Soil Microbial Ecology - Soil Molecular Ecology Laboratory

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Page 38 E X E R C I S E 5 SOIL MICROBIAL ENUMERATION: 2. DIRECT MICROSCOPIC COUNT OBJECTIVE Rapid quantification of microorganism numbers within a demarcated region of a slide known to have a certain volume of soil by using Fluorescein Isothiocyanate (FITC) for direct microscopic counting. METHOD: 1. Prepare staining solution (FITC) as follows: 1.3 ml of 0.5 M Na 2 C0 3 - NaHC0 3 mixed 1:1, pH 9.6 6 ml 0.01 M phosphate buffer pH 7.2 (2.8 ml 0.2 M monobasic sodium phosphate; 7.2 ml 0.2 M dibasic sodium phosphate; dilute to 20 ml with distilled water) 5.7 ml 0.85% NaCl 5.3 mg crystalline fluorescein isothiocyanate – Note: this is not a vital stain. 2. Shake staining solution and use immediately or store in dark at 4°C (6 hr only). 3. Prepare smears from soil dilution as follows: Mix 5 g of soil (agriculture or forest) with 45 ml of 0.1% agar solution and shake vigorously After 30 sec transfer 0.01 ml to a slide and spread evenly within a 1 cm 2 area previously marked on the slide with a marking pen Place slides on warmer to dry and then heat fix briefly. Note: dilution factor = 0.01/50. 4. Stain 3 min with FITC. 5. Wash slides thoroughly with 0.5 M Na 2 C0 3 - NaHC0 3 buffer.
Page 39 6. Mount smears immediately with glycerol (pH 9.6). Place cover slip over specimen. 7. Observe with fluorescence microscopy (in Room 2170 McCarty A): Count the number of bacteria within two microscope fields (normally at least 20 fields) at 400 X and record the mean. Mean count X No of fields in 1 cm 2 (8264) 1 No bacteria / g = ⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯⎯ X ⎯⎯⎯⎯⎯⎯⎯⎯ <strong>Soil</strong> dry weight Dilution factor QUESTION: How and why do direct counts differ from dilution plating? REFERENCES: Babiuk LA and EA Paul 1970 Can J Microbiol 16:57-62
Page 1 and 2: SOS 43 SOS4303C / SOS5305C Soil Mic
Page 3 and 4: Page 3 LABORATORY SCHEDULE 08/29 Fi
Page 5 and 6: Page 5 notebook. The instructor may
Page 7 and 8: Page 7 3. AUTOCLAVING Autoclaving (
Page 9 and 10: Page 9 Glomaceae Acaulosporaceae Gi
Page 11 and 12: Page 11 3. POT CULTURE AM mycorrhiz
Page 13 and 14: Page 13 hold a certain volume of so
Page 15 and 16: Page 15 METHOD This will be a demon
Page 17 and 18: Page 17 E X E R C I S E 3 SOIL MICR
Page 19 and 20: Page 19 Fungi constitute another ex
Page 21 and 22: Page 21 6. Continue this procedure
Page 23 and 24: Page 23 QUESTIONS: 1. Discuss the u
Page 25 and 26: Page 25 OBJECTIVE: Train to use saf
Page 27 and 28: Page 27 MICROSCOPE OPERATION Micros
Page 29 and 30: Page 29 METHOD 1. Place a small dro
Page 31 and 32: Page 31 GRAM-NEGATIVE AEROBIC RODS
Page 33 and 34: Page 33 2. OBSERVE SOIL FUNGI Exami
Page 35 and 36: Page 35 arthrospores or chlamydospo
Page 37: Page 37 SERIES ANNELLOSPORAE: First
Page 41 and 42: Page 41 E X E R C I S E 6 SOIL MICR
Page 43 and 44: Page 43 METHOD: EXAMINATION OF ROOT
Page 45 and 46: Page 45 E X E R C I S E 7 SOIL ALGA
Page 47 and 48: Page 47 The most probable number (M
Page 49 and 50: Page 49 MATERIALS: Soil samples (ag
Page 51 and 52: Page 51 E X E R C I S E 8 SOIL META
Page 53 and 54: Page 53 METHOD: RESPONSE TO CARBON
Page 55 and 56: Page 55 A BIOMETER VESSEL A G F B C
Page 57 and 58: Page 57 Further example of calculat
Page 59 and 60: Page 59 PROTOCOL • To the 2ml Bea
Page 61 and 62: Page 61 Mechanical lysis is introdu
Page 63 and 64: Page 63 HINTS AND TROUBLESHOOTING G
Page 65 and 66: Al-Agely and Ogram © 2004 Page 65
Page 67 and 68: Page 67 E X E R C I S E 10 SOIL RHI

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