Procedures

Fresh rumen fluid is collected in a pre-gassed Erlenmeyer flask from fistulated animals. Fresh feces from animals are collected into pre-gassed screw-cap tubes. Rumen liquor is immediately brought to the laboratory where it is strained through four layers of cheesecloth, and 0.5 ml of the serially diluted strained fluid is inoculated into each Hungate tube, which is incubated anaerobically at 39°C. In case of fecal samples, 1-2 gm of freshly collected feces are inoculated into 100 ml serum bottles containing 50 ml Orpin’s medium, containing antibiotics penicillin (0.10 mg/ml), streptomycin (0.10 mg/ml), and cellulosic substrate i.e. ball-milled wheat straw (1% w/v) for enrichment. Before inoculation, media are dispensed into 100ml serum bottles fitted with butyl rubber septa and then sterilized at 121°C for 15 minutes. The enrichment cultures are incubated for 5-7 days at 39°C, and subsequently, 0.5 ml of this enrichment culture is used to inoculate agar roll-tubes, from which anaerobic fungi are isolated as follows.

For isolation of rumen anaerobic fungi, the modified Hungate’s roll-tube technique involving series of dilutions of molten agar medium containing clarified rumen fluid and antibiotics is used. The carbon dioxide gas is first passed through heated (350°C) copper column of biosystem gassing manifold to make it free from oxygen. The composition of the anaerobic medium for isolating rumen fungi is given below. All media are prepared and dispensed under an atmosphere of CO2 and are autoclaved at 121°C for 15 min. Rumen liquor is strained through double layer of muslin cloth and clarified by centrifugation at 16,000 g for 20 min before being added to the medium. The fungi are isolated in pure culture from samples of rumen fluid diluted in anaerobic salts solutions including antibiotic control of bacterial contaminants. The roll tubes are kept in CO2 incubator at 39 ± 1°C and are regularly observed for the appearance of the fungal colonies (Hungate, 1969; Joblin, 1981). The distinct anaerobic rumen fungal colonies, appearing generally on third day, are sub-cultured in Orpin’s broth supplemented with anti-bacterial antibiotics.

1. Processing of rumen fluid for media preparation.
   The rumen liquor is collected in Erlenmeyer flask flushed with CO2, strained through four layers of muslin cloth, clarified through centrifugation at 12,000rpm for 10 minutes, and kept at –5°C before being added to the medium. The clarified rumen fluid is used to provide unidentified growth factors to the anaerobic fungus.
2. Media preparation
   Detailed composition and preparation of Orpin’s media is given below. With the exception of reducing agents (Sodium bicarbonate and L-cystein hydrochloride), all the quantified ingredients of the medium are taken in 250ml flask. The pH is adjusted to 6.9+0.1. The medium is boiled to free it from dissolved oxygen and cooled under the continuous flow of CO2, supplied through gassing manifold biosystem. Before solidification of media under continuous CO2 flow, the quantified reducing agents are added, and the media is autoclaved. Filter-sterilized antibiotics solutions are added to the sterile media under strict aseptic conditions. The slight yellowish appearance of media is indicative of perfectly anaerobic condition.
3. Preparation of anaerobic diluent
   The composition of the anaerobic diluent is given below. By following the anaerobic media preparation technique, 9 ml of anaerobic diluent is dispensed in pre-gassed 20 ml glass vial, capped and autoclaved. The anaerobic diluent is used for dilution of rumen liquor/ enriched fecal sample at the time of anaerobic fungal isolation.
4. Hungate’s roll-tube method
   For roll-tube method, the media (7 ml) is first dispensed into roll-tubes (7.5 x 1.5 cm; 20 ml), flushed with CO2 for 10-
   15 minutes under aseptic conditions, and finally the roll-tubes are fitted with butyryl rubber caps and autoclaved. Antibiotic solutions are added to the sterile roll-tubes containing molten agar media under strict aseptic conditions. These roll-tubes are then kept in water-bath (45-50oC) before being used for cultivation of anaerobic fungi. To these sterile roll-tubes containing molten agar media and antibiotic solutions, 0.5 ml of inoculum (serial dilutions of rumen
   liquor/ enriched fecal samples) is added, and the tubes are then rolled over roller-platform of cold water to solidify the media uniformly as a thin film on the inner wall of the roll-tubes. Then, the oxygen free CO2 is passed through the roll-tubes from Gassing Manifold Biosystem for 10-15 min and the tubes are kept in CO2 incubator at a temperature of 39 ± 1°C. During incubation, the roll-tubes are regularly inspected for the appearance of fungal colonies.
5. Sub-culturing of Fungal Colonies
   The anaerobic rumen fungal colonies, which appear in roll-tubes, are transferred to 15 ml screw cap tubes containing anaerobic broth up to the brim, in the presence of recommended dose of antibiotics. Precaution is taken during transfer, so that only single colony got transferred.
6. Purification of Culture
   The purity of the culture is examined regularly under microscope and confirmed by re-isolation on solid media until single type colony is obtained.
7. Culture maintenance
   Fungal cultures are maintained at 39°C by sub-culturing every 5 to 7 days in Orpin’s medium containing ball-milled wheat straw (1% w/v), depending on the rate of growth by removing small sections of the fungal colony which developed on the agar and transferring it to fresh medium.
8. Characterization
   Morphological identification of isolated fungal rhizoids is done upto genus level by fluorescence microscopy, based on the features of zoospores, types of sporangia and nature of rhizomycelia, using lactophenol cotton blue as staining solution. Identification of fungus is done up to the genus level on the basis of thallus morphology (monocentric or polycentric), shape of sporangia, rhizoid type (filamentous or a vegetative cell) and number of flagella per zoospore. Further characterization upto species level is done through biochemical parameters and through PCR-based molecular methods.

Solutions and recipes

Orpin’s medium

*Cellobiose is omitted if wheat straw (1% w/v) is used as substrate. If straw or other plant material is required, this is placed in the tube/bottle prior to adding the medium.
Cellobiose, yeast extracts and sodium hydrogen carbonate are dissolved in 500 ml of distilled water. To the above mixture, the mineral solutions I, II, clarified rumen fluid and resazurin solution are added, and volume is made up to 1 litre. The mixture is boiled to drive off any oxygen present using microwave on high temperature. The medium is then left to cool whilst being bubbled with CO2 for about 10 min., and cysteine-HCL is added. The pH is adjusted to 6.9 with NaOH or H3PO4 as required, and the medium is dispensed into roll-tubes or serum bottles (as required) and sealed
with butyl rubber stoppers. The medium is sterilized by autoclaving (121oC for 15 min.). Filter-sterilized antibiotic
solutions are added to the autoclaved medium at 0.1% concentration under strict aseptic conditions.

Mineral Solution I (per litre):

The above solute is dissolved in 1 litre of distilled water, and stored at 4oC.
 

Mineral solution II (per litre)

The above solutes are dissolved in 800 ml of distilled water. Separately CaCl2.2H20 is dissolved in 100 ml of distilled water. The solutions are then combined and made up to a final volume of 1 litre and stored at 4oC.
 

Joblin’s medium
Composition (per 100 ml):

With the exception of reducing agents (Sodium bicarbonate and L-cystein hydrochloride), all the quantified ingredients of the medium are taken in 250 ml flask. The pH is adjusted to 6.9+0.1. The medium is boiled to free it from dissolved oxygen and cooled under the continuous flow of CO2, supplied through gassing manifold system. Before solidification of media under continuous CO2 flow, the quantified reducing agents are added, and the media is autoclaved. Filter-sterilized antibiotics solutions are added to the sterile media under strict aseptic conditions. The slight yellowish appearance of media is indicative of perfectly anaerobic condition.
 

Anaerobic diluent

The mixture is warmed and bubbled with CO2 for 15-20 min. After bubbling, cystein-HCl is added and bubbling is continued till the solution turned colourless. 9 ml of the solution is then dispensed into pre-gassed screw-cap tubes and autoclaved.