ABSTRACT- Cells are always considered as a basic unit of life in every aspect, almost whole environment consists of various types of cells. Many scientists did research on such type of organisms and they concluded almost every functioning of how micro-organisms actually work in a proper manner. Every organism has a particular morphology which can be a factor to differentiate organism based on size but in this research, it has been found that how to measure the weight of single cell whether the prokaryotic or eukaryotic cell, independent of other factors. Thus, by finding the mass and relating with its size we can even find the density of organism, may be sometimes the size cannot be a factor to detect how heavy the cell is!. Hence, using this technique one can differentiate various organisms based on their single cell weight. Many applications can be found by the respective person if someone using this technique. One of application is one can isolate aerobic bacteria also using this technique and can sub-culture it.
Key-words- Cell size, Single cell weight, Differentiate organisms, Isolation of aerobes

INTRODUCTION- The cell is the basic structural, functional and biological unit of all known living organisms. A cell is the smallest unit of life that can replicate independently and cells are often called ‘the building blocks of life’.[1] Cells consist of cytoplasm enclosed within a membrane, which contains many biomolecules such as proteins and nucleic acids.[2] Organisms can be classified as unicellular or multicellular. While the number of cells in plants and animals vary from species to species. Cells are only visible under a microscope, with dimensions between 1-100 micrometers.[3]
There are two types of cells: Prokaryotic cell and Eukaryotic cell. A prokaryote is a unicellular organism that lacks a membrane-bound nucleus, mitochondria or any other membrane bound organelle [4]. In prokaryotes, all the intracellular water soluble components (proteins, DNA and metabolites) are located together in the cytoplasm enclosed by the cell membrane, rather than in separate cellular compartments. A eukaryote is an organism whose cell contains a nucleus and other organelles enclosed within membranes.[5]

Fig.1: Structure of Eukaryotic cell [6]



Fig. 2: Structure of Prokaryotic cell [7]

Silica gel is a granular, vitreous, porous form of silicon dioxide made synthetically from sodium silicate. Silica gel high specific surface area (around 800m²/g) allows it to adsorb water readily, making it useful as a desiccant. Silica gel is often described as “absorbing” moisture, which may be appropriate when the gel’s microscopic structure is ignored, as in silica gel packs or other products. However, material silica gel removes moisture by adsorption onto the surface of its numerous pores rather than by absorption into the bulk of the gel. [1]



Fig. 3: Silica Gel beads [8]

ESTIMATION OF MASS OF SINGLE CELL
AIM: To estimate the mass of single cell (Culture of interest) by Siddhesh’s Method.

PRINCIPLE: The silica gel bead contains pores on it which can be used to grow the microbes in it. This technique consists of dipping the beads in the Nutrient broth and allowing the beads to grow organisms in their pores which are then weigh and by applying calculations mass of single cell can be estimated.
In this technique, silica gel beads firstly dipped in Nutrient broth so that some essential proteins required for microbial growth gets entrapped in the pores of the beads. Then excess broth from the surface of the bead is removed with the help of ear cleaning cotton bud and a drop of specific microbial suspension whose weight is to be determine is pour on bead with the help of micropipette (Note: Pore size on the bead > The size of organism, if we are taking Staphylococcus aureus whose diameter is 1 µm then the pore size must be µm + n, where n= small increment in the diameter of pore size than organism’s max length or diameter so that it can enter in pore, choose beads accordingly they are available in various types and mesh size) and keep in incubator at 37^oC for 1 hr. After one hour, microbial growth can be seen in silica gel beads with naked eyes and then by weighing the silica gel beads and applying calculations the mass of single cell can be estimated which demonstrated in calculations portion.



Fig. 4: Principle of Mass of Single cell

REQUIREMENTS:

1. Sieved silica gel beads (2mm or as per requirement)
2. Microbial culture of interest
3. Ear cleaning cotton buds
4. Sterile plastic spoon
5. Nutrient broth (as per nutritional requirements of organism)
6. Petri plate dish
7. Micropipette
8. Distilled water
9. Sterile eppendorf tubes

PROCEDURE- Take sieved silica gel beads and keep in microwave for 5 mins at 120^oC so that the moisture present in the pore gets evaporated. Remove beads and weight to adjust ~1 gram as W₁. Dip these beads in the nutrient broth (liquid medium) in petri plate dish between burners (Beads should be totally sunk in broth). After 15 mins remove beads with the help of a plastic spoon and take in a clean petri plate dish and remove the excess nutrient broth on the surface of beads with the help of ear cleaning cotton bud. After cleaning, keep the beads for 5 mins between burners with a lid of dish open so that water molecules present on the surface gets evaporate. Take 5 µL culture suspensions and put on each bead and keep beads for 2 mins and remove excess suspension around beads with the help of ear cleaning bud. Close the lid of petri plate dish and kept in the incubator for 1 hr at 37°C. After 1 hr, microbes grow inside pores and beads turns white from transparent (Fig 5). Weigh the beads W₂. Take one bead and put in distilled water in the eppendorf tube, when bead put in water bead has the greater affinity towards water molecules and hence microbes present in pores gets displace by water molecules and do cell counting under the microscope (volume of distilled water take as per required for cell counting technique). Now apply calculation as per shown in sample calculation portion.

OBSERVATION-


Fig. 5: Silica gel beads
A: Normal silica gel bead (Transparent)
B: Microbial growth in bead (white)



Fig. 6: Six Microbial beads kept in sunlight denoted by 1, 2, 3, 4, 5, and 6 Observation at shadows of beads:
Bead 1: Total microbial growth throughout bead hence no light passed through it; Bead 2-4: Light passed through the nutrient broth (orange reflection in shadow) present in the centre of beads which resembles growth of microbes on the surface only
Bead 5: shows partial growth of microbes due to depletion of nutrient broth and hence white reflection can be seen in shadow
Bead 6: No microbial growth in centre
(NOTE: For this experiment, all beads taken must be like Bead no. 1 after microbial growth hence, necessary to check under light)

CALCULATIONS- W₁- Mass of dry beads- 1.000 gram –Let 10 beads weight for 1 gram
W₂ - Mass of beads after microbial growth- 1.010 gram Total microbial growth (TMG) in 10 beads= W₂ – W₁
                                               = 1.010-1.000= 0.01 gram
TMG in 1 bead= (TMG in 10 beads/No. of beads)= (0.01/10)=0.001 gram
After doing cell counting, there were 5 x 10⁶ cells present in 1 bead Then,
Mass of single cell= (TMG in 1 bead/No. of cells)= (0.001/5 x 10⁶ )= 2 x 10^-10 gram = 0.2 nanogram (ng) weight of single cell

CONCLUSION- The weight of single cell present in bead was 0.2 ng and hence using this technique weight of any organism can be found. If somebody wants to measure the density of particular single cell for example S. aureus whose diameter is 1µm, then the density of organism is given by (mass of single cell/ Volume of the sphere)= [m/(4/3) x p x r3]. Hence, by using this technique we can differentiate various organisms based on their single cell weight.

REFERENCES-

1. Available on website www.wikipedia.com, accessed on 30^th December 2016.
2. Cell Movements and the shaping of the vertebrate body Chapter 21 of Molecular Biology of the Cell 4^th Edition, edited by brucealberts (2002) published by Garland Science.
3. Campbell, Neil A; Brad Williamson; Robin J Heyden (2006). Biology: Exploring LifeBoston Massachusetts: Pearson Prentice Hall.
4. NC State University “Prokaryotes: Single-celled Organisms”.
5. Youngson, Robert M (2006) Collins Dictionary of Human Biology. Glasgow: HarperCollins.
6. Photo courtesy: Google image source available on getmededu.com accessed on 30 December 2016.
7. Photo courtesy: Google image source available on studyblue.com accessed on 30 December 2016.
8. Photo courtesy: Google image source available on silicagel.in accessed on 30 December 2016.
   
   

   International Journal of Life-Sciences Scientific Research (IJLSSR) Open Access Policy Authors/Contributors are responsible for originality, contents, correct references, and ethical issues. IJLSSR publishes all articles under Creative Commons Attribution- Non-Commercial 4.0 International License (CC BY-NC). https://creativecommons.org/licenses/by-nc/4.0/legalcode

   
   
   

   How to cite this article: Mahadik SM: Estimation of Mass of Single Cell. Int. J. Life. Sci. Scienti. Res., 2017; 3(2): 887-890. DOI:10.21276/ijlssr.2017.3.2.2 Source of Financial Support: Nil, Conflict of interest: Nil