6TH SEMESTER

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

LABORATORY MANUAL

SL NO EXPERIMENT PAGE NO

1 COLORIMERTIC ESTIMATION OF DNA 1

2 COLORIMETRIC ESTIMATION OF RNA BY ORCINOL METHOD 2

3 METHYLENE BLUE REDUCTION TEST (MBRT) 3

4 RESAZURIN DYE REDUCTION TEST 5

5 PHOSPHATASE TEST FOR RAW MILK 7

6 DIRECT MICROSCOPIC COUNT OF BACTERIA FROM MILK SAMPLE 9

7 AGAROSE GEL ELECTROPHORESIS OF DNA 12

8 ISOLATION OF BACTERIA AND FUNGI FROM SPOILED FOOD 16

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EXPERIMENT NO 1 : COLORIMERTIC ESTIMATION OF DNA

AIM: To estimate the amount of DNA present in the given unknown solution by diphenylamine method.

PRINCIPLE: When DNA is treated with diphenylamine under the acidic condition a bluish green colored complex is

formed which has an absorption peak at 595nm.This reaction is given by 2 deoxypentose in general. In acidic

solution deoxypentose are converted into a highly reactive β hydroxyl leavulinic aldehyde which reacts with

diphenylamine gives bluish green colored complex. The colour intensity was measured using a red filter at 595nm.

REAGENTS REQUIRED:

1. Stock Standard Solution: 50mg of DNA was dissolved in 50ml of Saline Sodium citrate buffer. Concentration 1mg/ml (400µg/ml). 2. Working Standard Solution: 5ml of stock solution. Solution was diluted to 50ml with distilled water. Concentration 100µg\ml (If Stock Standard Solution is used). 3. Diphenylamine Reagent: 10g of pure diphenylamine was dissolved with 25ml of concentrated Sulphuric acid which was made up to 1ml with glacial acidic acid the solution must be prepared freshly. 4. Buffered Saline ph 7.4: 0.14N Sodium chloride and 0.02M sodium citrate. 5. Unknown Solution: The given unknown solution is mad up to 100ml with distilled water(Refer the table). PROCEDURE:

i. Pipette out different volumes of DNA solution into test tubes and make it to 3 ml with distilled water as

shown in the table.

DNA (ml) Conc. of DNA (µg) Water ( ml) Reagent (ml) K

eep

in b

oili

ng

wat

er

bat

h f

or

10

min

ute

s OD @ 595nm

BLANK 00 3 5

0.2 80 2.8 5

0.4 160 2.6 5

0.6 240 2.4 5

0.8 320 2.2 5

1 400 2.0 5

UNKNOWN ? 2 5

ii. Add 5 ml of reagent to each tube.

iii. Mix well and keep it in boiling water bath for 10 minutes.

iv. After cooling the tubes,measure the optical density at 595 nm.

v. Plot a standard graph.

vi. Determine the concentration of DNA in a given sample using a standard graph and record the result.

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EXPERIMENT NO: 2 : COLORIMETRIC ESTIMATION OF RNA BY ORCINOL METHOD.

AIM: To estimate the amount of RNA present in the given unknown solution by Orcinol Reagent.

PRINCIPLE: It is a general reaction for pentoses and depends on the formation of furfural. When the

pentose is heated with concentrated HCl, the Orcinol reacts with furfural in the presence of ferric chloride

as a catalyst, giving a green colour. Only the purine nucleotide gives significant reactions.

REAGENTS REQUIRED:

1. Orcinol reagent: Dissolve 1gm of Ferric chloride in 1litre of Concentrated HCl and add 35ml of 6 %(w/v) Orcinol in

absolute alcohol.{total volume 1litre/35ml}.

2. Standard RNA: Prepare Standard RNA solution by dissolving 200µg /ml

PROCEDURE:

i. Pipette out different volumes of RNA solution into test tubes and make it to 2 ml with distilled water as

shown in the table.

RNA (ml) Conc. of RNA (µg) Water ( ml) Reagent (ml)

Kee

p in

bo

ilin

g w

ate

r b

ath

fo

r 1

0 m

inu

tes OD @ 665nm

BLANK 00 2 3

0.2 40 1.8 3

0.4 80 1.6 3

0.6 120 1.4 3

0.8 160 1.2 3

1 200 1 3

UNKNOWN ? 1 3

ii. Add 3 ml of reagent to each tube.

iii. Mix well and keep it in boiling water bath for 15 minutes.

iv. After cooling the tubes, measure the optical density at 665 nm.

v. Plot a standard graph.

vi. Determine the concentration of RNA in a given sample using a standard graph and record the result.

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EXPERIMENT NO:3:DYE REDUCTION TESS FOR MILK.

1. METHYLENE BLUE REDUCTION TEST (MBRT)

AIM: To check the quality of the given milk sample based on the difference in the microbial load milk

sample provided.

PRINCIPLE: Milk is a good medium for the growth of microorganism. A variety of microorganism can be found in both raw milk and pasteurized milk. These actively growing microorganisms reduce the oxidation reduction potential of the milk medium due to the exhausted oxygen by the microorganism. Normally the milk is contaminated with microorganisms such as Staphylococcus aureus, Streptococcus pyogenes,Pseudomonas aeroginosa, Enterobacter spp., Bacillus spp., Paenibacillus spp., etc. Contaminated milk is one of the important sources for transmission of diseases from animals to humans. The main reason for this contamination is the un-proper handling of milk. Normally milk is contaminated during the milking process by the microorganisms present in the exterior surface of the animals, pipelines such as udder and adjacent areas. Unsterilized dairy utensils such as milking machines, milk cans are also a good source of contamination by the microorganism. The formation of Methylene blue reductase is thus becoming a popular tool for determining the quality of the milk. The principle of methylene blue reduction test depends on the fact that the color imparted to the milk by adding a dye such as methylene blue will disappear more or less quickly, which depends on the quality of the milk sample to be examined. Methylene blue is a redox indicator, that lose its color under the absence of oxygen and is thought to be reduced. The depletion of oxygen in the milk is due to the production of reducing substances in the milk due to the enhanced rate of bacterial metabolism. The dye reduction time refers to the microbial load in the milk and the total metabolic reactions of the microorganism.

Important points

Disappearance of color with limited time indicates the absence of oxygen in milk. Refrigerated milk contains more oxygen than warm milk. At the time of milking process, it has more

oxygen content than the other cases. Rate of reduction depends on the nature of the organism present in the milk. The rate of reduction by

different microorganism is arranged in order below.

E.coli (coliforms) > Streptococcus lactis > faecal Streptococci > micrococci> Thermoduric organisms > psychrotrophic organisms

The presence of light fastens the reduction rate; hence the test tube under observation should be tightened properly.

Use uniform concentration of methylene blue dye in all test samples. Addition of more methylene blue dye will result in more reduction time.

Increased incubation time reduces the reduction time since the activity of some organism increases with increased incubation temperature.

Periodically invert the tubes at regular intervals during incubation time to improve the accuracy of the test result. Otherwise microorganisms may not be evenly distributed in the milk sample leading to wrong result interpretations.

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MATERIALS REQUIRED:

1. Milk samples to be analyzed 2. Screw-cap test tubes 3. Test tube rack 4. Pipettes(10 ml and 1ml) 5. Water bath (37o C) 6. Bunsen Burner

PROCEDURE:

i. Transfer 10 ml of each milk sample into appropriately labeled test tube.

ii. Add 1 ml of redox indicator, methylene blue to each test tube containing milk sample. iii. Tighten the test tube mouth with stoppers. Gently invert the tubes at about four or five times to ensure proper mixing of the methylene blue solution. iv. Keep the tubes in the water bath at 37 o C Note the incubation time. That is, the time elapsed for the color to turn whitish appearance. Stabilize the tubes for 5 minutes.

RESULT INTERPRETATION

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TIME TAKEN FOR REDUCTION GRADE

1 Reduction within 30 minutes Poor quality

2 Reduction occurring between 1 to 2 hours Fair

3 Reduction occurring between 3 to 4 hours Good quality

4 More than 4 hours Excellent quality

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EXPERIMENT NO:4: DYE REDUCTION TEST FOR MILK.-RESAZURIN DYE REDUCTION TEST

AIM: To Determine the quality of milk by conducting Resazurin reduction test.

PRINCIPLE: The resazurin test is conducted similar to the methylene blue reduction test with the

judgement of quality based either on the color produced after a stated period of incubation or on the

time required to reduce the dye to a given end-point.

MATERIALS REQUIRED:

1. Milk samples to be analyzed 2. Screw-cap test tubes 3. Test tube rack 4. Pipettes(10 ml and 1ml) 5. Water bath (37o C) 6. Bunsen Burner

PROCEDURE:

i.Prepare resazurin solution by dissolving one resazurin tablet (dye content/ tablet, approximately 11 mg,

certified by Biological Stain Commission) in 200 ml of hot distilled water as was done in the methylene

blue test.

ii. Place one ml of dye solution in a sterile test tube, then add 10 ml of sample. Stopper the tube, place in

the incubator and, when the temperature reaches 36o C,

iii. Invert to mix the milk and dye.

iv. Incubate at 37o C.

v. Tubes are examined and classified at the end of an hour in the "one-hour test" or at the end of three

successive hourly intervals in the "triple reading test."

The following relationships of color and quality are generally accepted:

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RESULT AND INTERPRETATION

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COLOR OF SAMPLE TIME QUALITY OF MILK

1 Slate Blue (no color change)

1 HOUR Normal

2 Mauve / Pink 1 HOUR Abnormal

3 Deep mauve to deep pink 1 HOUR Fair

4 Pink 30 MINUTES Grossly Abnormal

5 White 1 HOUR Bad-Leucocytes present in milk.

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EXPERIMENT NO:5: PHOSPHATASE TEST FOR RAW MILK.

AIM: To Determine the activity of Phosphatase enzyme present in raw milk sample.

PRINCIPLE:

When milk is pasteurised at 63ºC for 30 min in batch pasteuriser or 72ºC for 15 seconds in heat exchanger, continuous flow pasteurisers, All pathogenic bacteria are destroyed, there by rendering milk safe for human consumption. Simultaneously various enzymes present in milk, and which might affect its flavour, are destroyed. In order to determine whether or not milk has been adequately pasteurised, one of the enzymes normally present in milk phosphatase, is measured. A negative phosphatase result indicates that the enzyme and any pathogenic bacteria have been destroyed during pasteursation. If it is positive, it means the pasteurisation process was inadequate and the milk may not be safe for human consumption and will have a short shelf life.

MATERIALS REQUIRED:

Test tubes 5 ml pipettes 1 ml pipettes l00 ml volumetric flask 500 ml volumetric flask water bath at 37ºC

Note: All glassware must be rinsed, cleaned, rinsed in chromic acid solution and boiled in water for 30 min.

REAGENTS:

Buffer solution:Is mixed by 0.75g anhydrous sodium carbonate and l.75g Sodium bicarbonate in 500 ml distilled water.

Buffer-substrate solution:Place 0.l5 g of di-sodium paranitrophenylphosphate(the substrate)into a clean 100ml measuring cylinder.

Add the buffer solution to make to 100 ml mark.

Store this buffer-substrate solution in a refrigerator and protected against light. It should not be used after one week. Prepare a fresh stock.

PROCEDURE:

i. Pipette 5mls buffer-substrate solution into a test tube, stopper and warm the solution in the water bath at 37ºC.

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ii. Add to the test tube 1ml of the milk to be tested, stopper and mix well and place in water bath at 37ºC.

iii. Prepare a blank sample from boiled milk of the same type ( milk boiled for 3 minutes at 100ºC as that undergoing the test.

iv. Incubate both the test samples and the blank sample at 37ºC for 2hrs. After incubation, remove the tubes and mix them thoroughly.

vi. Place one sample against the blank in a Lovibond comparator" ALL PURPOSES" using A.P.T.W. disc and rotate the disc until the colour of the test sample is matched and read the disc number.

INTERPRETATION:

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Disc Reading after 2 hrs incubation at

37ºC

Remarks

0-10 Properly pasteurized

10-18 Slightly under pasteurized

18-42 UNDER PASTEURISED

> 42 NOT PASTEURISED

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EXPERIMENT NO: 6 : DIRECT MICROSCOPIC COUNT OF BACTERIA FROM MILK SAMPLE

AIM: To Determine the total count of Bacteria in a sample of milk by Breed’s method.

PRINCIPLE:

The Breed's Smear Method for Direct Microscopic Counts

This is a method in which the number of bacteria or yeasts in a sample (e.g. broth culture, cell suspension, milk) may be determined by direct microscopic examination. Since the staining procedure does not differentiate between living and dead organisms, the bacterial count obtained is known as a "total count". In the case of samples of milk and other foods with a high fat content it is necessary first to defat (e.g. with xylene). Newman's stain conveniently combines both defatting and staining processes although not allowing the determination of the Gram's staining reaction. The Breed's smear method is also used for counting the animal cells -predominantly leucocytes -which are found in much larger numbers in mastitis milk (see page 179) than in milk from healthy animals and so can be diagnostic of mastitis.

MATERIALS REQUIRED:

Glass slide

Glass marking pencil

Calibrated Inoculation loop (0.01ml)

Blotting paper.

Microscope

REAGENTS: Newman's stain

PROCEDURE:

i. One cm2 area is drawn on a glass slide

ii. A known volume (0'01 ml) of the sample or an appropriate dilution is then spread over a known area (I cm2), on a glass slide.

iii. The sample is allowed to dry, then stained with Newman's stain and examined microscopically.

iv. The average number of bacteria or cells per field is determined and it is then possible to calculate the number of bacteria or cells per ml of original sample.

Determination of the area of the microscopic field

1. Set up the microscope and, using the X 10 eye-piece and low power objective, adjust the stage micrometer so that the graduated scale (I mm divided into 100 units of 10ILm each) is in the centre of the field.

2. Place a drop of immersion oil on the stage micrometer and focus with the oil-immersion objective. Determine the diameter of the microscopic field in ILm, using the micrometer scale, adjusting the tube length of the micro- scope slightly if necessary. In subsequent observations this same tube length must be maintained.

3. Calculate the area of the microscopic field in mm for the oil-immersion lens and X 10 eye-piece using the formula

r2= Area of field

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where r = radius of field. Knowing the area of the microscopic field, it is then possible to determine the microscope factor (MF) by calculation:

(a ) MF = Number of fields in I cm2 (100 mm2)

Area of field in 1mm2

(b) Average number n of organisms or cells per field=

Number counted

Number of fields counted

(c) MF x n = Number of organisms or cells present in 1 cm2

(d) Since 0.01 ml of sample was spread over 1 cm2 ,

MF x n X 100 = Number of cells or organisms present in 1 ml of sample.

For most microscopes as used above, one organism per field represents approximately 500,000 organisms per ml of sample.

Preparation of smear

I. Thoroughly mix the sample to be examined. Prepare dilutions as necessary -to produce smears with no more than 20 organisms per field. (If pure cultures are being examined a barely detectable turbidity will be given by a suspension containing about 106 cells per mi.)

2. Deliver 0.01 ml of sample onto a clean glass slide and spread over an area of I cm2 using either a guide card or a marked slide. The sample may be delivered either (a) by means of a capillary pipette calibrated to deliver 0.01 ml or (b) by using a wire loop. A closed loop of 4 mm internal diameter will hold a drop of approximately 0.01 ml of sample, if withdrawn with the plane of the loop perpendicular to the surface.

3. Dry the smear immediately by placing on a warm level surface or in an incubator at 55°C. Drying should be complete within 5 min so as to prevent possible bacterial multiplication.

4. Stain by an appropriate method.

5. Examine, using the oil-immersion objective.

Counting

1. Count each single bacterium as one. Chains and clumps of bacteria are also counted as one.

In this way the "direct count" is also a "clump count" and bears a closer relationship to the probable plate count,

since each clump or chain would probably give rise to one colony only.

2. The precision of the count depends on the number of bacteria counted and, as in the plate count technique, maximum practical precision would be obtained by counting some 600 items (bacteria in this case, colonies in the case of the plate count). 3. The numbers of bacteria observed in each field summed, until the total number of bacteria observed is 150 or more.

4. Determine the average number of organisms per field and hence calculate the number per ml. or per g of sample.

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CALCULATION:

a) r2 = Area of Microscopic Field ( MF)

b) MF = No .of fields in 1 cm2 ( 100mm2) = 100 area of fields in mm2

c) Average No. ‘n’ of organisms or cells per field = No. counted No. of fields counted d) MF x n =No. of organisms or cells in cm2

e) Since 0.01 ml of sample was spread over 1 cm2

MF x n x 100 = No. of organisms or cells present in 1 ml of sample.

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EXPERIMENT NO: 7: AGAROSE GEL ELECTROPHORESIS OF DNA

AIM: To Perform Agarose gel electrophoresis of DNA restriction with E.CoRI and Hind III restriction

enzyme.

PRINCIPLE:

Electrophoresis is a technique used to separate and sometimes purify macromolecules especially proteins

and nucleic acids that differ in size, charge or conformation. When charge molecules are placed in an

electric field, they migrate either towards the positive or negative pole according to their charge. Shorter

molecules move faster and migrate farther than longer ones, because shorter molecules move faster

easily through the pores of the gel. In contrast to the proteins, which can have either a net positive or net

negative charge, nucleic acids have a consistent negative charge imparted by their phosphate backbone

and migrate towards the anode. Proteins and nucleic acids are electrophoresed within a matrix or gel.

Most commonly, the gel is cast in the shape of a thin slab with wells for holding the sample. The gel is

immersed within an electrophoretic buffer that provides ions to carry a current and some type of buffer

to maintain the pH at a relatively constant value.

Agarose is a polysaccharide extracted from seaweed. It is typically used at concentrations of 0.5 to 2%.

Higher the agarose concentration, stiffer the gel. Agarose gels are extremely easy to prepare. Agarose

powder is mixed with buffer solution, melted by heating and the gel is poured. It is also non toxic.

Agarose gels have a large range of separation, but relatively low resolving power. By varying the

concentration of agarose, fragments of DNA from about 200 to 50,000 bp can be separated using

standard eletrophoretic techniques. The more common dye used to make DNA or RNA bands visible for

agarose gel electrophoresis is Ehthidium bromide, usually abbreviated Etbr. It fluoresces under UV light

when intercalated into DNA or RNA. By running DNA through Etbr-treated gel and visualizing it with UV

light, any band containing more than 20bp DNA becomes distinctly visible.Etbr stained DNA is not visible

in natural light.DNA is mixed with negatively charged loading buffer before adding the mixture to the gel.

These are visible in natural light and they co sediment with DNA. Xylene cyanol and bromophenol blue are

common loading buffers. The sucrose (40%) or glycerol (80%) present in the loading buffer imparts

density on the gel which helps in sinking of the sample directly into the well.

REAGENTS AND BUFFERS

6X DNA LOADING DYE:

25mg Bromophenol blue (0.25%)

25 mg Xylene Cyanol (0.25%)

4 gram Sucrose(40%)

Adjust volume to 10 ml with water.

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Tris Borate EDTA buffer (TBE buffer: pH8.0; 5X)

53 g of Tris base

27.5 g of Boric acid 20 ml of 0.5M EDTA ( pH 8 )

1% Agarose Gel

Dissolve 1 g of agarose in 100 ml of 1X TBE buffer of pH 8

Ethidium Bromide ( 10mg / ml )

EQUIPMENT AND SUPPLIES

1) An Electrophoresis chamber and power supply

2) Gel casting trays which are available in a variety of sixes and composed of UV transparent plastic. The open

ends of the tray are closed with tape while the gel is being cast, then removed prior to electrophoresis.

3) Sample combs around which molten agarose is pored to form sample wells in the gel.

4) Electrophoretic buffer, usually Tris-Acetate-EDTA (TAE) or Tris –borate EDTA ( TBE).

5) Loading buffer, which contains something dense ( eg-glycerol) to allow the sample to fall into sample wells

and one or two tracking dyes, which migrate into the gel and allow visual monitoring of how far the

electrophoresis has progressed.

6) Ethidium bromide, a fluorescent dye used for staining nucleic acid.

7) Transilluminator (an ultra violet light box) which is used to visualize Ebr stained DNA in gel.

8) Micropipettes and tips ( 2 – 20 µl, 20-100µl,100 - 1000µl)

9) Lambda ( λ) DNA, E.cORI and H.ind III digested DNA sample.

PROCEDURE:

i. Seal the ends of a clean gel casting tray with cello tape and keep it on a flat surface. Weigh required amount

of agarose to get gel concentration of 1% and mix it with 1X TBE buffer. Heat in a microwave oven until

completely melted.

ii. Cool the gel solution to about 40OC and add Etbr to the gel (final concentration 0.5µg/ml).

iii. Pour the gel into the casting tray containing comb and allow it to solidify at room temperature ( 30 – 45

minutes).

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iv. Remove the comb after the gel has solidified, insert the gel horizontally with its casting tray into the

electrophoretic chamber such that the gel containing wells is kept near the negative electrode. Add buffer into

the chamber so as to just cover the gel with buffer.

v. Mix DNA samples with loading buffer , load 25µl into each well using micropipette.

vi. Replace the lid and connect the leads and apply a voltage ( 5 V) for first 10 minutes and the turn the voltage

to 100 V later.

vii. When the tracking dye has reached ¾th the length of the gel , stop the current and visualize the gel in the

UV trans illuminator.

DNA bands intercalated with Etbr appear as an orange colored fluorescent bands under UV light.

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OBSERVATION:

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EXPERIMENT NO:8: ISOLATION OF BACTERIA AND FUNGI FROM SPOILED FOOD

AIM: To Isolate spoilage microorganisms from fruits and vegetables.

INTRODUCTION Foods consumed by human beings have wide variety of nutritional substances such as carbohydrates,

Proteins, Fats, vitamins and minerals. However the composition of food varies from one food to another example:

vegetables are rich in amino acids, proteins, minerals, vitamins and fruits contain high content of Carbohydrates

(Glucose, fructose, sucrose etc.) and less proteins. The available water (aw) also varies from one food to another.The

growth of microorganisms in foods bring about several biochemical changes in the foods such production of acids, gases,

amines. Such biochemical changes include fermentation, deamination, Putrefaction, etc. The biochemical changes are

due to the enzymes produced by the micro-organisms such as pectinases, amylase, cellulases, proteases, lipase .

Spoilages are usually identified by visual symptoms on the surface of the food produce. Such changes include changes

include rots, slimy, rancidity, putrefactive, off odours, souring, changes in the colour, taint, phosphorescence. There are

several factors responsible for the spoilage of foods. These include-Handlers, contamination during harvesting,

transportation, storage area, shelf life, season of the year, temperature. Some of the food spoilages are common, some

are rare. Such foods are unfit for consumption.

MATERIALS REQUIRED

Food samples- Spoiled Fruits and Vegetables

Nutrient agar and Rose Bengal culture media plates. Spatula/Scalpel Diluent Test tubes, Inoculation loop Cotton swabs PROCEDURE: 1. Weigh 1 g of fruit / vegetable in a sterile container. ii. Transfer to 9ml diluents iii. Mix to homogenize the sample iv. A smear from the sample is made for direct microscopic observation. v. The sample is serially diluted. A dilution of 1;10, 1:100 or 1:1000 may be used for plating out.

vi. From the chosen dilution, 0.1ml of inoculum is transferred to the surface of nutrient agar and Rose Bengal culture plates.

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17 vii. Inoculum is spread uniformly over the surface of the medium using a sterile spreader, alternatively, on nutrient agar plates, the inoculums is streaked with a sterile loop over the surface of the medium. viii. Inoculated plates of nutrient agar are incubated at 37OC for 24 hours while Rose Bengal plates are incubated at 25Oc for 3-4 days. ix. The growth of bacterial colonies from nutrient agar are gram stained for observation and subsequent identification and the growth of fungi from the Rose Bengal plates are used for preparing teas mount for identification. x. The observation are recorded in the tables as given below. OBSERVATION OF BACTERIAL GROWTH ON NUTRIENT AGAR MEDIUM

TYPE OF COLONY COLONY CHARACTERS GRAM STAINING RESULT

OBSERVATION OF FUNGAL GROWTH ON ROSE BENGAL MEDIUM

TYPE OF COLONY MACROSCOPIC APPEARENCE MICROSCOPIC APPEARENCE

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