Rossi-Cholodny Technique

Nimerta Kumari Lecturer

Introduction
The distribution of microorganisms in soil is heterogeneous. Microbes need nutrients and water to survive and these resources are not evenly distributed in soil. The structure of soils is composed of particles of inorganic and organic matter and the pores in between these particles. The pore spaces may be filled with water or air. Bacteria are mostly found attached to particles growing in small micro-colonies wherever nutrients can be found. Filamentous organisms such as actinomycetes and fungi make up much of the biomass. When they grow around soil particles they help to cement them into aggregates. These aggregates have their own internal pore spaces. The filaments also stretch between aggregates.

One of the central questions a researcher asks when studying any bacterial system is how many bacteria are present in this sample? This applies to environmental samples where we want to learn about a native population as well as to lab cultures where we are measuring physiological functions. There are many ways to count bacteria and each of them has advantages and disadvantages. Techniques to detect form, pattern and arrangement in soil can be broken down into:
- Measuring the optical density (O.D.) by Spectrophotometer - Microscopic methods Direct Counting (Petroff-Hauser Chamber) Electron microscopy -Microscopic methods plus culturing Plate-Counts (Counting by Dilution Series) Direct culture methods Fluorescent antibody and related methods -Miscellaneous methods

It is often necessary to associate microorganisms with other structures or objects in soil. Plant roots, organic materials, mineral grains, fungal mycelium and arthropods are all colonized by particular types of bacteria and Actinomycetes. Often when soils are examined microbiologically, the native structure is destroyed and it is impossible to determine the original distribution of the microbes in their natural habitat. One way to visualize how microbes are distributed in soil is to use the soil contact slide method developed by Rossi (1928) and later Cholodny (1938) hence termed as Rossi-Cholodny Burried slide technique. In this technique, glass slides are buried in soil and allowed to incubate. Bacteria and fungi attach to the glass as though it was a mineral particle and grow on the surface. When the slide is carefully uncovered and stained, the microbes remain in their original positions and their associations with particles and each other can be seen. In this lab exercise, slides will be buried in soils that have received the following amendments: 1.6g of yeast extract, 0.2g of inorganic fertilizer, 1.6g of yeast extract and 0.2g of inorganic fertilizer, or no addition. All of the slides will be incubated for ~2 weeks.

Materials
Supplies staining racks and trays Pasteur pipettes and bulbs
Reagents acetic acid Phenolic rose Bengal stain (For 100 ml=5% aquous Phenol , 1gm Rose Bengal, 0.03 gm CaCl2 )

Cultures Microscope glass slides

Equipment - Light microscopes Phase contrast microscopes Bunsen burners

Procedures
Soil Slide Stain

2 3

Gently insert 2-4 clean microscope slides vertically in a beaker of garden soil leaving at least 2 cm (3/4 th is burried) protruding above the soil surface. Add enough water around the slide Do not water log the soil and incubate for over one week. Remove the slide by tilting it backwards so that the top of the slide is not scraped off.

Adapted from Environmental Microbiology: A laboratory Manual by Pepper, Gerba and Brendecke. 1995. Academic Press, New York.

4 Shake off any large soil particles and clean the bottom of the slide with a moist paper towel. Allow the slide to thoroughly dry. 5 Immerse the slide in 40% acetic acid for 1-3 min. (Handle the Acid carefully!). 6 Wash off the acid with water, being careful not to disturb the cells). 7 Flood the slide with phenolic rose bengal stain for 5-10 minutes. 8 Wash off the excess stain and allow the slide to dry. 9 View under the microscope. Look for fungal filaments, actinomycetes and bacterial microcolonies. 10 Record all observations.

Results
Name: Date:

Diagram: Observations: