How to determine iron oxidizing bacteria in a sample

Iron bacteria are aerobic, fresh water organisms appearing in a variety of forms. These organisms can cause taste and odour problems in water supply. Iron bacteria have an oxidative metabolism. They oxidise Ferrous Iron (Fe2+) to Ferric Iron (Fe3+) and deposit it as hydrated iron oxide. Iron bacteria are of concern because of their tendency to develop rapidly and form masses of growth which usually adhere to the inside surfaces of pipes and tanks. The precipitation of iron by the bacteria can build up in stagnant areas resulting in corrosion and inadequate water flow. The most troublesome iron bacteria are the filamentous types, such as Leptothrix and Crenothrix.

 

Iron oxidising bacteria found in cooling water systems can be classified into two nutritional groups viz.

a) Chemoautotrophic type eg Gallionella and

b) Chemoorganotrophic type eg. Leptothrix and Crenothrix.

These groups differ in their nutrition and hence are detected in two different media viz. Wolf’s medium and Muldar Van Vee’s medium respectively. Iron bacteria are also enumerated by Most Probable Number (MPN) technique.

The test procedure consists of inoculating the test sample in a proper medium. Visible brownish colour growth indicates presence of iron oxidizing bacteria.

 

Incubator

Autoclave

Pipettes (50 ml, 10 ml & 100 ml) Dilution flasks (250 ml & 100 ml)

Non Absorbent Cotton

Wolf’s Medium

Mulder Van Vee’s Medium

 

 

1. Wolf’s medium is prepared for chemoautotrophic iron bacteria such as Gallionella, as follows

a) Dissolve

1.0 gram Ammonium chloride, NH4Cl

0.2 gram Magnesium sulfate. Heptahydrate MgSO4.7H2O

0.1 gram calcium chloride dihydrate CaCl2. 2H2O.

0.05 gram Dipotassium hydrogen phosphate dihydrate K2HPO4 .2H2O in distilled water.

b) Add 0.5 ml of Formalin (40%)

c) Bubble CO2 as to adjust the pH of the medium to 6.0 + 0.1

d) Add 1.0 gram Ferrous Sulfite

e) Make up to 1000 ml with distilled water.

f) Sterilise the medium if formalin is not added, in an auto clave at 15 lbs pressure / inch2 at 121oC for 15 minutes.

g) Cool the medium and use it for experiment.

 

2. Mulder Van Vee’s Medium is prepared for chemoorganotrophic, iron bacteria such as Crenothrix and Leptothrix.

a) Dissolve in 1 ltr distilled water.

         2.0 gram Manganese Carbonate MnCO3

         1.0 gram Beef Extract

         0.075 gram yeast extract 

         0.15 gram sodium citrate traces of vitamin B12

         0.15 gram Ferrous Ammonium Sulfate

b) Adjust the pH in between 6.8 to 7.2

c) Dispense the medium in conical flask

d) Plug it with non absorbent cotton and wrap with paper.

e) Sterilise the medium in an auto clave at 15 lbs pressure / inch2 at 121oC for 15 minutes.

f) Cool the medium and use it for experience.

3. For the preparation of Double strength medium; dissolve the above ingredients of Wolf’s medium and Mulder Van Vee’s medium in 500 ml distilled water.

4. Aseptically add 50 ml of test water sample with the help of sterile 50-ml capacity pipette, in 250-ml capacity flask containing 50 ml Double Strength Wolf’s Medium.

5. Aseptically add 10 ml of test water sample, with the help of sterile 10 ml capacity pipette in 100 ml capacity flasks (5 Nos) containing 50 ml Normal Strength Wolf’s Medium.

6. Aseptically add 1ml of test water sample, with the help of sterile 1 ml. capacity pipette in 100 ml capacity flasks (5 Nos) containing 50 ml Normal Strength Wolf’s Medium.

7. Repeat Steps 4, 5, 6 using Mulder Van Vee’s Medium in place of Wolf’s Medium.

8. Incubate the flasks in an incubator as 37oC for 7 to 15 days.

9. Visible, brownish colored growth indicates presence of iron oxidizing bacteria. (IOB)

10. Use McCardy’s MPC table and number of positive tests to find out number of organisms.

11. Record the final result of iron oxidising bacteria (IOB) as Number of IOB / 100 ml.