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

LABORATORY MANUAL SEMESTER-V

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LABORATORY MANUAL FOR 5TH SEMESTER MICROBIOLOGY PRACTICAL –PAPER-G509.5P

SL NO EXPERIMENT PAGE NO 1 Demonstration of Anabeana Symbiotic Association in Azolla 2

2 Demonstration of Rhizobium (bacteria) from Root Nodules 4

3 Radial Immuno Diffusion 6

4 Sandwich Dot ELISA Test for Antigen- (Use of Teaching Kit) 8

5 Antibiotic Sensitivity Test-Kirby Bauer Method 13

6 Starch Hydrolysis Test 16

7 Nitrate Reduction Test 18

8 Qualitative Salivary Amylase Essay 21

9 Isolation of Plant Fungal Pathogens from Soil 24

10 Isolation and Observation of Bacteria from Mouth and Skin 28

11 Study Plant disease-i) Tobacco Mosaic Disease ii) Koleroga of Arecanut, iii)Blight of Rice and iv) Tikka Disease of Groundnut

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12 Agarose Gel Electrphoresis of Human Serum Proteins. 37

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EXPERIMENT NO: 1 DEMONSTRATION OF ANABEANA SYMBIOTIC ASSOCIATION IN AZOLLA.

AIM: To demonstrate the presence of symbiotic Anabaena in Azolla Plant.

Introduction: Azollaceae are small heterosporic free-floating aquatic pteridophytes with tropical and temperate

worldwide distribution. The deeply bilobed leaves (dorsal and ventral lobes) cover the entire rhizome, and the

cavities of the dorsal lobe harbour a colony of a filamentous heterocystous cyanobacterium Anabaena azollae and

several genera of bacteria. The Azolla species can be used as biofertiliser in rice culture, animal feed and

wastewater phytoremediation.

a) MATERIALS REQUIRED:

i. Azolla ( whole plant)

ii. Slides and cover slips

iii. Fast green

iv. Microscope

b) Procedure:

i. Take a drop of fast green on a clean slide.

ii. Place a leaf of Azolla in the drop of the stain.

iii. Place a cover slip over the leaf.

iv. Crush the leaf gently from back of pencil or needle.

v. Blot the excess stain.

vi. Mount the slide on stage of microscope and observe under lower (10X) power and high power dry

(95X) objective.

vii. A short filament of an Anabaena appears as a bright green fragment within the crushed leaf cells.

viii. Note the morphology and heterocyst within the filament of Anabaena.

ix. Record the observation.

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c) Observation and Result: Small fine filaments o Anabaena are observed in crushed leaf with

intercalary heterocyst.

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EXPERIMENT NO: 2 DEMONSTRATION OF RHIZOBIUM (BACTERIA) FROM ROOT NODULES.

AIM: To demonstrate the presence of Rhizobium in root nodules of Leguminous plants.

Introduction: Root nodule symbiosis enables nitrogen‐fixing bacteria to convert atmospheric nitrogen into a form that is directly available for plant growth. Biological nitrogen fixation provides a built‐in supply of nitrogen fertiliser for many legume crops such as peas, beans and clover. Legumes (Fabales) interact with single‐celled Gram‐negative bacteria, collectively termed rhizobia. Nitrogen fixation normally takes place within specialized bacteroid cells enclosed within organelle‐like cytoplasmic compartments termed symbiosomes.

a) MATERIALS REQUIRED:

i. Root nodules

ii. Nylon mesh

iii. Test tubes

iv. Glass rod

v. Glass marking pencil and Inoculation loop

vi. Slide

vii. Stains -Crystal violet, Safranine and Reagents Grams Iodine ,Acetone alcohol

viii. Blotting Paper

b) Procedure:

i. Procure healthy root nodules of a young leguminous plant by loosening the soil around roots and

uprooting the plant with intact roots.

ii. Wash the nodules thoroughly first with tap water and then with sterile distilled water by keeping over

a nylon mesh under aseptic condition to remove contaminants and adhering soil particles.

iii. There after immerse root nodules in 0.1% acidified HgCl2 for 5 minutes.

iv. Transfer root nodules to a sterile beaker containing 10 ml of 95% ethanol and wait for 2-3 minutes

v. Wash nodules thoroughly for 5 minutes with a sterile water.

vi. Transfer one or two nodules to a clean test tube containing 1ml of distilled water.

vii. Crush the nodule with a sterile glass rod to release the bacteriods in to suspension.

viii. Prepare a wet mount and a Gram smear from the suspension.

ix. Observe the wet mount under high power dry objective and Gram smear under oil immersion

objective.

x. Record the observation made from wet mount and Gram smear.

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a) Observation and Result:

i) In wet mount preparation, bacteroids could be seen as a small organisms suspended in a suspension.

ii) In Gram smear, pleomorphic gram negative bacteriods could be seen.

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EXPERIMENT NO:3 RADIAL IMMUNO DIFFUSION

AIM: To learn the techniques of Radial immunodiffusion.

Introduction: Single radial immunodiffusion (RID) is used extensively for the quantitative estimation of

antigens. The antigen-antibody precipitation is made more sensitive by the incorporation of antiserum in

the agarose. Antigen (Ag) is then allowed to diffuse from wells cut in the gel in which the antiserum is

uniformly distributed. Initially, as the antigen diffuses out of the well, its concentration is relatively high

and soluble antigen-antibody adducts are formed. However, as Ag diffuses farther from the well, the Ag-

Ab complex reacts with more amount of antibody resulting in a lattice that precipitates to form a

precipitin ring. Thus, by running a range of known antigen concentrations on the gel and by measuring the

diameters of their precipitin rings, a calibration graph is plotted. Antigen concentrations of unknown

samples, run on the same gel can be found by measuring the diameter of precipitin rings and

extrapolating this value on the calibration graph.

a) MATERIALS REQUIRED:

i. Agarose

ii. 10x assay buffer

iii. Standard Antigens (A,B,C &D)

iv. Test Antigen(1 &2)

v. Antiserum

vi. Gel punch with syringe

vii. Glass plate

viii. Template

b) PROCEDURE:

i. 10ml of 1.0% Agarose (0.1g/10ml) in 1x Assay buffer was prepared and heated slowly till Agarose

dissolves completely. Take care not to scorch or froth the solution.

ii. Molten Agarose was allowed to cool to 55°C.

iii. 120 µl of antiserum to 6ml of Agarose solution was added. Mix by gentle swirling for a uniform

distribution of antibody.

iv. Agarose solution containing the Antiserum was poured onto a grease free glass plate and set on a

horizontal surface. Leave it undisturbed to form a gel.

v. Cut wells using a Gel Puncher using the template provided.

vi. 20 µl of the given Standard Antigens and Test Antigens were added to the wells.

vii. The gel plate were kept in a moist chamber (box containing wet cotton) and incubate overnight at room

temperature.

viii. Mark the edges of the circle and measure the diameter of the ring.

ix. Plot a graph of diameter of ring (on Y-axis) versus concentration of antigen (on X-axis) on a semi-log

graph sheet.

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x. 10.Determine the concentration of unknown by reading the concentration against the ring diameter

from the graph.

c) RESULT: The Concentration of antigen of the test samples were found to be…………………

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EXPERIMENT NO:4 SANDWICH DOT ELISA TEST FOR ANTIGEN- (USE OF TEACHING KIT)

AIM: Objective: To perform sandwich Dot ELISA test for antigen.

INTRODUCTION AND PRINCIPLE: Dot-ELISA (Enzyme Linked Immunosorbent Assay) is an extensively used

immunological tool in research as well as analytical/diagnostic laboratories. In sandwich Dot-ELISA, the

antigen is sandwiched directly between two antibodies which react with two different epitopes on the

same antigen. Here one of the antibodies is immobilized onto a solid support and the second antibody is

linked to an enzyme. Antigen in the test sample first reacts with the immobilized antibody and then with

the second enzyme-linked antibody. The amount of enzyme linked antibody bound is assayed by

incubating the strip with an appropriate chromogenic substrate, which is converted to a coloured,

insoluble product. The latter precipitates onto the strip in the area of enzyme activity, hence the name

Dot-ELISA. The enzyme activity is indicated by intensity of the spot, which is directly proportional to the

antigen concentration.

Kit Description:

In this kit, ELISA strips are supplied having three well defined zones:

• Negative control zone that is blocked with an inert protein.

• Test zone having an antibody immobilized on it and then blocked with an inert protein.

• Positive control zone having the antibody immobilized on it, blocked with inert protein and has a

specific antigen bound to the immobilized antibody.

These strips will be used to detect the antigen in the test serum samples supplied, by using a secondary

antibody conjugated to Horse radish perxoidase (HRP). HRP is then detected using hydrogen peroxide as a

substrate and Tetramethylbenzidine (TMB) as a chromogen. HRP acts on hydrogen peroxide to release

oxygen, which oxidizes the TMB to TMB oxide. The TMB oxide is deposited wherever enzyme is present

and appears as a blue spot.

If the test sample does not contain the antigen specific to the antibody, there will be no enzyme reaction

and no spot develops.

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The schematic representation of the reaction is given below:

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a) MATERIALS REQUIRED: Glassware: Test tubes or 1.5 ml vials.

Reagent : Distilled water.

Other Requirements : Micropipette, Tips

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b) Procedure:

ii. 1. Take 2 ml of 1X Assay Buffer in a test tube and add 2 µl of the test serum sample. Mix thoroughly by

pipetting. Insert a Dot-ELISA strip into the tube.

iii. 2. Incubate the tube at room temperature for 20 minutes. Discard the solution.

iv. 3. Wash the strip two times by dipping it in 2 ml of 1X Assay Buffer for about 5 minutes each. Replace

the buffer each time.

v. 4. Take 2 ml of 1X Assay Buffer in a fresh test tube, add 2 µl of HRP conjugated antibody to it. Mix

thoroughly by pipetting. Dip the ELISA strip into it and allow the reaction to take place for 20 minutes.

vi. 5. Wash the strip as in step # 3 for two times.

vii. 6. In a collection tube (provided in the kit) take 1.3 ml of TMB/H2O2 and dip the ELISA strip into this

substrate solution.

viii. 7. Observe the strip after 5 - 10 minutes for the appearance of a blue spot.

ix. 8. Rinse the strip with distilled water.

Observation: Record your observations as follows:

c) Interpretation:

• Spot in positive control zone and no spot in the negative control zone indicates proper performance of

test.

• Spot in test zone indicates presence of specific antigen in the sample. Note: Intensity of the spot will

vary depending upon the test sample used.

• No spot in the test zone indicates the absence of specific antigen in the sample.

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EXPERIMENT NO: 5. ANTIBIOTIC SENSITIVITY TEST-KIRBY BAUER METHOD

Aim: To determine bacterial sensitivity to a given antibiotic by paper disc diffusion method.

INTRODUCTION Certain bacteria can display resistance to one or more antibiotics. Determining bacterial

antibiotic resistance – whether a bacterium can survive in the presence of an antibiotic - is a critically

important part of the management of infectious diseases in patients. The Kirby-Bauer (K-B) disk diffusion

test is the most common method for antibiotic resistance/susceptibility testing. The results of such

testing help physicians in choosing which antibiotics to use when treating a sick patient. The Kirby-Bauer

(K-B) test utilizes small filter disks impregnated with a known concentration of antibiotic. The disks are

placed on a Mueller-Hinton agar plate that is inoculated with the test microorganism. Upon incubation,

antibiotic diffuses from the disk into the surrounding agar. If susceptible to the antibiotic, the test

organism will be unable to grow in the area immediately surrounding the disk, displaying a zone of

inhibition (see figure below). The size of this zone is dependent on a number of factors, including the

sensitivity of the microbe to the antibiotic, the rate of diffusion of the antibiotic through the agar, and the

depth of the agar. Microorganisms that are resistant to an antibiotic will not show a zone of inhibition

(growing right up to the disk itself) or display a relatively small zone.

a) MATERIALS REQUIRED:

i. Test tube rack

ii. Forceps

iii. Sterile swabs

iv. Two Mueller-Hinton agar plates

v. Antibiotic disks-(i) TWO different antibiotics (ii) Antibiotic-free disks (BLANK)

vi. Stock broth cultures of: - Escherichia coli (Gram negative) - Staphylococcus epidermidis (Gram

positive).

b) Procedure:

i.Label the agar plates with the usual 5 items. Also mark, using dots, where you will put the antibiotic

disks and the BLANK (antibiotic-free) disk.

a. Disks should be a minimum of 20 mm apart.

b. Disks should not be placed near the edge of the plate

ii.Inoculate one plate with your first bacterium as follows:

a. Using aseptic technique, wet a swab with the bacterial broth culture.

b. Thoroughly swab the surface of the plate, making sure to cover the entire surface.

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c. Do NOT get more culture.

d. Turn the plate approximately 60 degrees and repeat the

previous step (2nd swabbing).

e. Do NOT get more culture.

f. Repeat the previous step (3rd swabbing).

iii. Discard the swab in a bleach-containing beaker.

iv. Place one antibiotic disk onto the surface of the agar, using

aseptic technique as follows:

a. flame the tips of the forceps by passing over the flame for

5-10 seconds.

b. Cool the forceps by waving them in the air for about 10

seconds.

c. Carefully pick up your test disk with the forceps, and gently place it in the appropriate spot on the agar

surface,

d. To ensure that the disk is flat on the agar, gently push it down with the forceps.

e. Reheat the tips of the forceps as above to kill any bacteria.

v. Repeat the procedure with the second antibiotic disk.

vi. Repeat the procedure with the BLANK disk.

vii. Repeat steps 1 – 6 on a new agar plate with your second bacterium.

viii. Wait until the surface of the plates has completely dried (it may help to leave the lid slightly open for

3 - 5 minutes).

i. Incubate both plates at 37 C. for 18-24 hours.

c) Observation and Result:

1. Observe both agar plates.

2. If present, measure the diameter of the zone of inhibition in mm (see the figures below), and record your results

in the Table .

3. If there is no zone present, record your result as 0 mm.

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d. Results Table – Sizes of zones of inhibition (in mm)

Antibiotic & Concentration

→ Blank Disc

E.coli

S.epidermidis

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EXPERIMENT NO:6. STARCH HYDROLYSIS TEST

Aim: To determine if the organism is capable of breaking down starch into maltose through the activity of the

extra-cellular α-amylase enzyme.

Introduction: Starch is a polysaccharide made up of a-D-glucose subunits. It exists as a mixture of two

forms, linear (amylose) and branched (amylopectin), with the branched configuration being the

predominant form. The a-D-glucose molecules in both amylose and amylopectin are bonded by 1,4-a-

glycosidic (acetal) linkages (Figure 6-83). The two forms differ in that the amylopectin contains

polysaccharide side chains connected to approximately every 30th glucose in the main chain. These side

chains are identical to the main chain except that the number 1 carbon of the first glucose in the side

chain is bonded to carbon number 6 of the main chain glucose. The bond is, therefore, a 1,6-a-glycosidic

linkage. Starch is too large to pass through the bacterial cell membrane. Therefore, to be of metabolic

value to the bacteria it must first be split into smaller fragments or individual glucose molecules.

Organisms that produce and secrete the extracellular enzymes a-amylase and oligo-l,6-glucosidase are

able to hydrolyze starch by breaking the glycosidic linkages between the sugar subunits. Although there

usually are intermediate steps and additional enzymes utilized, the overall reaction is the complete

hydrolysis of the polysaccharide to its individual a-glucose subunits (Figure 6-83). Starch agar is a simple

plated medium of beef extract, soluble starch and agar. When organisms that produce a-amylase and

oligo-Le-glucosidase are grown on starch agar theyhydrolyzethe starch in the medium surrounding the

bacterial growth. Because both the starch and its sugar subunits are soluble (Clear) in the medium, the

reagent iodine is used to detect the presence or absence of starch in the vicinity around the bacterial

growth. Iodine reacts with starch and produces a blue or dark brown color; therefore, any microbial

starch hydrolysis will be revealed as a clear zone surrounding the growth.

a) MATERIALS REQUIRED:

i. Starch agar plate

ii. Gram iodine

iii. Organisms: Bacillus subtilis and

Staphylococcus aureus

iv. Inoculation loop

Composition of Starch Agar

Ingredients Gms / Litre Meat Extract 3.000 Peptic digest of animal tissue 5.000 Starch, soluble 2.000 Agar 15.000 Final pH( at 25°C) 7.2±0.1

b) Procedure:

i. Using a marking pen, divide the starch agar plate into three equal sectors. Be sure to mark on the

bottom of the plate.

ii. Label the plate with the organisms' names, your name, and the date.

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iii. Spot inoculate two sectors with the test organisms.

v. Invert the plate and incubate it aerobically at 35°C for 48 hours.

c. Observation and Result:

i. Remove the plate from the incubator and note the location and appearance of the growth before adding

the iodine. (Growth that is thinning at the edge may give the appearance of clearing in the agar after

iodine is added to the plate.)

ii.. Cover the growth and surrounding areas with Gram iodine. Immediately examine the areas surrounding

the growth for clearing. (Usually the growth on the agar prevents contact between the starch and iodine

so no color reaction takes place at that point. Beginning students sometimes look at this lack of color

change and incorrectly judge it as a positive result. Therefore, when examining the agar for clearing, look

for a halo around the growth, not at the growth itself.)

iii. Record your results in the table provided.

Organism Result Symbol Interpretation

Clearing around growth

B.subtilis

S.aureus

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EXPERIMENT NO:7 NITRATE REDUCTION TEST

Aim: To determine the ability of bacteria to reduce Nitrate.

Introduction : Nitrate reduction test is used for the differentiation of members of Enterobacteriaceae on

the basis of their ability to produce nitrate reductase enzyme that hydrolyze nitrate (NO3–) to nitrite

(NO2–) which may then again be degraded to various nitrogen products like nitrogen oxide, nitrous oxide

and ammonia (NH3) depending on the enzyme system of the organism and the atmosphere in which it is

growing.

Principle :Heavy inoculum of test organism is incubated in nitrate broth. After 4 hrs incubation, the broth

is tested for reduction of nitrate (NO3–) to nitrite (NO2

–) by adding sulfanilic acid reagent and α-

naphthylamine.

1. If the organism has reduced nitrate to nitrite,

the nitrites in the medium will form nitrous

acid. When sulfanilic acid is added, it will react

with the nitrous acid to produce diazotized

sulfanilic acid. This reacts with the α-

naphthylamine to form ared-colored

compound. Therefore, if the medium turns red

after the addition of the nitrate reagents, it is

considered a positive result for nitrate

reduction.

2. If the medium does not turn red after the

addition of the reagents, it can mean that the

organism was unable to reduce the nitrate, or the organism was able to denitrify the nitrate or

nitrite to produce ammonia or molecular nitrogen.Therefore, another step is needed in the

test. Add a small amount of powdered zinc. If the tube turns red after the addition of the zinc, it

means that unreduced nitrate was present*.Therefore, a red color on the second step is a

negative result.

*Note: The addition of the zinc reduced the nitrate to nitrite, and the nitrite in the medium formed nitrous

acid, which reacted with sulfanilic acid. The diazotized sulfanilic acid that was thereby produced reacted

with the α-naphthylamine to create the red complex.

3. If the medium does not turn red after the addition of the zinc powder, then the result is called a

positive complete. If no red color forms, there was no nitrate to reduce. Since there was no nitrite

present in the medium, either, that means that denitrification took place and ammonia or

molecular nitrogen were formed.

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a. MATERIALS REQUIRED:

i. Media: Nitrate Broth with inverted Durhams tube

ii. Reagents: Sulphalinic acid reagent, Alpha napthylamine reagent, zinc dust

iii. Others: Inoculating loop, burner, dropper.

b ) PROCEDURE:

i.Inoculate nitrate broth with a heavy growth of test organism using aseptic technique.

ii. Incubate at an appropriate temperature for 24 to 48 hours

iii. Add one dropperfull of sulfanilic acid and one dropperfull of a α-naphthylamine to each broth.

A. At this point, a color change to RED indicates a

POSITIVE nitrate reduction test. If you get a red

color, then you can stop at this point.

B. No color change indicates the absence of nitrite.

This can happen either because nitrate was not

reduced or because nitrate was reduced to nitrite,

then nitrite was further reduced to some other

molecule. If you DO NOT get a red color, then you

must proceed to the next step.

iv. Add a small amount of zinc (a toothpick full) to each

broth. Zinc catalyzes the reduction of nitrate to nitrite.

A. At this point, a color change to RED indicates a

NEGATIVE nitrate reduction test because this means

that nitrate must have been present and must have

been reduced to form nitrite.

B. No color change means that no nitrate was present. Thus no color change at this point is a

POSITIVE result.

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c) Result and Interpretation

1. Nitrate Reduction Positive: (Red after sulfanilic acid + alpha-naphthylamine; no color after zinc)

2. Nitrate Reduction Negative: (No color after sulfanilic acid + alpha-naphthylamine followed by Red

after zinc).

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EXPERIMENT NO: 8 QUALITATIVE SALIVARY AMYLASE ASSAY

Aim: To demonstrate the hydrolytic activity of salivary amylase on starch.

INTRODUCTION AND PRINCIPLE:

Amylase is a hydrolytic enzyme which breaks down many polysaccharides such as starch. Starch is a

polymer of D- glucose units linked by α-1, 4 glycosidic bonds. The starch is made up of two

polysaccharides: Amylopectin (branched-chain polysaccharide) and amylose (unbranched-chain

polysaccharide

The hydrolytic effect of amylase on starch results in yielding maltose (composed of two D-glucose

molecules ) as the end product. There are two broad groups of amylases: α and β- amylases.The β-

amylases rapidly hydrolyse the amylose portion of starch to maltose by acting on residues at the non

reducing terminals. They hydrolyse α-1, 4 glycosidic links in polysaccharides so as to remove successive

maltose units from the non-reducing ends of the chains. The α amylases, in contrast to the β- amylases,

cause a rapid loss of the capacity of amylase to give a blue colour with iodine, also the rate of appearance

of maltose is much slower in the α amylases catalysed reaction than in the β- amylases catalysed one.

Types of amylase: -human amylase - α amylases,β- amylases, bacteria and plant amylase

Source of amylase: 1- Pancreas, 2- Salivary gland. Secretion: Serum or urine.

The Assay

In any enzyme assay, the rate of the reaction can be known by measuring the amount of substrate (s) that

is utilized or the amount of product (s) that is formed in unit time, it also called enzyme unit.

In the case of amylase, the substrate is starch (colourless), the product is maltose (colourless).They can

be converted to coloured products by specific chemical reactions.

Starch + Amylase maltose (colourless)

Starch + Amylase +Iodine blue colour ( Qualitative)

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At a pH of about 6-7 and in the presence of chloride ions, α amylases catalyses the hydrolysis of starch to

maltose with the intermediate formation of various dextrins. Dextrins are polysaccharide produced by the

hydrolysis of starch.

Starch+ Cl+ + α amylase maltose + intermediate formation of dextrins.

Steps of reaction:

1. Starch +α amylase + Higher dextrins Iodine blue colour

2. Intermediate dextrins Iodine reddish brown colour

3. Lower dextrins + maltose Iodine Colorless- This point is known as Achromatic Point ( time

taken to reach the point at which the reaction mixture no longer gives colour with iodine).

a) MATERIALS REQUIRED

i. Test tubes

ii. Amylase enzyme-saliva is the best and easily available source of enzyme.Rinse the mouth

approximately with 25 ml of water for 15 seconds, the saliva is collected in a clean beaker.

iii. Iodine solution

iv.0.02 M phosphate buffer of pH 6.7

v. Glass plate

vi. Glass rod

vii. Starch soluton: I gram of soluble starch is mixed with 20 ml of phosphate buffer and the solution is

slowly poured into 150ml of boiling phosphate buffer. The solution is stirred and boiled for 1-2 minutes

and then cooled tp roo temperature tp make the volume to 200ml with the phosphate buffer.

b) PROCEDURE:

i. Pipette out 5 ml of starch solution into 2 test tubes ,labelled “T“ ( Test)and “C” ( Control)and then add

1ml of 1% Nacl solution to both tubes.

ii. Mix well and keep the tubes at 37OC for 10 minutes.

iii. Add 1 ml of 1:20 diluted saliva solution to tube labelled ‘T”. Mix and rotate noting the time .

Meanwhile , place a two rows of series of iodine drops on a clean glass plate and Mark top row as

control and bottom row as test.

iv. At intervals of 1 minute, take a drop of reaction mixture from tube marked “T” with a glass rod and

mix with a iodine drop placed in a test row. Similarly, a drop from the tube marked “c” is mixed with

Iodine placed in control row.

Iodine drop mixed with solution from “C” tube shows blue colour while the drop from the tube “T” may

fail to show blue colour after a few minutes.

v. Note the time required to achromatic spot to develop in a test row of iodine.

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vi. The time taken for the achromatic spot indicates the complete hydrolysis of starch by salivary amylase

enzyme .

c) RESULT AND INTERPRETATION:

Development blue colour indicate presence of starch and achromatic spot indicates hydrolysis of starch.

Appearance of blue color from the control tube indicates the reaction of starch and iodine in the absence

of amylase enzyme while in the test mixture, starch would be hydrolyzed by salivary amylase and

therefore the absence of starch no colour develops when it is mixed with iodine.

CONTROL

TEST

ACHROMATIC SPOT

TIME IN MINUTES 1 2 3 4 5 6

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EXPERIMENT NO:9 ISOLATION OF PLANT FUNGAL PATHOGENS FROM SOIL.

Aim: To isolate plant pathogens using selective media and identify them.

Soil is a reservoir of numerous microorganisms including fungi, bacteria and actinomycetes.

Arzording to their habit they may be saprophytes or pathogens. However, the fungi pathogens grow

In soil and survive in the form of dormant propagules. Under favourable conditions fungal pathogens

Proliferate by utilizing organic material and increase their biomass. They infect the living plants of

Economic importance and cause serious diseases such as damping off of seedlings, seedling blight (

species of Pythium, Phytophthora), root rots (Macrophominaphaseolina, Sclerotium rolfisii, Rhizoc-

tonia solani, Verticillium spp.), vascular wilts (Fusarium spp, Verticillium spp.), and the other root

diseases (Armillaria mellea, Gaemannomyces graminis triticz). These soil-borne plant pathogens are

isolated by different techniques; for example, PCNB (Pentachloronitrobenzene) agar plates (for iso-

lation of Fusarium spp. from soil), Richard’s agar plate (for Rhizoctonia solani), CMR agar plates

for Macrophomina phaseolina), P10 VP agar plates (fo risolation of Phytophthora spp. from soil

and infected plants).

Isolation of Fusarium spp. from Soil

Fusarium is a saprophyte and plant pathogen which lives in soil. It survives by chlamydospores

for varying periods, However, certain species cause wilting in plants and also associated with

mycotoxins in stored food grains.

a) REQUIREMENTS

i.Soil sample from agricultural fields supporting wilt disease

ii. PCNB (Pentachloronitrobenzene) agar plates (for isolation of Fusarium spp. from soil)

iii.Pipettes (1 ml, 10 ml)

iv. Bunsen burner

v.Flasks containing 90 ml sterile distilled water

vi. Erlenmeyer flask (250 ml)

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(B) PROCEDURE

i. Weigh 10 g of soil sample and transfer into a flask containing 90 ml sterile distilled water.

ii. Prepare suspension by agitating for 20 minutes on a magnetic stirrer.

iii. Serially dilute the suspension to get 10-1, 10-2 and 10-3 dilution.

(iv) Pour aliquots of 1 ml suspension of final dilution over the surface of PCNB agar and gently

shake the plates so as to spread water suspension properly.

(v) Incubate the plates at 25d: 1°C for 5-7 days.

(C) RESULTS

Observe the plates for discrete growth of fungal colonies on the surface of medium. Colonies of

Fusarium appears whitish or pinkish forming aerial cottony mycelial mat. Identify them.

II. ISOLATION OF MACROPHOMINA PHASEOLINA FROM SOIL

M. phaseolina is a dangerous fungal pathogen causing charcoal rot/dry rot in about 500 plants

including herbs, shrubs and trees. It can be isolated by using selective medium of Meyer et al.

(modified by Filho and Dhingra (1980) as described below:

(A) REQUIREMENTS

i.Soil samples from field where plants suffer from charcoal rot disease

ii.CMR (Chloroneb or Demosan-mercuric chloride-rose Bengal) Agar* plates

iii.Spirit lamp

iv.Incubator

*CMRA medium (for isolation of Macrophomina phaseolina):

Molten rice agar medium l000ml

Weigh 10 g of polished rice and boil in 750 ml of distilled water for about 30 minutes.

water extract and make the volume to 1 litre. Add 20 g of agar and autoclave at 121°C for about 30

minutes. When this basal medium is cooled down to 50-5 5°C add the following

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Chloroneb or Demosan 65WP 312 mg

HgCl2 8.5 mg

Rose Bengal 112 mg

Streptomycin sulphate 40 mg

Penicillin as potassium salt 60 mg

(B) PROCEDURE

i. Collect soil samples from agriculture field where plants suffer from charcoal rot disease and

bring to the laboratory.

ii. Powder the soil in sterile pestle and mortar.

iii. Sprinkle 50 mg of soil onto surface of CMRA medium solidified in plates.

iv. Incubate the plates in inverted position in dark at 30°C for 7 days.

(C) RESULTS

Greyish to black colonies appears on plates. However, if no colonies appear, sprinkle more soil

(70-80 mg) on CMRA plates as above. Count the colonies and record result as CFUs/g dry soil.

III. ISOLATION OF PHYTOPHTHORA SP. FROM SOIL .

Phytophthora sp. is the soil-borne fungal pathogen belonging to the order Peronosporales of the

class Oomycetes. It is commonly called as downy mildew fungus and the disease as downy mildew.

Some of the important species are: R undulatum, R cinnamomi, R infestans (causal agent of late

blight of potato), etc. They survive in soil by dormant structures called oospores/sclerotia/chlamy-

dospores.

A) REQUIREMENTS

i. PIOVP agar medium*

ii.Rhizosphere soil from diseased plant

iii.Sieve (pore size 1-2 mm)

ivWater agar (0.1%)

v.Incubator

vi Pipette (1 ml, I0 ml)

vii.Shaker

viii.Sterile Petri dishes (3)

27

*P,0 VP agar medium:

Potato (peeled and sliced) 200 g

Dextrose 20 g

Agar 20 g

Distilled water 1 litre

Pimarin 10 ppm

Vancomycin 200 ppm

Pentachloronitrobenzene (PCNB) 100 ppm V

(Add pimarin, vancomycin and PCNB after

autoclaving and just before pouring when

tempera~

ture is about 40°C)

(B) PROCEDURE

(i) Collect rhizosphere soil of a plant suffering fi"om Phytophthora spp. and screen through a

sieve of 1-2 mm pore size.

(ii) Dilute the soil in 0.1% water agar and make the fmal serial dilution of 10_3.

(m) Pour 0.5 ml soil samples in the sterilised Petri dishes, and dispense about 15 ml PIOVP

medium.

(iv) Gently rotate the dishes to spread the soil particles.

(v) Incubate the plates at i 1°C for about a week. Q

(C) RESULTS

Colonies of Phytophthora sp. grow on the surface of agar medium. Identify them under microscope.

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28

EXPERIMENT NO:10 :ISOLATION AND OBSERVATION OF BACTERIA FROM MOUTH AND SKIN.

Aim: To isolate normal flora of bacteria from skin and mouth and identify them.

INTRODUCTION: Microbes, like bacteria and fungi (which includes yeast), are prevalent on and in

particular regions of the body and are considered the normal flora (or microflora). Many of these

microbes serve beneficial roles by preventing pathogens from growing and/or by producing products that

the human body needs. An example of a beneficial product is the production of menaquinone (vitamin K)

in the intestinal tract by Escherichia coli.

The Normal Flora: Skin: The human skin is home to about 1012 microbes! There are four main groups of

bacteria that predominate almost everywhere on the skin: Diphtheroids (e.g. corynebacteria like

Corynebacterium diphtheria; Propionibacterium acnes was once classified as a Corynebacterium is

considered part of this group), micrococci (which include the staphylococci such as Staphylococcus

epidermidis), streptococci (either alpha (α) or gamma (γ) hemolytic), and the Enterococci. Besides bacteria

the skin also is the home to yeast (like Candida) and fungi. The populations of microbes vary over the

body’s skin due to differences in pH, oxygen, water, and secretions. Certain groups, such as the

diphtheroids, are found mainly in the groin and armpits. The densities of microbes vary considerably. The

armpit is home to about 500,000 bacteria per square inch; the forearm - about 12,000 bacteria per square

inch.

Oral Cavity (Mouth And Teeth): Microbes normally found on the mouth and teeth include staphylococci,

streptococci (particularly Streptococcus mutans), Lactobacillus acidophilus, Actinomyces odontolyticus,

anaerobic spirochetes, and vibrios (comma-shaped bacteria).

ISOLATION FROM MOUTH

The widely accepted test for determining the susceptibility to dental caries is the Snyder test.

This test is meant for measuring the amount of acid produced by the action of lactobacilli on glucose.

The differential medium contains Snyder agar with glucose (pH 4.7) and bromcresol green (pH

indicator). The saliva containing lactobacilli ferments the glucose and produce acid. Due to the formation

of acid, colour of the medium turns from green to yellow within 24-48 hours. That indicates the

presence of lactobacilli in saliva and shows the susceptibility of the host towards dental caries. If

there is no change in colour, it indicates that the host has low susceptibility.

29

(A) REQUIREMENTS

i.Saliva

ii.Snyder test agar medium*

iii.Deep tubes

iv.Bunsen burner

v.Block of parafihi

vi.Pipette

vii.Test tubes

*Snyder test agar medium

Tryptone 20.00 g

Dextrose 20.00 g

Sodium chloride 5.00 g

Bromcresol green 0.02 g

Agar 20.00 g

Distilled water 1 litre

pH 4.8

(B) PROCEDURE

(i) Prepare Snyder agar medium, transfer into culture tubes and autoclave, transfer in tubes :

45°C. Put a block of a paraffin piece in mouth for about 3 minutes. Take care not to swalit: P

the saliva or piece of paraffin.

(ii) Alter few minutes of chewing, saliva will develop; collect it in a separate test tube.

(m) Shake it vigorously and mix with Snyder agar in tube .

(iv) Mix both the contents thoroughly by rolling the tube between the palms of the hands.

(v) Keep the tube in ice containing beaker for rapid cooling and incubate at 37°C for 72 hours.

30

C ) OBSERVATION AND RESULT:

Examine the tubes for a change the change medium and interpret the result. The uninoculated tube of

the medium is considered as control.

If color of tube turns yellow, it is considered positive and susceptibility to dental caries and if the

colour remains green negative.

**********

ISOLATION OF MICROFLORA FROM HUMAN SKIN

The resident flora of the skin is determined by using blood agar medium which allows the

hemolytic bacteria to grow. A mannitol salt agar plate for the isolation of staphylococci and Saburaud

agar plate for the isolation of yeast and molds are used.

(a) MATERIAL REQUIREMENTS

i. Blood agar plates

ii. Mannitol salt agar plate

iii. Chocolate agar plate

iv Hinton Sabouraud agar plate

v. Sterile saline test tubes

vi. Crystal violet

vii. Graln’s iodine

viii.Ethanol

ix .Lactophenol

x.Cotton blue

(B) PROCEDURE

(i) Prepare one plate each of blood agar, Mannitol salt agar and Sabouraud agar and label them

accordingly.

(ii) Use sterile cotton swab moistened in sterile saline and rub on the skin of your palm

repeatedly.

iii) .Inoculate a tube of sterile saline with the swab and mix it thoroughly.

(iv) . From this tube inoculate and streak on all the three plates by means of a four-way streak

inoculation.

(v) Incubate the Sabouraud agar plate at 25°C, and the other plates at 37°C for a period of 48

hours.

31

(C) OBSERVATIONS

Examination of blood agar plate. The blood agar plate cultures show zones of haemolysis. The

lysis of RBC with reduction of haemoglobin to methaemoglobin results in a greenish halo around the

bacterial growth indicates alpha-haemolysis while complete destruction of RBC results in a clea:

zone surrounding the colonies is due to beta-haemolysis.

Examination of Sabouraud agar plate: Cottony growth in scattered patches reveals the

presence of mold.

Examination of mannitol salt agar plate: Presence of bacterial colonies indicates the

staphylococci.

Select isolated colonies and prepare smear from each plate. Observe the Gram-stained cocc:

32

Note their shape and cell arrangement for identification of the individual. Prepare slides from the

Sabouraud agar, in cotton blue plus lactophenol. Afier identification, make a suitable diagram of the

fungal culture in the record.

Yeast Colonies on Sabouraud Agar Wet Mount of Yeast

&&&&&&&&&&&&&&&&&&&&&&&

33

Experiment No:11 STUDY PLANT DISEASE-I) TOBACCO MASIAC DISEASE PLANT,II). KOLEROGA OF

ARECANUT,III)BLIGHT OF RICE AND IV) TIKKA DISEASE OF GROUNDNUT.

AIM:To understand disease pattern ,symptoms and control of plant diseases.

INTRODUCTION: Plants, whether cultivated or wild, grow and produce well as long as the soil provides them with sufficient nutrients and moisture, sufficient light reaches their leaves, and the temperature remains within a certain “normal” range. Plants, however, also get sick. Sick plants grow and produce poorly, they exhibit various types of symptoms, and, often, parts of plants or whole plants die. It is not known whether diseased plants feel pain or discomfort. The agents that cause disease in plants are the same or very similar to those causing disease in humans and animals. They include pathogenic microorganisms, such as viruses, bacteria, fungi, protozoa, and nematodes, and unfavorable environmental conditions, such as lack or excess of nutrients, moisture, and light, and the presence of toxic chemicals in air or soil. Plants also suffer from competition with other, unwanted plants (weeds), and, of course, they are often damaged by attacks of insects. Plant damage caused by insects, humans, or other animals is not usually included in the study of plant pathology. Uncontrolled plant diseases may result in less food and higher food prices or in food of poor quality. Diseased plant produce may sometimes be poisonous and unfit for consumption. Some plant diseases may wipe out entire plant species and many affect the beauty and landscape of our environment. Controlling plant disease results in more food of better quality and a more aesthetically pleasing environment, but consumers must pay for costs of materials, equipment, and labor used to control plant diseases and, sometimes, for other less evident costs such as contamination of the environment. a) MATERIAL REQUIRED: i. Specimen of diseased plants

b) PROCEDURE:

I. Examine the specimens provided thoroughly and compare with a healthy plants.

II. Note the changes in the colour, shape, texture and size of plant and parts.

III. Record all visible changes in case sheet.

1. Tobacco Mosaic Disease:

Etiology:RNA Virus

Symptoms: Affected plants show leaves with mottling or mosaic pattern of light green and dark-

green areas.

Primary symptoms appear on newly formed young leaves as vein

clearing, greenish yellow mottling.

Infection on young plants results in stunted growth, malformation,

distortion and puckering of leaves. Darkgreen blisters and sometime

enations (leafy growth) appear on the dorsal side of the leaf.

34

Control:

One of the common control methods for TMV is sanitation, which includes removing infected

plants, and washing hands in between each planting.

Crop rotation should also be employed to avoid infected soil/seed beds for at least two years.

Planting of resistant strains against TMV may also be advised.

2.Koleroga of areca nut

Etiology: Phytophthora meadii (major species), P. arecae, P.heveae

Symptoms:

The first symptom appears as dark green/yellowish water soaked lesions on the nut surface usually near the soft inner perianth region and infected nuts lose its natural green luster

The lesions gradually spread covering the entire nuts (before or after shedding) which consequently rot and shed.

A felt off white mycelial mass envelopes on entire surface of the fallen nuts.

As the disease advances the fruit stalks and the axis of the inflorescence rot and dry, sometimes being covered with white mycelial mats.

Infected nuts are lighter in weight and possess large vacuoles. Dark brown radial strands on kernel make them unfit for chewing.

CONTROL:

Prophylactic spray of 1% Bordeaux mixture with stickers once before the onset of south west

monsoon followed by second and third applications at 40-45 days interval

Cover bunches with polythene sheets before monsoon rains

Collect and destroy all fallen and infected nuts

Remove and destroy all completely affected inflorescence and immature bunches

35

3) Tikka Diseae of Ground nut:

Etiology: The causal organism of tikka disease are Cercospora arachidicola Hori (perfect stage of the

pathogen:Mycosphaerella arachidicola ) and Cercosporidium personatum (perfect stage of the

pathogen: Mycosphaerella berkeleyii ).

Symptoms:

The primary symptoms of the disease are appearing in 35 to 60

days old plants.

The tikka disease occurs as two distinct types of lea spots caused

by two species of Cercospora. C. personatumcauses small (1-6 mm),

almost circular and dark coloured spots on the leaves, stipules, petioles

and stem which may coalesce to form a large dark brown to black

irregular patch.

There may be few to many spots on each leaf. The severe infection

or spotting on the leaves causes premature dropping.

The disease is more severe at the time between flowering and harvesting, when the climatic

conditions are favourable.

The leaf spots caused by Cercospora arachidicola are almost circular to irregular, large (1-10 mm),

surrounded by bright yellow haloes and dark brown centre.

The conidia are formed on upper surface of leaf while C. personatum produced conidia on lower

surface of leaves with concentric rings.

Control:

Plant disease debris should be burnt.

Seed dressing with a suitable fungicide like Benlate and Vitavax (2 gms/kg of seed).

Foliage spray with Bordeaux Mixture (4:4:50), Dithane M-45 (0.2%), Benlate and Bavistin (0.1%) gives good results.

Early maturing and spreading type varieties are less liable to attack of the disease.

36

4) Blight of Rice:

Etilogy: Xanthomonas oryzae pv. oryzae -is a bacterium.

Symptoms:

Symptoms appear on

the leaves of young plants as

pale-green to grey-green,

water-soaked streaks near

the leaf tip and margins.

These lesions

coalesce and become

yellowish-white with wavy

edges.

The whole leaf may

eventually be affected,

becoming whitish or greyish and then dying.

Leaf sheaths and culms of more susceptible cultivars may be attacked.

Systemic infection results in wilting, desiccation of leaves and death, particularly of young

transplanted plants.[6]

In older plants, the leaves become yellow and then die.

Control: Management of bacterial leaf blight is most commonly done by planting disease resistant

rice plants.

**********************************

37

EXPERIMENT NO:12 AGAROSE GEL ELECTRPHORESIS OF HUMAN SERUM PROTEINS.

Aim: To separate and identify the serum proteins by Agarose Gel Electrophoresis Method.

INTRODUCTION AND PRINCIPLE: Serum protein electrophoresis is used to identify patients with multiple myeloma and other serum protein disorders. Electrophoresis separates proteins based on their physical properties, and the subsets of these proteins are used in interpreting the results. Plasma protein levels display reasonably predictable changes in response to acute inflammation, malignancy, trauma, necrosis, infarction, burns, and chemical injury. A homogeneous spike-like peak in a focal region of the gamma-globulin zone indicates a monoclonal gammopathy.

In a serum protein electrophoresis, blood serum is run through an agarose gel across an electrical current. This forces separation of the different protein components of plasma into several distinct visible bands,

shown to the right.

The leftmost lane of the gel is called the serum protein electrophoresis. This lane is impregnated with a stain that will give all proteins a blue color nonspecifically. The dark band on top is albumin, one of the most common and abundant proteins in the bloodstream. The bands below each have names; alpha-1, alpha-2, beta, and gamma as marked in the figure.

The leftmost lane of the gel is called the serum protein electrophoresis. This lane is impregnated with a stain that will give all proteins a blue color nonspecifically. The dark band on top is albumin, one of the

most common and abundant proteins in the bloodstream. The bands below each have names; alpha-1, alpha-2, beta, and gamma as marked in the figure.

Different blood components tend to aggregate in the different

protein bands. For example, the HDL type of cholesterol runs in

the band labeled alpha-2. All of the immunoglobulins run in the

broad gamma band. The reason that the band is wide and fuzzy

is that there are many different types of immunoglobulin proteins of different shapes and sizes, ranging

from the small IgG subtype to the larger IgM subtype, and each runs slightly differently on the gel.

a) MATERIALS REQUIREMENT:

1. Freshly prepared human serum

2. Horizontal electrophoresis apparatus

3. Electrophoresis Power pack

4. Microscopic slides and cover slips

5.Agarose

6.Buffer:Tris Glycine -0.05MTris & 0.01M Glycine in litre of distilled water.

7.Dye solution: Coomassie blue- 1 g, Methonol 90ml,Acetic acid 10ml.Final volume 100ml.

38

8.Destaining solution-100ml-Methonol 50ml,Acetic acid 7ml and distilled water 43ml and made to final

volume of 100ml.

b. PROCEDURE:

1.Place two thoroughly cleaned microscopic slide on a level table.

2.Weigh 100mg of agarose dry powder and put in a dry test tube.

3. Add 10ml of Tris glycine powder to the test tube and keep it in a boiling water bath..

4. Agarose powder dissolves in the buffer on boiling and solution becomes clear.

5.Cool it for some time and pour 3.5ml of agarose solution carefully onto each slide using a pipette.

6. Allow 15-20 minutes for the gel solution to forma gel. The gel appears translucent.

7. Pipptte few drops of undiluted serum on a clean slide. Drop a pinch of bromophenol blue (BPB)powder

over it. Mix the serum with a cover slide and stamp on to the gel at one end of the slide.Serum sticking to

the slide is now transferred to the gel as a thin narrow line . A different serum can be applied to the other

gel slide.

8. Placethe slides over the bridges of the apparatus ( Sample application point should be near cathode –

negative , end).

9. Pour 75-100ml of buffer to each reservoir.

10. Cut 3.0 x 2.5 cm Whatman filter paper. Make a 1.0cm fold. Wet the paper in the buffer and place it on

each end of the gel slide. The other end of the paper wick should touch the buffer as shown in the

illustration.

11. Connect the apparatus to the power supply. Turn the power on. Turn selector switch towards

constant voltage mode. Slowly increase the voltage to 100V.

12. Continue to run for 1 to 2hours till the blue colour marker dye reaches the anodic (+) end of the gel.

13. Decrease the voltage ,stop the power and disconnect the power pack from the apparatus.

14. Take the slides out carefully and place them in a tray.

15. Add the dye solution drop wise to cover the entire gel. Close the tray and allow to stain the gel for 15

to 30 minutes. During this process proteins are fixed and stained simultaneously.

16. Drain the dye solution. Cut Whatman1 papers to the size of the gel slides ,wet them in the destaining

solution and layer onto the gel.

17.Keep the slides under the fan for drying.

18. Remove the filter paper and it comes out when the gel is dries. Whereas, the gel has become a thin

film sticking on the glass slide plate. Bands can be observed .

19. Place the slides in a petri dish and add the distaining solution to remove the background dye if any.

20. Take the slides out and dry them. Examine the bands on the white light Transilluminator. They can be

photographed.

PASS WORD:LB5

39

40

LID

SLIDE

SAMPLE

PAPER WICK

BUFFER RESERVOIR

BRIDGE

ANODE (+) CATHODE (-)

c) RESUL AND INTERPRETATION:

The dark band on top is albumin, one of the

most common and abundant proteins in the

bloodstream. The bands below each have

names; alpha-1, alpha-2, beta, and gamma

Abnormal serum protein electrophoresis pattern in a patient with multiple myeloma. Note the large spike in the gamma region.

*********************

41