Delhi Technological University

(formerly Delhi College of Engineering)

EN205 – MICROBIOLOGY LAB FILE

Submitted by: Submitted to:

Aastha Singh Ms. Sonam Taneja
2K20/EN/02

Department of Environmental Engineering

Delhi Technological University
Bawana Road, Delhi – 110042
EXPERIMENT - 1
Aim: To determine the pH of water.

Apparatus: pH meter, beakers

Reagents Used: pH buffer solutions 4.0, 7.0 and 10.0, water sample,
distilled water

Theory: pH is the concentration of hydrogen ions in the solution. A

solution containing more H+ ions remains acidic while the solution
containing more OH-ions remains alkaline. pH value of solution ranges
from 0 to 14.

H2O = H+ + OH-

pH = -log[H+]

[H+] = 10-pH

For every one unitary pH, it represents a ten-fold increase or decrease

in the hydrogen ion concentration

A pH metre is used to measure the pH for more quantitative

measurement in place of universal indicators. It shows the pH and
temperature of solutions. A pH metre consists of a pH electrode
which consists of a reference electrode and an internal electrode.

The internal electrode is made of a glass/thin sheet of pH-sensitive

glass and is filled with KCl or buffered with KCl at a fixed pH of 7 so
it is neutral in nature. It is further coated with a silver plate (AgCl)
which comes in contact with the solution. A potential is developed
between the internal electrode and the sample and due to hydrogen
ions present in the sample. The other electrode, the reference
electrode, is dipped in KCl solution with a fixed molar concentration.
As KCl ions are fixed, Cl- ions are fixed, the potential is fixed that
making the electrode independent of pH as there is no gradient.

Procedure:

(1) Remove the electrode cap and rinse the electrode in distilled water
in a beaker to avoid spillage
 (2) Dip the electrode in the pH 7 buffer solution and note the readings

Note: If the pH metre shows different reading like 7.02, calibrate it

using the up and down button or the calibration norm and set it to pH
7 as the buffer solution added is of pH 7.

(3) Rinse the electrode in distilled water and wipe it and repeat the
same above steps for pH 4 and pH 10 buffer solution

(4) Now as the pH metre is properly calibrated we wash the electrode

and dip it in the sample and take 2-3 readings for better accuracy

(5) After taking the readings, keep the pH metre in distilled water so
as to prevent dehydration or dryness of the electrode

Results:

The pH value of the solution is ________

Precautions:

Don’t let the electrode touch the bottom of the beaker of sample
solution as it may lead to inaccurate readings

Rinse the electrode in distilled water after every reading Handle the
instruments with care.

EXPERIMENT - 2
Aim: To determine the DO of water.

Apparatus:

Burette, pipette, incubation bottle, conical flasks, beakers, measuring

cylinder
Reagents Used: Manganese sulphate solution, Alkali-iodide azide reagent,
Sulphuric acid, Starch solution, Sodium thiosulphate titrant, water sample,
distilled water

Theory:

THE WINKLER METHOD FOR DO DETERMINATION

If no oxygen is present, a pure white precipitate is formed when MnSO4

and alkali-iodide reagent (NaOH + KI) are added to the sample.
Mn2+ + 2OH- -> Mn(OH)2

White precipitate

If the sample has some oxygen, Mn2+ is oxidized to Mn4+ and precipitates
brown hydrated oxide.
Mn2+ + 2OH- +0.5O2  MnO2 + H2O

Brown Hydrated Precipitate

The oxidation of Mn2+ to MnO2 is called fixation of the oxygen, occurs

slowly at low temperature
Mn(OH)2 + 0.5O2  MnO2 + H2O

After shaking the sample for a time sufficient to allow all oxygen to
react, the floc is allowed to settle so as to leave 5 cm of clear liquid
below the stopper; then sulfuric acid is added.

MnO2 + 2I- + 4H+ -> Mn2+ + I2 + 2H2O

I2 + I- ↔ I3-
Now the sample is ready for titration with thiosulphate solution.

The Azide Modification

Because of nitrites present–

Azide modification to overcome nitrite interference : use of sodium azide

(NaN3), which is incorporated in the alkali-KI reagent. When sulfuric acid
is added, following reactions happen
 Procedure:

1. Take 300ml of water sample and fill it without forming the bubbles.

2. Now put the pipette in the MnSO4 solution and pipette out 1ml of MnSO4
(Winklers A).

3. Now take the pipette and dip the pipette below the surface of water and put
1ml of MnSO4 in water.

4. Take 1ml of alkali iodide azide reagent (Winklers B) and dissolve it in the
water sample like done previously with MnSO4.

5. Now cover the water sample with the topper and shake it by inverting the
bottle.

6. Wait for 10-15 mins for the precipitate to settle down.

7. Once the precipitate has settled, take 1ml conc H2SO4 and pour it in the
water sample.

8. Again shake the water sample (solution created) until the precipitate
dissolves.

9. Take 100ml sample of the solution in an Iodometric Flask

10. Take a burette and fill it with 0.025N Na2S2O3.

11. Now pour 1-2 drops of 0.025N Na2S2O3 in the iodometric flask.

12. Put starch indicator in the flask and cover it.

13. Colour changes to dark blue.

14. Keep adding Na2S2O3 in the flask till the end point is reached, which is
when dark blue changes to colourless.

Reactions:

  Mn2+ + 2OH-  Mn(OH)2 White precipitate

  Mn2+ + 2OH- +0.5O2  MnO2 + H2O Brown Hydrated Precipitate

  Mn(OH)2 + 0.5O2  MnO2 + H2O

  MnO2 + 2I- + 4H+  Mn2+ + I2 + 2H2O

  I2 + I- ↔ I3-

Calculations:

D.O mg/litre = (B.R X Normality of Na2S2O3 X 8 X 1000)/(Vol. Of

sample) = 3.7 x 0.025 x 8 x 1000/100
= 7.4 mg/litre

Result:

DO of water is 7.4 mg/litre

Precautions:

1. Drawing tubes and sample flasks must be free of bubbles.

2. Do not allow air to trap while sampling water during BOD analysis.

3. Dip the tip of the pipette just at the bottom of the BOD bottle and
gently release the reagents.

4. Take care that the chemicals do not flow out from the bottle while
shaking/swirling.

EXPERIMENT - 3
Aim: To determine the TDS and TSS of water

Theory:

 Solids may be classified as settleable, suspended, dissolved, volatile

and fixed.
 Total Dissolved Solids (TDS): Substances present in dissolved form in
water collectively is called TDS and are present in ionic form like Na+,
K+, Ca+ etc.

 Total Suspended Solids (TSS): Solids that can be filtered and are
removed by using filters are TSS.

 It is necessary to find the TDS as it affects the physical and chemical

properties it water and can alter its taste and also be toxic in nature.

 The maximum permissible limit of TDS for drinking water is 500 mg/l
and the maximum permissible limit for other uses is 2000 mg/l

 Total Solids (TS): TDS + TSS

 We use gravimetric analysis to measure TDS and TSS. This method

analyses the mass/weight of the analyte to determine the TDS of water.
There is a three-step process. The first step is filtration in which we
take a filter paper, weigh it and we will then use it in a filtration
assembly.

 To calculate the volatile component and fixed component of TSS, we

use the gravimetric analysis of evaporation

Procedure:

Part 1

 Take a filter paper and weigh it

 Filter the water sample through and oven-dry it at 104 degrees celsius once
the filtration is done and then weigh the sample present on the filter paper

 As TSS is filtrable it will be present on the filter paper

 After filtering it, we get a filtrate, a clear solution that will contain all the
TDS.

o The TDS is measured preferably using a multimeter.

o Another method is drying it again in the filter paper at 104° celsius and
calculating the TDS

Part 2
 Take a crucible and weigh it
Put the filter paper having suspended solids on the crucible

 Place the crucible in a muffle furnace at 550° celsius then cool it and weigh
it again

Observation:

Where,

A = Weight of filter paper after oven-dry

B= Initial weight of filter paper

Where, C= Weight of empty crucible

D= Weight of the crucible after muffle furnace

E= Weigh crucible+ filter paper

Results:

The TDS & TSS of water is ____ & ___ respectively

Precautions:

 Water or waste water samples which contain high concentrations of

calcium, chloride, magnesium or sulfate can rapidly absorb moisture
from the air.

• Such samples may need to be dried for a longer period of time, cooled
under proper desiccation and weighed rapidly in order to achieve a
reasonable constant weight.
• We should be aware prolonged drying may result in loss of constituents,
particularly nitrates and chlorides.

• Volume of sample should be adjusted to have residue left after drying

as 100 to 200mg. It is mainly to prevent large amount of residue in
entrapping water during evaporation.

• Samples with high concentrations or bicarbonate require additional

drying at 180oC to ensure that all of the bicarbonate is converted to
carbonate.

EXPERIMENT - 4
Aim: To determine the Hardness of water
 Apparatus: Burette, pipette, conical flasks, beakers, measuring
cylinder

Reagents Used: EDTA solution (0.01M), Eriochrome Black-T

indicator, NH4Cl NH4OH buffer solution, water sample, distilled
water

Theory:

 Total hardness = Temporary + Permanent hardness

 Temporary hardness refers to carbonate and bicarbonate hardness which
can be removed by boiling.
 Non-carbonate hardness + Carbonate hardness = Total hardness
 Carbonate hardness is referred to total alkalinity.
 When hardness is equal to or less than the sum of carbonate and
bicarbonate alkalinity, all hardness is carbonate hardness and non-
carbonate hardness is absent.
 The experiment is based on complexometric titration where EDTA is used
as a complexing agent which forms complexes with calcium and
magnesium present in water which is the basic cause of hardness in water
to remove them.
 As using Erichrome Black T as an indicator makes the solution slightly
acidic, the ammonium buffer solution is used to balance out the indicator
and maintain constant pH.

Procedure:

1) Take distilled water in a conical flask

2) Add 0.5 ml of Ammonium Buffer solution along with a pinch of indicator
and after adding the indicator (Erichrome Black T), the solution turns
slightly pinkish
3) After this, we titrate the solution against the titrant (EDTA) till the solution
turns steel blue
 Observations:

The volume of EDTA used will indicate the amount of calcium and
magnesium ions present in the solution

Reactions:

Results:

The total Hardness of water is ____________

Precautions:

 Always read lower meniscus in case of colourless solution and upper

meniscus in case of coloured solutions.

 Shaking of the titration flask should be continuous during adding the

solution from the burette.

 Handle the instruments with care.

EXPERIMENT - 5
Aim: To determine the Chloride content of water
 Apparatus: Burette, Pipette, conical flasks, measuring cylinder

Reagents Used: Silver nitrate solution (0.0141N), Potassium chromate

indicator, water sample, distilled water

Theory:

It is a titration based experiment used to determine the permissible limit of

Chloride in water.

Chlorides make the water hard and corrode the piping and sewage
systematically. It is harmful to plants and lifeforms. The permissible limit
Cl- ions is 250 mg/l for drinking purposes
Sources of chloride ions include coastal areas and anthropogenic sources
like agricultural discharge and industrial effluent.

During titration, chloride ion is precipitated as white silver chloride. All

silver ions consume Cl- ions to form AgCl which then forms white
precipitates
When Cl- ions are consumed, excess Ag+ ions combine with Potassium
chromate that forms a reddish-brown precipitate and which indicates the
endpoint.

Also, 0.0141N silver nitrate solution is used for ease of calculations as -

1ml of 0.0141N silver nitrate solution = 1mg chloride in solution

Procedure:

1) Add distilled water in a conical flask

2) Add 2-3 drops of Potassium Chromate solution (Used as an indicator)
which turns the solution yellow
3) Titrate the solution with Silver Nitrate present in Burette
4) After some time we see the formation of milky layers due to the
concentration of AgCl which forms the white precipitate
5) After some time we observe reddish-brown colour which is the endpoint of
the experiment

Reactions:

White Precipitate

Reddish Brown Precipitate

 Observations:

Result: The chloride ion concentration in water is _________

Precautions:

 A single person should complete the experiment and caution should be made
to notice indicator colour change as it can vary person-to-person. The usual
range is 0.2 to 0.4 mL of titrant.
 Keep pH strips handy. The pH must be in the range of 7 to 8 as silver ions
are precipitated as AgOH at high pH levels and the chromate ions are
converted to Cr2O7 at low pH values
 A definite amount of indicator must be used to provide a certain
concentration of chromate ions, otherwise silver chromate may form too
soon or not soon enough.