Biochemistry

ninhydrin_test_qualitative_analysis_for_amino_acids

PRINCIPLE :

Ninhydrin is a powerful oxidising agent which reacts with the aminoacids to form a purple coloured complex. Imino acids like proline give yellow colour with ninhydrin.

MATERIALS :

Ninhydrin reagent

Aminoacids

METHOD : Take 2ml of test solution and add 1ml of ninhydrin reagent. mix the contents well and boil the test tube in waterbath for 2 minutes and then pull it.

osazone_test_qualitative_analysis_of_carbohydrates

PRINCIPLE :

Compounds containing carbonic group reacts with phenyl hydrazine to give osazone crystals. These crystals have characteristic shapes and melting points which are used for identification of reducing sugars.

MATERIALS :

Glacial acidic acid

Sodium Actetate

Phenyl Hydrazine Carbohydrates

METHOD :

Take 5ml of test solution 10 drops of glacial acidic acid. To this add few drops of phenyl hydrazine and a little sodium acetate. mix thoroughly and boil the test tube for 20-30 minute., until the crystals are formed. Cool it to room temperature. Examine the crystals under microscope.??

iodine_test_qualitative_analysis_of_carbohydrates

PRINCIPLE :

Iodine forms blue coloured complex with polysaccharides.??

MATERIALS :

Iodine Solution

Carbohydrates

METHOD :

Take 1ml of test solution in test tube and add drops of iodine solution. Observe the colour change.

seliwanoffs_test_qualitative_analysis_of_carbohydrates

PRINCIPLE :

This is the test used for ketoses. Ketoses are dehydrated more rapidly than aldoses, to give furfurals. These furfurals react with resorsenal to form cherry red coloured complex.

MATERIALS :

Selwanoff’s reagent

Carbohydrates

METHOD :

Add 1ml of test solution to 3ml of ??the reagent. Boil it in a boiling water bath and observe the colour change.

Methylene Blue Reduction Test

Milk is extremely susceptible to spoilage by microorganisms and microbiologist play a major role in the dairy industry in quality control of milk. Methylene blue reduction test is a method to quantify viable cells based on Methylene blue dye reduction in the sample.

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Method

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  • Sterilize all glassware and rubber stoppers either in an autoclave or in boiling water. Be sure all glassware is chemically clean.
  • Measure 1 ml of the Methylene blue thiocyanate solution into a test tube. Add 10 ml of milk and stopper.
  • Tubes may be placed in the water bath immediately or may be stored in the refrigerator at 0o??to 4??o??C for a more convenient time of incubation. When ready to perform the test, the temperature of the samples should be brought to 35??o??C within 10 minutes.
  • When temperature reaches 36??o??C, slowly invert tubes a few times to assure uniform creaming. Do not shake the tubes.
  • Record the time at the beginning of the incubation period. Cover to keep out light.
  • Check samples for decolorization after 30 minutes of incubation. Make subsequent readings at hourly intervals thereafter.
  • After each reading, remove decolorized tubes and then slowly make one complete inversion of remaining tubes.
  • Record reduction time in whole hours between last inversion and decolorization. Interpretations of the results are based on the classification list below.

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Comparison of Raw milk and Pasteurized milk

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  • Label two tubes as raw milk and pasteurized milk.
  • Transfer 10 ml of each milk sample in a 20 ml test tube appropriately labelled.
  • Add sufficient Methylene blue solution to milk to produce a final dye concentration of one part in 300,000 parts of milk.
  • Cover with the stopper and shake and mix the content well.
  • Place test tube containing mixture in a water bath at 37?? C. Record the time of incubation.
  • Allow the tubes to stabilize for 5 minutes, remove them from the water bath, invert them gently once, and place again in the water bath.
  • Observe periodically every 10 minutes for the first half hour and then every 30 minutes after-until complete reduction of litmus is observed.
  • Record the time required for reduction in both the milk samples.
  • Based on the observations, determine and record the quality of the milk sample as very poor, poor, fair or good from the list given below.

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Classification??

Grade 1: Excellent, not decolorized in 8 hours.

Grade 2: Good, decolorized in less than 8 hours but not less than 6 hours.

Grade 3: Fair, decolorized in less than 6 hours but not less than 2 hours.

Grade 4: Poor, decolorized in less than 2 hours.

Nitrate Reduction Test

Bacterial species may be differentiated on the basis of their ability to reduce nitrate to nitrite or nitrogenous gases. The reduction of nitrate may be coupled to anaerobic respiration in some species.??

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Nitrate reduction medium

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Ingredients???????????????????????????????????????????????????????????????????? Amounts

Beef (meat) extract???????????? ???????????????????????????????????? 3.0 g

Gelatin peptone?????????????????????? ???????????????????????????????????? 5.0 g

Potassium nitrate (KNO3)?????????????? ???????????? 1.0 g

Deionized water?????????????????????? ???????????????????????????????????? 1,000 ml

Final pH 7.0 ?? 0.2 at 25??C

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Reagents

Sulfanilic acid solution (Reagent A)

Dissolve 8 g of sulfanilic acid in 1 litre of 5N acetic acid. Reagent A can be stored at room temperature for up to 3 months in darkness.??

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??-Naphthylamine solution (Reagent B)

Dissolve 6 g of N,N-Dimethyl-1-naphthylamine in 1 litre of 5N acetic acid. Reagent B can be stored at room temperature for up to 3 months in darkness.

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Method

  • Dissolve 9 g of Nitrate Broth in 1 litre of distilled water. Dispense 10 ml aliquots of the broth into tubes fitted with Durham tubes. Sterilize by autoclaving at 121??C for 15 minutes at 15 lbs.
  • Inoculate the tubes heavily with a fresh culture of the suspect organism. Inoculate at least 1 ml sample in a tube or take a big part of a colony with an inoculating loop. Do not forget a negative control without any bacteria.
  • Incubate the tubes at 35 to 37??C (bacilli at 30??C) for 24 to 48 hours in an incubator with or without supplemental carbon dioxide.??
  • The first obvious product of reduction to look for is reduction to Nitrogen gas called denitrification, within the Durham tube.
  • A visible gap or bubble can be seen in the Durham tube as a result of collection of N2 gas into it. This is looked for first before any reagents are added.
  • Put a 5 drops of Sulfanilic acid solution and 5 drops of ??-Naphthylamine solution into the tube containing culture to be tested.
  • Shake the tube well to mix reagents with medium. A distinct red or pink color, which should develop within a few minutes, indicates nitrate reduction,??positive result.
  • If no red color develops a small amount of zinc powder is added.
  • If a red color develops, nitrate is present in the broth and the test is negative.
  • If no red color develops, nitrate has been reduced and the test is positive.

Preparation Of Nuclear Extracts

All buffers, autoclave then add HEPES or filter sterilize
Nuclei extract buffers: Dignam et al., Nuc. Acid Res. 11, 1475-1489
A: 10 mM HEPES (pH7.9 at 4??C) 1 ml 1 M
1.5 mM MgCl2 150 ??l 1 M
10 mM KCl 500 ??l 2 M
[Add fresh 0.5mM DTT]
TVf = 100mls
B: 0.3 M HEPES 7.9 30 mls 1 M
1.4 M KCL 70 mls 2 M
0.03 M MgCl2 3 mls 1 M
TVf = 100 mls
C: 20 mM HEPES 7.9 2 mls 1M
25% v/v glycerol 50 mls 50%
0.42 M NaCl 10.5 mls. 4M
1.5 mM MgCl2 150 ??l 1M
0.2 mM EDTA 40 ??l 0.5 M
[Add PMSF to 0.5mM fresh]
[0.5 mM DTT fresh]
TVf = 100mls
D: 20 mM HEPES 7.9 2 mls 1 M
20% v/v glycerol 40 mls 50%
0.1 M KCL 5 mls 2 M
0.2 mM EDTA 40 ??l 0.5 M
[0.5 mM DTT]
[Add DMSF to 0.5 mM fresh]
TVf = 100 mls
Stocks:100x DTT = 50 mM or 200x = 100 mM = 15.4 mg/1 ml
100x PMSF = 50 mM 200x = 100 mM = 77 mg/ml
1M HEPES 7.9 = 23.8 g/100mls 0.01 M = .238 g/100mls
4 M KCL = 29.82 g/100mls
50% glycerol need 100mls
1 M MgCl2 = 20.33 g/100mls
PBS
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(FOR HELA CELLS)
TEN MAXI PLATES–~2-2.5 x 108 HeLa cells
Pellet ~2.5mls
Buffer: A – 20 mls
C – 1 ml
D – 200 mls
B – 1 ml
1. Wash cells w/PBS, ice cold.
2. Scrape in 5 mls PBS/Plate.
3. Pellet at 2000 RPM 10′ clinical centrifuge.
4. Resuspend in 5 x vol. Buffer A, 4??C, ice 10′ ~12mls.
5. Pellet at 2000 RPM 10′, 4??C.
6. Resuspend in 2 x vol. Buffer A ~5mls.
7. Homogenize ~20 strokes.
8. Check lysis on microscope.
9. Pellet at 2000 RPM 10′,4??C.
From this point, treat the pellet and supernatant separately.
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A. Pellet = nuclei
1. Transfer to ultracentrifuge tubes.
2. Pellet at 25,000g 4??C = 17,000 RPM.
3. Resuspend in 0.6 mls buffer C.
4. Homogenize, Vf~1.2-1.5 ml.
5. Stir w/magnet 30′ at 4??C.
6. Pellet at 25,000g, 30′.
7. Dialysis SUPER (~1ml) against buffer D.
for 5 hours
8. Pellet at 25,000g, 20′
9. SUPER = nuclear extract
freeze
expect 6-8 mgs/ml
B. Super = cytoplasmic extract
1. Add to Super 0.11 vol. buffer B (e.g., 6 mls + 0.66 ml buffer B)
2. Spin 100,000 g, 1 hr. at 4??C.
3. Dialysis of Supernatent (~4-5 mls) against buffer D (200 mls) for 5-8 hours.
4. This is S100 fraction. Freeze

Abo Blood Grouping

To find the blood group of given sample by ABO bloodgrouping methos was done by using forward grouping technique.

PRINCIPLE :

A suspension of RBC’s of the specimen is reacted with known reagent antisera – anti A and anti – B. positive agglutination indicates the presence of corresponding Antigen on the red cells.

Apart from forward group there ??is a another method called reverse grouping. This forward each other in determining the blood group of an individual incase of any deviation from the normal pattern, possible technical error, presence of subgroup of other unusual circumstances should be considered.

REQUIREMENTS :

GLASSWARES :

Slides, testtubes and pasteur pipettes.

REAGENTS :

Sample, anti-A and anti-B antisera and other standard labwares.

PROCEDURE :

SLIDE TEST :

A microscopic slide was taken and marked as A, B and control.

Added one drop of reference cell suspension(10%) onto the respective circles.

Add two drops of test serum on the cell suspension on each circle, their content was mixed well.

Result was observed.

TUBE TEST :

Three small test tube were takne and marked as A,B and control.

One drop of anti-A and anti-B and saline was added in respective tube.

Two drops of saline suspension was added in the tubes and mixed gently.

Tubes were centrifuged at 2500 rpm for about a minute.

OBSERVATION :

Little clumps of red cells were seen floating in a clear liquid marked as A.

No agglutination was visible in solution marked as B and control.

Isolation Of Plasmid Dna

To isolate the plasmid DNA from bacteria.

PRINCIPLE :

Alkali lysis methos is a predominantly used method for plasmid isolation. Glucose prevents the sudden lysis of cell membrane. SDS denatures the protein in cellwall and creates a pore through which the DNA is released along with other components. During the process plasmid DNA reaches first the supernant slowly followed by the chromosomal DNA.

REAGENTS REQUIRED :

SOLUTION 1 : 50 mM of glucose. 25 mM tris Hcl(pH 8) 10 mM EDTA (pH 8) solution1 is prepared and autoclaved and stored at 4 C.

SOLUTION 2 : 0.2 N of NaoH, 1% SDS

SOLUTION 3: 3 M sodium acetate, glacial acetic acid 11.5 ml, water-28.5ml.

SOLUTION 4 : 10mM TE buffer and 10mM tris and 4 mM EDTA ( pH-8).

PROCEDURE :

1.5 ml of bacteria culture was taken in a microfuge tube, centrifuged at 10,000 rpm for 30 sec at 4 C. The supernatant was discarded and the bacteria pellet was resuspended in 100micro litre ??od ice cold solution 1 by vigorous vortexing. 200 micro liter of freshly prepared solution2 was added, the tube was closed tightly. The content of the tube were mixed by inverting the tube rapidly 5 times( store the tube in ice). To this 15 micro liter icecold solution 3 was added, the testtubes are closed tightly and inverted gently to disperse the solution3, to the viscous bacterial lysate. The solution from the above is centrifuge at 10,000 rpm for 5min at 4 C in a microfuge tube, the supernatant was transfered to a fresh tube. Double stranded DNA was precipitated with 2 volume of 95% ethanol at room temperature. Contents of the tube were mixed by inverting the tube 5 times. Centrifuge at 10,000 rpm for five times at 4 C. The supernatant was allowed to stand in an inverted position on a paper towel to allow all the fluid to drain away. The pellet of double stranded DNA was rinsed with 1ml of 70% ethanol at 4C. Supernatant was removed and the pellet of nucleic acid was allowed to dry in the air for 10 min. The nuclear pellet was redissolved in 20 micro liter of TE buffer of pH 8 and allow to stand at 20 C. The isolated plasmid DNA was analysed agarose gel electrophoresis(0.8%).

Separation Of Proteins By Sds Page

To separate the proteins by SD-PAGE

PRINCIPLE :

Acrylamide (CH2=CH-CONH2) is copolymerised with the cross linking agent NN-methyl bis acrylamide[CH2-(NHCOCH=CH2)2] in the presence of the catalyst accelerated by chain intiation mixture. This mixture contained freshly prepared ammonium per sulphate (APS) as a catalyst together with the suitable base N,N,N’,N’=tetramethylene ethylene diamine as an initiator. Gelation is due to vinyl polymerisation. The porosity of the gel is determined by relative proportion of acrylamide monomers to the cross linking agent. Gel may be defined both in terms of total percentage (T) of acrylamide monomers as bis acrylamide present, where T is equal to,

?? ?? ?? a+b

T = _____A * 100

?? ?? ?? ?? m

??and by the amount of cross linking agent as the percentage of total acrylamide concentration,, where

?? ?? ?? ?? ?? ?? ?? ?? ?? ?? ?? ??b

??C not equal to ??__ +b *100

?? ?? ?? ?? ?? ?? ?? ?? ?? ?? ?? ?? a

where

?? ?? a = acrylamide

?? ??b = cross linking agent

?? ??m= volume of buffer (cm3).

PAGE is an important technique for determining molecular weight of proteins. The macromolecules have to be first treated with SDS, to produce uniform charge : mass ratio, so that subsequent separating gel will be achieved by treating the protein with the mixture of ethanol with sulpheric acid and sulphur in it. 2- mercaptoethanol which breaks the disulphide bonds and SDS which is an anionic reagent assists in solubilization and more inportantly attack on anionic group at regular intervals along the polypeptide chain.

PROCEDURE :

The 7 % resolving gel solution was poured between the glass plates in the gel apparatus. The gel is then allowed to polymerize. The resolving gel is overlapped with 3% of stacking gel. The comb was inserted without any air bubbles in between the plates containing stacking gel solution. The stacking gel was allowed to polymerize. After polymerization the comb was removed and the wells were used for loading the sample. The sample was mixed with solubilizing buffer and heated in a boiling waterbath for 20 minutes. The resolving buffer was filled in the lower and upper tank of the apparatus. Electrical conncetions were made and electrophorosis was started at 100 V. After the dye front moves to the ??resolving gel, the voltage is increased to 150 V. When the dye front reaches the bottom of the gel. the power was disconnected. The gel plates were removed the gel apparatus and a spatchula was used to separate the plates. A small cut was made at one end close to the first well as a marker.

GEL STAINING :

0.25% of coomassie brilliant blue (CBB) in acetic acid, ethanol (CH3OH) and water in 4:1:5 ratio. The electrophorosed gel was soaked in the staining solution overnight after which it is transferred to another tray contianing destaining solution where the proteins appeared as discrete bands in the gel.