5. MICROBIOLOGICAL INVESTIGATIONS

5.1 TOTAL BACTERIAL COUNT

To estimate the natural microbial population, all the cells present in the sample were counted under the microscope. For this, membrane filters (pore size less than 0.3 um), millipore filtration unit, phenol, erythrosin and ocular grid (area 25–35 mm², 25 sections) were required. A known volume of sample collected from pond water is filtered using a millipore filtration unit or a glass funnel with suction pump. However, a glass syringe and a small membrane filter unit may also be used for the purpose. Normally, the volume of water filtered is about 10–24 cm³ for oligotrophic ponds, 5–10 cm³ for mesotrophic ponds and 2–5 m³ for eutrophic ponds (when differential staining is not attempted, the water samples can be fixed with formaldehyde at 2% final concentration for storage). After filtration the membrane filter is dried (the micro-organisms on the filters are fixed in formaldehyde vapour or by heating at 70°C for 20 min) and stained.

The membrane filter is placed on filter paper soaked in 3% erythrosin solution in 5% phenol. Staining requires 12–24 h at room temperature or about 20 min at 65°C in the desiccator. The stained filters are decolourized by placing them on the surface of moistened filter paper to a weak pink colour (the erythrosin staining can be contrasted by subsequent staining with diluted carbol fuchsin heated to 60°C).

For microscopic examination, filters are put on a drop of immersion oil placed on the surface of a slide and covered with coverslip. A grid having 25 sections with an area 25–35 mm³ is placed inside one of the oculars and counts are taken over random fields.

The number of bacteria (N) in the sample is calculated using the formula:

To count the microflora of the bottom sediment, the Winogradsky method modified by Kuznetsov and Romanenko (1963) is used. A sample of the sediment (0.5 cm³) is taken with a sterile glass tube and transferred to a flask with 50–100 cm³ of 0.005 N potassium hydroxide. For sediment containing lime, the sample is treated with 2% hydrochloric acid. It is shaken for several minutes and allowed to settle for 1 min. Then, 0.1–0.2 cm³ of the suspension is placed on a slide that is cleaned with alcohol and ether. A drop of 0.5% agar solution previously filtered through millipore filter is added. The mixture is spread over the slide surface on an area of 6 cm². The preparation is dried, fixed by flaming or by absolute alcohol and stained. The carbolic erythrosin solution is poured into the slide preparation and heated until slight vaporization takes place. The dye is washed off and diluted carbol fuchsin (1/300) is poured on it. After heating again, the preparation is washed and dried. The microscopic examination is done under oil immersion and bacterial counts are made using an ocular grid. Calculations are made in a similar way as for the water sample.

5.2 PREPARATION OF MEDIA FOR BACTERIAL CULTURE

Before inoculation, 30 ml of 1% solution of sodium sulphide (Na₂S) in 5% sodium carbonate (Na₂CO₃) should be added to each 100 cm³ of the medium, and pH to be adjusted to 7 with HCl.

Methane-producing bacteria are heterotrophic in nature, and act upon organic carbon reducing it into methane. The abundance of these bacteria in the sediment indicates the extent of the reduced compounds in the sediment.

C. For oligocarbophilic bacteria

1. A medium for oligocarbophilic bacteria is prepared by dissolving 15 g of agar in 1 000 cm³ of pond water.

2. Another medium for the oligocarbophilic bacteria is prepared by dissolving 15 g agar agar in 1 000 cm³ of interstitial water collected by squeezing pond soft sediment.

3. Nutrient agar used in hygienic microbiology is quite inadequte for counting the autochthonous, slowly-growing water inhabiting bacteria. Sodium-caseinate is a better substitute for this and the medium is prepared with sodium-caseinate agar.

D. For rapidly growing bacteria

This is the commonly used nutrient medium for bacterial culture.

E. For ammonifyers

Peptone agar is provided for obtaining the growth of ammonifyers in water and sediment of a water body. The process of ammonification is carried out by heterotrophic bacteria.

The medium is sterilized in an autoclave at 15 psi pressure (121°C) for 20 min and inoculated with samples in Petri dishes.

F. For urea decomposers

Urea hydrolyzing bacteria decompose urea into ammonia which is alkaline. This change in the pH can be detected with the indicator, bromothymol blue, which turns from olive green to blue:

Urea, glucose and bromothymol blue are steamed for sterilization on three days. The pH should be 6.8–6.9 as indicated by the olive-green colour of the indicator. When the bacteria release ammonia the colour of the medium turns blue.

G. For aerobic nitrogen fixation

Nitrogen-fixing bacteria play a great role in the replenishment of nitrogen compounds in the water. The dominant bacterial species involved in aerobic nitrogen fixation belong to the genus Azotobacter.

Colonies of Azotobacter appear as droplets after incubation on the inoculated medium in the Petri dishes.

H. For anaerobic nitrogen fixation

Among the anaerobic nitrogen fixing bacteria, Clostridium spp. is important. One bacillus has spindle-shaped cells and contains oblong spores.

Before sterilization, calcium carbonate is added to the medium at about 4 g/100 cm³. The medium is sterilized at 110°C for 30 to 40 min.

After inoculation, the tubes are incubated anaerobically by introducing 1 cm³ each of Pyrogallol (20%) and sodium carbonate (20%) on the inserted cotton plugs and closing with rubber corks.

I. For nitrification

(a) NH₃-NO₂: Nitrosomonas spp.

In the first phase of nitrification, ammonia is oxidized to nitrite by Nitrosomonas. The modified Winogradsky medium is used for counting the bacteria.

Calcium carbonate is sterilized separately and added to the medium which is distributed in about 25 cm³ quantities in 100 cm³ Erlenmeyer flasks.

The disappearance of ammonia and appearance of nitrite in the culture medium indicates the presence of nitrifying bacteria. It is checked at weekly intervals.

Nessler's reagent for ammonia: 50 g potassium iodide is dissolved in 50 cm³ undistilled water to which is added a hot concentrated solution of mercuric chloride until the red precipitate formed stops disappearing. The liquid is filtered. A solution of 150 g potassium hydroxide in 400 cm³ distilled water and several cm³ of mercuric chloride are added. When the reagent cools, the volume is brought to 1 000 cm³ using distilled water. The reagent is stored in a cold environment. On reaction, it yields an orange-yellow colour, and when the concentration is high the colour is red-brown. A few drops of reagent are placed on a white porcelain plate and a drop of the culture liquid is added, mixed, and the colour is noted.

(b) (NO₂NO₃: Nitrobacter spp.)

The principal bacteria involved in this phase, Nitrobacter spp., have oval cells (1.1 × 0.45 μm), are ear-shaped and show poor staining reaction.

To purify the commercial product of sodium nitrate, 500 g of salt is dissolved in 750 cm³ distilled water by heating. The salt solution is filtered, the filtrate is concentrated until crystals begin to appear, and then cooled.

After the medium is distributed in 25 cm³ quantities in 100 cm³ Erlenmeyer flasks, it is sterilized and inoculated with samples. The disappearance of nitrite and appearance of nitrate are checked at weekly intervals.

Griess reagent for nitrate: 1 g α-naphthylamine, 10 g sulphuric acid and 89 g tartaric acid are mixed and ground in a mortar to a fine powder. This is kept in a dark glass bottle with a ground glass stopper. Instead of tartaric acid, 50 g succinic or oxalic acid may also be used. Then, 7–10 mg of dry reagent is placed in the depression of a clean porcelain plate; 4–5 drops of culture are added and mixed with a glass rod. A pink colour appears immediately if a significant amount of nitrate is present and gradually if small quantities are present. An accurate determination of nitrate may be made using a colorimeter.

Detection of nitrate: 0.2 g of Pyrogallol is mixed with 10 cm³ of culture medium in a test tube; 2 cm³ of concentrated sulphuric acid is added using a pipette and 0.1 g of sodium chloride is added. Boiling begins round the edge and a purple ring appears, indicating the presence of nitrate.

J. For microbial denitrification

When mass development of denitrifying bacteria occurs, salts dissolved in water and vital for phytoplankton development may be destroyed, i.e., converted into molecular nitrogen and therefore inaccessible to algae. With the presence of nitrates, reduced oxygen content and presence of organic matter favour the process.

If citric acid is used, it should be mineralized with 10% potassium hydroxide solution, with phenolphthalein as the indicator. Solutions A and B are mixed and diluted to 1 000 cm³ with distilled water.

The media are inoculated anaerobically in test tubes. The cotton plugs are cut, inserted into the tubes, covered with 1 cm³ each of phyrogallel (20%) and sodium carbonate (20%) and closed with rubber areas.

K. For precipitated inorganic phosphate solubilizing bacteria

(a) All the ingredients except for the insoluble inorganic phosphatic source, are put in a small flask and autoclaved.

(b) Tricalcium phosphate (any phosphatic source) is thoroughly powdered in mortar, 0.5 g of gum arabic is again similarly pulverized. The two are then mixed and sterilized with 100 cm³ distilled water and autoclaved separately.

Inoculation: 1 cm³ of the thoroughly shaken tricalcium phosphate suspension in gum arabic was added aseptically to each Petri dish and thoroughly mixed before solidification of agar and incubated at 30°C for 7–10 days.

L. For organic phosphorus mineralizing bacteria

Pokorni suggested preparing the medium without lecithin and sterilizing for 30 min at a pressure of 10⁵ Pa. The lecithin is stored separately and dissolved previously in sulphuric acid. Lecithin, 0.03 g, is distributed with a pipette on the surface of each agar plate of the Petri dish together with the inoculum and spread with a glass triangle. Incubation is at 25°C for 7 days and the colonies with halos can be counted.

M. For protein decomposers producing sulphide

Proteins are used as energy and nutrient sources by a group of anaerobic bacteria releasing sulphide highly poisonous for the fish. Their number can be detected by the black colour of the colonies.

The medium is poured onto the inoculum in the upper cover of the Petri dish and covered with the lower one, taking care that no air is trapped inside. This agar medium can be taken in test tubes and inoculated before the agar solidifies. The medium and the inoculum are well mixed and kept for incubation under anaerobic conditions with alkaline pyrogallol.