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

Cultural Methods Bacterial numbers can be determined by direct counts or by culturable counts. 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

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?

------------- Culturable counts (CFU/g) ---------- Effect of medium on culturable counts ------------- Culturable counts (CFU/g) ---------- Depth (m) Saturation status AODC (cells/g) PTYG agar 5% PTYG agar SSA 0.3 1.6 3.8 7.4 7.9 U topsoil U vadose S Consolidated 1.9 (0.5) x 109 2.9 (0.9) x 108 8.2 (4.0) x 106 1.2 (0.2) x 107 4.8 (3.8) x 106 9.3 (1.0) x 106 2.2 (0.6) x 104 4.8 (1.1) x 103 5.1 (3.0) x 106 1.6 (0.2) x 107 3.1 (0.2) x 104 3.5 (0.8) x 104 1.0 (0.1) x 107 1.3 (0.2) x 107 8.4 (1.0) x 104 4.2 (0.4) x 104 1.1 (0.2) x 107 U = unsaturated S = saturated Consolidated = mostly nontransmissive zone beneath aquifer From: Bone and Balkwill, 1988, Microb. Ecol. 16:49-64

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

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

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

You count an average of 76 colonies on the 10-5 dilution plates. 76 x 105 CFU/g soil or 7.6 x 106 CFU/g soil 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. Moisture content = moist wt - dry wt dry wt 0.2 = 1 g – dry wt dry wt = 0.833 g Therefore: 7.6 x 106CFU/g soil = 9.12 x 106 CFU/g dry soil 0.833 g dry soil/g soil

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. Most probable number (MPN) (see Fig. 10.4) Uses successive dilution of the sample to reach a point of extinctions. Replicate dilutions (3-10) are scored as positive or negative and results are analyzed using a statistical table

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. Direct counts are performed using the following techniques: fluorescent staining and epifluorescence microscopy. acridine orange (AODC) 4’6’-diamino-2-phenylindole (DAPI) fluorescein isothiocyanate (FITC) Molecular probes fluorescent antibodies electron microscopy (virus) particle counter (coulter counter, flow cytometer)

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

Comparison of culturable and direct counts in soil and marine water Sample Soil Direct counts Cells/g Culturable counts CFU/g Marine water Cells/ml CFU/ml A B C 5.0 x 108 1.1 x 109 2.0 x 109 3.1 x 107 6.2 x 107 1.7 x 108 2.2 x 103 8.2 x 104 1.3 x 106 1.3 x 101 7.6 x 102 2.1 x 104

r2 = 0.97

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