English: Bacteria as indicators of quality and state of water from an ecological perspective

- Determining aerobic heterotrophic bacteria:

1. Using a sterile pipette, 1 ml aliquots of the sample and/or desired dilution are to be transferred in a sterile Petri dishes. Cooled liquid nutrient medium is to be poured over the inoculum and homogenization is to be achieved with gentle circular movements of the plate.

2. The solid plates are to be inverted and incubated at 20 о С for 5-7 days in order to determine aerobic heterotrophic psychrophilic bacteria and at 37 ^оС for 24-48 hours in order to determine aerobic heterotrophic mesophilic bacteria.

RESULTS: The plates should show growth of aerobic heterotrophic bacteria. This group ofbacteria represent indicators of water quality in respect to the degree of organic contamination. High counts of aerobic heterotrophic bacteria is indicative of water with relatively large amounts of organic matters subject to bacterial degradation.

- Determining presence of Clostridium Sp.:

1. Using a sterile microbiological needle, inoculum from the sample is to transferred in deep agar by piercing the surface of the deep agar.

2. The test tubes are to be incubated at 37 о С for 48 hours.

RESULTS: Presence of Clostridium sp. is to be determined by the vertical column of bacterial growth as Clostridia are an anaerobic group of bacteria.

- Determining facultative oligotrophic bacteria:

1. Using a sterile pipette, 1 ml aliquots of the sample and/or desired dilution are to be transferred in a sterile Petri dishes. Cooled liquid nutrient medium is to be poured over the inoculum and homogenization is to be achieved with gentle circular movements of the plate.

2. The solid plates are to be inverted and incubated at 22-26 ^оС for 5-7 days in order to determine facultative oligotrophic bacteria.

RESULTS: Plates should show growth of facultative oligotrophic bacteria, indicative of water with low organic matter content. These bacteria are able to grow in media with low nutrient concentrations, and may adapt to environments with higher nutrient concentrations just as readily.

- Determining proteolytic bacteria:

1. Nutrient agar with gelatin is prepared and sterilized by autoclave at 121 degrees centigrade for 15 minutes.

2. Using spread plate equipment, an aliquot of the sample is to be spread on the previously sterilized and solidified plates.

3. Plates are to be incubated at 22-26 ^оC for 5-7 days.

4. After incubation, formed colonies are to be directly stained using a proteolytic reagent.

RESULTS: Only colonies which show a halo zone after application of the proteolytic reagent can be considered colonies of proteolytic bacteria. The reagent (dissolved mercury II chloride) causes protein precipitation of the proteins present in the medium. Proteolytic bacteria secreting proteases cause nearby gelatin to hydrolyse and cause discoloration of the reagent as there are scarce proteins to precipitate. Only those colonies in which an illuminated zone appeared around them, are counted.

- Determining lipolytic bacteria:

1. Nutrient agar with tributirine or Tween 80 is prepared and sterilized by autoclave at 121 degrees centigrade for 15 minutes.

2. Using spread plate equipment, an aliquot of the sample is to be spread on the previously sterilized and solidified plates.

3. Plates are to be incubated at 22-26 ^оC for 5-7 days.

RESULTS: Only those colonies demonstrating a murky white halo can be classified as lipolytic (the white halos form as a result of the interaction of calcium soap byproducts).

- Determining amylolytic bacteria:

1. Starch agar is prepared and sterilized by autoclave at 121 degrees centigrade for 15 minutes.

2. Using spread plate equipment, an aliquot of the sample is to be spread on the previously sterilized and solidified plates.

3. Plates are to be incubated at 22-26 ^оC for 5-7 days

4. After incubation, formed colonies are to be directly stained using Lugol’s reagent.

RESULTS: The medium stains blue due to the present starch reacting to the added Iodine. Visible discoloured zones form around the colonies of amylolytic bacteria due to the activity of excreted amylase. The difference in color of these zones varies and depends on the activity of the particular amylase secreted as well as the size of the colony, varying from brown shades indicative of the presence of small dextrine chains, to colorless.

- Determining Nitrogen-fixing bacteria:

1. Ashby agar is prepared and sterilized by autoclave at 121 degrees centigrade for 15 minutes.

2. Using spread plate equipment, an aliquot of the sample is to be spread on the previously sterilized and solidified plates.

3. Plates are to be incubated at 22-26 ^оC for 5-7 days

RESULTS: As the medium has no available nitrogen, only nonsymbiotic nitrogen fixing bacteria may survive and form colonies. Any colonies present after the incubation period are of nitrogen fixing bacteria.

- Determining Pseudomonas spp.

1. Cetrimide agar is prepared and sterilized by autoclave at 121 degrees centigrade for 15 minutes.

2. Using spread plate equipment, an aliquot of the sample is to be spread on the previously sterilized and solidified plates.

3. Plates are to be incubated at 22-26 ^оC for 5-7 days

RESULTS: Plates should show the growth of green to light green colonies.

- Determining cellulolytic bacteria:

1. Cellulolytic agar is prepared and sterilized by autoclave at 121 degrees centigrade for 15 minutes. 2. Using spread plate equipment, an aliquot of the sample is to be spread on the previously sterilized and solidified plates.

3. Plates are to be incubated at 22-26 ^оC for 5-7 days

RESULTS: Colonies of cellulolytic bacteria usually appear lemon green, dark yellow, orange or red-brown in coloration.

- Determining nitrificators:

1. Differential media for nitrifying bacteria is prepared and sterilized by autoclave at 121 degrees centigrade for 15 minutes.

2. Using spread plate equipment, an aliquot of the sample is to be spread on the previously sterilized and solidified plates.

3. Plates are to be incubated at 22-26 ^оC for 5-7 days

RESULTS: Oxidation of NH 3 + to NО 2 - and NО 3 - , causes discoloration of the media from a red shade to a yellow shade around colonies actively causing the process of nitrification. Only those colonies or test tubes if done with liquid media can be considered as positives.

- Determining denitrificators

1. Differential media for denitrifying bacteria is prepared and sterilized by autoclave at 121 degrees centigrade for 15 minutes.

2. Using spread plate equipment, an aliquot of the sample is to be spread on the previously sterilized and solidified plates.

3. Plates are to be incubated at 22-26 ^оC for 5-7 days.

RESULTS: The production of ammonia and other by-products of denitrifying bacteria cause discoloration of the medium from green to an intense blue shade. Gas bubbles may also form and rest at the surface of the medium.

Planned and performed by Sofija Kostandinovska, Institute of Biology, FNSM, Ss. Cyril and Methodius University, Skopje, Macedonia.