quality control of packed food and canned food

QUALITY CONTROL OF PACKED FOOD AND CANNED FOOD

AIM: To analyze the food as per the quality control procedure for detection of their contamination level

INTRODUCTION:

Spoilage of heat-processed, commercially canned foods is confined almost entirely to the action of bacteria that produce heat-resistant endospores. Canning of foods normally involves heat exposure for long periods of time at temperatures that are adequate to kill spores of most bacteria. Particular concern is given to the processing of low-acid foods in which Clostridium botulinum can thrive to produce botulism food poisoning. Spoilage occurs when the heat processing fails to meet accepted standards. This can occur for several reasons: (1) lack of knowledge on the part of the processor (usually the case in home canning); (2) carelessness in handling the raw materials before canning, resulting in an unacceptably high level of contamination that ordinary heat processing may be inadequate to control; (3) equipment malfunction that results in undetected under processing; and (4) defective containers that permit the entrance of organisms after the heat process.

In this experiment you will have an opportunity to become familiar with some of the morphological and physiological characteristics of organisms that cause canned food spoilage, including both aerobic and anaerobic endospore formers of Bacillus and Clostridium, as well as a non-spore-forming bacterium.

MATERIALS REQUIRED:

Samples of canned food, hammer solder and soldering iron plastic bags gummed labels and rubber bands, can opener, punch type, small plastic beakers, Parafilm, gram-staining kit, spore-staining kit

PROCEDURES:

1. Label the sample container and In addition, place a similar label on one of the plastic bags to be used after sealing of the cans.

2. With an ice pick or awl, punch a small hole through a flat area in the top of each can. This can be done easily with the heel of your hand or a hammer, if available.

 3. Pour or take 1 ml or 1 g of a small amount of the liquid or solid food sample from the can.

 4. Use an inoculating needle to inoculate each can sample in to the saline.

5. Serial dilutes the sample from 10^-3 up to 10^-8 and transfer the diluted sample in the appropriate nutrient agar plate.

6. Tranfer 1 ml of serially diluted sample in to the nutrient agar plate which should be labeled their corresponding dilution.

7. Incubate the inoculated nutrient agar plates in the room temperature such as 37^o C for 24 hrs.

8. The same procedure can be repeated for detection of fungal load in the canned food. SDA Media can be used instead of nutrient agar and incubation can be extended up to 3 – 5 days to get fungal mycelia.

9. Record your observations on the report sheet on the demonstration table.

AGAROSE GEL ELECTROPHORESIS

AGAROSE GEL ELECTROPHORESIS

AIM:

To demonstrate Agarose gel electrophoresis in the microbiology laboratory

PRINCIPLE:

Agarose gel electrophoresis is a routinely used method for separating proteins, DNA or RNA. Nucleic acid molecules are size separated by the aid of an electric field where negatively charged molecules migrate toward anode (positive) pole. The migration flow is determined solely by the molecular weight where small weight molecules migrate faster than larger ones. In addition to size separation, nucleic acid fractionation using agarose gel electrophoresis can be an initial step for further purification of a band of interest. Extension of the technique includes excising the desired “band” from a stained gel viewed with a UV transilluminator. In order to visualize nucleic acid molecules in agarose gels, ethidium bromide is commonly used dye. Illumination of the agarose gels with 300-nm UV light is subsequently used for visualizing the stained nucleic acids. Furthermore, samples can be recovered and extracted from the gels easily for further studies. Another advantage is that the resulting gel could be stored in a plastic bag and refrigerated after the experiment. Depending on buffer during electrophoresis in order to generate a suitable electric current and to reduce the heat generated by electric current can be considered as limitations of electrophoretic techniques.

Materials Required:

Buffers and Solutions:

Agarose solutions, Ethidium bromide, Electrophoresis buffer.

Nucleic Acids and Oligonucleotides: DNA samples & DNA Ladders.

·         An electrophoresis chamber and power supply.

·         Gel casting trays, Sample combs, Electrophoresis buffer, usually Tris-acetate-EDTA (TAE) or Tris-borate-EDTA (TBE).

·         Loading buffer, which contains something dense (e.g. glycerol) to allow the sample to "fall" into the sample wells, and one or two tracking dyes, which migrate in the gel and allow visual monitoring or how far the electrophoresis has proceeded.

·         Ethidium bromide, a fluorescent dye used for staining nucleic acids.

·         Transilluminator (an ultraviolet light box), which is used to visualize ethidium bromide-stained DNA in gels.

 BUFFER AND AGAROSE GEL PREPARATION:

1.       Prepare a 50x stock solution of TAE buffer in 1000m of distilled H₂O:

For this weigh 242 g of Tris base in a chemical balance. Transfer this to a 1000ml beaker.
Prepare EDTA solution (pH 8.0, 0.5M) by weighing 9.31g of EDTA and dissolve it in 40ml distilled water. EDTA is insoluble and it can be made soluble by adding sodium hydroxide pellets. Check the pH using pH meter. Make the solution 100ml by adding distilled water.
Pipette out 57.1 ml of glacial acetic acid.Mix the Tris base, EDTA solution and glacial acetic acid and add distilled water to make the volume to 1000ml

2.       Prepare sufficient electrophoresis buffer (usually 1x TAE ) to fill the electrophoresis tank and to cast the gel:

 For this we take 2ml of TAE stock solution in an Erlenmeyer flask and make the volume to 100ml by adding 98ml of distilled water. The 1x working solution is 40 mM Tris-acetate/1 mM EDTA

3.      Prepare a solution of agarose in electrophoresis buffer at an appropriate concentration:

 For this usually 2 grams of agarose is added to 100ml of electrophoresis buffer.

Agarose Concentration in Gel (% [w/v])	Range of Separation of Linear DNA Molecules (kb)
0.3	5-60
0.6	1-20
0.7	0.8-10
0.9	0.5-7
1.2	0.4-6
1.5	0-2-3
2.0	0.1-2

4.      Loosely plug the neck of the Erlenmeyer flask. Heat the slurry in a boiling-water bath or a microwave oven until the agarose dissolves.

5.      When the molten gel has cooled, add 0.5µg/ml of ethidium bromide. Mix the gel solution thoroughly by gentle swirling. 

6.      While the agarose solution is cooling, choose an appropriate comb for forming the sample slots in the gel.

7.      Pour the warm agarose solution into the mold and the gel should be between 3 - 5 mm thick. 

8.       Allow the gel to set completely, then pour a small amount of electrophoresis buffer on the top of the gel, and carefully remove the comb. Pour off the electrophoresis buffer. Mount the gel in the electrophoresis tank.

9.      Add just enough electrophoresis buffers to cover the gel to a depth of approx. 1mm.

10.  Mix the samples of DNA with 0.20 volumes of the desired 6x gel-loading buffer.

11.  Slowly load the sample mixture into the slots of the submerged gel using a disposable micropipette or an automatic micropipettor or a drawn-out Pasteur pipette or a glass capillary tube. Load size standards into slots on both the right and left sides of the gel.

12.  Close the lid of the gel tank and attach the electrical leads so that the DNA will migrate toward the positive anode (red lead). Apply a voltage of 1-5 V/cm.

13.  Run the gel until the bromophenol blue have migrated an appropriate distance through the gel.

14.   The gel tray may be removed and placed directly on a transilluminator. When the UV is switched on we can see orange bands of DNA. 

CONGLUSION:

Schematic illustration of a typical horizontal gel electrophoresis setup for the separation of nucleic acids.

Nucleic acids running on an electrophoresis can be detected by staining with a dye and visualized under 300-nm UV light. Staining and visualization of DNA are conducted by using either ethidium bromide. Ethidium bromide can be used to detect both single- and double-stranded nucleic acids (both DNA and RNA). In fact, most fluorescence associated with staining single-stranded DNA or RNA is attributable to binding of the dye to short intrastrand duplexes in the molecules. The banding pattern of DNA resolved through the gel by recorded images. Images of ethidium bromide stained gels may be captured by using transmitted or incident UV light.

However, the amount of nicking of the DNA is much lower at 302 nm compared to 254 nm. If SYBR Green used instead of ethidium bromide another 10-20-fold increase in the sensitivity using conventional image taking techniques is in the range of possibility. Detection of DNAs stained with this dye requires the use of a yellow or green gelatin or cellophane filter with the camera along with the illumination with 300-nm UV light.

Gel electrophoresis based image analysis. Agarose gels, stained by Ethidium bromide under the UV illuminator

SUCROSE GRADIENT PROCEDURE

DENSITY GRADIENT CENTRIFUGATION-SUCROSE GRADIENT

AIM:

 To prepare a density gradient centrifugation for the separation of molecules from the sample solution

PRINCIPLE:

Sucrose density gradient ultracentrifugation is a powerful technique for fractionating macromolecules like DNA, RNA, and proteins. For this purpose, a sample containing a mixture of different size molecules is layered on the surface of a gradient whose density increases linearly from top to bottom. During centrifugation, different sized molecules sediment through the gradient at different rates. The rate of sedimentation depends, in addition to centrifugal force, on the size, shape, and density of the molecules, as well as on the density and viscosity of the gradient. In this way, molecules are separated by size with larger ones sedimenting towards the bottom and lighter ones remaining close to the top of the gradient. The method has been particularly successful in the size fractionation of large molecules.

MATERIALS REQUIRED:

-50%, 40%, 30%, and 25% (w/v) Sucrose solution

-Ultracentrifuge, Centrifuge tubes, Micropipette tips, Beakers

PROCEDURE:

1.      The gradient is prepared by layering progressively less dense sucrose solutions upon one another; therefore the first solution applied is the 50 % sucrose solution.

2.      Firstly a tube is held upright in a tube stand.

3.      Next a 200 μl pipettor tip is placed on the end of a 1000 μl pipettor tip.  Both snugly fitting tips are held steady by a clamp stand and the end of the 200 μl tip is allowed to make contact with the inside wall of the tube. 

4.      Now sucrose solutions can be placed inside the 1000 μl tip and gravity will feed the solutions into the tube slowly and steadily, starting with the 50 % solution.

5.      Once the 50 % solution has drained into the tube, the 40 % solution can be loaded into the 1000 μl tip which will then flow down the inside of the tube and layer on top of the 50 % solution. 

6.      This procedure is continued with the 30 % and 25 % respectively. 

7.      Once the sucrose gradient is poured discrete layers of sucrose is visible.

8.       Centrifugation should begin as soon as possible.

9.      However all centrifugation procedures require a balanced rotor therefore another tube containing precisely the same mass must be generated. 

10.  In practice this means 2 gradients must be prepared although the second gradient need not contain an experimental sample but could contain 0.5 ml water in place of the 0.5 ml of sample.  

11.  The tubes are centrifuged in a swinging bucket type rotor at 37 500 rpm for 16 hours 30 minutes at 4^oC.

12.  Immediately after the run the tube should be removed from the rotor, taking great care not to disturb the layers of sucrose.

13.  For fraction collection the tube should be held steady and upright by a clamp stand.  A tiny hole should be introduced into the very bottom of the tube using a fine needle.  The hole should be just big enough to allow the sucrose solution to drip out at approximately 1 drop per second. 

14.  Fractions of equal volume are then collected in eppendorf tubes below the pierced hole.  The fractions can now be stored at – 80^oC.

RESULT:

When separating a sample, discrete layers were observed.

The diagram below demonstrates this process in the collection of different fraction.

Collecting fractions from sucrose density gradient

PRODUCTION OF BIOFERTILIZER-mass cultivation of AZOSPIRILLUM

PRODUCTION OF BIOFERTILIZER-AZOSPIRILLUM

AIM:

To isolate and mass cultivate the biofertilizer Azospirillum.

INTRODUCTION:

Azospirillum was first described as Spirillum lipoferum by beijerinck in 1925 as a nitrogen fixing bacterium. Tarrand et al., (1978) renamed this organism as Azospirillum (N-fixing Spirillum). Azospirillum have been found to be associated with the roots of rhizosphere of many members of the Gramineae particularly in the tropics. Digitaria, maize, sorghum, rice, sugarcane, wheat and forage grasses are most frequently cited as hosts. Azospirillum species are characterized as micro aerobically nitrogen fixing bacteria. Diverse nitrogen fixing organisms contribute to the soil nitrogen pools. The major biological nitrogen fixation systems include cyanobacteria and photosynthetic bacteria that inhibit flood waters and soil surface and heterotrophic bacteria present in the rhizosphere and bulk soil. Azospirillum is recognized as a ubiquitous soil organism capable of colonizing effectively not only the roots of a wide variety of plants but also their above ground portions forming apparently an associative symbiosis

MATERIALS REQUIRED: 

Crystal violet, Safranin, Malate Semisolid Medium, BMS Agar, Lignite, CaCO_3, Polythene bags, Nfb medium (Nitrogen Free Bromothymol Blue (Nfb) Medium)

PROCEDURE:

Collection of soil samples:

  1. The soil samples were collected at 0.30 cm soil depth.

 2. Collected samples were mixed and placed in sterile polythene bags that was sealed and then brought to the laboratory and kept at 5-10⁰c.

  3. To 9 ml sterilized distilled water in a test tube 1 g of soils were added and mixed thoroughly from this 1 mL of diluted sample was transferred into another tube.

4. This process was continued upto 10^-9 dilution from the above diluted samples 1mL of 10^-5, 10^-6, 10^-7, 10^-8, were inoculated into NFb semisolid medium and incubated into 48 hours of 28± 2⁰c in the incubate.

 Isolation of Azospirillum:

  5. Isolation from soils 25 mL test tubes with 5 mL of NFb semi-solid medium were inoculated with one gram rhozophere soil were inoculated with malate semisolid medium.

  Bacterial smears preparation:

  6. Flame heat fixed smears prepared from light suspension of cells, flooded with crystal violet solution for 1 minute and washed for 30 seconds in the running water.

7. Rinse off excess water and flooded in iodine solution for 1 min and again washed in running water for 30 seconds.

8. Slides were passed through iodinated alcohol solution for removing excess stain and washed with tap water for five seconds.

9. The excess water removed and flooded with counter stain (safranin) for 1 minute and again washed in tap water, air dried and examined under microscope.

 Mass inoculums production:

 10. The isolated strains were used for large scale multiplication. The isolated strain selected for the preparation of Azospirillum inoculums.

11. These strains were inoculated into BMS agar slants. They are called starter culture.

12. These starter cultures were transferred into 100mL NFb liquid medium containing 250mL conical flask.

13. These cultures were incubated at 28±2⁰c for 4 days with occasional shaking of the conical flasks, for proper aeration.

14. After 4 days, the cultures were mixed with carrier materials. Lignite was used as carrier for Biofertilizer production, 4kg lignite used per liter of broth culture for mixing.

15. After mixing 2% CaCO3 was added and the mixed inoculums covered by polythene sheets for curing for 24 hours. After 24 hours the carrier based inoculums were pocketed into polythene bags.

Media Preparation:

Nfb medium (Nitrogen Free Bromothymol Blue (Nfb) Medium)

DL-malic acid: 5.0 g
K₂HPO₄: 0.5 g
MgSO₄ • 7H₂O: 0.2 g
NaCl: 0.1 g
CaCl₂ • 2H₂O: 0.02 g
Micronutrient solution: 2 ml
Bromthymol blue solution (0.5% in 0.2N KOH): 2 ml
Fe(III) EDTA (1.64%): 4.0 ml
Vitamin solution: 1.0 ml
Distilled water: 1.0 L

Adjust pH to 6.8
For semisolid medium, add 0.5 g of agar; for solid medium, add 15 g of agar. Autoclave at 121°C for 15 min.

Micronutrient solution:
CuSO₄ • 5H₂O: 0.4 g
ZnSO₄ • 7H₂O: 0.12 g
H₃BO₃: 1.4 g
Na₂MoO₄ • 2H₂O: 1.0 g
MnSO₄ • H₂O: 1.5 g
Distilled water: 1.0 L
Vitamin solution:
Biotin: 10 mg
Pyridoxol HCL: 20 mg
Distilled water: 0.1 L

BMS AGAR (POTATO INFUSION AGAR MEDIUM)

Components gms/litre

Potatoes 200 g

Mallic acid 2.5

KOH 2.0

Cane sugar 2.5

Biotine 2.1

 Cook washed potatoes and filter through cotton. Prepare potassium malate by dissolving 2.5g malate in 50ml water adding 2 drops of Bromothymol blue (0.5% ethanol) and 2 g of KOH. Adjust pH to green colour. Add malate, sugar, biotine to potato filtrate and dilute to 1000ml. Solid media is obtained by adding 2% agaragar.

Production of biofertilizer-Phosphobactria

PRODUCTION OF BIOFERTILIZER-PHOSPHOBACTERIA

AIM:

To produce biofertilizer Phosphobacteria by mass cultivation.

INTRODUCTION:

Biofertilizer  is a substance which contains living micro organisms which ,when applied to the seed ,plant surfaces or soil colonizes the  rhizosphere or the interior of the plant and promotes growth by increasing the supply or availability of primary nutrients to the host plant. Bio-fertilizers add nutrients through the natural processes of nitrogen fixation, solubilizing phosphorus, and stimulating plant growth through the synthesis of growth-promoting substances. Bio-fertilizers can be expected to reduce the use of chemical   fertilizers and pesticides. Bio-fertilizers provide eco-friendly organic agro-input and are more cost-effective than chemical fertilizers. Since a bio-fertilizer is technically living; it can symbiotically associate with plant roots. Involved microorganisms could readily and safely convert complex organic material in simple compounds, so that plants are easily taken up. It maintains the natural habitat of the soil. It increases crop yield by 20-30%, replaces chemical nitrogen and phosphorus by 25%, and stimulates plant growth. It can also provide protection against drought and some soil-borne diseases.

Phosphobacteria means microbial inoculants capable of phosphate solubilizing nature. Commonly used Phosphobacteria is Bacillus megaterium. Phosphobacteria is suitable for all crops. Phosphorus besides to  nitrogen  is one of the most important element in crop production.It makes about 0.2% of  the  total dry weight of the  plants. It is a plant nutrient that is essential for food synthesis, flower formation, fruit setting and seed setting. When Phosphobacteria is added in soil it produces organic crops and makes it to function well in alkaline soils. It is recommendable to crops of all categories

MATERIALS REQUIRED: 

     The Pikovskayas Media, conical flask, Petri plates, Inoculation loops, Cotton plugs, L rods

PROCEDURE:

1. The soil samples were collected from various fields and serial dilutions were done.

2. The organism was isolated by the analysis of the characteristics according to the Morphological and Biochemical characteristics.

3. The various biochemial tests conducted were citrate utilization, catalase, urease, indole, methyl red, vogues prokauer,H2S and nitrate reduction test  were performed and confirmed.

4. Then using the specific medium Pikovskayas medium for phosphobacter was used to grow the organism for the mass production. 

Mass production:

5. For mass production of Phosphobacteria, is isolated from various regions and grown on slants for preservation as per need culture from slant were transferred to liquid broth of selective as well as optimized medium in the rotary shaker for 4 days to prepare starter culture.

6. Later on the starter cultures is transferred to the fermenter in batch culture mode with proper maintenance of 30⁰C and continuous agitation for 4-9 days.

7. When cell count reached to 10⁸- 10⁹ cells/ml, the broth used as inoculants.

8. For easy handling, packing, storing and transporting broth is mixed with an inert carrier material which contains sufficient amount of cells. After proper mixing carrier containing inoculant was left for 7days and above formulated microbial inoculants used as biofertilizer.

RESULT AND DISCUSSION:

MUSHROOM CULTIVATION PROCEDURE

BUTTON MUSHROOM CULTIVATION

AIM: To cultivate the button and oyster mushroom

INTRODUCTION

A mushroom is described as the “fruiting body of a fungus plant that typically appears above the ground and contains spores”. It is this fleshy bracket (fruiting body) that is commonly eaten and which reproduces by dispersing spores in the same way that other plants disperse seeds. Instead of drawing nutrients through the roots, fungi are sustained by a network of fine, microscopic threads known collectively as the “mycelium”. This network can extend over vast distances, implanting into rotting wood, soil, or other preferred medium.

Fungi are more akin to molds and yeasts than to vegetable plants. Although mushrooms are technically part of the plant kingdom, they are very different organisms since they do not contain chlorophyll or have a root system. Mushrooms must also rely on organic material for their nutrition and do so in three ways:

• as saprophytes (living on dead wood or dead tissue of living trees or dung)
• as parasites (attacking living plant or animal tissue), or
• as mycorrhizae (having a symbiotic relationship with plants).

To separate some of the confusion as to what is a simple fungus and what is a mushroom, scientists now generally use the term 'mushroom' to encompass fungi of either the order Agaricales or the order Boletales.

MATERIALS REQUIRED:

Spawn, PDA media, paddy straw, Calcium carbonate, polypropylene cover, autoclave, gloves, jude threads, Cotton

SPAWN PREPARATION:            Procedure Wash the sorghum/paddy straw grains in water thoroughly to remove chaffy and damaged grains. Cook the grains in an autoclave / vessel for 30 minutes just to soften them. Take out the cooked grains and spread evenly over a dry platform to remove the excess water. Mix Calcium carbonate (CaCO3) thoroughly with the cooked, dried grains/paddy @ 20 g / kg. Fill the grains in polypropylene bags up to ¾ th height (approximately 300-330 g/bag), insert a PVC ring, bold the edges of the bag down and plug the mouth tightly with non-absorbent cotton wool. Cover the cotton plug with a piece of waste paper and tie tightly around the neck with a jute thread. Arrange the bags inside an autoclave and sterilize under 20 lbs. pressure for 2 hours. Take out the bags after cooling and keep them inside the culture room and put on the UV light. After 20 minutes put off the UV light and start working in the culture room. Cut the fungal culture into two equal halves using a inoculation needle and transfer one half portion to a bag. Similarly, transfer another half portion of the culture to an another bag. Incubate the inoculated bags in a clean room under room temperature for 10 days for further use to prepare bed spawn. MUSHROOM BED PREPARATIONS The cultivation of oyster mushroom is usually carried out in transparent polythene covers. The size of the cover should be 60 x 30 cm, with a thickness of 80 gauges. Procedure Wash hands thoroughly with antiseptic lotion. Take the polythene cover and tie the bottom end with a thread and turn it inwards. Shade dry steam sterilized straw to get a uniform moisture level in all areas. Take out a well-grown bed spawn, squeeze thoroughly and divide into two halves. (Two beds are prepared from the single spawn bag) Fill the straw to a height of 3” in the bottom of polythene bag, take a handful of spawn and sprinkle over the straw layer , concentrating more on the edges.    Fill the second layer of the straw to a height of 5” and spawn it as above.  Repeat this process to get five straw layers with spawns.   Gently press the bed and tie it tightly with a thread. Put 6 ventilation holes randomly for ventilation as well as to remove excess moisture present inside the bed. Arrange the beds in side the thatched shed, (Spawn running room) following Rack system or hanging rope system. Maintain the temperature of 22-25° C and relative humidity of 85-90 % inside the shed. Observe the beds daily for contamination, if any. The contaminated beds should  be removed and destroyed. Similarly, observe regularly for the infestation of insect pests viz., flies, beetles, mites etc., If noticed, the pesticide like Malathion should be sprayed in side the shed @ 1 ml per litre of water. The fully spawn run beds can be shifted to cropping room for initiation of buttons. OYSTER MUSHROOM CULTIVATION             The fully spawn run beds should be transferred to cropping room in the thatches shed, where the diffused light and good ventilation are necessary for the button development. There are different methods to handle the spawn run bed to initiate button development. They are Open bed method, wherein the polythene cover is completely removed and allowed for cropping. Closed bed method, wherein the polythene cover is intact and buttons will come out through the holes made on the cover. Half cover open method, wherein the one half of the polythene cover is removed for cropping and second half after first harvest. Stripe method, wherein the polythene cover as longitudinal strips of 5-cm breadth at 4-5 places in the bed. Tear method, wherein the polythene cover is teared longitudinally at several places. Round opening method, wherein the round shaped openings of 5 cm diameter are made at random. However, among all the methods of opening of beds, complete removal of the polythene cover is found to give more yields than others. The steps followed in full opening of bed are described below Procedure:  Use a new blade and cut the polythene covers and remove fully. Allow the bed to dry for a day, as freshly opened beds contain more moisture. Spray water on the beds from second day of opening using an atomizer. Observe the beds regularly, if any bed  showing contamination should be removed. Two to three days after opening pinheads of mushroom button develop which will be ready for harvest with in another 4 days Harvest the entire bunch of mushroom gently in the early hours of morning. Remove the straw bits adheres to the mushroom and cut off the bottom portion of the stalk. Pack neatly in a polythene cover @ 200 g per bag and put a few ventilation holes. Keep them in an icebox and send to sales unit immediately. After harvest, scrap out the mushroom bed with a new comb to remove dried and rotten buds of mushroom. Spray the beds daily, based on the conditions of the beds two to three sprays may be needed.( Second harvest can be done 7-10 days after the first harvest) After second harvest, scrap out the outer layers as above and spray water regularly. (Third harvest can be had after a week or ten days). Dispose the beds after third harvest as it is uneconomical to keep the beds further. RESULT AND DISCUSSION: