DEPARTMENT OF MICROBIOLOGY,ST ALOYSIUS COLLEGE …DEPARTMENT OF MICROBIOLOGY,ST ALOYSIUS COLLEGE...

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DEPARTMENT OF MICROBIOLOGY,ST ALOYSIUS COLLEGE (AUTONOMOUS),MANGALURU LABORATORY MANUAL 4TH SEMESTER

Transcript of DEPARTMENT OF MICROBIOLOGY,ST ALOYSIUS COLLEGE …DEPARTMENT OF MICROBIOLOGY,ST ALOYSIUS COLLEGE...

DEPARTMENT OF MICROBIOLOGY,ST ALOYSIUS COLLEGE (AUTONOMOUS),MANGALURU

LABORATORY MANUAL 4TH SEMESTER

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SL NO EXPERIMENT PAGE NO 1 Estimation of Dissolved Oxygen in Water. 2

2 Estimation of Carbon dioxide in Water. 5

3 Estimation of Chlorine. 6

4 Estimation of Alkalinity of Water 8

5 Standard Qualitative Microbial Analysis of Water. 12

6 Microscopic Observation of Root Colonization by VAM Fungi. 16

7 Production of Ammonia from Organic Compounds (Ammonification). 17

8 The Study of Winogradsky Column. 19

9 Study of Microbial Interaction. 21

10 Isolation of Fungi from Air. 23

11 Study of Air Samplers. 25

12 Demonstration of Microflora of Skin, Mouth and Nose. 26

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EXPERIMENT NO.1 . ESTIMATION OF DISSOLVED OXYGEN IN WATER.

AIM:To Estimate the Amount of Dissolved Oxygen in the Given Water Sample.

INTRODUCTION: AND PRINCIPLE: Organic material discharged into a water source serves as a food source for the

bacteria present there. Bacteria will break down these material to produce less complex organic substances and

eventually carbon dioxide and water. Bacteria will multiply using up the available dissolved oxygen as they do so.

If the bacterial uptake of water is faster than the rate which dissolved oxygen is replaced from the atmosphere

and from the action of photosynthesis, the water will become depleted in oxygen. This causes anaerobic

condition in which offensive products such as H2Sand ammonia are produced. The depletion of oxygen may

result in the other undesirable effects such as fish kills.

An iodine thiosulphate titration can be used to measure the dissolved oxygen present in a water sample.Because

the dissolved oxygen does not directly react with the redox reagent, an indirect procedure was developed by

Winkler.

A known amount of oxidizing agent i.e; manganese sulphate is added to the water to be estimated. Manganese

sulphate reacts with oxygen present in the water. During the reaction, the oxygen is bound to manganese , this

process is called fixing the oxygen. For this process to work, the solution must be at a high pH. Potassium iodide

is added to function as a dye which later react with sulphuric acid.

Manganese sulphate reacts with (KOH-KI) alkali iodide solution of high pH to produce a white flocculant

precipitate of manganese hydroxide.

MnSO4 +2KOH Mn(OH)2 + K2SO4

If there is any dissolved oxygen in the water , a second reaction between the manganese hydroxide and

dissolved oxygen occurs immediately to form a brownish manganic oxide.

2 Mn(OH)2+ O2 2MnO (OH)2

The addition of sulphuric acid dissolves the precipitate, the manganic sulphate immediateky reacts with

potassium iodide liberating the number of moles of iodine equivalent to the number of moles of oxygen present

the sample. During the reaction, potassium iodide is oxidized from I- to I2-. The absence of iodine imparts a

brown coloration to the water , typical of

2MnO ( OH)2 + H2SO4 2 Mn(SO)4 + 6H2O

2 Mn(SO)4 + 4KI 2 MnSO4 2K2SO4 + 2I2

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When sodium thiosulphate is added , the sodium separates from the thiosulphate ion and reacts with iodine

molecule available in water. When the iodine molecules reacts , they break up into iodine ions which are

colourless. Iodine is titrated by sodium thiosulphate solution using starch as an indicator.

MATERIALS REQUIRED

1. BOD bottle

2. Burette

3. Pipette

4. Conical flask

5. Water sample

REAGENTS:

1. Sodium thiosulphate solution-(0.025N):-Dissolve 205 grams of sodium thiosulphate in distilled water and

make up the volume to 1000ml.

2. Alkali Iodide(KOH-Ki) Solution: Dissolve 100mg of KOH and 50 grams of KI in 200ml of preboiled distilled

water.

3. Manganese Sulphate solution : Dissolve 100grams of manganese sulphate in 200ml of distilled water and

heat to dissolve.

4. Starch indicator:dissolve 1 gram of starch in 100ml of warm distilled water.

5. Concentrated Sulphuric Acid.

PROCEDURE:

i. Fill the water sample in a glass stopped BOD bottle of known volume (300ml) carefully avoiding any kind

of bubbling.

ii. Add 2ml of manganese sulphate and 2 ml of alkali iodide solution(KOH-KI) well below the surface of the

bottle through water.

iii. A precipitate will appear .Place the stopper and shake the contents well by inverting the bottle

separately. Keep the bottle for sometime to allow the precipitate to settle.

iv. Add 2ml of conc. Sulphuric acid (H2SO4) and it will dissolve the precipitate.

v. Take 50 ml of sample in a conical flask for titration. Prevent any bubbling to avoid further mixing of

oxygen.

vi. Titrate the contents against sodium thiosulphate solution taken in a burette the colour changes to pale

yellow.

vii. Add 1ml of starch solution, the solution turns to bluish or brownish and continue titration slowly adding

solution drop wise till the colour disappears.

viii. The disappearance of the colour in the solution is the end point. Stop titration and note the volume of

Sodium thiosulphate required for the titration.

ix. Calculate the amount of dissolved oxygen using the formula given below.

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The Amount of dissolved oxygen present per litre in mg= A x N x 8 x 1000

V2 V1-V

V1

Where:

A= Volume of Sodium thiosulphate

N= Normality of sodium thiosulphate=0.025N

V1= Volume of contents used for carrying out the reaction = 300 ml ( water)

V2 = Amount of substance taken for titration

V= Volume of manganese sulphate and alkali iodide solution ( KOH-KI) = 4 ml.

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TRIAL NO BURETTE READING

INITIAL FINAL

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EXPERIMENT NO:2 ESTIMATION OF FREE CARBONDIOXIDE IN WATER.

AIM: To Estimate the amount of Free Carbon dioxide in the Given Water Sample.

INTRODUCTION AND PRINCIPLE:

Carbon dioxide is an oxidative colourless gas produced during the respiration cycle of animals, plants and

bacteria. All animals and many bacteria use oxygen and release carbon dioxide . Green plants in turn

absorb carbon .Carbon dioxide quickly combine in water to form carbonic acid, a weak acid. The presence

of carbonic acid in water will be good or bad depending upon water pH and alkalinity ...If the water is

alkaline, carbonic acid will act to neutralize it. But if water is already quite acidic the carbonic acid will act

to make the things worse by making it even more acidic.

Free carbon dioxide in water rarely exceeds 20mg per liter and therefore most fishes are able to tolerate

this carbon dioxide level without bad effects. If carbon dioxide is 1mg per liter , fishes avoid this water.At

12 mg/ liter affects the survival of fresh water fishes. At 30mg/liter ,most of the sensitive fishes are killed

immediately and at about 40mg/liter eggs will not not hatch..

MATERIALS REQUIRED:

1.Burette

2.Conical flask

3.Measuring cylinder

REAGENTS:

1.Sodium hydroxide solution-0.05N:Dissolve 20 grams of sodium hydroxide in 1 ml of water.

2.Phenophthalein indicator Dissolve 0.5 wt % 100ml of methyl alcohol and 100 ml of distilled water.

PROCEDURE: I. Collect 50 ml of water sa mple. ii. Add 2-3 drops of phenolphthalein indicator to it. iii. If the colour turns pink, free carbon dioxide is absent. iv. If the sample remains colourless, titrate against 0.05N of sodium hydroxide till the permanent pale pink colour appears. v.Calculate the amount of free carbon dioxide present in water using the formula given below: The amount of free carbon dioxide present per liter in mg= T x N x 44 x 1000 Volume of the sample Where T= Titrant used and N = Normality of Sodiumhydroxide.

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EXPERIMENT NO:3: ESTIMATION OF CHLORINE

AIM: To determine the chloride ion concentration of a solution by titration with silver nitrate.

PRINCIPLE: This method determines the chloride ion concentration of a solution by titration with silver nitrate.

As the silver nitrate solution is slowly added, a precipitate of silver chloride forms: Ag+ (aq) + Cl - (aq) → AgCl(aq)

The end point of the titration occurs when all the chloride ions are precipitated. Then additional chloride ions

react with the chromate ions of the indicator, potassium chromate, to form a red-brown precipitate of silver

chromate. 2 Ag+ (aq) + CrO4 2- (aq) → AgCrO4(s) This method can be used to determine the chloride ion

concentration of water samples from many sources such as seawater, stream water, river water, and estuary

water.

MATERIALS REQUIRED: Burette Pipette Graduated cylinders 250 mL Erlenmeyer Reagents AgNO3(aq) solution- 0.10 M K2CrO4(aq) solution -0.10 M PROCEDURE :

i.Take 50 mL distilled water and

ii. Add 2 mL of chromate indicator to the Erlenmeyer flask.

iii. Titrate the sample with 0.10 M silver nitrate solution. Although the silver chloride that forms is a white

precipitate, the chromate indicator initially gives the cloudy solution a faint lemon-yellow colour. The endpoint of

the titration is identified as the first appearance of a peach colour of silver chromate.

iv. Repeat the titration with further samples of the diluted sample until concordant results (titrations agree

within 0.10 mL) are obtained.

Notes: 1. Silver nitrate solution will stain clothes and skin. Any spills should be rinsed with water immediately. 2.

Residues containing silver ions are usually saved for later recovery of silver metal. Dispose of them in the waste

container provided.

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OBSERVATION

CALCULATION: Chlorine (mg/L) = Vol. of AgNo3 solution x 1000 x 355 Vol. of Water sample used (50)

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EXPERIMENT NO:4: ESTIMATION OF TOTAL ALKALINITY OF WATER

AIM: To Determine the Alkalinity in a Given Water Sample.

INRODUCTION AND PRINCIPLE: Alkalinity is a measure of the capability of water to absorb H+ ions without significant

change of pH. In other words, alkalinity is a measure of the acid buffering capacity of water. The determination of

alkalinity of water is necessary for controlling the corrosion, to calculate the amount of lime and soda needed for

water softening; in conditioning the boiler feed water, etc.

Alkalinity of a sample of water is due to the presence of OH– (hydroxide ion), HCO3– (bicarbonate ion) and CO3

2–

(carbonate ion) or the mixture of two ions present in water. The possibility of OH– and HCO3– ions together is not

possible since they combine together to form CO32– ions.

The alkalinity due to different ions can be estimated separately by titration against standard acid solution, using

selective indicators like phenolphthalein and methyl orange.

The neutralization reaction upto phenolphthalein end point shows the completion of reactions (i) and (ii) (OH–

and CO32–) and (CO3

2– and HCO3–) only. The amount of acid used thus corresponds to complete neutralization of

OH– plus half neutralization of CO32–. The titration of water sample using methyl orange indicator marks the

completion of the reactions (i), (ii) and (iii). The amount of acid used after phenolphthalein end point

corresponds to one half of normal carbonate and all the bicarbonates. Total amount of acid used represent the

total alkalinity due to all ions present in water sample.

MATERIALS REQUIRED :Burette, pipette, conical flask, beakers, burette stand and clamp

CHEMICALS: Dry Na2CO3, concentrated 12(N) HCl, phenolphthalein and methyl orange indicator

PROCEDURE:

1.Primary standard solution of Na2CO3 (0.1N) is provided.

2. Secondary standard solution of HCl and the water sample are provided.

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3. Standarization of HCl solution by primary standard Na2CO3 solution– Pipette out 10 mL of Na2CO3 solution in a conical flask, add 2 drops of methyl orange indicator, fill up the burette with (N/10) HCl solution and titrate till the color of the solution changes from yellow to red. 4. Analysis of water sample i) Pipette 20 mL of the sample of water into a 100 mL conical flask and 2 drops of phenolphthalein indicator was added and titrated against (N/10) HCl till the color of the solution changes from pink to colorless. Corresponding burette reading indicates the phenolphthalein end point (V1). ii) Again pipette out 20 mL of the water sample in a conical flask, add 2 drops of methyl orange indicator. Color of the solution becomes yellow. Continue the titration against the (N/10) HCl solution till the color changes to red. This burette reading corresponds to the methyl orange end point (V2).

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DISCUSSIONS: i) Phenolphthalein alkalinity (P) = 0; that means the volume of acid used till the completion of reaction (i) and (ii) is 0. This can only happen when both OH– and CO32– ions are not present in water. Alkalinity is present due to HCO3– ion only which can be determined using methyl orange indicator and called methyl orange alkalinity (M).

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ii) P = ½ M; indicates that only CO32– ions are present. Using phenolphthalein indicator neutralization reaches upto HCO3– but using methyl orange indicator the complete neutralization of HCO3

– takes place. iii) P > ½M; implies OH– ions are also present along with CO3

2– ions. Upto phenolphthalein alkalinity OH– ions will be neutralized completely where as CO32– will be neutralized upto HCO3– ion. But using methyl orange indicator HCO3

– will be completely neutralized along with OH– and CO32–.

iv) P < ½ M; indicates that beside CO32– ions HCO3

– ions are also present. The volume of acid required for the neutralization upto phenolphthalein end point correspond half neutralization of CO3

2– (equation ii). Neutralization using methyl orange indicator corresponds to HCO3

– obtained from CO32– and HCO3

– originally present in the water sample. v) P = M; indicates only OH– ions are present. Precautions: i) All the glass apparatus should be washed thoroughly with distilled water before use ii) The burette and pipette should be rinsed with solution to be taken in it. iii) There should not be any leakage in the burette. iv) The conical flask should be placed on white paper or board to identify the colour change at the end point.

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EXPERIMENT NO: 5 : STANDARD QUALITATIVE MICROBIAL ANALYSIS OF WATER. Part A: Presumptive Test: Determinatin of the Most Probable Number of Coliform Bacteria. AIM:i.To determine the presence of coliform bacteria in a water sample,ii. To obtain some index as to the possible number of organisms present in the sample under analysis. PRINCIPLE: The presumptive test is specific for detection of coliform bacteria. Measured aliquots of the water to be tested are added to a lactose fermentative broth containing an inverted gas vial ( Durham tube). Because these bacteria are capable of using lactose as carbon source, their detection is facilitated by use of this medium. In this experiment the lactose fermentation broth also contains a surface tension depressant, a bile salt, which is used to suppress the growth of organisms other than coliform bacteria. Tubes of this lactose medium are inoculated with 10 ml , 1 ml and 0.1 ml aliquots of the water sample. The series consists of at least three groups, each composed of five tubes of specified medium. The tubes in each group are then inoculated with the designated volume of the water sample as desirable under “procedure”. The greater numbers of tubes per group, the greater the sensitivity of the test. Development of gas in any of the tubes is presumptive evidence of the presence of coliform bacteria in the sample. The presumptive test also enables the microbiologist to obtain some idea of the numbers of the coliform bacteria present by means of the most probable number test (MPN). The MPN is estimated by determining the number of tubes in each group that show gas following the incubation period. MATERIALS REQUIRED: Culture Water sample to be tested. Media: Lauryl Tryptose broth-Double strength and single strength. Equipment: Bunsen burner, test tubes, test tube racks, sterile 10 ml, 1 ml and 0.1 ml pipettes, labels. PROCEDURE: I. Set up three series consisting of three groups , a total of 15 tubes per series, in a test tube rack, for each tube label the water source and volume of sample to be inoculated. ii. Flame bottle and then, using a 10ml pipette , transfer 10 ml aliquots of water sample to the five tubes labeled LB2X-10 ml. iii. Flame bottle and then, using a 1ml pipette , transfer 1 ml aliquots of water sample to the five tubes labeled LB1X-1 ml. iv. Flame bottle and then, using a 0.1ml pipette , transfer 0.1 ml aliquots of water sample to the five tubes labeled LB1X-0.1 ml. v. Incubate all tubes for 48 hours at 37ᴏ C. PART-B-CONFIRMED TEST. AIM: To confirm the presence of coliform bacteria in a water sample for which the presumptive test was positive. PRINCIPLE: The presence of a positive or doubtful presumptive test immediately suggests that the water sample is non potable. Confirmation of these results is necessary , since positive presumptive tests may be the result of organisms of non coliformorigin that are not recognized as indicators of faecal pollution. The confirmed test requires that selective and differential media such as Eosine Methylene Blue (EMB) or Endo agar be streaked from a positive lactose broth tube obtained from the presumptive test. Eosine Methylene Blue contains the dye methylene blue , which inhibits the growth of gram-positive organisms. In the presence of an acid environment ,EMB forms a complex that precipitates out into the coliform colonies,

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producing dark centres and a green metallic sheen. This reaction is characteristic for Escherichia coli, the major indicator of faecal pollution. Endo agar is a nutrient medium containing the dye fuchsion, which is present in the decolorized state. In the presence of acid produced by the coliform bacteria, fuchsion forms a dark pink complex that turns the E.coli colonies and the surrounding medium pink. MATERIALS REQUIRED: Cultures One 24 hour –old positive lactose broth culture from each of the three series from the presumptive test. MEDIA: Eosine Methylene Blue agar plates or Endo agar plates. EQUIPMENT: Bunsen burner, label and inoculation loop. PROCEDURE: I. Label the covers of the three EMB plates or Endo agar plates with source of water sample. ii. Using a positive 24 hour lactose broth cultures from the presumptive test ,streak the surface of one EMB agar or one Endo agar plate to obtain discrete colonies. iii. Incubate all plate cultures in an inverted position for 24 hours at 37ᴏ C. PART-C-COMPLETE TEST AIM: To confirm the presence of coliform bacteria in a water or if necessary, to confirm a suspicious but doubtful result of the previous test. PRINCIPLE: The complete test is the final analysis of the water sample. It is used to examine the coliform colonies that appeared on the EMB or Endo agar plates used in the confirmed test..An isolated colony is picked from the confirmatory test plate and inoculated into a tube of lactose broth and streaked on a nutrient agar slant to perform a Gram stain. Following inoculation and incubation, tubes showing acid and gas in the lactose broth and presence of gram-negative bacilli on microscopic examination are further confirmation of the presence of E.coli and they are indicative of a positive completed test. MATERIALS REQUIRED: Cultures One 24 hours coliform positive EMB or Endo agar culture from each of the three series of confirmed test. MEDIA: Nutrient agar slants and Lactose fermentation broths. REAGENTS: Crystal Violet, Grams iodine, decolorizer, Safranine. EQUIPMENT: Bunsen burner, staining tray, inoculation loop, blotting paper, microscope. PROCEDURE: I.Label each tube with the source of water sample. ii. Inoculate one broth and one nutrient agar slant from the same isolated E.coli colony obtained from an EMB agar or Endo agar plate. iii. Incubate all tubes for 24 hours at 37ᴏ C.

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OBSERVATION AND RESULT: PART-A PRESUMTIVE TEST-MPN TEST

MPN / 100 ml REFER MPN CHART.

PART-B-CONFIRMED TEST COLONIES ON EMB PLATE FROM PRESUMTIVE POSITIVE TEST

PART-C-COMPLETE TEST-GROWTH ON NUTRIENT AGAR SLANT AND GAS IN LACTOSE BROTH.

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VOLUME OF SAMPLE 1 2 3 4 5

A/G A/G A/G A/G A/G

1Oml 2X LTB

1ml 1XLTB

0.1ml 1X LTB

VOLUME OF SAMPLE 1 2 3 4 5

E.COLI COLONIES

1Oml 2X LTB

1ml 1XLTB

0.1ml 1X LTB

VOLUME OF SAMPLE 1 2 3 4 5

GROWTH/ GAS

1Oml 2X LTB

1ml 1XLTB

0.1ml 1X LTB

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EXPERIMENT NO:6 : MICROSCOPIC OBSERVATION OF ROOT COLONIZATION BY VAM FUNGI.

AIM: To study and observe the root colonization of VAM by staining root crush Method.

INTRODUCTION: Plant roots get associated with various types of fungi establishing an association between roots

and fungi which is known as mycorrhiza. Depending on the nature of mycorrhiza association, different types of

mycorrhiza are identified. One of the types of mycorrhizal association is called vesicular Arbuscular Mycorrhiza in

which fungi penetrate into root cells and form fine branched structures called arbuscles and thin small sac like

structures known as vesicles.The cleaned root fragments when crushed on slides ,stained and observed under

microscope shows the presence of thin hypahe and vesicles of fungi within root cells.

MATERIALS REQUIRED:

Root tips,FAA ( 5ml formaline,5 ml acetic acid38-40% and 90 ml ethano l70%),KOH,HCl,TRyphan blue,

Lactophenol ( 250ml lacticacid ,300 grams Phenol,250ml Glycerine and 30 ml water for several hours), Glycerine,

Slide andMicroscope.

PROCEDURE: i.Collect roots, cut into small pieces ( 5-10cm long), wash thoroughly and fix in FAA.

ii.After removing fixative by washing with water, immerse roots in 10% KOH ,heat at 90 ᴏC for 1-2 hours. The time and temperature depends upon the thickness of the roots .Delicate roots need less time and temperature. iii. If the roots are darker in color , immerse them in a alkaline H2O2 solution ( 3 ml ammonium hydroxide , 30 ml 10 % H2O2 and 567 ml water ). iv. Wash roots with water 3-4 times to remove the traces of H2O2 and then treat with 1% HCl for a period of 3 minutes.

v.Pour off acid and add 0.05% Tryphan blue in lactophenol. vi.Boil roots in stain for 3 minutes. Vii .Pour off stain, add lactophenol and leave for a night destain the tissue.

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EXPERIMENT NO: 7: PRODUCTION OF AMMONIA FROM ORGANIC COMPOUNDS (AMMONIFICATION).

AIM: To Demonstrate the Production of Ammonia from Organic compounds.

INTRODUCTION:

The nitrogen in most plants and animals exist in the form of organic molecule mostly proteins derived from the

decomposition of dead plant and animal tissue. During nitrogen cycling, plant and animal proteins are broken

down to amino acids, which deaminate and release ammonia, This is a step wise process when an organisms dies

, its proteins are attacked by proteases of soil bacteria which produce peptides and amino acids .This process is

called as peptonization, then the amino groups on amino acids are removed by the process called deamination

producing ammonia ( NH3).

In most soils , the ammonia dissolves in water to form ions (NH4).

The process of production of ammonia from organic compounds is called ammonification. In addition to the

ammonification of amino acids, other groups such as nucleic acids, urea, uric acid go through the ammonification

process. The bacteria that accomplish it (Bacillus, Clostridia, Proteus, Psedomonas, Streptomyces) are called

ammonifying bacteria. Ammonification of organic compounds is a very important step in which the cycling of

nitrogen in soil .Since most autotrophs are unable to assimilate amino acids, nucleic acids, urea,uric acidsand use

them for their own enzyme and protoplasm construction.

To demonstrate the existence of this process, the peptone broth is inoculated with a sample of soil ,incubated

for a week for ammonium production. After seven days of incubation, it will be tested for ammonium

production.

MATERIALS REQUIRED:

Peptone broth Cultures of bacteria Soil Spatula Flask Test tubes Nesseler’s Reagent: Potassium iodide-50g, Mercury Chloride (saturated) 35 ml, Distilled water (Ammonia free) 25 ml, Potassim hydroxide ( 50%) 400ml. PROCEDURE:

i. Prepare a peptone broth (4%) that contains an organic nitrogen substrate.

ii. In broth for 24 hours and add 0.5 g of soil.

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iii. Incubate further for 7 days at 37ᴏC .

iv. Add Nesseler’s reagent to the tubes.

OBSERVATION AND RESULT: The development of yellow to brown precipitate after adding Nesseler’s reagent

indicates formation of ammonia by bacteria through decomposition of proteins present in the broth.

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EXPERIMENT NO:8: THE STUDY OF WINOGRADSKY COLUMN.

AIM: To Demonstrate Succession of Microbial Populations and Changes in the Environment brought about by

Certain Microbial Groups.

INTRODUCTION:

The Winogradsky column is a simple device for culturing a large diversity of microorganisms. Invented in the

1880s by Sergei Winogradsky, the device is a column of pond mud and water mixed with a carbon source such as

newspaper (containing cellulose), blackened marshmallows or egg-shells (containing calcium carbonate), and a

sulfur source such as gypsum (calcium sulfate) or egg yolk. Incubating the column in sunlight for months results

in an aerobic/anaerobicgradient as well as a

sulfide gradient. These two gradients

promote the growth of different

microorganisms such

as Clostridium, Desulfovibrio,Chlorobium, C

hromatium, Rhodomicrobium,

and Beggiatoa, as well as many other

species of bacteria, cyanobacteria, and

algae.

The column provides numerous gradients,

depending on additive nutrients, from

which the variety of aforementioned

organisms can grow. The aerobic water

phase and anaerobic mud or soil phase are

one such distinction. Because of oxygen's low solubility in water, the water quickly becomes anoxic towards the

interface of the mud and water. Anaerobic phototrophs are still present to a large extent in the mud phase, and

there is still capacity for biofilm creation and colony expansion, as shown in the images at right. Algae and other

aerobic phototrophs are present along the surface and water of the upper half of the columns. Green growth is

often attributed to these organisms.

MATERIALSREQUIRED: Samples collected from various sources ( fresh water ponds, salt marsh, compost pile,lake , river, field soil, beach sand). Amendments ( news paper bits, glucose ,leaves)

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Glass column or Cylinder Calcium sulphate or Sodium sulphate Calcium carbonate Distilled water Analytical balance Beakers, jars. PROCEDURE: i. Label column with name and date. ii. Choose materials to use to pack the column and prepare them as a slurry having the consistency of a milkshake .Materials may also be added as layers. iii. Weigh approximately 0.5 grams of Calcium sulphate or sodium sulphate per 100 gram of sediment to be added to column and mix with sediment. iv. Weigh approximately 0.25 grams of Calcium carbonate per 100 gram of sediment to be added to column and mix with sediment. v. Remove twigs and other large debris from the slurry as they float to the surface. vi. Add any other chosen amendments to the sediment mixture. vii. Incubate the column where it will receive day light or artificial light . The temperature may be chosen accordingly. viii. Observe the column over the next severalweeks for development of layers, small, colours and zones etc. OBSERVATION: The column contains at least two steep gradients. The water column at the surface is in contact with atmosphere and is therefore aerobic but it becomes increasingly anaerobic with depth. The surface layers of column may produce an aerobic liquid air biofilm ( pellicle) that can be sampled by dipping a cover slip into the column and lifting a portion of the film from water. In the highly anaerobic base of the column, decomposition and the activity of sulphate reducing bacteria results in the production of hydrogen sulphide gas. The hydrogen sulphide gradient decreases towards the top of the column. These two gradients acting in opposite directions create a great range of habitats selective for a variety of microorganisms. The column is illuminated by sunlight or by artificial light. In aperfectly uniform column one might expect a variety of phototrophs to be selected in specific zones with in the column, but such uniformity is rarely obtained. Typically the lower portion of column are colonized by phototrophic green and purple sulfur bacteria.The aerobic surface of the column is occupied by oxygenic cyanobacteria. Just below the surface, phototrophic purple non-sulfur bacteria predominate. A great variety of heterotrophs also can be found in these columns including obligate anaerobes such as Clostridia and methonogenic bacteria. The majority of the bacteria are located in a thin film between the solid mud substrate and the container wall. When using plastic containers such as two liter pop bottles , these bacteria can be sampled by inserting a needle through the container wall. *********************************

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EXPERIMENT NO: 9: STUDY OF MICROBIAL INTERACTION.

AIM: To Study the Microial Antagonisms activity Between microbes such as Fungi versus Bacteria, Fungi versus

,Bacteria versus Bacteria.

INTRODUCTION: Environment comprises of a wide variety of microbial populations. Most of them live under

certain relationships among each other. The relationship between microbes could be of different types such as

commensalism, synergism, antagonisms.

Antagonism is a phenomena is in which growth and activity of an organism is inhibited by the growth and activity

of another organism. Antagonism effect might be due to competing for nutrients or depleting nutrients required

for growth of certain organisms..Certain microbes might produce toxic substances which creates an environment

in which the other organisms cannot grow. Microbial antagonism in the laboratory is tested by dual culture

method on various media using plate method.

MATERIALS REQUIRED:

Antagonistic between Bacteria and fungi: specific culture media ( Saboroud’s agar ,n Nutrient agar ,Rose Bengal agar media). Scalpel ,Inoculation loop Straight wire. PROCEDURE: I. Prepare plates with specific media depending on growth requirement –Saboraud’s agar, Nutrient agar, media and Rose Bengal agar for Fungi versus Bacteria i, Bacteria versus Bacteria and Fungi versus fungi respectively. ii. For fungi versus fungi, Trichoderma culture is swabbed on the surface of Rose Bengal agar medium and the culture of Aspergillus is inoculated in the centre of media. iii. For fungi versus bacteria, the culture of Penicillin is inoculated in the centre of Saboraud’s agar media. A fresh culture of Staphylococcus is spread over the rest of the plate. iv. For bacteria versus bacteria , the culture of Staphylococcus is spread over the entire plate and the culture of Bacillus is streaked in the centre of the plate. v. The plates are incubated at the appropriate temperatures-Fungi Vs Fungi plate at 25ᴏ C for 3 to 4 days, Fungi - Vs Bacteria plate at 25ᴏ C for 2 to 3 days and bacteria Vs Bacteria plate at 37 ᴏ C for 24 hours. vi. The observation are made after the incubation period.

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OBSERVATION AND RESULT:

INTERACTING ORGANISMS GROWTH (+) INHIBITION (-) OBSERVATION

ASPERGILLUS Vs TRICHODERMA-FUNGI Vs FUNGI

+ -

BACILLUS Vs STAPHYLOCOCCUS BACTERIA Vs BACTERIA

+ -

PENCILLIUM Vs STAPHYLOCOCCUS FUNGI Vs BACTERIA

+ -

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EXPERIMENT NO :7: ISOLATION OF FUNGI FROM AIR.

AIM: To Isolate fungal spores from Air by Gravity Settle Plate Method.

INTRODUCTION: Settle plate sampling is a direct method of assessing the likely number of microorganisms

depositing on the product or surface in a given time. It is based on the factthat is absence of any kind of influence

airborne microorganisms typically attached to the large particles that will deposit onto open culture plates. The

average sized microbial particles will deposit by gravity on the surfaces at a rate of approximately 1cm/s. In

settle plate sampling, petriplates are opened and exposed for a given period of time . This allows spores to

deposit onto them. Petri plates which are 90cm in diameter are most commonly used. Spores that are

deposited on surface of culture medium in the petri plates are allowed form colonies on incubation. Later, tease

mount of fungal culture and identification of the fungi are done under microscope.

MATERIALS REQUIRED: Rose Bengal culture plates, Glass slides and cover slips Needle Lactophenol Cotton blue. Microscope. Blotting paper. PROCEDURE: I. Prepare required number of Rose Bengal agar culture plates. ii. Label the plates with date , place and duration of exposure. iii. Keep the culture plated exposed to air for 10 minutes by holding at the height of about 1 meter from the surface. iv. Place the lid on the exposed culture plates and keep for incubation at room temperature for 3-4 days. v. Prepare a tease mount of the fungal colonies using a lacto phenol cotton blue stain. vi. Observe the tease mounted slides under low power and dry objectives of microscope. v. Identify the t referring types of spores of fungi by referring to illustrated manual. vi Record the microscopic and macroscopic characters of the isolated fungi from air. OBSERVATION:

SL.NO FUNGI MACROSCOPIC APPERENCE MICROSCOPIC APPEARENCE

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FUNGUS MACROSCOPIC APPEARENCE

MICROSCOPIC APPEARENCE

FUNGUS MACROSCOPIC APPEARENCE

MICROSCOPIC APPEARENCE

MUCOR

CLADOSPORIUM

RHIZOPUS

HELMINTHO SPORIUM

ASPERGILLUS FLAVUS

TRICHODERMA

ASPERGILLUS NIGER

YEAST

ASPERGILLUS FUMIGATUS

FUSARIUM

PENICILLIUM

ALTERNARIA

CURVULARIA

SYNCEPHALASTRUM

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EXPERIMENT NO: 11: STUDY OF AIR SAMPLER AIM: To Understand the Construction and working of Anderson Sampler, Rotorod and Burkard Trap. INTRODUCTION: Various types of air samplers are available for trapping and study of suspended biopollutants of fungi. The most common among them are Anderson Sampler, Rotorod and Burkard Trap. These are convenient and offers ease of use work under different circumstances. Each one of them has its own advantages and disadvantages. i. ANDERSON TRAP: This is most commonly used air sampler in which air is drawn through a circular orifice and then through a

succession of six circular plates, each perforated with hundred holes. The air passes through the perforation and the [article get impacted on to sterile medium on petri dish .The succession of the plates has progressively smaller holes so that the largest particles are impacted onto the first dish and the smallest on the sixth. Thus, the Anderson sampler is a device for size grading on solid culture media. The observation are made after incubation of culture plates from the sampler. ii. ROTOROD: This instrument consists of a pair of thin rods of square cross section which are

rotated at a constant speed by battery operated motor. The outer edge of each rod carries a strip of cello tape smeared with glycerin jelly. After exposure the strip is removed ,cut into pieces, mounted on amicroscope slide and observed. The Rotorod is highly useful as a portable spore trap. Iii .BURKARD TRAP:

This trap works on the principle of suction. It has a built in vacuum pump which draws 10 liters of air per minute, through an orifice of 14 x 2 mm. The orifice is protected from rain by a horizontal sheet . The trap is also provided with a wind vane which directs orifice towards the direction of wind. The particles drawn in along with air are impacted on an adhesive coated transparent cello tape mounted on a clock driven drum. The drum completes one rotation in 7 days . the tape is changed once in a 7 days regularly .the drum moves at 2 mm / hour . Hence, tape is

divided into 7 parts to represent one day catch of the air borne particles. The tape is mounted on a slide with glycerin jelly and scanned microscopically for pollen and fungal spores.

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EXPERIMENT NO:11 DEMONSTRATION OF MICROFLORA OF SKIN, MOUTH AND NOSE

AIM: To Isolate and Identify the Common Microflora from Human Skin, Mouth and Nose. INTRODUCTION: Normal microflora is regularly found in specific areas of the body. The microflora on the human body depends on environmental factors such as pH,oxygen concentration, amount of moisture present and types of secretions associated with each anatomical site. Native microflora are broadly located as follows: On Skin: Staphylococcus, Streptococcus,Diptheroid bacilli and Yeast. In Mouth: Anaerobic spirochetes and Vibrios, Fusiform bacteria, Staphylococci, are wide spread in nature, although they are mainly found on the skin, skin glands and mucus membranes of mammals and birds .The coagulase positive species Staphylococcus aureus , is well documented as an human opportunistic pathogen. Stap hylococci have the unique ability to clot plasma continues to be the most widely used and accepted criteria for identification of pathogenic Staphylococci associated with acute infections. Isolation of coagulase positive Staphylococci on phenol red mannitol agar supplemented with7.5% Nacl was studied by Chapman. The resulting mannitol salt agar is recommended for the isolation of coagulase positive Staphylococci from cosmetics, milk and other specimens. Staphylococcus aureus ferments mannitol and produces yellow colored colonies surrounded by yellow zones. Coagulase negative strains of Staphylococcus aureus are usually mannitol non fermenter and therefore produce pink to red colonies surrounded by red purple zones. Presumptive coagulase positive yellow colonies of Staphylococcus aureus should be confirmed by performing the coagulase test. MATERILS REQUIRED: Culture media-Mannitol Salt agar, Nutrient agar ,Ctton swabs, Inoculation loop. PROCEDURE: i. Divide the culture plate into 2 parts using a glass marking pencil. ii. Moisten the swabs with steril water. iii. Rub on the surface of the skin from where the microflora has to be isolated. iv. Smear the swab on surface of culture media in one part. v. Similarly, rub a fresh swab in the inner cheek and smear that swab over the surface of culture media . vi. Label the plates and incubate at 37ᴏ C for 18 to 24 hours. vii. Observe the plate for growth of typical colonies of bacteria and record the observations.

LOCATION MACROSCOPIC APPEARENCE MICROSCOPIC APPEARENCE

SKIN

NOSE

MOUTH

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