There are an enormous number of cells in the human body and in all organisms. Including an exception for numerous other bacteria and other microorganisms lower down the complication order. The basis of all these cells are two processes namely mitosis and meiosis. To be more specific in humans the fertilized egg is the source of all types of cells. From the fertilized egg in the humans to the innate process of mitosis and meiosis in the smallest of organisms all undergo cell differentiation. This process in defined as bringing in the characteristic of a specific cell to it specific function. These cells basically develop into various types of cells from their first stage of interphase in mitosis. But to remember that the zygotic stage never determines the specific function of the cell. Not only the newly formed cells undergo differentiation but the adult cells or the adult stem cells to be more specific undergo differentiation to form specific tissues and then organs and later to form a whole organism. The adult stem cells after their process of differentiation transfer their characteristics to their daughter cells in such a way that the daughter cells also exhibit the same characteristics as their parental generation. The cell differentiate along these lines that the whole morphology of the cell like the cell size, cell structure, membrane potential and even its response to signals alter. On the contrary cell death also plays a major part in cell differentiation and organ development. For example the process of metamorphosis in butterfly from larvae to the completely metamophosized butterfly or from the tadpole to a frog. The deaths of many cells are involved in this process, but very specific cells. Apoptosis is the word given for a programmed process of cell death without which the development of organs or any higher organism is most unlikely to happen. All the processes mentioned above are the consequences of gene manipulation within the cell. These are controlled by specific genes within a cell giving out certain signals as and when needed for different processes. Determining the genes that are involved in these processes is called as genetic regulation. Genetic regulation or gene evaluation plays a vital role in the field of medicine.
This article discusses about the use of Caenorhabditis elegans, a transparent nematode worm as a specimen for determining the genetic regulation of organ development and programmed cell death or apoptosis. This specific species was short listed among many others as it had a very short time spanned cell cycle (Wood and William, 1988). Which consisted of only 959 adult cells in its generation cells making it very easy to analyse and determine the genetic regulation (Brenner, 1974). On the whole three scientists worked on determining the genetic regulation of apoptosis and organ development.
Sir John Sulston was the first one among the three to initiate the experiment starting with developing all the techniques to study cell division in the nematode worm from its stage of a fertilized egg to a completely mature adult stage of the worm. (Sulston and Horvitz, 1977).
Dr. H. Robert Howitz continued the work of Sir John Sulston by putting forward the question whether there was a genetic code for all death and development processes taking place in an organism. A specific genetic programmed that he suggested might be and determined the genetic regulations for the same processes in the worm (Jonathan and Robert, 1978).
Dr.Sydney Brenner played his part by proving the work done by the previous scientists on determining the specific genes. He mutated those specific genes involved in the processes by using EMS or Ethyl Methane Sulphonate. This landed up on the result that, when these genes are mutated the organ development does not take place and consequentially lead to the death of the organism (Brenner, 1973) (Jonathan and Sydney Brenner, 1978).
The work of all the three scientists helped in landing up in a theory and experimental proof of genetic regulation of apoptosis and organ development and also that there is a major connection between both the processes for the survival of the organism. The use of nematode worm was considered because it is difficult to determine the same in higher animals. The genes like Ced-3 and Ced-4 were primarily determined to be the genetic regulators of apoptosis and the proteins which codes for the initiation of these genes were used for degrading the DNA after apoptosis. Also making an understanding on how the dead cell is eliminated after the process of apoptosis. It was proposed that the same regulations also take place in higher organisms including humans as one of them with the help of homologous proteins like Apaf-1 in humans replacing CED-4 in C.elegans (Hua and William, 1997).
The male and the hermaphrodite are differentiated by the morphology or by their internal organs. The male nematode is supposed to have 959 cells in its mature adult stage and makes it very easy to determine the genetic regulation. The picture above gives the lateral view of dissected C.elegans. The lateral dissected view clearly shows the simplicity of the organism and also on why the organism was narrowed down to study upon. (Sulston and Horvitz, 1977)
This report mainly concentrates on the determination of genes involved in the process and how the two processes of development and death are linked in the complete life cycle of an organism. Determining the genetic regulation of the same plays a vital role in curing a hand full of dreadful diseases like Cancer, AIDS and Myocardial Infarction (Thompson, 1995). The unstoppable growth of cells inhibiting the process of apoptosis in the case of cancer (Morgan et al., 2006) and the death of cells inhibiting the process of development and initiating the process of apoptosis by activating all the available pathways (Explained in detailed in the proceeding pages) for the process of apoptosis in the case of AIDS (Perez et al., 2008). Determining the genes involved or genetic regulation has a major role in the treatment of these diseases. As genes and gene coding are the bases of every live organism in this universe.
Genetic Regulation of Apoptosis and Organ Development: To just basically explain about genetic regulation before getting deep into the degree of the paper, genetic regulation is the process of turning on or turning of the genes that are needed and those of which are not needed respectively. The first ever gene regulation developments were on the lac and the trp operon model. Basically it is a system used for saving up the enzymes and using them whenever necessary and not wasting them by accumulating them by continuously producing them on the contrary. These are helped by the genes. And for better understanding s schematic is given below of the overall process of genetic regulation.
Introduction to Apoptosis Apoptosis is derived from the Greek language meaning “dropping off” or “falling off” of parts. This I suppose does not give the appropriate meaning but the term was coined according to the preliminary discoveries of researchers regarding the same. The term was titled to fit the process as there were findings and literature that stated the dropping of all organelles (not literally) of the cell after the depletion of the cell wall in the continuum of processes of apoptosis. The term generally means programmed cell death, which is defined as the well timed suicide of the cells by gene regulation as and when needed by the organism. This is the exact process that takes place in all organisms from a single celled to a multicellular complex organism. Apoptosis regulates certain morphological features of the cell leading to its death in the coming cycles of apoptosis. The morphological changes include bleebing, loss of cell membrane, asymmetry of the cell, fragmentation of the DNA and many other structural and functional changes (Alberts et al., 2008).
Atrophy is caused in the final stages of apoptosis which can lead to the complete destruction of the cells. The process was primarily considered to be incomplete as it was not known how the cells were dissolved after their death as the organelles after the cell death would cause to create an unwanted mass in the organism. But latter it was determined that the cells after the process of apoptosis ended up in creating apoptotic bodies which were engulfed by other cells using pseudo arms and then were dissolved using the proteins that code for the gene to regulate the process of apoptosis (Walker and Sikorska 1994). During the years of the primary experimentation this process was written along with the process of necrosis which was the premature death of cells due to external effects like toxins, hazardous chemicals or radiations. But further experimentation and trials proved that apoptosis was a self-inducing factor of cells for their suicide in order that the following processes of organ development takes place without obstructions. When human trials are concerned it is to be noted that about 50 to 70 billion cells die each day in the same process, where at the same time daughter cells are generated.
Cell death plays an important role in organ development and tissue homeostasis. The regulation of cell proliferation by Programmed Cell Death (Apoptosis) contributes to organ and tissue development and differentiation to a great extent. Depending upon the time and clinical impacts many genes change their expression during organ development. The success of organ development completely depends upon the interaction between the maintenance of cell survival and cell death. Cell death plays a significant role in promoting growth and tissue development of an organism. Generally when cell death occurs, the following points are to be taken into account,
Development of normal tissues and cell death.
Regulation of cell cycle and expression of genes.
Determining the cell death pathway.
The pathway acts as a molecular target for therapy (Prof. Dr. M. Nurhalim Shahib, 2001).
When cells die, the contents of the cell are released in the surrounding and it causes inflammation or swelling which is termed as necrosis. When cells die during normal development or tissue homeostasis, they tend to condense and shrink and the dead cells are phagocytised by neighbouring cells before the contents of the cell get leaked in the surrounding. They do not induce any inflammatory response unlike the necrosis. This process is termed as Apoptosis or Programmed Cell Death (Kerr et al., 1972).
Apoptotic Pathways Extracellular and intracellular pathways are the two terms that are concerned when there is a death of a cell taking place or a death of group of cells. Extracellular pathways are the subjecting of cells to toxin or hormones or growth factors which can in turn lead to the death of the cell. This not of much concern in our review as we are concentrating on the pathways of apoptosis. To get a brief knowledge about the death of the cells we should know that the death of the cells can take place by sensoring the signals from inside the cell or from the outside of the cell. Apoptosis mainly takes place by transducing signals from the inside of the cell. This process can be put into two main categories namely positive induction and negative induction. Induction means the inhibition of the reaction and hence positive induction refers to the undergoing apoptosis reaction.
To give a brief about the extracellular and intracellular processes. The intracellular processes will be explained in detail below and the extracellular process is simply the transducing of signals in case of unstable environment or the sequential crossing of the plasma membrane.
Intracellular signalling has many different pathways which follow a definite sequence of steps. These including some like the binding of receptors by glucorticoids in the presence of high calcium concentration. All processes in the cells are initiated by enzymes specific enzymes that code for specific genes to be activated for the following reaction to take place. Hence in the contrary these proteins or enzymes can also be used to inhibit the reaction by finding out the genes which code for the enzyme to be produced. The process of apoptosis is mainly targeted to the mitochondria of the cell which then ceases almost all the functions the cell.
For explaining in detail the intracellular processes that are taking place we need some specific pathways for a deep understanding. Many pathways are proposed and on literature which we are going to discuss in brief in the following pages of my report.
Mitochondrial Regulation of Apoptosis Briefly explaining about mitochondria, they are the power house of the cell and supplies energy for the complete survival of the cell without which the cell is not in existence. Obviously needless to say that without the functionality of the mitochondria the cell is just another non living organism without any appropriate use. This vivid function of the bacteria is used by the apoptotic pathways. There are two pathways by which the cells are forced to death by inhibiting the function of the mitochondria. They are the intrinsic and extrinsic pathway. They both vary in the pathways but the end product is always the death of the cell. (Susin et al., 1999).
The intrinsic pathway is basically the swelling up of the mitochondria by the formation of pores. This is formed by the binding of Cytochrome C to the apoptotic protease activating factor and then followed by the process of apoptosis.
On the other hand the extrinsic pathway is the decrease of the membrane potential of the mitochondrial membrane by the action of nitric oxide and also the increase of the permeability causing a leak of enzymes inside the mitochondria. This causes the uncontrollable swelling of the cell and also causes blebbing. When the cells continue to swell it leads to the damage of the cellular membrane that eventually leads to cell death. These are reported to be caused by the SMAC’s which are stated to be secondary mitochondria derived activation of caspases. This can be proved to be the right opposite to the intrinsic pathway as this process deactivates the pathway that inhibits apoptosis by binding to the inhibition of apoptosis proteins or namely IAP rather than the intrinsic pathway that directly induces apoptosis. (Mayer and Oberbauer, 2003). When we get to compare both the processes we tend to end up in a result which states that the damage of the cell is indistinguishable between the two.
2.2.2 Caspase Independent Pathway
Caspase in considered as the most important protein in the proper functioning of the apoptosis cycle. But it is proved that the apoptosis can also take place without the activity of the caspase protein. This takes place by a binding factor known as Apoptosis Inducing Factor (Cande et al., 2002) which only needs transduced signals rather than the caspase binding (Magali et al., 1999)
2.2.3 Signal Transduction
Signal transduction is generally described as the process of transferring signals. The same as mentioned above this also involves both extracellular and intracellular signals. This process is also called as the shape shifting process as the morphology of the cell is completely changed after this process takes place. There are two different pathways TNF and Fas pathways namely.
Figure 4: TNF and FAS Pathway within the cell. The diagram gives an easier explanation for the Fas pathway including the caspase binding reaction. It also shows many other proteins involved in programmed cell death. The TNF and the Fas pathways are two completely different pathways with the same functionality as to programmed cell death. They assist mainly in transferring signals not only from the inside the cells to the point of pathway commencement but also from the exterior of the cell to the important signal transducers inside the cell. (Philip, 2004).
The TNF pathway or the Tumor Necrosis Factor pathway. The pathway is commenced by the binding of TNF R1 and TNF R2 which in turn leads to the initiation of caspase activating pathway which then eventually leads to the death of the cell. Both the pathways to be mentioned are results of binding. This pathway was also proved to be leading to one too many activations of transcription factors, the cause of numerous diseases (Chen and Goeddel, 2002).
The Fas pathway also follows the same binding pathway as the TNF pathway. Only that the activation of transcription factors is unlikely to happen and the binding here takes place in caspase 8 and caspase 10. Binding of Fas and Fas L takes place along with the Death Inducing Signaling Factors or DISC’s (Wajant H, 2002).
2.3 Extracellular Control of Apoptosis
Programmed cell death or apoptosis have found to be activated or suppressed by extracellular signals from other cells apart from the apoptotic cells by controlling the mechanism. These extracellular signals are sent majorly to prevent programmed cell death. (Raff, 1992). There are a few examples to demonstrate how the signals activate or suppress apoptosis.
Most of the vertebral cells tend to undergo apoptosis when cultured at a low density with the secretion of extracellular signals on their own. For Example- Blastomeres have the ability to survive and divide even in the absence of extracellular signals. The cells from tissues that are made of only single cell types have the ability to produce self-survival signals (Biggers et al., 1971).
In some cells, a combination of several signals from different cells is required for their survival in a long term aspect. Example- The vertebral neurons during development compete for signals for their survival which are secreted by the target cells that they innervate. In this process only half of the neurons get enough signals to survive and the rest undergoes programmed cell death. Therefore, the normal death can be prevented by injecting exogenous Nerve Growth Factors (NGF) to the neurons. Similarly when the genes that code for NGFs are inactivated or by addition of anti-NGF antibodies, all the neurons undergo Apoptosis. Usually both inactivation and injection of NGFs are carried in a neuron to provide a balance between the number of neurons innervating the target cell and the unwanted neurons that target inappropriate cells (Levi, 1987).
Some cells are triggered by programmed cell death inducing signals which suppresses the action of the signals that are responsible for the survival of the cell. Example- In amphibians like tadpoles, a systematic induction occurs at the metamorphosis stage where the cells in the tail undergo apoptosis due to the increase of thyroid hormone in the blood and this facilitates the resorption of the tail (Kerr et al., 1974).
2.4 Overall Process of Apoptosis- Morphological Concern
The figure above gives us an easier understanding of the morphology of the cells in the course of Apoptosis. The diagram gives us a clear cut explanation about how normal cells receive signals and then followed by cell shrinkage and nuclear collapse leading to death and formation of apoptotic bodies to the complete lysis of the cell. (Philip, 2004).
Observation Causes Cell shrinkage and rounding
Breakdown of proteinaceous cytoskeleton by caspase.
Density of Cytoplasm
Signal transduction by TNF pathway or Mitochondrial regulation
Tight packing of organelles
Signal transduction by TNF pathway or Mitochondrial regulation
Chromatin shrinkage against nuclear envelope- Phykonosis
Condensation of Nucleus
Karyorrhexis – Degradation of DNA
Breaking of Nucleus
Blebbing (Mathew et al).
Localized decoupling of the cytoskeleton from plasma membrane
Phagocytosis or engulfing of dead cells
Usually present on the cytosolic surface but spread by scramblase
Table 1: Tabulated format of the observations during Apoptosis of the cell and their primary causes.
2.5 Role of Inhibitory or Promoter genes in Apoptosis
The cells after undergoing apoptosis in all tissues and animals appear similar and this cell death gets involved in many operations that are active and intracellular which can be promoted or inhibited by physiological or pathological stimuli.
Regulation in Caenorhabditis elegans
The genes responsible for apoptosis was first identified in a nematode, Caenorhabditis elegans, related to cell death and its control (Horvitz et al., 1982; Ellis and Horvitz, 1986).
Initial genetic studies in C.elegans led to the identification of a gene called Ced-3, a promoter gene responsible for programmed cell death to occur during the development of the worm (Ellis et al., 1991).
The Ced-3 gene codes for an enzyme which is Cysteine Protease (Yuan et al., 1993).
The gene cleaves the substrate after every active and specific aspartic acid sites and they get activated by cleaving at the aspartic acid sites. These are now referred to as “Caspases” (Alnemriet et al.,1996)
The caspases mediate the apoptosis by cleaving at specific intracellular proteins that are of high selectivity and these proteins in turn activate the apoptosis process (Chinnaiyan and Dixit, 1996).
Similarly there are many genes that inhibit the apoptosis process in the nematode, Caenorhabditis elegans. One such gene is Ced-9, which belongs to the same family as the Ced-3 and this gene inhibits apoptosis in the nematode (Hengartner and Horvitz, 1994).
If Ced-9 is activated by disruption or any mutation, the worm dies at a very early stage when compared to the usual growth. Therefore Ced-9 is necessary to prevent programmed cell death if the cell has to survive in the developing worm. (Hengartner et al., 1992)
Regulation in Mammalian cells
Similar to the nematode there are many genes that act as inhibitors or promoters of programmed cell death or apoptosis in mammalian cells also. They in turn contribute to organ and tissue development.
Certain genes like Bcl- 2 and Bcl- XL act as inhibitors that inhibit programmed cell death.
Genes like Bax and Bak act as promoter genes. They promote programmed cell or apoptosis.
The average ratio of the inhibitors to the promoters determines the capacity of a mammalian cell to undergo apoptosis (Korsmeyer, 1995).
Bcl- XL, in three dimensional structures is noted to function as a pore forming protein in the intercellular membrane where the genes are actually present. (Muchmore et al, 1996)
When the Bcl- 2 and Bcl- XL is disrupted in a mammal like mice, the animal tends to die either as an embryo itself of in the post natal stage due to excessive programmed cell deaths in particular organs.
When Bax is disrupted, normal programmed cell death or apoptosis process itself fails to occur (Deckwerth et al, 1996).
Although proteins are required for the apoptosis process, inhibitors of RNA or protein synthesis often inhibit apoptosis indicating that transcription and translation are required to activate the programmed cell death process.
2.6 Importance of Programmed Cell Death (Apoptosis)
In the mutant nematodes, where the apoptosis process is deficient, it is found to have a normal life span. But whereas apoptosis process deficient flies are found to die at an early stage. The vertebrates exhibit results similar to that of the flies. This difference is due to the inhibitory and the promoter genes in the different organisms and their relation to organ development and tissue homeostasis.
2.7 Consequences of Defective Pathways
The consequences caused (diseases) are only caused by the defective apoptotic pathways. Where normal apoptosis does not take place. The only way by which the flow of apoptosis is disrupted is by deferring the signal. When the pathway is inhibited the growth of the cell continues and the cells live more that they are supposed to actually live and differentiation of these cells also transfer the fault to their progeny. Which most probably leads to cancer. The inhibitor or the suppressor as we call it here binds to caspase preferably 8, 9 or 10 here in this case and stops the cell death.
AIDS: This specific viral protein deactivates the anti-apoptotic Bcl-2 and triggers the mitochondrial regulation pathway to progress the reaction at a higher pace. FAS mediated apoptosis is increased and the death rate also increases (Perez et al., 2008).
Cancer: This disease is a causative of inhibition of apoptosis. Where the X-linked inhibitor of apoptosis protein plays a vital role. When the cells do not die at the specified time a tumor is produced leading to cancer (Ott et al., 2006; John and Kerr, 2006).
Having mentioned about the disadvantages of the defective apoptotic pathways, the advantages of apoptosis is none other than the development of organs by programmed suicide of the cells As I have mentioned before, there are uncountable processes that are taking place inside an organism every milli second. The process of apoptosis has its own significance amidst all the other. It can also be rightly named the mother of all processes as apoptosis is the cause of organ development and also the root cause of development of any organism.
3. Organ Development Organ development is also known as organogenesis. For our clear understanding we can explain it as the budding of organs from the growth cycle beginning from the form of a zygote. This routine is followed in all organisms which is just the consequence of death of cells in a programmable manner. The cells from the stage of division are never pre-determined the cells later are differentiated to form certain organs or organ systems. This programmed process of cell death coupled with organ development is the most important course of action in any organism as it determines the growth or death of the same. Then which can be followed by all the regular functions of the gene. To give a brief description about how organ development takes place, it is the proceedings of the ectoderm, endoderm and mesoderm to develop organs and organ systems. The embryo is at its weakest in this stage of development which may lead to anomalies or discretion.
The soul reason behind this process on paper to progress in a timely manner is the proper differentiation of cells by gene coding. Which can literally imprint an impact on the cells to develop only to certain organs by coding them. Stem cells play the most vital part. Being the cells that can undergo easy differentiation combined with a superiorly faster rate of proliferation when compared to normally proliferating cells.
Figure 9: Development of Endoderm, Mesoderm and Ectoderm into specified organs by genetic coding. The layers are distinctively separated and differentiated into specific organs and organ systems by genetic regulation. This differentiation is defined by the genes that regulate the development.
Endoderm ïƒ Forms the tissue within the lungs and pancreas (Anne and Douglas, 2000).
Mesoderm ïƒ Function of forming muscles and also the tissues of kidneys.
Ectoderm ïƒ Primordial function of formation of tissues with the epidermis and most important characteristic of formation of neurons.
3.1 Causes of improper organ development
There are many conditions that can cause improper organ development. Some of them like Toxicity, high amounts of radiation in the form of zygote or even in the higher order in the development line can cause permanent shift from the frame of reference. Other prime movers for this plight are some like tobacco and alcohol and other brain stimulating drugs. These obstructions of organ development due to mutation can been experiment on Arabidopsis thaliana and proved to cause the same effect on humans (Stein et al., 2004). This is because human field trials on this has been stated illegal all around the globe. Irregular apoptotic pathways can also be mentioned as some the reasons for the cause of improper organ development. But the root cause of everythig always lies in its beginning that is the primary infection of exposure to hazrdous materials or drugs. This can interfere in the primary pathway, leading inturn to the defective pathway of apoptosis. The defective pathways in apoptosis can be the major cause of the irregular or improper organ development. May be considered as one of the most important reason for improper organ development.
One of the most important reasons for improper organ developent is the cause of genetic mutations. The genetic mutations are caused by the radiotions or hazardous toxins that I have mentioned before. But what leads to improper organ development is genetic modification or mutations in the gene. This can lead to permanent damage of the cell or the gene. This gene damaged, specifies to an organ. Finally the organ is completely damaged due to the mutation of the gene that tends to regulate that specific organ of the organism. Also considering the fact that the cells can also die due to mutations causing permanent damage in the organ development phase of the organism.
4 Applications 4.1 Clinical applications
Many diseases like cancer, auto immune diseases, neuro-degenerative diseases do not either inhibit apoptosis or leads to inappropriate activation of apoptosis. They do not completely eliminate harmful cells which lead to loss of all the essential cells that prevent the oncoming of these diseases. Therefore, potential therapeutic strategies must be incorporated by including small molecules that either inhibit or activate certain target proteins that are responsible for apoptosis (Murphy et al., 2003).Generally there occurs a natural delay in the activation of Caspases after any injury and this delay allows enough time for treating the molecules that target Caspases. They are said to show therapeutic applications in preclinical studies (Reed, 2000; Nicholson, 2000). Bcl-2, another inhibitor gene of apoptosis, plays a vital role in the mitochondrial pathway and is regulated in many cancer cells. Introduction of an antisense Bcl-2 oligo molecule has shown promising results in preclinical trials in SCID mice and phase III clinical trials (Reed, 2000; Nicholson, 2000). There is something called, Inhibitors of Apoptosis (IAPs) that are of potential therapeutic targets for treatment of diseases. Some cancers over expresses the IAPs that is associated with the genes responsible for the resistance of apoptosis. One such gene is called Survivin (an IAP) which is involved in cancer cells. By eliminating this Survivin, the cancer cells become more sensitive to drugs that initiate apoptosis (Nicholson, 2000).
4.2 Immunoblotting techniques
Cytochrome C, an indicator of apoptosis is attached to the apoptotic cells along with the presence of genes responsible for apoptosis and immunoprecipitates were formed. By addition of anti-Cytochrome C or anti-Bc
Microbial Contamination on Toothbrush Storage
The study of microbial contamination on storing a toothbrush in a bathroom with a toilet
Aims: To examine the microbial contamination of storing a toothbrush in the bathroom with a toilet among the Colony Forming Unit (CFU) of microbial groups.
Methods and Results: The results are analyzed by the counting of CFU of agar plates
Conclusion: The toothbrush storing in a bathroom with a toilet or without toilet is being contaminated and it is a bad place for storage
Introduction Escherichia coli (E.coli) is recognized as a coliform bacterium which is gram negative, anaerobically developed and shaped like a rod. It is generally found in the intestine of warm-blooded animals such as humans. In addition, E.coli is able to discharge into the environment with fecal substance under airborne condition especially by the flushing of toilet and the bacteria can grow numerously in fresh fecal substances aerobically for short periods of time. The bacterial aerosols by the flushing can move as far as six to eight feet away from the toilet. A humid, warm surface permit more bacteria to grow and the bristles of the toothbrush would increase the surface area for microbial adhesion, hence, the fecal-oral transmission can be used as a major route via which pathogenic strain of bacteria to bring into oral diseases. Apparently, the occurrence of fecal coliforms in water is not straightforwardly harmful and does not essentially express the presence of feces (Doyle, M. P., and M. C. Erickson. 2006).
Five types of plates were used with the microbial groups in this experiment.
Chocolate blood agar, CBA, is an unselective and a medium with enriched development used for fastidious bacterial isolation. It is a alternative of the blood agar plate which contains red blood cells lysed by moderate heating to 80 °C.
Reasoner’s 2A agar, R2A, is used for the isolation of heterotrophic bacteria from treated drinkable water (Sandle, T, 2004). These bacteria is likely to grow slowly and would rapidly be restrained by speedy-growing species on a rich medium.
Diagnostics Pseudomonas Isolation Agar, PYO, is used for the isolation and differentiation of Pseudomonas aeruginosa selectively by raising the pyocyanin production. Pyocyanin production is indicated as a bluish-green, water-soluble pigment that gives a greenish color into the media (Bodey, G.D., et al., 1989). Malt Extract Agar, ME, is used for the cultivation and isolation of yeasts and molds by suitable nutrient supply (7). The pH is modified to nearly 5.5 for upgrading the fungi growth and to lightly hinder bacterial growth which usually determined as environmental contaminants.(6) Lauryl Sulfate Broth, LTA, is used for the isolation of coliforms in water and foods. The coliform groups involves aerobic and facultative anaerobic, gram-negative and the bacilli without spores which is able to progress the fermentation of lactose and generate acid and gas at 35°C. Additionally the LTA44°C makes faecal coliforms to be enumerated and the Sodium Lauryl Sulfate demonstrates excellent inhibition of organisms except the coliforms.
The technique of spreading plates is used for the isolation and enumeration of microorganisms in a mixed culture by even allocation. This method can measure the bacteria easier by using a sterilized spreader and applying a small quantity of bacteria suspension on the surface of plate. The plate is required to be dry before incubation so the bacteria can be absorbed into the agar rapidly. The plate counting approximate the amount of cells depend on the capability to produce colonies under particular states of nutrient medium, temperature and time. The counting of CFU believes that every colonies is aggregate and observed by a single viable cell. Moreover, CFU/mL of the original suspension is figured out mathematically and then factored in the quantity plated and the dilution factor. This study aims to examine the microbiological contamination of toothbrush to prove whether a bathroom with a toilet is a good place to store a toothbrush or not.
Material and methods Sample collections and suspensions preparation of samples
Group 1, Stored normally at the sink in a bathroom which contained a toilet situated 0.5 – 2 m away from the sink. Group 2, not stored near a toilet. Brush either stored in bedroom or in bathroom devoid of toilet.
The samples of plaque, toothbrush, sink and tap water were collected by respective sterile method. The plaque sample was put into a 2.5ml Ringer’s solution and sonicated for 10 minutes. Then the weight of plaque sample is determined by weighing the bijou bottle and the toothpick is removed by aseptic techniques. The toothbrush sample is put into the 10ml Ringer’s solution into a Universal bottle and the toothbrush head is cut and removed aseptically. The swab originated from 2 x 2 cm square of the sink sample is put into 2.5 Ringer’s solution of the bijou bottle and is vortexed for approximately 3 minutes. Then the swab is taken out by sterile techniques and the suspension is ensured to squeeze out of the swab. The sample of tap water is put into the 15ml of sterile tube.
10-fold dilutions and plates spreading
Each of the samples were processed with orders of plaque, toothbrush, sink and tap water. Different dilutions of samples are prepared by the 10-fold dilutions expect the tap water samples. 0.5ml of undiluted original suspension (100) is removed and inoculated into 4.5 ml of another Ringer’s solution aseptically and mixed well to be a 10-1 dilution. All the samples are prepared down to 10-5dilution.
Four plates of CBA, R2A, ME and two plates of PYO, LTA37 and LTA44 of each samples were collected and labelled. For the plaque sample, 2 PYO, 2 LTA37 and 2 LTA44 plates with 100, 2ME plates with 10-1, 2ME plates with 10-2, 2 CBA and 2 R2A plates with 10-4, 2 CBA and 2 R2A plates with 10-5 were inoculated. For the toothbrush sample, 2 PYO, 2 ME, 2 LTA37 and 2 LTA44 with 100, 2 ME plates with 10-1, 2 R2A with 10-2, 2 CBA and 2 R2A plates with 10-3, 2 CBA plates with 10-4 were inoculated. For the sink sample, 2 PYO, 2 LTA37 and 2 LTA44 plates with 100, 2 ME plates with 10-1, 2 ME and 2 R2A plates with 10-2, 2 CBA and 2 R2A plates with 10-3, 2 CBA plates with 10-4 were inoculated. For the tap water sample, all 18 plates were inoculated with 100 undilutedsuspension. 0.2ml of relative dilutions were spread across the surface of the agar plates with aseptic methods. All the plates were allowed to dry before inoculation. 2 LTA44 plates were put in the incubator with 44„ƒ, 2 PYO plates, 2 LTA37 and 4 CBA plates were put in the incubator with 37„ƒ, 4 R2A and 4 ME plates were put in the incubator with 25„ƒ (United States Pharmacopeia, 2015).
Data collection The inoculated plates were collected and colonies were counted to determine the Colony Forming Units (CFU) by multiplying the average number of colonies by 5 to convert from 0.2ml to 1ml and is multiplied by the dilution factor. The best number of colonies (30-60 colonies) were counted for the plates which have two dilutions plated onto them. The CFU was calculated as the CFU ml-1 100 suspension and needed subsequent conversions except for the tap water sample. For the plaque, CFU ml-1 100 suspension was multiplied by 2.5 and divided by the weight of plaque to determine the CFU g-1 in 2.5ml bijou bottle. For the toothbrush, CFU ml-1 100 suspension was multiplied by 10 to determine the CFU head-1 in the 10ml Universal bottle. For the sink surface, CFU ml-1 100 suspension was multiplied by 2.5 and divided by 4 to determine the CFU cm-2 in the 2.5ml bijou bottle from a 4cm2 square.
Results Mean CFUs per unit with SD : a measure that is used to quantify the amount of variation or dispersion of a set of data values. A low standard deviation indicates that the data points tend to be close to the mean (also called the expected value) of the set, while a high standard deviation indicates that the data points are spread out over a wider range of values.
Average with SD (Plaque CFUs per gram)
Group 1 (with toilet)
Group 2 (without toilet)
4.16 12 x 108
4.71 16 x 108
1.2 3.41 x 108
1.96 3.55 x 108
8.34 23.5 x 102
7.41 38.5 x 107
4.7 23.4 x 102
8.49 4.33 x 103
2.55 13.2 x 102
3.35 8.78 x 104
9.51 49.2 x 105
ME (fil fungi)
1.72 6.2 x 104
2.95 5.71 x 103
Average with SD (toothbrush CFUs per head)
Group 1 (with toilet)
Group 2 (without toilet)
1.92 3.52 x 106
1.51 5.76 x 107
2.35 5.71 x 106
1.12 4.60 x 107
1.27 4.07 x104
2.78 14.4 x 108
1.84 5.94 x104
2.54 12.9 x 106
1.18 5.88 x 104
4.85 24 x 104
2.10 9.78 x 104
3 13.6 x 105
ME (fil fungi)
2.59 11.8 x 104
1.34 2.26 x 102
Average with SD (sink per square cm)
Group 1 (with toilet)
Group 2 (without toilet)
8.91 22.7 x 104
6.33 24.7 x105
2.56 11.5 x 105
7.82 36.7 x105
1.22 2.98 x 102
4.65 16 x103
4.55 12.4 x102
5.73 16.9 x103
4.24 14.5 x10
1.54 6.19 x102
4.84 1.57 x 102
3.13 7.55 x103
ME (fil fungi)
1.40 1.76 x 102
4.71 18.5 x102
Average with SD (tap water CFU per mL)
Group 1 (with toilet)
Group 2 (without toilet)
1.53 2.14 x 10
1.5 7.69 x104
9.04 21.6 10
3.49 14.4 x104
8.46 30.7 10-1
1.85 9.62 x10-1
3.7 19.2 x10-2
1.15 5.88 10-1
ME (fil fungi)
p-value. Two-tailed independent samples t-test (Group 1 vs Group 2) – 95% = P<0.05 is significant
ME (fil fungi)
From the table, the CFU in the plaque sample collected from storing the toothbrush with and without toilet are observed. For the CBA and R2A, the CFU in group 1(with toilet) is counted as 4.16 12 x 108 CFU g-1 and 1.2 3.41 x 108 respectively, and the CFU in group2 (without toilet) is marginally greater than group1 which resulted as 4.71 16 x 108 and 1.96 3.55 x 108 respectively. For the PYO, LTA37, LTA44 and ME (yeasts), The CFU of group 2 are slightly more than the group 1 except the fewer CFU (2.95 5.71 x 103) without toilet than in which with toilet (1.72 6.2 x 104) observed in ME (fil fungi). It is found that the fewest CFU is on LTA44 or even observed as 0 CFU per unit since 44„ƒis over the optimal temperature and the bacteria can be inactive. Besides, for the other microbial groups, toothbrush, sink and the tap water samples, they showed the similar results as the plaque sample that the average CFUs per unit of the without toilet sets were slightly more than the sets with toilet on the CBA, R2A, PYP, LTA37, LTA44 and ME (yeasts) and the CFUs per unit of the without toilet sets were slightly less than the toilets sets. However, the samples in the tap water evaluated some differences from the other groups was that the small quantity of average CFUs per ml within each samples were indicated since the presence of fecal coliforms in water might not be directly harmful and did not necessarily indicate the presence of feces.
Overall, the CFU of the plaque sample collected without toilet is slightly greater than which with toilet but probably there is no obvious difference between two groups by the examination of p value.
P value is used to compare the significance of the tests. Significant difference between two groups can be determined if the p value is smaller than 0.05 and the null hypothesis is not be supported. From the table of two-tailed independent sample t-test, it indicated that all p values among the different agar plates of the 4 microbial groups are greater than 0.05 which showed that the difference between 2 groups for any sample type (with toilets and without toilets) or medium types are not significant. Although the tables showed large variability within a given sample, this is common for environmental samples and hence the null hypothesis is supported.
Discussion The toothbrush, the plaque and the sink samples collected with or without the toilet sets, except the tap-water, were observed to contain large quantity of coliforms and it proved the presence of pathogenic bacteria, E.coli. The toothbrushes contamination acts as a crucial role in the expansion of various diseases such as respiratory infection and oral diseases (M. B. Dayoub, D. Rusilko, and A. Gross, 1977). Toothbrushes are often stored in the toilets and disclosed to contamination as it is a microbial atmosphere with the occurrence of pathogenic bacteria which is spread by aerosols and the flushing of toilets (Taji SS, Rogers AH, 1998). Moreover, the presence of the E.coli is correlated with the uncleaned bristles or the storage of brushes with high humidity (the sink) and warm environment which are near to the bristles and this can effectively enhance the spread and growth of bacteria than those stored in aerated conditions by 70%. (R. T. Glass, 1992).
However, the experimental results showed the colony forming units in the bathroom without toilets sets were greater than the sets with toilets which is apparently difference from the hypothesis. P-values of Two-tailed independent samples t-test can evaluate the situation. Although the large variability within a given sample was observed, this is common for environmental samples. Because of the large variability, there is no significant difference between the two groups for any sample type or medium type. The p values is greater than 0.05 which showed 95% level of confidence that the two parameters are not the same and there is no enough difference within the samples to conclude a difference so the null hypothesis is accepted. It is conclude that toothbrush would get contaminated regardless of storing near or far from the toilet.
There are some limitations existed in this experiment. Firstly, there is manageable amount of data for the analysis which can affect the precision of the results. In this experiment, only 26 samples and 27 samples for each groups were examined which were not excessive enough to evaluate the hypothesis. More sample sizes are suggested and hence sufficient statistical power to the final results can be determined. Another error would be the dilution error since the pipetting for 10- fold dilutions could lead to a considerable departure from the expected identity and inaccurate results. It is suggested that the precise pipetting from 10-1 down to 10-5 and proper vortex with vibration of suspension within each dilutions is required. Additionally, more time allowed for the first lab practical and appropriate aseptic techniques could be suggested to ensure the accuracy of results.
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