1. Chapter 10 - Cultural Methods Objectives 1.Understand the terms enrichment, selective, and differential medium. 2.Understand the difference between culturable counts and direct counts. 3.Understand the difference between MPN and culturable counts 4.Given a soil or water sample be able to describe a dilution scheme. 5.Be able to convert soil wet weight results to soil dry weight results.

2. Culturable counts Plate counts are performed using dilution and plating The choice of plating medium is important - enrichment media allow one to enrich for certain populations - selective media contain components that select against certain populations - differential media contain components that allow certain populations to stand out on the plating medium Bacterial numbers can be determined by direct counts or by culturable counts. Cultural Methods

3. Rose bengal agar – for fungi low pH rose bengal (dye) streptomycin neomycin What antibiotic would you add to a general medium to select for bacteria ?

4. ------------- Culturable counts (CFU/g) ---------- Depth (m) Saturation status AODC (cells/g)PTYG agar5% PTYG agarSSA 0.3 1.6 3.8 7.4 7.9 U topsoil U vadose S Consolidated 1.9 (0.5) x 10 9 2.9 (0.9) x 10 8 8.2 (4.0) x 10 6 1.2 (0.2) x 10 7 4.8 (3.8) x 10 6 9.3 (1.0) x 10 6 2.2 (0.6) x 10 4 4.8 (1.1) x 10 3 5.1 (3.0) x 10 6 0 1.6 (0.2) x 10 7 3.1 (0.2) x 10 4 3.5 (0.8) x 10 4 1.0 (0.1) x 10 7 0 1.3 (0.2) x 10 7 8.4 (1.0) x 10 4 4.2 (0.4) x 10 4 1.1 (0.2) x 10 7 0 From: Bone and Balkwill, 1988, Microb. Ecol. 16:49-64 Effect of medium on culturable counts U = unsaturated S = saturated Consolidated = mostly nontransmissive zone beneath aquifer

5. Dilution series 0.1 ml Dilution = 10 -4 10 -5 10 -6

6. Example: you are asked to determine the number of heterotrophic bacteria and the number of 2,4-D degraders in a soil sample contaminated with the herbicide 2,4-D. What do you do? Step 1: choose media Step 2: collect soil sample, air-dry, sieve, weigh out a 10 g sample saline dilution Step 3: place 10 g soil into 95 ml 10 -1 1 ml 9 ml 10 -2 1 ml 9 ml 10 -3 1 ml 9 ml 10 -4 1 ml 9 ml 10 -5 Plate 0.1 ml 10 -6 CFU = 1 x number of colonies dilution factor

7. Incubate under appropriate conditions Always count plates with 30 to 300 colonies. Less is not statistical and more is too numerous to count. Dilution = 10 -4 10 -5 10 -6

8. This is for a moist soil. For comparison with other samples, express CFUs in terms of the dry weight of the soil. To determine dry weight, weigh a sample, dry it in an oven, reweigh it and then calculate the % moisture. If you weigh 1 g of soil, dry it and find it then weighs 0.8 g, it has a 20% moisture content. Therefore: 7.6 x 10 6 CFU/g soil = 9.12 x 10 6 CFU/g dry soil 0.833 g dry soil/g soil Moisture content = moist wt - dry wt dry wt 0.2 = 1 g – dry wt dry wt dry wt = 0.833 g 76 x 10 5 CFU/g soil or 7.6 x 10 6 CFU/g soil You count an average of 76 colonies on the 10 -5 dilution plates.

9. Most probable number (MPN) Uses successive dilution of the sample to reach a point of extinction. Replicate dilutions (3-10) are scored as positive or negative and results are analyzed using a statistical table.

10. Direct counts Give an estimate of the total number of bacteria in a sample. This is useful for a variety of habitats and has no specific bias for particular genera. On the other hand, it is hard to distinguish living from dead cells. fluorescent antibodies Direct counts are performed using the following techniques: fluorescent staining and epifluorescence microscopy. acridine orange (AODC) 46-diamino-2-phenylindole (DAPI) fluorescein isothiocyanate (FITC) Molecular probes electron microscopy (virus) particle counter (coulter counter, flow cytometer)

11. Giardia (left) and Cryptosporidium (right)– Fluorescent Antibody Staining H.D.A. Lindquist, USEPA

12. SampleSoil Direct counts Cells/g Soil Culturable counts CFU/g Marine water Direct counts Cells/ml Marine water Culturable counts CFU/ml ABCABC 5.0 x 10 8 1.1 x 10 9 2.0 x 10 9 3.1 x 10 7 6.2 x 10 7 1.7 x 10 8 2.2 x 10 3 8.2 x 10 4 1.3 x 10 6 1.3 x 10 1 7.6 x 10 2 2.1 x 10 4 Comparison of culturable and direct counts in soil and marine water

13. r 2 = 0.97

14. Culturing viruses Living cells are required to detect virus replication whether they are animal or bacterial cells. Usually a lawn or a confluent layer of cells is prepared. The sample (presumably containing virus) is added. The plates are incubated and evaluated for: CPE (cytopathogenic effects) PFU (plaque forming units) CPE Infected culture Normal culture