immersion oil
Gram stain Kit

3.0 Utilities Required

LPG
Electricity

4.0 Procedure

4.1 Cultural Characteristics

4.1.1 Select the unknown culture plate (microbial flora observed in sterile area environment).
4.1.2 Examine the plate for Colony characteristic as per Table - I and record the characteristic.
Table-I Colony Characteristics

4.2 Gram Staining

4.2.1 Prepare a thin smear of microbial culture observed in the sterile environment, dry in air and fix by gentle heat.
4.2.2 Hold the slide in slide rack near to ss tray and flood with Gram’s Crystal Violet (HiMedia S012) for 1 minute.
4.2.3 Wash with water and flood with Gram’s Iodine (HiMedia S013) for 1 minute.
4.2.4 Wash with water and decolourize with Gram’s Decolorizer (S032) until no further violet colour comes off.
4.2.5 Wash with water and counterstained with 0.5% safranin (HiMedia S027) for about 1 minute.
4.2.6 Wash with water, dry and observe under oil immersion objective.
4.2.7 Those bacteria that appear purple or violet color are referred as Gram Positive; those appearing pink color are described Gram Negative.
Related: Identification of Microorganisms to Species Level

Table-II Morphological Characteristic

4.2.8 After observing morphological characteristics record the observation.

5.0 Precaution

5.1 All preparation should be carried out under LAF of Microbiology room.
5.2 Use nose mask and hand gloves while working with microbial cultures.
5.3 After completion of work wash your hand with disinfectant solution.

6.0 Frequencies

Daily or as soon as colony observed in sterile area environment.

7.0 Abbreviation

LAF: Laminar Air Flow
LPG: Liquid Petrolium Gas