DESC:FIELD OF THE INVENTION:
The present disclosure relates to a portable device for estimating microbial content in water. More specifically it relates to a novel portable spread plate (NPSP) device for estimating microbial content in water under aseptic conditions, in the absence of a laminar airflow cabinet and a method for estimating microbial contamination in water under aseptic conditions in the absence of a laminar airflow cabinet using the same.

BACKGROUND OF THE INVENTION:
A Petri dish, invented by Julius Richard Petri, also known as a Petri plate is a shallow transparent dish with a lid, used by the biologists to hold growth medium in which cells are cultured. Petri dishes are usually cylindrical and vary in diameters from 30 to 200 mm. They have been traditionally made of glass so that they can be sterilized. The lids in this case are loose fitting. Subsequently, disposable Petri plates have been made from transparent plastics such as Polystyrene. The lids in this case can be loose fitting to delay the drying of the contents. Small holes could be made round the rim, or ribs may be provided on the underside of the cover. This allows air flow over the culture and prevents condensation of water. In some cases, grids could be printed on the bottom to help measure the density of cultures. A bacterial culture when spread on the medium placed on the petri plate, leads to bacterial growth which is seen as colonies.
Petri plates are widely used to cultivate microorganisms such as bacteria, yeasts, and molds and are most suited for microorganisms that thrive on solid or semisolid surfaces. The culture medium is often an agar layer a few mm thick, containing nutrients on which the microorganism can grow, including but not limited to salts, carbohydrates, protein hydrolysates, amino acids and optionally dyes, indicators, and medicinal drugs. The constituents are dissolved in warm water or water at room temperature and heat sterilized at 121°C for 15 minutes at 15 lbs pressure. After sterilization cycle, the molten (~40°C) medium is poured into the dish and left to cool down. Once the medium solidifies, a sample of the microorganism is inoculated under aseptic conditions, normally in a laminar airflow cabinet. The plates are then left undisturbed for the microorganisms to grow, in an incubator if necessary. They are usually covered to minimize contamination due to airborne spores. The colonies are counted at the end of the growth phase.
The performance of Petri plates depends on the composition and preparation of the agar gel. The ability of the agar gel to set depends on the concentration of the agar powder, type and content of sugar, type and content of other nutrients, pH and temperature. These parameters and storage conditions determine the duration for which the gel can be stored without losing its integrity and performance. Moisture loss leads to the agar drying out while moisture condensation on the lid and dripping onto the agar surface leads to contamination.
Various compositions of the agar gel have been reported to meet the experimental objectives.
Hoben and Somasegaran (1982), compared Pour, Spread, and Drop plate methods for enumeration of Rhizobium spp. in inoculants made from presterilized Peat. The three plating methods were found to be interchangeable. The drop plate method was preferred because of its economy in materials and labor.
Reasoner and Geldreich (1985) reported two new media, for assessing the aerobic heterotrophic plate count of treated drinking water and for subculture of aquatic bacterial isolates, their characterization and identification.
Milagres et al (1999) described a modification in the chrome azurol sulfonate (CAS) - agar assay for detection of siderophores in solid medium so that it could be applied to a wide range of microbial species, including fungi and Gram-positive bacteria.
Microbial cultivation technologies require highly subdivided multi well plates as well as methods of sorting encapsulated microcolonies. Ingham et al (2007) reported a micro-Petri dish, a million-well growth chip for the culture and high-throughput screening of microorganisms. A range of disposable, surface-culture, microbial growth chips, or ‘‘micro-Petri dishes,’’ were fabricated using a micro-engineered mechanical systems (MEMS) approach to facilitate growth compartments on top of porous aluminium oxide (PAO), which act as the surface on which a large number of microbial samples were grown, assayed, and recovered.
Kasana et al (2008) reported an improved method for the detection of extracellular cellulase production by microorganisms using plate assay. Carboxymethyl cellulose (CMC) plates were flooded with Gram’s iodine instead of 1% hexadecyltrimethylammonium bromide or with 0.1% Congo red followed by 1 M NaCl. Gram’s iodine formed a bluish black complex with cellulose but not with hydrolyzed cellulose, giving a sharp and distinct zone around the cellulase-producing microbial colonies within 3 to 5 minutes.
Sanders (2012) described the plating methods used in the laboratory to isolate, grow, and enumerate microorganisms. More particularly the operation and performance of pour plate and spread plate was described. According to the author, spread-plate technique has an advantage because the colonies that form using this technique are evenly distributed across the surface of the agar medium and cells from individual colonies can be readily isolated.
Apart from use of Petri plates for wide ranging applications in microbiology / biotechnology illustrated above, Pour plates and Spread plates have been routinely used in the past for the estimation of microorganisms in water especially to ascertain the effectiveness of water treatment techniques.
Taylor et al (1983) analysed water samples collected during flushing of dead-end mains for bacterial count by using both pour plate and spread plate procedures. According to the authors pour plate procedure was neither as accurate nor as precise as the spread plate procedure for enumerating the heterotrophic plate count population.
Reasoner and Geldreich (1985) compared several new media for pour and spread plate methods. The standard pour plate procedure yielded the lowest bacterial counts. These results confirmed earlier findings of Means et al (1981). It was concluded that the spread plate method was more accurate than the pour plate method and had additional advantages, including colony development and appearance and the option of preparing plates well in advance of sample processing.
Terrones-Fernandez et al (2023) compared the performance of the pour plate method and the spread plate method. The spread plate technique was considered time and material-consuming and susceptible to human error, and could compromise the accuracy of the enumeration. The authors described the limitations of the pour plate method and suggested a modification wherein it was demonstrated that separating agar from the other components of commonly used media during sterilization and reducing the agar concentration to 10 g/L enhanced microbial growth. Lower concentration of agar also facilitated the visualization of the embedded colonies. It was also quoted that the method also improved the culturability of reticent bacteria and slow-growing bacteria from natural environments. Modified Sabouraud dextrose agar (SDA) yielded better growth for Saccharomyces cerevisiae. The modification of tryptic soy agar (TSA) resulted in significantly higher counts of Staphylococcus aureus, Salmonella typhimurium, and Candida albicans. Improved microbial growth and selectivity resulted by reducing the agar concentration of the culture medium and separating the nutrients and gelling agent during sterilization.
It may be noted that all the investigations here to far need to be carried out in the laboratory under aseptic conditions, normally in a laminar air flow cabinet.
However, there are numerous situations wherein the microorganism content needs to be estimated under conditions where such provisions are not readily available. These include but are not limited to evaluation of wastewaters, drinking water in public places, in remote areas where facilities for testing under aseptic conditions are not available, process water used in industrial establishments, inspection of water bodies etc. More specifically the presence of microorganisms in cooling tower plants leads to biofilm formation which results in fouling and deterioration of cooling tower performance.
Thus, there is a need for a device and a method to estimate microorganism loading in such water samples on site under aseptic conditions in the absence of a laminar air flow cabinet.

SUMMARY OF THE INVENTION:
The inventors of this disclosure have devised a novel portable spread plate (NPSP) that enables carrying out microorganism estimation in water samples under aseptic conditions in places where the laminar airflow cabinets are not available. It also discloses a method for estimating the microorganism content using the NPSP.
In an embodiment of the invention the device is portable.
In an embodiment of the invention, the device is a novel portable spread plate (NPSP).
In an embodiment of the invention, the NPSP comprises a Petri dish, a lid, the spreader bar, syringe filter and the gel.
In an embodiment of the invention the NPSP is made up of materials selected from glass, borosilicate glass, polystyrene, polycarbonate, polysulfone, polyethersulfone, cyclic olefin copolymers, transparent polypropylene, poly methyl methacrylate and styrene acrylonitrile copolymers.
In an embodiment of the invention, the lid of the NPSP houses a spreader which is rotatable from outside the lid, without opening the spread plate device.
In an embodiment of the invention, the lid and the body of the NPSP are snug fitting.
In an embodiment of the invention, the lid and the body of the NPSP are sealed using an adhesive tape.
In an embodiment of the invention, the lid and the body of the novel portable spread plate (NPSP) are sealed using Parafilm.
In an embodiment of the invention, the lid and the body of the NPSP are made of the same material.
In an embodiment of the invention the body of the NPSP is circular in shape.
In an embodiment of the invention the diameter of the NPSP is selected from 30 mm to 200 mm.
In an embodiment the length of the spreader attached to the lid of the NPSP is selected from 12 mm to 95 mm.
In an embodiment of the invention the height of the spreader bar above the lid is 10 mm to 25 mm.
In an embodiment of the invention the spreader bar is made of a material selected from polyethylene, polypropylene, high impact polystyrene and glass.
In an embodiment of the invention the vertical movement of the spreader bar is controlled by the bush and lock mechanism. (Figure 1E, 1F)
In an embodiment of the invention, the lid and the body of the NPSP improved spread plate are made not of the same material.
In an embodiment of the invention, the lid and the body of the NPSP improved spread plate are made of the same material.
In an embodiment of the invention, the NPSP enables carry out microorganism estimation in water under aseptic conditions. in the absence of a laminar air flow cabinet.
In an embodiment of the invention, the NPSP enables estimation of “heterotrophic bacteria” which includes all bacteria that use organic nutrients for growth.
The term "heterotrophic bacteria" includes primary and secondary bacterial pathogens including, but not limited to Escherichia, Klebsiella, Enterobacter, Citrobacter, Pseudomonas.
In an embodiment of the invention, the microorganisms that can be estimated are selected from Escherichia coli, Salmonella enterica, Staphylococcus aureus, Shigella sonnei, Pseudomonas aeruginosa, Enterobacter cloacae, Enterococcus faecalis, Vibrio cholerae.
In an embodiment of the invention, the differential / selective growth media used in the NPSP are selected from MacConkeys Agar, Eosin Methylene Blue Agar, HiCromeTM Coliform Agar, HiCromeTM MacConkey Sorbitol Agar Base, Xylose Lysine Deoxycholate (XLD) Agar, Salmonella-Shigella Agar, Wilson Blair Agar, Mannitol Salt Agar, Cetrimide Agar, Pseudomonas Agar Base, Enterobacter Sakazakii Agar, Enterococcus Selective Agar, Potassium Tellurite Agar and Thiosulfate-Citrate-Bile Salts-Sucrose Agar.
In an embodiment of the invention, the common growth media used in the NPSP are selected from Soybean casein digest agar and Nutrient agar,
In an embodiment of the invention the growth medium used in the NPSP is a blend of the common growth media and the differential / selective growth media
In an embodiment of the invention the number of media is selected from 1 to 6.
In an embodiment of the invention, the contaminating microorganism is identified.
In an embodiment of the invention, the biocide for the contaminating microorganism is identified to treat the contamination.

BRIEF DESCRIPTION OF DRAWINGS:
The objectives and advantages of the present invention will become apparent from the following description read in accordance with the accompanying drawings wherein,
FIG. 1 depicts the components of the Novel portable spread plate (NPSP) of the present invention;
FIG. 2 shows the completely assembled Novel portable spread plate (NPSP) of the present invention;
FIG. 3 shows images of E. coli NB22 colonies on SCDA media, 1. Control (un inoculated); 2,3 and 4 are triplicates of inoculated sample of stock executed in the laminar air flow (LAF) cabinet using conventional spread plate method;
FIG. 4 shows E. coli NB22 colonies on SCDA media, 1. Control (un inoculated), 2,3 and 4 are triplicates of inoculated sample of stock executed in LAF cabinet using NPSP (coloured colonies due to reduction of TTC dye);
FIG. 5 shows a) Top row: E. coli NB22 colonies on SCDA medium, 1) control 2), 3), 4) triplicates tests executed in LAF unit; b) Middle row: 5) control 6), 7),8) triplicates of NPSP on SCDA with TTC dye executed in close room but not LAF unit. Sample 7 shows a single colony, indicating no contamination from outside; c) Bottom row 9) control 10, 11,12, triplicates of spread plate on SCDA executed in open conditions (lid kept open) Sample 10 shows colonies other than E. coli, indicating contamination from outside;
FIG. 6 shows E. coli NB22 colonies on SCDA media with TTC dye, 1. Control (un inoculated), 2 (found minute colonies as contaminants, 3 and 4 are triplicates of inoculated sample of stock executed in open area (cooling tower site);
FIG. 7 shows E. coli NB22 colonies on SCDA media with TTC dye with, 1. Control (un inoculated), 2, 3 and 4 are triplicates of inoculated sample of stock executed in open area (cooling tower site) using NPSP;
FIG. 8 shows a petri plate of 150 mm diameter accommodated with 7 different media at left side (1) and at right side (2) having growth of specific microbes representing consortium when inoculated
FIG. 9 shows images of petri plates having various media wherein on left side all media are treated as a control (Un-inoculated), on right side,
1A, represents Mannitol Salt Agar (MSA), HiCromeTM Staph Agar (HSA) showed growth of Staphylococcus aureus 2592
2A, represents HiCromeTM Coliform agar (HCA) and MacConkey’s Agar (MCA) showed growth of Escherichia coli 11775
3A, represents Brain Heart Infusion Agar (BHI), Enterococcus Agar Base (EAB) showed growth of Enterococcus faecalis 19433
4A, represents Enterobacter Sakazakii Agar (ESA) and MacConkey’s Agar (MCA) showed growth of Enterobacter cloacae 13047
5A, represents Centrimide Agar (CA), showed growth of Pseudomonas aeruginosa 10145 but not on Pseudomonas Agar Base (PAB)
6A, represents Deoxycholate Agar (DCA), Wilson Blair Agar (WBA) showed growth of Salmonella enterica 4931;
FIG. 10A shows effect of ferroin indicator on growth of E. coli NB22;
FIG. 10B shows effect of TTC indicator on growth of E. coli NB22;
FIG. 10C shows effect of resazurin indicator on growth of E. coli NB22;
FIG. 10D shows effect of INT indicator on growth of E. coli NB22;
FIG. 10E shows a control SCDA plate with no indicator added;
FIG. 11 shows a 150 mm Petri plate having six medium in equal sections. (A) Sample inoculated in Laminar air flow cabinet on spread plate, (B) Sample inoculated with syringe filter in open environment on NPSP, (C) Sample inoculated (open lid) in open environment on spread plate, (D) Control plate- Un-inoculated with sample;
FIG. 12 shows a 150 mm Petri plate having six equal sections with different concentrations of cetrimide evaluated for (A) P. aeruginosa (B) E. coli (C) P. aeruginosa + E. coli with CSP and NPSP performed in Laminar air flow cabinet as well as open conditions; and
FIG. 13 shows analysis of different market samples under LAF (Left column), Open-NPSP (middle column) and Open (lid open) conditions using NPSP of present invention.

DESCRIPTION OF THE INVENTION:
References in the specification to "one embodiment" or "an embodiment" means that a particular feature, structure, characteristic, or function described in connection with the embodiment is included in at least one embodiment of the invention. The appearances of the phrase “in one embodiment” in various places in the specification are not necessarily all referring to the same embodiment.
References in the specification to “preferred embodiment” means that a particular feature, structure, characteristic, or function described in detail thereby omitting known constructions and functions for clear description of the present invention.
The foregoing description of specific embodiments of the present invention has been presented for purposes of illustration and description. They are not intended to be exhaustive or to limit the present invention to the precise forms disclosed and obviously many modifications and variations are possible in light of the above teaching.
The Novel Portable Spread Plate is fabricated as follows.
A hole (2 mm-5 mm dia.) is drilled in the centre of the lid of the petri dish. If the petri dish material is glass, the hole is drilled by glass blowing. If the material is a plastic, the hole is drilled using a drill machine or a heated rod. It is ensured that the hole has a smooth finish.
Another hole (2 mm-5 mm dia.) is drilled near the periphery of the lid such that the centre of the hole is 5 mm to 15 mm from the periphery of the lid.
Syringe filters of diameter 13 mm to 33 mm and filter pore diameter 5µ are procured from the market.
The syringe filter is mechanically snug fitted in to the hole drilled near the periphery of the lid of the petri dish and optionally further sealed using an adhesive.
The adhesive for sealing the syringe filter is selected from an epoxy, acrylate, and polyvinyl acetate adhesive.
The membrane filter of the syringe filter is made of a material selected from nylon, polyvinylidene fluoride, cellulose and polysulfone.
For the spreader bar, cell spreaders, more particularly L shaped cell spreaders can be procured from labware suppliers i.e. Sigma Aldrich, Labfriend India and Bio Plas USA.
The height of the cell spreader is 10 mm-25 mm.
The long arm of the cell spreader is cut such that when inserted in the lid of the petri dish through the hole drilled in the centre, the distance between the tip of the long arm and the edge of the lid is 4-8 mm.
The spreader bar, optionally along with the bush lock, is inserted in the lid of the petri dish through a flexible bushing inserted in the hole drilled in the centre so as to create a seal, allowing rotation of the spreader rod along its axis.
The flexible bushing is fabricated from a material selected from silicone, nylon, polyolefins and elastomers. (See Figure 1D)
Petri dish lid fitted with the spreader bar and syringe filter and the petri dish container are sterilized by autoclaving at 121°C, 15 psi for 15 minutes and cooled to room temperature or alternatively sterilized by ? irradiation or ethylene oxide.
The sterilized components are transferred to laminar air flow cabinet.
The petri dish container is filled with the agar gel to a thickness in the range 3 mm - 12 mm.
The concentration of agar in the gel is in the range 1-3% wt / vol.
The agar gel is selected from MacConkeys Agar, Eosin Methylene Blue Agar, HiCromeTM Coliform Agar, HiCromeTM MacConkey Sorbitol Agar Base, Xylose Lysine Deoxycholate (XLD) Agar, Salmonella-Shigella Agar, Wilson Blair Agar, Mannitol Salt Agar, Cetrimide Agar, Pseudomonas Agar Base, Enterobacter Sakazakii Agar, Enterococcus Selective Agar, Potassium Tellurite Agar and Thiosulfate-Citrate-Bile Salts-Sucrose Agar.
Petri dish lid fitted with the spreader bar and syringe filter is snug fitted on the Petri dish filled with the Agar gel and is sealed using a sealant.
The sealant is selected from 1) Polyvinyl acetate adhesive, acrylate adhesive, epoxy adhesive 2) a flexible polyolefin film 3) Parafilm.
The sealed Petri dish is then flushed with inert gas such as nitrogen and carbon dioxide.
The syringe filter is plugged using a sterile cotton plug or a sterile plastic plug which is snug fitted in to the syringe filter.
The Novel Portable Spread Plate is now ready for use.
The method of use of the Novel Portable Spread Plate is illustrated below with examples, which are illustrative in nature and do not limit the scope of the invention in any manner.

EXAMPLES:
Only a few examples and implementations are disclosed. Variations, modifications, and enhancements to the described examples and implementations and other implementations can be made based on what is disclosed.
Examples are set forth herein below and are illustrative of different amounts and types of reactants and reaction conditions that can be utilized in practicing the disclosure. It will be apparent, however, that the disclosure can be practiced with other amounts and types of reactants and reaction conditions than those used in the examples, and the resulting devices various different properties and uses in accordance with the disclosure above and as pointed out hereinafter.
Example 1:
0.2 mL water sample to be analyzed was drawn into the syringe. The syringe was then connected to the syringe filter, attached on the top of the lid of NPSP of the present invention. After dispensing the sample, the spreader bar attached to the centre of the lid of NPSP was pushed down on the surface of the medium and rotated around across the surface 3-4 times. The spreader bar was then pulled up and returned to its original position, using optionally the bush lock (Figure 2). The NPSP was then incubated in an incubator for 24 hrs at 37 °C. The growth of the E. coli colonies was observed as a dark red coloured colony against the background of the light-yellow colour media. Number of colonies was counted and CFU/mL (colony forming units) was calculated according to the following formula.
Cfu/ml of sample= Number of colonies counted*5*dilution factor
Note: The 0.2 mL sample was taken from the test sample which was diluted appropriately by a suitable factor so as to get the number of colonies which can be counted. This represents the dilution factor.
Same assay procedure was carried out for the conventional standard spread plate except that the sample was inoculated directly on agar.
Two samples were examined as per the procedure mentioned above.
IEMBL-01
Sample was from tap water i.e. municipal water supply (visibly found less turbid)
IEMBL-02
Sample was from tap water municipal water supply (visibly found turbid)
The samples were doped with E.coli NB22 at two different concentration levels.
Table 1: Number of colonies of E coli on NPSP
Dilution Factor Count Range
(No. of cells on plate) Number of colonies counted on NPSP
Sample
(IEMBL-01) Sample
(IEMBL-02)
Step I
Dilution factor 1 01-40 20 Beyond Range
Step II
Dilution factor 10
41-400 - 35
Step III
Dilution factor 100
- - -

Dilution factor 10: (1 mL of original sample made up to 10 mL using sterile water)
Dilution factor 100: (1 mL of original sample made up to 100 mL using sterile water)
Table 2: Number of E coli of sample on NPSP (CFU/mL)
Dilution Factor Counted colonies On plate Colony count
(CFU/mL)
Sample
(IEMBL-01) Sample
(IEMBL-02)
Step I 20 - 100
Step II - 35 17500

Example 2:
1 to 2 mL of glycerol stocks of pure cultures of E. coli NB 22 and P. aeruginosa NB14 were reconstituted in 100 mL of sterile soybean casein digest broth (SCDB). These were further incubated for 24 hours at 37 °C at 120 rpm under shaking condition.
After 24 hrs, a loop full culture taken on nichrome loop was streaked over agar plate containing soybean casein digest agar (SCDA) medium. Pure and single colony culture of E. coli and P. aeruginosa on SCDA medium was selected as a starting material for further experimental work. Single colony transfer was carried out again in fresh SCDB medium and incubated for 24 hours at 37 °C at 120 rpm under shaking condition.
This broth culture was transferred to sterile centrifuge tubes. The broth was then centrifuged at 8000 rpm at 4 °C for 20 min and the pellet isolated, was collected. Pellet was reconstituted again with sterile saline (0.95% NaCl) and centrifuged at 8000 rpm at 4 °C for 20 min. This procedure was repeated two times. Finally the pellet was reconstituted into 100 mL sterile saline (Stock A, see Table 3). The optical density of the stock solution at 620 nm was measured using UV-Vis spectrophotometer. The stock was further examined for CFU counts using serial dilutions method (101 to 109).
On the basis of microorganism content of the stock (CFU/mL) obtained as above, the dilutions in steps of 1 × 107, 1 × 106, 1 × 105, 1 × 104, 1 × 103 and 1 × 102 were prepared. 0.2 mL of a sample for each dilution was then spread over SCDA medium plates and incubated at 37 °C for 24 hrs. Number of bacterial colonies were counted and expressed as CFU/mL.
The value of microorganism count (CFU/mL) obtained for each dilution was compared with serial dilutions prepared. The results obtained validated consistent growth behaviour of microorganism. All experimental work was carried out under aseptic conditions and standard protocols of microbiology were followed.
Table 3: Optical density and initial count of stock of E. coli NB22 and P. aeruginosa NB14
Parameters Batch I Batch II Batch III
Escherichia coli NB22
Optical Density at 620 nm 2.07 2.04 2.06
Initial count of stock A (CFU/mL)
350 x 107 630 x 107 580 x 107
Pseudomonas aeruginosa NB14
Optical Density at 620 nm 2.01 2.01 2.02
Initial count of stock A (CFU/mL) 570 x 107 795 x 107 685 x 107

Table 4: Validation of colony forming units of E. coli NB22
Dilutions Batch I Batch II Batch III
No. of colonies ×dilution Count
CFU/mL No. of colonies ×dilution Count
CFU/mL No. of colonies ×dilution Count
CFU/mL
1 x 107 156 × 104 0.7 × 107 166 × 104 0.94 × 107 205 × 104 1.02 × 107
1 x 106 20 × 104 1 × 106 131 × 103 0.83 × 106 158 × 103 0.8 × 106
1 x 105 32 × 103 1 × 105 19 × 103 1.0 × 105 189 × 102 0.94 × 105
1 x 104 25 × 102 1 × 104 123 × 101 1.2 × 104 25 × 102 1.2 × 104
1 x 103 156 0.7 × 103 166 1.3 × 103 19 × 101 0.95 × 103
1 x 102 24 1 × 102 10 1.3 × 102 19 0.95 × 102

Table 5: Validation of colony forming units of P. aeruginosa NB14

Dilutions Batch I Batch II Batch III
No. of colonies ×dilution Count
CFU/mL No. of colonies ×dilution Count
CFU/mL No. of colonies ×dilution Count
CFU/mL
1 x 107 235 × 104 1.1 × 107 29 × 105 1.4 × 107 31 × 105 1.5 × 107
1 x 106 185 × 10 3 0.92 × 106 115 × 10 3 0.6 × 106 249 × 10 3 1.2 × 106
1 x 105 30 × 10 3 1.5 × 105 197 × 10 2 1.0 × 105 253 × 10 2 1.2 × 105
1 x 104 37 × 10 2 1.8 × 104 21 × 10 2 1.0 × 104 191 × 10 1 0.95 × 104
1 x 103 235 1.1 × 103 15 × 10 1 0.75 × 103 241 1.2 × 103
1 x 102 43 2.1 × 102 15 0.75 × 102 30 1.5 × 102

The validations were individually carried out in three different experiments at different times. The results expressed as CFU/mL for various dilutions prepared were consistent within acceptable limits of variation (Table 4 and 5).
These experiments established the validity of the assay protocols.
Example 3:
Concentration of agar in the medium was varied in the range 1% to 3 % w/v to evaluate the effect of agar concentration on colony counts. Respective media varying in agar content were sterilized at 121 °C for 15 min. After sterilization cycle, each medium was plated in the conventional standard Petri plate (CSP) and NPSP, and was allowed to solidify under aseptic conditions in a laminar air flow cabinet.
SCDA plates preparation, inoculums preparation and inoculation activity assay were performed in the laminar air flow cabinet. Inoculum stock was prepared according to the procedure explained in the example 2. The dilution was made in such way that the 0.2 mL would give countable colonies.
The colony formed counts (in CFU/mL) were obtained according to method described in example 1. The results are summarized in Table 6 and 7.
Table 6: Effect of different concentration of agar powder in SCDB medium on CFU/mL of E.coli NB22 (Inoculums used, 0.5 × 107 CFU/mL)
Agar powder
(%) wt/vol Trial I Trial II Trial III
CSP NPSP CSP NPSP CSP NPSP
1.0 0.56 × 107 0.84 × 107 0.60 × 107 0.48 × 107 0.38 × 107 0.39 × 107
1.5 0.22 × 107 0.85 × 107 0.27 × 107 0.49 × 107 0.32 × 107 0.33 × 107
2.0 0.05 × 107 0.80 × 107 0.35 × 107 0.49 × 107 0.56 × 107 0.35 × 107
2.5 0.11 × 107 0.88 × 107 0.23 × 107 0.41 × 107 0.26 × 107 0.32 × 107
3.0 0.32 × 107 0.82 × 107 0.24 × 107 0.46 × 107 0.32 × 107 0.37 × 107

Table 7: Effect of different concentration of agar powder in SCDB medium on P. aeruginosa NB14 colony count (CFU/mL) (Inoculums used, 0.5 × 107 CFU/mL)
Agar powder
(%) wt/vol Trial I Trial II Trial III
CSP NPSP CSP NPSP CSP NPSP
1.0 0.16 × 107 0.89 × 107 0.82 × 107 0.80 × 107 0.56 × 107 0.63 × 107
1.5 0.80 × 107 0.82 × 107 0.90 × 107 0.82 × 107 0.32 × 107 0.53 × 107
2.0 0.15 × 107 0.97 × 107 0.75 × 107 0.73 × 107 0.04 × 107 0.52 × 107
2.5 0.19 × 107 0.89 × 107 0.75 × 107 0.79 × 107 0.50 × 107 0.48 × 107
3.0 0.19 × 107 0.72 × 107 0.80 × 107 0.66 × 107 0.44 × 107 0.54 × 107

Results obtained showed no significant difference when the concentration of agar in the medium was varied in the range investigated and the results obtained for NPSP of the present invention were consistent with those obtained using the conventional spread plate method.
Example 4:
The quantity of sterilized SCDA medium required to provide a thickness in the range 3 -12 mm was poured in the conventional spread plate and NPSP of the present invention, the concentration of agar in the medium in all cases was 1.5% w/v. All the plates were plated with 0.2 mL of inoculums (5 million CFU/mL count).
SCDA plates preparation, inoculums preparation and inoculation activity measurement was performed in the laminar air flow cabinet. Inoculum stock was prepared according to the procedure explained in the example 2. The dilution was made in such way that the 0.2 mL would give countable colonies. The estimation was carried as in example 1 and the results are summarised in Table 8 and 9.
Table 8: Effect of thickness of SCDA on E.coli NB22 count (CFU/mL)
Media
Thickness (mm) Trial I Trial II Trial III
CSP NPSP CSP NPSP CSP NPSP
3.0 0.24 × 107 0.81 × 107 0.47 × 107 0.48 × 107 0.32 × 107 0.39 × 107
6.0 0.33 × 107 0.75 × 107 0.53 × 107 0.59 × 107 0.31 × 107 0.35 × 107
9.0 0.55 × 107 0.66 × 107 0.05 × 107 0.59 × 107 0.04 × 107 0.39 × 107
12.0 0.72 × 107 0.75 × 107 0.57 × 107 0.55 × 107 0.37 × 107 0.42 × 107

Table 9: Effect of thickness of SCDA on P. aeruginosa NB14 count (CFU/mL)
Media
Thickness (mm) Trial I Trial II Trial III
CSP NPSP CSP NPSP CSP NPSP
3.0 0.36 × 107 0.98 × 107 0.85 × 107 0.84 × 107 0.61 × 107 0.58 × 107
6.0 0.42 × 107 0.91 × 107 0.86 × 107 0.86 × 107 0.58 × 107 0.55 × 107
9.0 0.04 × 107 0.91 × 107 0.08 × 107 0.63 × 107 0.06 × 107 0.49 × 107
12.0 0.83 × 107 0.92 × 107 0.66 × 107 0.73 × 107 0.53 × 107 0.37 × 107

Example 5:
Performance of NPSP of the present invention was compared with that of the conventional spread plate method under three different conditions 1) In Laminar Air Flow cabinet, 2) Close room condition but not in laminar air flow cabinet, 3) Open environment. The open environment in present case was the cooling tower site in a chemical plant.
The test samples used were E. coli NB22 spiked in demineralised water (DM) as to give a count of 10 million CFU/mL.
0.2 mL of this stock solution was injected into the conventional spread plate as well as NPSP of the present invention. The E. coli NB22 count was estimated as described in example 1 at the end of 24 hrs and 48 hrs. In addition the contamination of any other microorganism was examined by observing morphology of the colonies. The results are summarised in Table 10 and 11.

Under laminar air flow conditions:
Table 10: CFU counts of E coli NB22 and contamination presence (Figures 3 and 4)
CSP
(SCDA Medium) E. coli NB22 CFU/mL Remark
24 hrs 48 hrs No observation of cross contaminations till 48 hrs incubation at 37 °C.
1.76 × 107 1.76 × 107
NPSP
(SCDA Medium) 1.65 × 107 1.65 × 107 No contaminations were observed for 48 hrs at 37 °C.

In closed room but not in laminar air flow cabinet:
0.2 mL sample was drawn from sterile container having E. coli using sterile syringe and fitted on the 5 µm syringe filter fitted over NPSP Figure1. Sample was allowed to pass from filter and spread using spreader attached over the lid of NPSP. For Spread plate method, normal Petri dish lid was opened to add 0.2 mL sample and spread with sterile spreader while lid was still opened and exposed to the room environment. The subsequent assay was carried out as an example 1.
Table 11: Counts of E coli NB22 and contamination presence
CSP
(SCDA Medium) E. coli NB22 CFU/mL Remark
24 hrs 48 hrs 3 different colonies (contaminations) were observed after 48 hrs at 37 °C.
1.6 × 107 1.6 × 107
NPSP
(SCDA Medium) 1.4 × 107 1.4 × 107 No contaminations were observed for 48 hrs at 37 °C.

In this condition, the results were observed for 24 hrs and 48 hrs of incubation. In the case of conventional spread plate method three different colonies were observed at the end of 48 hrs. However, no such contaminations were observed in the case of NPSP of present invention. This shows that NPSP of the present invention when used to assay E. coli NB22 in close room but in the absence of laminar air flow cabinet does not allow contaminations (Figure 5).
In open environment conditions (cooling tower site):
The experiment was carried out using 0.2 mL sample having 2.5 million CFU/mL of E. coli NB22. The experimental procedure was carried as described in the previous section.
Table 12: E coli counts NB22 (CFU/mL) and contamination presence (Figure 6 and 7).
Incubation Time (Hrs) E. coli NB22 (SCDA medium) Remark
In Laminar Air Flow cabinet In Open Environment
CSP NPSP CSP NPSP
24 hrs 0.21 × 107 0.13 × 107 0.23 × 107 0.13 × 107 For CSP under open environment conditions 8 colony morphologies other than those of E coli. were observed
48 hrs 0.21 × 107 0.13 × 107 0.23 × 107
8 colonies
observed 0.13 × 107 Contamination was not observed in case of NPSP

Under these conditions, both the CSP and NPSP of the present invention showed identical counts of E.coli in the laminar air flow cabinet and under open environment. However, in the case of conventional spread plate, eight different colonies were observed apart from the E. coli when the assay was carried out in open environment, whereas, no contamination was observed in case of NPSP of the present invention (Table 12). This illustrates that the NPSP of the present invention can be satisfactorily used for monitoring the growth of E. coli in an open environment in the absence of laminar air flow cabinet without the possibility of contaminations by other microorganisms.
Example 6:
Standard bacterial cultures viz, Salmonella enterica 4931, Escherichia coli 11775, Staphylococcus aureus 25923, Pseudomonas aeroginosa 10145, Enterobacter cloacae 13047, Enterococcus faecalis 19433 were acquired.
Following media viz, HiCromeTM Coli form Agar, MacConkeys Agar, Eosin Methylene Blue Agar, Mannitol Salt Agar, HiCromeTM Selective Staph Agar, Deoxycholate Agar, Wilson Blairs Agar, Enterobacter Sakazakii Agar, Brain Heart Infusion agar, Enterococcus Agar Base and Cetrimide Agar were selected to monitor the growth of above organism.
To monitor the growth, the cultures were first grown using soybean casein digest broth (SCDB) as a nutrient rich medium. The ATCC (American type culture collection) lyophilized cultures were re-constituted in SCDB and incubated for 24 hrs at 37 °C. After 24 hrs each culture was collected by centrifuging the medium and washed with sterile saline (0.95% NaCl). The wash cycles were repeated for two times and final pellets were reconstituted in 100 mL of sterile saline.
The stock solutions containing 5 million CFU/mL of each microorganism were prepared.
The assay procedure used was the same as in example 2 and the results are shown in Table 13.
Table 13: Growth studies of ATCC cultures on different growth media
Culture Media Colony count CFU/mL
CSP NPSP
Escherichia coli 11775 HiCromeTM Coliform Agar 0.56 × 107 0.38 × 107
MacConkeys Agar 0.39 × 107 0.51 × 107
Eosin Methylene Blue Agar 0.27 × 107 0.50 × 107
Staphylococcus aureus 25923 Mannitol Salt Agar 0.36 × 107 0.48 × 107
HiCromeTM Staph Agar 0.29 × 107 0.38 × 107
Salmonella enterica 4931 Deoxycholate Agar 0.17 × 107 0.38 × 107
Wilson Blair Agar 0.70 × 107 0.68 × 107
Enterobacter cloacae 13047 Enterobacter Sakazakii Agar 0.29 × 107 0.33 × 107
Enterococcus faecalis19433 Brain Heart Infusion Agar 0.73 × 107 0.47 × 107
Enterococcus Agar Base 0.24 × 107 0.32 × 107
Pseudomonas aeruginosa 10145 Centrimide Agar 0.10 × 107 0.11 × 107

On differential/selective media, NPSP and CSP showed nearly same counts. (See USP-NF 2022)
This validates the use of specific media on NPSP to check water samples for specific microorganisms
Example 7:
Stocks of six microorganisms viz, Escherichia coli, Salmonella enterica, Pseudomonas aeruginosa, Enterobacter cloacae, Staphylococcus aureus and Enterococcous feacalis at 5 million CFU/mL were prepared in demineralized water. 1 mL of each of these was added together to prepare concoction containing all these microorganisms. NPSP of the present invention was split into six sections of equal area and six media viz, HiCromeTM Coliform Agar, Enterobacter Sakazakii Agar, Brain Heart Infusion Agar, Centrimide Agar, Mannitol Salt Agar and Deoxycholate Agar were coated on each of the surface. The NPSP of 150 mm diameter was used. The experiment was carried out in the laminar air flow cabinet.
Table 14: Growth of six different microorganisms as a consortium in specific medium sectioned on single 150 mm NPSP (Injection volume 1.2 mL).
Media NPSP CFU/mL Microorganisms found
HiCromeTM Coliform Agar 0.15 × 107 E. coli
Enterobacter Sakazakii Agar 0.16 × 107 E.cloacae
Brain Heart Infusion Agar 0.22 × 107 E. feacalis
Centrimide Agar 0.12 × 106 P.aeruginosa
Mannitol Salt Agar 0.58 × 104 S. aureus
Deoxycholate Agar 0.70 × 106 S. enterica
*HiCromeTM Coliform Agar, Enterobacter Sakazakii agar, Brain Heart Infusion agar and Deoxycholate agar may show variation or mix colonies. Hence colonies were identified here on basis of morphological characteristics (Table 14).
The method used was the same as in example 1. The results showed that NPSP of the present invention can be used to monitor the growth of more than one microorganism from the concoction of microorganism using corresponding selective media in the same experiment (Figure 8).
Example 8
In this example each microorganism was grown on two different growth media deposited on a single NPSP of the present invention.
HiCromeTM Coliform agar and MacConkey’s Agar were selected for Escherichia coli 11775, Enterobacter Sakazakii Agar and MacConkey’s Agar were selected for Enterobacter cloacae 13047, Brain Heart Infusion Agar and Enterococcus Agar Base were selected for Enterococcus faecalis 19433, Centrimide Agar and Pseudomonas Agar Base were selected for Pseudomonas aeruginosa 10145, Mannitol Salt Agar and HiCromeTM Staph Agar were selected for Staphylococcus aureus 2592, Deoxycholate Agar and Wilson Blair Agar were selected for Salmonella enteric 4931. Single colony of each culture was transferred to SCDB media and incubated for 24 hrs at 37 °C at 120 rpm. The grown broth was centrifuged after 24 hrs at 8000 rpm at 4 °C, the procedure was reproduced thrice. Finally the pellets were reconstituted in 100 mL sterile saline (0.95% NaCl). The stock was assayed for microorganism count (CFU/mL) and the final count was adjusted to 5 million CFU/mL. 0.2 mL of the solution was inoculated on sectioned NPSP having two different growth media. Subsequently the assay was carried out as in example 1.
Table 15: Growth of individual microorganisms on two different growth media sectioned on single NPSP (Figure 9).
Microorganisms Growth Media NPSP, CFU/mL
Escherichia coli 11775 HiCromeTM Coliform agar 0.40 × 107
MacConkey’s Agar
Enterobacter cloacae 13047 Enterobacter Sakazakii Agar 0.37 × 107
MacConkey’s Agar
Enterococcus faecalis 19433 Brain Heart Infusion Agar 0.33 × 107
Enterococcus Agar Base
Pseudomonas aeruginosa 10145 Centrimide Agar 0.40 × 106
Pseudomonas Agar Base
Staphylococcus aureus 2592 Mannitol Salt Agar 0.41 × 107
HiCromeTM Staph Agar
Salmonella enterica 4931 Deoxycholate Agar 0.40 × 107
Wilson Blair Agar

All microorganisms showed growth in their respective growth medium (Table 15). This demonstrated growth of a given microorganism over two different growth media. This finding helps in confirming the presence of a specific microorganism in different samples of water / liquids / and clinical samples.
Example 9:
This example shows that use of redox indicators helps develop coloured microbial colonies over creamy agar plates. Among redox indicators, 2,3,5-triphenyltetrazolium chloride (TTC) dyes is largely used for the enumeration of microorganisms on solid culture medium (Senyk et al, 1987). Tetrazolium salts (including TTC) and resazurin have been also used to established viability of microorganism in culture medium. (Gabrielson et al, 2002). In particular, tetrazolium dyes detect the oxidative enzyme system present in microbes. Tetrazolium dye is colourless in oxidized state and is coloured when reduced. Resazurin is a coloured compound that is blue in oxidized state and becomes pink in reduced state.
Two tetrazolium dyes used in this example were, TTC and 2-[4-iodophenyl]-3-[4-dinitrophenyl]-5-phenyltetrazolium chloride (INT). In addition to resazurin, ferroin solution was also used in view of its use in biochemical and waste water applications respectively (Table 16). All the dyes were tested at concentration of 50, 100, 150, 200, 250, 300 and 350 ppm. Stocks of all indicators were prepared in sterile water except DPA, the stock for which was prepared in sterile 2% methanol. Sterilized soybean casein digest agar (SCDA) medium was used to evaluate the growth response of E. coli NB22 in the presence of indicators. To achieve the desired concentrations of indicators in the NPSP, the respective indicator stocks was added into individual flasks of SCDA medium which was kept at 40 °C temperature. This stock was mixed quickly and poured into the empty sterile NPSP of the present invention. The media was then allowed to solidify at room temperature in agar plates. 0.2 mL of E. coli NB22 at 500 CFU/mL was inoculated on each NPSP containing respective indicators. The E. coli assay was carried out as described in example 1. A similar experiment was carried out using a control which was without any indicator added.
Table 16: Different dyes and their application in microbial analysis
Dye/
Indicator Application Reaction Reference
Ferroin Microbial chelating assays Red (Excess Fe²?) to colorless. (Excess Fe3?) Dobbin et al, 1995
Diphenylamine Molecular biology Blue colour formation when bind with Purine nucleotides in nucleic acid. Webb and Levy, 1955
INT Cell viability
Reduction of INT to Formazan by viable cell. Yellow to Purple/ pink color. Smith and McFeters, 1997
MTT Cell viability
Reduction of MTT to Formazan by viable cell. Yellow to Purple/ pink color. Wang et al, 2010
Resazurin Microbial assays of milk and products Non-fluorescent resazurin to fluorescent reaction product resorufin by viable cells Van den Driessche et al, 2014
TTC Cell viability Reduction of TTC to Formazan by viable cell. Yellow to Purple/ pink color. Gabrielson et al, 2002

Table 17: Effect of redox indicator dyes
Concentration
(PPM) E. coli NB22 CFU/mL
Ferroin TTC Resazurin INT
50 210 270 305 255
100 310 230 265 200
150 260 210 270 225
200 235 270 270 310
250 205 320 300 230
300 230 270 265 185
350 220 355 220 255
Control 265

TTC showed consistent results at all concentrations when tested with E. coli (Table 17) and no inhibition was noted. TTC dye did not affect the media colour when added but forms coloured colonies (Figure 10B). In contrast, ferroin, resazurin and INT changed the colour of medium and did not inhibit. (Figure 10A, 10C and 10D) when compared with control (Figure 10E).
Example 10
Different sterile media were poured separately on different sections of NPSP of the present invention as to enable simultaneous assays. Water samples from cooling tower site were collected in sterile containers. 1.2 mL sample was drawn by using a sterile syringe and sample was spread evenly under close lid condition in NPSP. A conventional spread plate was used as a control. The experiment was carried out under three different conditions viz, laminar air flow cabinet and open environment but not laminar air flow. The samples were incubated for 37 °C for 24 hours. The results were noted for growth on particular medium in both cases.
Non pathogenic spore forming Bacillus sp. NB3 (1400 CFU/mL) was sprayed using spray bottle to check if this shows up as a contaminant during the analysis carried out in the open environment.
Table 18: Analysis of cooling tower water in 150 mm NPSP and CSP
Media CFU/1.2mL*
In Laminar Air flow Cabinet In open environment
CSP NPSP CSP*
HiCromeTM Coliform Agar 161 95 TNTC
Enterobacter Sakazakii Agar 106 160 202
Brain Heart Infusion Agar 141 196 TNTC
Centrimide Agar 6 3 14
Mannitol Salt Agar 4 1 TNTC
Deoxycholate Agar 109 63 77

* Sample inoculated by opening lid of spread plate.
*Results expressed as CFU/1.2 mL since six growth media were used, TNTC- Too numerous to count
Results for NPSP were similar to those for CSP when analysis was carried out in the laminar air flow cabinet. Under open lid condition, CSP shows higher CFU count as compared to that when carried out in the laminar air flow cabinet and also NPSP. (Table 18, Figure 11). Identification of the contaminating microorganism in NPSP using a specific medium is useful to treat water with the biocide which will kill the microorganism.
In this experiment colony morphology and growth characteristics on specific media suggested the presence of Klebsiella sp. (enterobacteriaceae family) in water sample. A biocide for Klebsiella sp. can be used to treat water in the cooling tower for controlling Klebsiella sp
Example 11
Brown and Lowbury (1965) used cetrimide to achieve selective growth of pyocyanin producing P. aeruginosa. For evaluation of growth of P. aeruginosa NB14, cetrimide was tested at various concentrations. Stock solution of cetrimide was prepared in sterile water and added into 500 mL flask containing 100 mL of sterile molten SCDA medium (at 40 °C) to obtain solutions of 20, 50, 80 and 100 ppm concentration. Medium was then fortified with TTC dye to obtain coloured colonies. 150 mm sterile empty Petri plates were then prepared with five sections. Inoculum of E. coli NB22 and P. aeruginosa NB14 were prepared according to example 2. Inoculum size of 100 CFU/mL was used to get the countable colonies for both cultures. Three inoculums viz. E. coli NB22, P. aeruginosa NB14 and mixtures of both were used at LAF and open environment conditions. Non pathogenic spore forming Bacillus sp. NB3 (1400 CFU/mL) was sprayed using spray bottle to check if this shows up as a contaminant during the analysis carried out in the open environment.
Evaluation of Pseudomonas aeruginosa NB14 with CSP and NPSP
Table 19: Cultures Cetrimide concentration
(PPM) CFU/0.2 mL*
Under laminar air flow cabinet In open environment
CSP NPSP CSP*
P. aeruginosa NB14 0 78 58 TNTC
20 62 51 TNTC
50 53 20 53
80 8 13 46
100 6 4 49

E. coli NB22 0 49 23 TNTC
20 28 35 TNTC
50 NIL NIL 26
80 NIL NIL 36
100 NIL NIL 42

P. aeruginosa NB14 + E. coli NB22 0 64 35 TNTC
20 19 9 TNTC
50 10 2 TNTC
80 6 2 12
100 3 NIL 10
* Sample inoculated by opening lid of spread plate,
*Results expressed as CFU/0.2 mL of individual ppm concentration of cetrimide,
TNTC- Too numerous to count, NIL- No growth
The use of cetrimide in NPSP showed selective growth of P. aeruginosa when inoculated with mixed culture (Table 19). When analysis was carried out in the laminar flow cabinet, results for NPSP were similar to those for CSP. In contrast, when the assay was carried out under open conditions, CSP showed contaminations due to other cultures, while the assays carried out using NPSP did not (Figure 12).
Example 12
Packaged mineral water bottles/pouches and loose samples were purchased from local market. Loose samples were collected in the sterile containers. These loose samples were diluted with sterile saline to obtain the countable colonies. All samples were analyzed under two conditions viz, under laminar air flow cabinet and open environment on CSP and NPSP containing sterile SCDA medium. 0.2 mL water sample was inoculated on the medium and spread. All the samples were incubated at 37 °C for 24 hours. Assay was carried out as described in example 1. Results were calculated as CFU/mL.
Non pathogenic spore forming Bacillus sp. NB3 (1400 CFU/mL) was sprayed using spray bottle to check if this shows up as a contaminant during the analysis carried out in the open environment
Table 20: Microbial analysis of different market samples with CSP and NPSP (CFU/mL)
Samples In laminar air flow cabinet In open environment
CSP NPSP CSP*
Packaged mineral Water 1 (Bottle/Brand 1) NIL NIL 1000
Packaged mineral Water 2 (Pouch/local) NIL 5 900
Packaged mineral Water 3 (Bottle/Brand2) NIL NIL 1000
Packaged mineral Water 4 (Bottle/Brand 3) NIL NIL 1100
Lemon Juice NIL 5 395
Butter milk 180000 220000 380000
Sugarcane juice 105000 100000 290000
Pani puri water 5000 30000 55000
* Sample inoculated by opening lid of spread plate, NIL- No Growth
Results for NPSP were comparable to those with the CSP in the laminar air flow cabinet. In open lid condition CSP showed a greater number of CFUs as compared to those when the analysis was carried out in the laminar flow cabinet and also NPSP (Table 20, Figure 13). NPSP is thus useful to test the food and water sample from market under aseptic conditions even in the absence of a laminar air flow cabinet.
The embodiments were chosen and described in order to best explain the principles of the present invention and its practical application, to thereby enable others, skilled in the art to best utilize the present invention and various embodiments with various modifications as are suited to the particular use contemplated.
It is understood that various omission and substitutions of equivalents are contemplated as circumstance may suggest or render expedient, but such are intended to cover the application or implementation without departing from the scope of the present invention.

,CLAIMS:We claim,

1. A portable device for estimating microbial contamination in water under aseptic conditions in the absence of a laminar air flow cabinet.
2. The novel portable spread plate (NPSP) comprising 1) at least one gel layer deposited on the surface of the Petri dish, 2) a lid fitted with a syringe filter near the periphery of the lid, 3) a rotatable spreader bar inserted in to the lid through a hole provided at the centre of the lid so as not allow the entry of the microorganism wherein the said petri dish and the said lid are fastened together so as not to allow the entry of the microorganism into the novel portable spread plate (NPSP) .
3. The novel portable spread plate (NPSP) as claimed in claim 1 wherein novel portable spread plate is circular having a lid diameter in the range 30 mm to 200 mm.
4. The novel portable spread plate (NPSP) as claimed in claim 1 wherein the material for the lid is selected from glass, borosilicate glass, polystyrene, polycarbonate, polysulfone, polyethersulfone, cyclic olefin copolymers, transparent polypropylene, poly methyl methacrylate and styrene acrylonitrile copolymers.
5. The novel portable spread plate (NPSP) as claimed in claim 1 wherein the material for the Petri dish is selected from glass, borosilicate glass, polystyrene, polycarbonate, polysulfone, polyethersulfone, cyclic olefin copolymers, transparent polypropylene, poly methyl methacrylate and styrene acrylonitrile copolymers.
6. The novel portable spread plate (NPSP) as claimed in claim 1 wherein the material for the Petri dish and the material for the lid is the same.
7. The novel portable spread plate (NPSP) as claimed in claim 1 wherein the material for the Petri dish and the material for the lid is not the same.
8. The novel portable spread plate (NPSP) as claimed in claim 1 wherein the material for the rotatable spreader bar is selected from polyethylene, polypropylene, high impact polystyrene and glass.
9. The novel portable spread plate (NPSP) as claimed in claim 1 wherein the length of the rotatable spreader bar is in the range 12 mm to 95 mm.
10. The novel portable spread plate (NPSP) as claimed in claim 1 wherein the height of the rotatable spreader bar above the lid is in the range 10 mm to 25 mm.
11. The novel portable spread plate (NPSP) as claimed in claim 1 wherein the spreader bar is inserted in to the lid through flexible bush fitted in the hole provided at the centre of the lid.
12. The novel portable spread plate (NPSP) as claimed in claim 1 wherein the spreader bar vertical movement of the spreader bar is controlled by the bush and lock mechanism.
13. The novel portable spread plate (NPSP) as claimed in claim 10 wherein the flexible bush is made of a material selected from Teflon, polyurethane, polyvinylidene fluoride, polyolefins and elastomers.
14. The novel portable spread plate (NPSP) as claimed in claim 1 wherein the number of gel layer deposited on the surface of the Petri dish is in the range 1-6.
15. The novel portable spread plate (NPSP) as claimed in claim 1 wherein the thickness of gel layer deposited on the surface of the Petri dish is in the range 3 mm-12 mm.
16. The novel portable spread plate (NPSP) as claimed in claim 1 wherein the material for the gel layer deposited on the surface of the Petri dish is selected from MacConkeys Agar, Eosin Methylene Blue Agar, HiCromeTM Coliform Agar, HiCromeTM MacConkey Sorbitol Agar Base, Salmonella-Shigella Agar, Wilson Blair Agar, Mannitol Salt Agar, Xylose Lysine Deoxycholate (XLD) Agar, Cetrimide Agar, Pseudomonas Agar Base, Enterobacter Sakazakii Agar, Enterococcus Selective Agar, Potassium Tellurite Agar, Thiosulfate-Citrate-Bile Salts-Sucrose Agar and Soybean Casein Digest Agar.
17. The novel portable spread plate (NPSP) as claimed in claim 14 wherein the material for the gel layer deposited on the surface of the Petri dish is mixed with redox indicators selected from 1) 2-[4-iodophenyl]-3-[4-dinitrophenyl]-5-phenyltetrazolium chloride and 2) 2,3,5-triphenyl tetrazolium chloride.
18. The novel portable spread plate (NPSP) as claimed in claim 14 wherein the material for the gel layer deposited on the surface of the Petri dish is mixed with dyes selected from Ferroin, Diphenylamine, MTT and Resazurin.
19. The novel portable spread plate (NPSP) as claimed in claim 1 wherein the concentration of material selected for the gel layer in the gel layer is 1-3 % wt./vol.
20. The novel portable spread plate (NPSP) as claimed in claim 1 wherein, the lid of the Petri dish and the Petri dish on which the gel is deposited are fastened together by sliding the lid over the petri dish as the two are snug fitting.
21. The novel portable spread plate (NPSP) as claimed in claim 1 wherein, the lid of the Petri dish and the Petri dish on which the gel is deposited are fastened together by an adhesive.
22. The novel portable spread plate (NPSP) as claimed in claim 1 wherein, the lid of the Petri dish and the Petri dish on which the gel is deposited are fastened together by an adhesive selected from an epoxy resin, an acrylate resin and polyvinyl acetate.
23. The novel portable spread plate (NPSP) as claimed in claim 1 wherein, the lid of the Petri dish and the Petri dish on which the gel is deposited are fastened together by Parafilm.
24. The novel portable spread plate (NPSP) as claimed in claim which enables carry out microorganism estimation under aseptic conditions, in the absence of a laminar air flow cabinet.

Dated this 31st day of August 2023.

For Ion Exchange India Ltd

MAHURKAR ANAND GOPALKRISHNA IN/PA-1862
(Agent for Applicant)