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 counts4. 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.

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

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) ----------

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

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

0

1.6 (0.2) x 107

3.1 (0.2) x 104

3.5 (0.8) x 104

1.0 (0.1) x 107

0

1.3 (0.2) x 107

8.4 (1.0) x 104

4.2 (0.4) x 104

1.1 (0.2) x 107

0

From: Bone and Balkwill, 1988, Microb. Ecol. 16:49-64

Effect of medium on culturable counts

U = unsaturatedS = saturatedConsolidated = mostly nontransmissive zone beneath aquifer

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 dilutionStep 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

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

• 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 106CFU/g soil = 9.12 x 106 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 105 CFU/g soil or7.6 x 106 CFU/g soil

You count an average of 76 colonies on the 10-5 dilution plates.

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.

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)4’6’-diamino-2-phenylindole (DAPI)fluorescein isothiocyanate (FITC)Molecular probes

• electron microscopy (virus)

• particle counter (coulter counter, flow cytometer)

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

Sample Soil

Direct counts

Cells/g

Soil

Culturable counts

CFU/g

Marine water

Direct counts

Cells/ml

Marine water

Culturable counts

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

Comparison of culturable and direct counts in soil and marine water

0

5

10

15

20

0 20 40 60 80 100 120

Culturable counts (CFU/g x 106)

Dir

ect

coun

ts (

cells

/g x

108 )

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