Planorbid snails were collected from the Manchester surrounding areas and were stored in refrigerator. They were supplied with some food such as cabbage leaves and grass.
Planorbidae, fresh water air breathing snails, is used for the study the properties of lectins and the the effects in haemagglutination. To collect the haemolymph from the planorbidae, the body of the snails was been punctured. Inorder to collect more haemolymph from the snails, the visible part of the snails body was been poked which makes the snail to contract its' body. Using a sterile disserting needle, the snails shell is punctured and the haemolymph is
The above ingrediants were mixed with 1 litre of distilled water which gave a litre solution of PBS. By using pH meter, the PBS solution was adjusted to 7.3 by addition of NaOH or HCl. If the pH of PBS is more than 7.3, then HCl was added to the buffer to decrease and if the pH is below 7.3, then NaOH is added to increase.
collected by 10µl micropipettes into sterile eppendorf tubes. After the collection into eppendorf tubes, these were kept into beaker containing ice cubes to avoid aggregation of cells. Care is taken to avoid more freezing which causes the lysis of cells( Dr.G.Ingram ).The snail haemolymph are subjected to centrifugation of 2000rpm for 5 min before we use for the experimental procedures.
DILUTION METHOD TO PREPARE RED BLOOD CELL SUSPENSION
Blood from Horse, guinea pig, rabbit and sheep were been received from Alisevers' solution from TCS Biosciences limited, UK.
Firstly the blood from these animals were been washed by series of centrifugation.
3 ml of blood from Horse, Guinea pig, Rabbit and Sheep were been pipetted
out separately into graduated tubes and labelled as H, P, R, S respectively.
Then these graduated tubes were been centrifuged at 2000 rpm for 7 minutes.
The serum from each of the graduated tubes were discarded after centrifugation.
3 ml of PBS Buffer was added to the left pellet and centrifuged for about 7 minutes at 2000 rpm followed by discarding the serum.
To the above left pellet, 3 ml of PBS Buffer was added and centrifuged . This whole process is repeated for 3 times.
After the centrifugation process, 1 ml from each of the labelled tubes were been pipetted out into separate beakers and labelled.
To that separate beakers, 5 ml of PBS buffer was been added into each H, P, R and S blood samples which gave the whole amount to 6 ml solution.
Next step is to calculate the absorbance of respective animal samples by using spectrophotometer.
For every 0.1 ml of animal sample, 1.4 ml of distilled water must be added to the cuvvette as suggested by Dr.G.Ingram.
0.2 ml from each animal samples was been taken and 2.8 ml of distilled water was added to the cuvvette.
Using the spectrophotometer, the absorbance of each respective animals' blood samples were noted by setting the wavelength at 540 nm.
The respective absorbance reading of each animals H, P, R and S blood samples was noted.
Horse = 0.224
Pig = 0.189
Rabbit = 0.210
Sheep = 0.306
According to the Kabat's (1971) formula:
Final volume (washed cells) = Initial volume x Absorbance
0.70
VF = V0 x A
0.70
Here,
VF = Final volume of erythrocytes
Vo = Total volume of erythrocytes (6 ml)
A = Absorbance Readings
Horse VF = 1.92
Pig VF = 1.62
Rabbit VF = 1.26
Sheep VF = 2.62
This gives the 5% concentration. Separate both final and original volumes of each respective animals. Centrifuge the resulted amounts and label it separately which can be used in future.
In order to make 1 % concentration, 1 ml from each blood samples were been taken separately from 5 % concentration and 5 ml of PBS Buffer was added to it followed by labelling. This makes 1 % concentration.
After the preparation of 1% concentration of each H, P, R and S blood samples, 0.1 % concentration was been prepared by the addition of 1 ml from each blood samples with 9 ml of PBS Buffer. This gave me the 0.1 % concentration.
Addition of equal amounts of blood samples and PBS Buffer gives the 0.05 % concentration.
1 ml of PBS Buffer was added to 1 ml of 0.1 % concentration of each blood sample and labelled. This gives 0.05 % concentration of blood samples.
So blood samples of different concentrations 1%, 0.1% ,0.05% were prepared for different animal species H, P, R and S respectively.
PROCEDURE FOR SELECTED PHYSIO-CHEMICAL TREATMENTS
Various physical and chemical treatments were performed on both of these haemolymph and ER samples to study the effects of haemagglutination and the properties of lectins.
INFLUENCE OF TEMPERATURE
In this temperature treatment, haemolymph from planorbidae are heated in a waterbath at different temperatures to find out the optimum temperature at which the samples ends the haemagglutination. 25µl of haemolymph is subjected to heating at different temperatures at 25, 30, 35, 40, 45,50, 55, 60, 70, 80, 90 and 100. The haemolymph is heated for 45 min. Later the samples were examined for haemagglutination activity. Controls for all these samples were taken as PBS with ER.
PH-EFFECT
To find out the end point of the titre at different PH values, this PH treatment is done on the samples by haemaggltination. So for this treatment, different range of PH values starting from 3 to 10 is prepared. The PH values can be altered by the addition of 1M of Hydrochloric acid or 1M of Sodium hydroxide solution to the prepared PBS buffer. Then the haemagglutination assay is carried out on the samples.
EFFECTS OF METAL IONS (Ca2+ and Mg2+)
The presence of haemolymph metal ions Mg2+ and Ca2+ in the PBS affected HGN activity was determined through this method. In this procedure, the snail haemolymph and the metal ions were mixed in an eppendorf tube with equal amounts and are subjected to two-fold dilutions on the agglutination plate. To this plate, same amount of ER of different samples were placed. PBS with respective ER were used as the controls.
INHIBITION OF CARBOHYDRATE AND GLYCOPROTEIN
The effects of sugar inhibition in haemagglutination was examined by this wxperimental procedure. Various types of carbohydrates and two glycoproteins were examined. Carbohydrates includes different types such as monosaccharides, amino sugars, deoxy sugars, oligosaccharides and polysaccharides supplied from Sigma Limited, UK.
Following carbohydrates and glycoproteins were used.
MONOSACCHARIDES USED:
D - Mannose
D - Xylose
D - Galactose
D - Glucose
L - Sorbose
β- D -fructose
DEOXY SUGARS USED:
2- Deoxy -D-ribose (2-dRib)
2- Deoxy -D-Glucose (2-dGlc)
6- Deoxy -L-mannose (= α-L- rhamnose)
6- Deoxy -L- galactose (=α-L (-)-fucose
AMINO SUGARS USED:
D (+) - Mannosamine ( ManN )
N- Acetyl-D- galactosamine ( GalNAc )
N- Acetyl-β-D- mannosamine ( ManNac )
N- Acetyl-D- glucosamine (GlcNAc )
OLIGOSAACCHARIDES USED:
Maltose {α-D-Glu (1-4) D-Glu}
D (+) - Trehalose {α-D-Glu (1-1) α-D-Glu}
D (+) - Melazitose
Sucrose
Raffinose
D - (+) -Melibiose
β - Lactose
POLYSACCHARIDE USED:
Lipopolysaccharide
GLYCOPROTEINS USED:
α- Amylase
β- Glycosidase
Preparation of sugar samples:
Almost all the sugars involved in this experimental procedure are taken in 200mM concentration as recommended by Ingram. Before the haemolymph and sugar dilutions, respective sugar concentrations were prepared separately and labelled. Molecular weights of each sugar were noted.
The sugar concentration that has to be taken for 1000ml of buffer is calculated as follows:
For glucose 200 x 180
1000
=36 gm in 1000ml buffer
=0.18 gm in 5ml of buffer
Molecular weight of Glucose is 180.
Similarly the calculations for all the sugars were calculated based on there respective molecular weights.
In the haemolymph dilution procedure, 3µl of PBS buffer is placed followed 1:2 dilution of haemolymph. To this dilution mixture, 2µl of respective sugar is added. This is followed by incubation for about 30min. Later 5µl of respective ER of animal is added followed by incubation for about 30min. Similarly all the ER samples were tested separately. Controls includes ER of animal with PBS buffer solution.
Later experimental procedures includes the sugar dilution. In this procedure, 3µl of buffer is placed on the wells followed by 1:2 dilution series of sugars. Later 2µl of haemolymph is added. 5µl of ER samples are added separately before incubating. Controls includes the ER of animal with PBS buffer.
ENZYME INHIBITION
In this experimental procedure, several enzymes received from Sigma Limited, UK , were used namely :
Trypsin (7.5 pH)
Chymotripsin (7.8 pH)
Pepsin (7.3 pH)
Lipase (7.5 pH)
Fungal protease (7.6 pH)
β - glucosidease (7.2 pH)
α - glucosidease (7.2 pH)
Neuraminidase (5.5 pH)
Preparation of enzyme solutions:
The enzyme solution is prepared by mixing 1 mg of enzyme with 1 ml of PBS buffer as suggested by Ingram. So 5 mg of trypsin is added to 5ml of buffer and mixed properly followed by altering PH to 7.5 .Similarly this method is followed to other enzymes for respective stock.
Equal volumes of ER samples were treated with equal amounts of enzyme samples followed by incubation at room temperature for 30 min.
