Paper 1: Introduction to Microbiology, Viruses, Bacteria, Cyanobacterta and Phycology ISOLATION OF BACTERIA FROM SOIL BY POUR PLATE METHOD Introduction: Soil is the rich repository of microorganisms, The type of media used their population Size depends on the type of soil, its moisture content, Ph and organic matter present. The micro flora of the soil includes bacteria, fungi, algae, protozoans, viruses and mycoplasma. These organisms increase the fertility of soil and support the growth of higher plants Originally developed by Robert Koch, in this technique successive dilution of the inoculums are poured into sterile petriplates to which molten agar at 45°C is added and thoroughly mixed by rotating the plates and then allowed to solidify. The plates are incubated in inverted position for 48 hours at 37°C and examined for the appearance of colonies of different microorganisms (bacteria/fungi). The microorganisms can be tentatively identified based on colony characteristics Requirements: > MEDIA: a. Nutrient Agar media for isolation of bacteria b. Martin Rose Bengal Agar for isolation of fungi (Note: Add streptomycin after autoclaving to avoid growth of bacterial colonies) > Soil sample Sterilized distilled water blanks a. Test tube with 10 ml D.W b, 6 test tubes with 9 ml D.W numbered from 2-7 > Sterilized 1 ml pipette-7, Sterilized petriplates-14 > Spirit lamp or Bunsen burner, Marker pen v Procedure: > Arrange the D.W blanks in attest tube rack and label them 1-7 Add 1.0 g soil sample to the test tube containing 10 ML sterile D.W, shake well to get a uniform soil solution, this is the stock solution » Transfer 1 ml of stock solution to the test tube containing 9 ml of sterile D.W. using a sterile | ml pipette. Shake well, this is 107 Repeat this step as under 3 for making dilution 107, 10°, 10° ,10°,10°,10" > ‘Transfer 1 ml of each dilution to sterile petriplates using respective pipette * Pour approximately 20 ml of molten and cooled NA/MRBA into each plate and rotate the plate gently to ensure uniform mixing of inoculums and the medium Allow the medium to solidify Incubate NA plate for 24-48 hrs at 37°C in inverted position for growth of bacterial colonies Incubate MRBA plates for 3-4 days at 28°C for growth of fungal colonies Vv vy aa Department of Botany MES CollegeFood Paper 1: Introduction to Microbiology, Viruses, Bacteria, Cyanobacteria and Phycology E [Ss oe OUndulate 72 Febate D Berle 9D Filamerdeus, TOY Sceleaceles 21 D Rhigeidal — | §) Gimake H Gove a7] | 74 AGED ON ELevaTION BASED ON SHAPE 2 ae , ss BASED ON MARGIN 9 Flak ©) Raised 9 Convex | DUmbenate | 6 Puluine — | — | | S| ee | hc 17 Department of Botany MES College one. ecm RARAAAARAAA AKA A A eo oo oe ee se ee KS Se eeeae Paper 1: Intreduction to Microbiology, Viruses, Nacterta, Cyanobacteria and Phycology Colony characters are described in the following manner: Shape: Circular, round, punctiform, and irregular Margin: Filamentous, rhizoidal, ciliate, irregular, entire, undulate/ lobate, branched Surface texture: Smooth, entire, rough, wrinkled, concentric, dry, powdery, mucoid and granular, Consistency: Viscous, rubbery, Opacity: Opaque, transparent, fluorescent. Pigmentation: Bluish, green, black, white, yellow, orange, grey, pink and brown Elevation: Raised, flat, convex, undulate and growing into the media. SL |Shape [Colour | Elevation | Margin | Opacity |Gram’s | Possible |NO Character | organism 1 "| Circular | Creamish | Raised Entire [Opaque | G+veRods | Bacillus 2 | Punctiform | White/yellow | Flat Entire | Opaque Gre ‘Staphyllococeus | Cocciin | cluster 3 [irregular | Fluorescent, | Flat Lobate | Transparent |G-veRods | Pseudomonas | green! purple 4 | Cirewlar | White Flat Entire | Transparent |G- ve Shon | E.Coll rods 3 | Gireular | Light orange | Raised Entire | Opaque Give ‘Streptococci Cocei in chain ale As, Department of Botany MES College