Cell disruption technique - grinding with sand

 

 Aim

To demonstrate the breaking up of cell structures using abrasives like sand particles.

Principle

 In order to release microbial cell contents during downstream processing, a number of methods can be used for cell disruption to disintegrate the extremely tough cell walls. Any potential method of disruption must ensure that liable materials are not denatured by the process or hydrolyzed by enzymes present in the cell. The use of combination of different techniques release products from specific locations within yeast cell. Although many techniques are available which are satisfactory at laboratory scale, only limited number have been proved to be suitable for large-scale applications, particularly for intracellular enzyme extraction. Types of cell disruption methods are physical, mechanical and chemical.

Grinding with sand is a mechanical cell disruption method used to break microbial, plant, or animal cells to release intracellular components such as enzymes, proteins, nucleic acids, pigments etc. This technique tears up the structures of a cell using abrasive sand particles. Abrasion and shear force produced by grinding cells with fine sand particles mechanically rupture the cell wall and cell membrane. This helps to achieve the release of cytosolic contents, and allows some degree of breakup of physical structures. Physical structures are further removed by isopycnic centrifugation.

Grinding with sand is commonly used for laboratory-scale extraction from microorganisms and plant tissues. Usually, the process is carried out at low temperature to prevent denaturation of cellular components.

 

Materials Required

1.Yeast culture

2.Mortar and pestle

3.Sterile fine Sand

4. Sabouraud's dextrose Agar

5. Routine microbiological facilities

 

 

Procedure

1. Five ml of yeast suspension was taken into sterile test tube. One test tube was kept as control.

2. The yeast suspension was taken in a sterile mortar, and cloth filtered fine sand was added to it.

3. It was then ground with pestle several times and serially diluted.

4. 1 ml of suspension from 10 ^-4 and 10^-5 dilutions were plated on Sabouraud's dextrose agar and 

    CFU was counted

5.1 ml of the suspension from the control tube was also serially diluted and CFU was counted.

 

Result

Sabouraud's dextrose Agar plated with the sample ground with sand particle showed a fall in colony count, which indicate fall in cell count. 

 

Conclusion

Grinding with sand is a mechanical cell disruption technique in which the abrasive action of sand particles breaks the cell wall and membrane, releasing intracellular materials.

 

Cell disruption technique by repeated freezing and thawing

 

Aim
To demonstrate the extent of cell disruption by freezing and thawing. 

Principle
Microorganisms are protected by extremely tough cell walls. In order to release their cellular contents during downstream processing a number of methods are available for cell disruption. Cell disruption can be done by physical or chemical methods.

 Repeated freezing and thawing is a physical cell disruption method used to break cells and release intracellular materials. The technique works on the principle that ice crystals are formed during freezing and their melting during thawing damage the cell membrane and cell wall, leading to cell lysis. During freezing treatment, water inside cells forms ice crystals which expand and puncture cellular structures during thawing. Rapid melting causes osmotic shock and membrane rupture.  Repeated cycles increase the extent of cell disruption. The intracellular contents comes out which can be easily separated by centrifugation. By plating microbial suspension after centrifugation the number of viable cells can be obtained.

 It is crucial that cell disruption methods do not denature labile materials present in the cell. Repeated freezing and thawing is a simple, inexpensive, and chemical-free cell disruption technique that efficiently releases intracellular biomolecules while preserving many heat-sensitive and biologically active compounds. It is commonly used for laboratory-scale extraction of biomolecules from microbial and animal cells.

Materials  required
1. Overnight grown culture of E.coli

2. Sterile distilled water
3. Sterile saline
4. Nutrient Agar
5.Routine Microbiological facilities

 

Procedure:
1.10 ml of distilled water and saline were taken in sterile test tubes.
2. Test tubes were inoculated with 0.5 ml of overnight broth culture of E coli.

3. One test tube each of distilled water and saline were kept at 5°C for one hour. 
4. The same pair of test tube with distilled water and saline was  kept at room temperature for next 1 hour. This process was repeated for 4 times. Another set was kept at 5°C, as control throughout the experiment

5. 0.1 ml sample were taken from each tube and proper dilution was plated.

6. The plates were incubated at 37 °C overnight and colonies were counted and compared with control

Result

A fall in cell count was obtained in tubes subjected to freezing and thawing

 

 

 

Ammonium Sulfate precipitation

Aim

To precipitate out dissolved egg white protein by ammonium sulfate precipitation

salting out

Principle

Salts such as ammonium sulphate and sodium sulphate ae used for the recovery and fractionation of proteins. As the salt concentration of a solution increases, more of the bulk water become associated with salt ions. The salts remove water from the surface of the protein revealing hydrophobic patches which come together causing the proteins to precipitate. Proteins which exhibit hydrophobic interactions aggregate and precipitate from the solution, which is also called salting out. This is a vital step in downstream processing of proteins. Precipitation relies on the principle that when compounds of higher affinity are added to the protein solution in a solvent, the protein get separated as a precipitate.

 Materials Required

Egg white solution, 20% ammonium sulphate solution, glass rod, routine microbiological facilities

Procedure

1. 20 % ammonium sulphate solution was gently added to the solution of egg white with constant stirring, till precipitation was maximum.

2. The mixture was filtered and washed with ammonium sulphate solution in a funnel over filter paper

Result

The egg white protein was precipitated.

 

Isolation of microbial flora of fermented milk

Aim

To isolate and enumerate bacteria of fermented milk.

Principle

Fermentation with certain microorganisms is necessary in the preparation of foods such as cheese, curd, yoghurt etc. Milk is fermented with lactic acid bacteria to make curd. Thus, the presence of microorganisms in food though considered harmful in some cases, it is definitely beneficial in other cases.

Materials Required

Curd, sterile tubes, nutrient agar, petri plates, Bunsen burner, pipettes, L-rod.

Procedure

1. Sterile nutrient agar plates containing 20 ml of medium were prepared.

2. Curd sample was diluted by serial dilution technique to obtain dilutions of 10^-4, 10^-5, 10^-6, 10^-7.

3. The plates were labelled corresponding to the dilutions.

4. Using sterile pipettes 0.1 ml from each dilution was placed in respective nutrient agar plates.

5. The L-shaped glass rod was sterilized with alcohol followed by flaming. The rod was cooled and gently placed on the surface of agar.

7. Petri plate was rotated in both clockwise and anticlockwise direction to uniformly spread the sample over agar surface. The plates were incubated in inverted position for 24 - 48 hours at 37⁰ C.

8. The number of colonies were counted and the total microorganisms per ml of original sample was calculated.

Observation

Each of the dilution plates were observed for colonies of bacteria. The number of colonies were counted.

Result

The number of organisms per ml was found to be  2.6 x 10⁷

 

Observation (on left side)

 Enumeration of Bacteria from Curd

Organism	Dilution	Number of Colonies	Number of Organisms per ml
BACTERIA	10-5	26	26 x 105    = 2.6 x 107 0.1
	10-6	4	4 x 106    = 4 x 107 0.1

 

The number of organisms per ml of curd can be calculated by applying the formula,

Number of organisms per ml of curd = Number of colonies ×dilution factor

                                                                           Amount plated

 

Pellicle formation

 Aim

To demonstrate pellicle formation in a broth culture 

Principle

Homogenous suspended microbial growth in a liquid medium will aid in availability of oxygen and other nutrients. This is ensured by using a rotary shaker or impeller driven bioreactor that keeps the cell, product, substrate and oxygen well mixed. When agitation stops, the culture remains static and the dissolved oxygen in the culture broth gets quickly used up, restricting growth and encouraging anaerobic physiology. At the surface of the broth, due to oxygen availability from the above gaseous phase, cells continue to multiply forming a well textured physical mat known as pellicle at the liquid surface. This further cuts off any oxygen diffusing from the top, pushing the lower part to further anaerobiosis.

Pellicle formation can result in low yields in a bioprocess industry due to unintended channelling of the substrate and efforts since unwanted by-products will be formed and will complicate the downstream processing.

 Materials Required

1. Culture: Aspergillus niger

2. Media: Doelger Prescott broth

3. Routine microbiological facilities

 Procedure

1. Aspergillus niger was inoculated in 300 ml of Doelger Prescott media.

2. It was incubated for 6 hours in a rotary shaker and then at room temperature undisturbed for about 7 days.

3. After incubation, the culture broth was observed for pellicle formation.

Observation and Result

A thick mat of microbial growth was observed on the surface of the liquid media.

Production and estimation of Citric acid

Aim

To demonstrate and estimate citric acid production by Aspergillus niger

 

Principle

Citric acid is the key intermediate of tricarboxylic acid cycle. Citric acid is used as acidulant in food and pharmaceutical industry for the production of carbonated beverages and used as plasticizer. Commercially, citric acid is produced by surface fermentation using Aspergillus niger. Beet molasses medium containing 10-20% sucrose is used in the commercial production. Doelger Prescott medium is used in this experiment to produce citric acid using Aspergillus niger. 

Estimation of citric acid is done by titration against standard NaOH using phenolphtahelin indicator. The amount of citric acid produced is expressed in gram per 100ml.

 Materials Required

1. Culture: Aspergillus niger

2. Media: Doelger Prescott broth

3. Routine microbiological facilities, Burette, pipette, conical flask, measuring cylinder

4. Reagents: 0.1 N NaOH, Phenolphthalein

 Procedure

1. 300 ml of Doelger Prescott media was prepared. pH was adjusted to 4.0 using 0.1 N HCL.

2. The medium was autoclaved and inoculated with a loopful of Aspergillus niger culture.

3. The flask was incubated at room temperature for 7 days.

4. After incubation for 7 days, the pH of the medium was noted.

5. The amount of citric acid in the culture broth was estimated by titration.

Estimation of Citric acid by titration

1. 1 ml of phenolphthalein indicator was added to 200 ml hot boiled water in a 250 ml flask.

2. 1 ml of culture filtrate was added to the above flask and titrated against 0.1  NaOH to the endpoint, in a well illuminated white background.

3. The initial and final readings were noted to calculate the volume of standard NaOH

 Result

The pH of the media was 4 before inoculation of Aspergillus niger culture and it decreased to 2 after 7 day incubation. This is due to the production of citric acid by the fungal cylture.

The amount of citric acid was estimated to be 0.30 g/100ml

  

Observation (left side)

1 N NaOH stock solution was prepared by dissolving 40 g NaOH in distilled water. From this stock solution, 0.1 N NaOH was prepared by mixing 50 ml stock solution with 450 ml distilled water.

Citric acid estimation

Conical flask: 200 ml hot boiled water + 1 ml of phenolphthalein + 1 ml of culture filtrate

Burette: 0.1 N NaOH

Endpoint: Appearance of distinct pale pink colour

	1	2	3
Initial burette reading	50	48	46
Final burette reading	49.5	47.6	45.5
Volume of 0.1 N NaOH rundown (ml)	0.5	0.4	0.5

 

Calculation (left hand side)

Citric acid (g/100ml) =

Volume of 0.1N NaOH × Normality × Equivalent molecular weight of citric acid ×100

Volume of sample taken

0.47 × 0.1 × 0.064 × 100/1

Citric acid (g/100ml) =         0.30 g/100 ml

 

Doelger and Prescott Medium (pH 4.0)

Sucrose 140 g

Ammonium nitrate 2.23 g

MgSO4 0.23 g

Distilled water 1000 ml

Isolation of Cellulase producing organisms from soil

 Aim

To isolate cellulose producing organisms (cellulolytic) from the soil

 Principle 

Cellulose is a predominant component of higher plants and most abundant biopolymer and polysaccharide found on earth. It forms the main constituent of cell wall of plants, most algae and some fungi. Cellulose is a linear homologous polymeric chain consisting of D-glucose residues that make up to 10,000 glucose residues, linked by β-1,4 glycosidic bonds. 

Though cellulose is plentiful, it is not utilized completely due to the fact of being insoluble. It is the food for ruminant mammals and termites which have bacteria that secrete cellulose digesting enzymes-cellulases. A cellulosic enzyme system consists of three major components: endo-ß-glucanase (CMCase) exo-ß-glucanase (cellobiohydrolase) and ß-glucosidase (cellobiase). The co-operative action of these three enzymes result in the complete hydrolysis of cellulose to glucose.

 The organisms which produces cellulase are known as cellulolytic organisms. Soil contains numerous microorganisms which produce cellulase. Eg. Cellulomonas, Clostridium, Pseudomonas and Trichoderma, Aspergillus etc. Fungi are the main cellulase-producing microorganisms, though a few bacteria and actinomycetes have also been recently reported to produce cellulase. 

Cellulase production can be detected using agar containing cellulose. Cellulose agar is a medium where if we grow microorganisms which produce cellulase, a clear zone develops around the growth, which indicates cellulolysis.

 Cellulose is the most abundant and renewable feedstock for energy source and can be used for the production of alternative fuels by the conversion of lignocellulosic biomass to biofuel. It has a wide range of applications such as food and beverage, pulp and paper, textile, animal feed, detergent and agriculture. 

Materials Required

 Soil sample, Petriplates, Carboxymethyl cellulose agar,  Tubes for serial dilution, L- rod

 

Procedure

1.      1g fertile soil was dissolved in 100 ml sterile distilled water to obtain 10^-2 dilution. It was then serially diluted to obtain dilutions till 10^-6.

2.    Aliquots of I ml from 10^-4, 10^-5, 10^-6 dilutions were transferred into Carboxymethyl cellulose plates and spread plate performed.

3.      Inoculated Carboxymethyl cellulose plates were then incubated at 37⁰C for 2-5 days.

4.      After incubation, the plates were flooded with Gram’s Iodine and observed

 

Observation

Presence of colourless zones or clear halo was detected around cellulolytic colonies on the plates, which indicated cellulose hydrolysis.

Result

A clear zone around cellulase producing colonies indicated the presence of cellulase producers which were isolated from the soil sample.

 

Media Composition (Don't write in record sheet)

 Carboxymethyl Cellulose Agar (pH 7.0)

Carboxymethyl Cellulose 10 g

NaCl 0.1 g

Yeast extract 0.4 g

NH 4 H 2 PO 4 0.1 g

MgSO 4 7H 2 O 0.2 g

Agar 15 g

Distilled water 1000 ml

Isolation and screening of amylase producing microorganisms from soil

 Aim

To isolate bacteria producing amylase, capable of degrading the polysaccharide starch from the soil

Principle

Polysaccharides are large molecular weight polymers of monosaccharide units joined by glycosidic bonds. Polysaccharides can be structural and nutritional in nature. Nutritional polysaccharides are formed by both plant and animal cells and they are used as reserve food supplies. Glycogen is the reserve polysaccharide of animal cells and starch serves a similar function in plant cells.

Starch is a high molecular weight polysaccharide containing single monomeric units of glucose. Starch contains two forms of glucose polymers- amylose and amylopectin. Amylose consists of long unbranched chains of D-glucose units connected by α-1,4 glycosidic linkages. Amylopectin is branched with one branch for every thirty glucose units. The monomeric glucose units are linked by α-1,4 glycosidic linkages in the unbranched region and α-1,6 glycosidic linkages in the branched regions in amylopectin.

Starch is not capable of permeating the cell membrane because of its high molecular weight. To assimilate starch for energy and catabolic reactions, it must be degraded into basic glucose units by starch hydrolyzing enzyme, amylase. Starch is converted to dextrins and then maltose, which is broken down to soluble glucose molecules that can be transported into the cell for energy production, through glycolysis. Since enzymes are catalysed and are not consumed in chemical reactions, a few molecules of enzymes are used over and over to break down a great deal of starch.

Amylases are commercially used in bread making, brewing, for production of sugar syrups, for mash preparations, in spot removers, for the removal of silver from used photographic films, in instant hot breakfast cereals and also in dry cleaning process.

Amylases give a deep blue colour and amylopectin gives a red to brown colour when they react with iodine depending on the source of starch. Once starch is completely hydrolyzed the oligo and disaccharides do not form a colour complex with iodine. The zone of hydrolysis is evident by the lack of colour.

Materials Required

Starch agar medium, Gram’s Iodine solution, sterile petridish, inoculation loop, Bunsen burner

Procedure

1. 1 gm soil was suspended in 100 ml sterile distilled water in a conical flask

2. Serial dilution was performed in tubes containing 9 ml sterile distilled water.

2. Aliquots of  0.1 ml was transferred from 10^-5 and 10^-6 into starch agar plates and spread plate performed

3. The inoculated plates were incubated at 37^oC for 24-48 hours

4. After incubation, the plates were analyzed with Gram’s Iodine solution and then observed for starch hydrolysis

Observation

Presence of colorless hydrolysis zone around the colony after treatment with Gram’s Iodine solution is positive evidence for degradation of starch. Absence of clear zone indicate a negative reaction.

Result

A clear zone around amylase producing colonies indicated the presence of amylase producers which were isolated from the soil sample.

 

Media Composition (dont write in the record sheet)

 

Starch Agar (pH 7.0)

Peptone 5 g

Beef extract 3 g

Soluble Starch 2 g

Agar 15 g

Distilled water 1000 ml