MATERIALS AND METHODS 2.1. Toys The toys that were used vary in material. This is particularly important when comparing and contrasting the types of bacteria that may grow on different surfaces and therefore which type of surface is more culpable for harbouring bacteria. Toys varied from being plastic, wooden or soft and were roughly all of similar size. When obtaining samples of microorganisms for comparison a reproducible method best used in order that comparisons can be made between one site and another between different toys. Typically, a 2cm square was produced allowing an area of 4cm2 to be swabbed. In doing so, the square cut-outs can be transferred and taped to other surfaces required for testing. 250 ml of 0.1% peptone water (Difco ) was then prepared by mixing 25g of the dehydrated peptone into distilled water. The glass jar is then shaken and autoclaved to sterilise the culture media. The peptone water, otherwise known as Bacto Peptone, is used as an organic source of nitrogen, in a form that is readily available for bacterial growth and in microbiological culture media for cultivation of a wide variety of bacteria and fungi. Bacto Peptone is an enzymatic digest of animal protein. Sterile cotton swabs were used in order to obtain microorganisms from the test surfaces. The swabs were carefully moistened with peptone water allowing better collection of microorganisms from the test surface. Once a swab is taken the cotton buds are inoculated in 5ml of peptone water in self-standing, sterile tubes with conical bottoms and were kept overnight at 37oC to allow incubation. ~ 11 ~
Once the sterile tubes have been incubated at 37oC, serial dilutions are then undertaken. Serial dilutions are an accurate method of producing solutions of low molar concentrations. The cotton swabs inoculated in sterile tubes, or stock solutions provide a molarity that is greater than that which is required therefore dilutions are necessary in order to obtain more accurate results when plating the medium on to nutrient agar. Since measuring minuscule volumes of solution is prone to too much experimental error, a series of dilutions are performed in order to progressively reduce the concentration of the solution from that of the stock solution allowing one to quantifiably and readily identify the colonies of bacteria present. Once completed, the relevant samples can be pipette at 1ml onto nutrient agar and spread using a bent glass rod which allows equal distribution of the suitable dilutions across the agar. 2.2. Autoclaving Autoclaving is a technique used in the sterilization and disinfection of media and equipment by use of steam under intense pressure. It is an extremely effective method in the removal of microorganisms and reducing the levels of infective organisms, while not necessarily destroying them all, to a safe level. Furthermore, it should be noted that the process can be rendered ineffectual if the materials to be sterilized are spoilt by heat or moisture, or if the steam is unable to penetrate the material successfully. To begin, the inner container found within the autoclave is removed and water levels are brought up to just above small ledges embedded at the base. Any media or materials, for example, pipette tips or glass jars of agar, are carefully placed into the bench autoclave and lids of glass jars are not fastened completely in order to allow steam to penetrate. The lid is ~ 12 ~
fastened carefully by use of the wing nuts and the steam exhaust is left open. The autoclave can now be turned on and allowed to reach 3psi, at which point, the steam exhaust valve is screwed tightly shut to allow the build-up of pressure. Sterilization takes place at 15psi above the atmospheric pressure for approximately 15-20 minutes. In doing so, the saturated steam at this pressure causes the temperature within the autoclave to rise up to 121 C due to the removal of air within the apparatus. If air remains trapped within the autoclave then at a particular gauge pressure the temperature of the air/steam mixture will be lower than that of pure steam at the same pressure. After sterilization for 15 minutes at 121 C the autoclave can be switched off and the pressure within the apparatus is allowed to return slowly to the atmospheric pressure or 0psi. Extreme care must be taken in the handling and removal of the autoclave and its contents when the process is complete. Gloves must be worn at all times as the medium from within the apparatus may still be at an incredibly high temperature when it is removed soon after pressures have returned to ordinary atmospheric. For a 500ml glass jar of, for instance, nutrient agar medium, this is incredibly important as it is a fairly large volume. 2.3. Nutrient agar Agar is a gel at room temperature, remaining solid at temperatures as high as 65 C (Selby and Selby, 1959). At approximately 85 C however it begins to melt, while at 32-40 C it solidifies, a property known commonly as hysteresis. Nutrient agar plates can be made very straightforwardly in the laboratory. It is the phenomenon of hysteresis that yields solid agar when autoclaving has taken place. ~ 13 ~
The nutrient agar (Oxoid ) prepared involved dissolving 14 grams of granules in 500ml of distilled water. In doing so, enough of the agar is made up in order to pour out approximately 20 plates. Once dissolved, the agar contained within a 500ml glass jar is autoclaved in order to sterilize the medium and prevent contamination from microorganisms other than those found from the surface of the toys. When the pressure within the autoclave has been allowed to reach 0psi and the contents of it carefully removed then the glass jar can be transferred into a water bath set to 50oC allowing it to cool but remain in a liquid state. The nutrient agar is now ready to be poured and when removing the cap from the glass jar the neck of the jar must be passed through a bunsen flame to sterilize it, then begin to pour carefully. The agar medium will then cool and set like gelatin when at room temperature. 2.4. Gram staining Gram staining is possibly the most important staining technique in microbiology. It is used to make a distinction between bacteria based on their different cell wall constituents. (Bergey et al, 1994). The Gram stain method distinguishes between the two large groups of Gram positive and Gram negative bacteria by coloring these cells pink/red or dark blue/violet. Gram positive bacteria will stain blue/violet due to the presence of a thick layer of peptidoglycan found within their cell walls, which retains the crystal violet these cells are stained with after iodine fixation and alcohol washing. On the other hand, Gram negative bacteria will stain red, which is attributed to a thinner peptidoglycan wall, which does not retain the crystal violet during the decolouring process and requires counterstaining by safranin (Cappuccino and Sherman, 2001). ~ 14 ~
A miniscule quantity of each unknown bacterial sample is obtained with a sterile inoculating loop and then spread on a sterile glass side into 20µl of distilled water. The slide is then heat fixed by use of a Bunsen flame. The procedure involves staining the cultured, unknown bacterial cells obtained from the surfaces of the toys with crystal violet dye. Subsequently, a Gram's iodine solution is added to form a complex between the crystal violet and iodine which is insoluble in water. Alcohol is then used as a decolouriser and is added to the sample, which dehydrates the peptidoglycan layer causing it to shrink and hence tighten. In doing so, the large crystal violet-iodine complexes that are formed are not able to penetrate this tightened peptidoglycan layer, and is thus trapped in the cell in Gram positive bacteria allowing the dark blue/violet colour to be retained. Conversely, the outer membrane of Gram negative bacteria is degraded and the thinner peptidoglycan layer of Gram negative cells is not capable of retaining the crystal violet-iodine complex and for that reason the colour is lost. (Cappuccino and Sherman, 2001) A counterstain, such as safranin, is then finally added to the sample, staining it a characteristic red. The glass side can now carefully be dried using blotting paper and examined under a light microscope using oil immersion techniques. (Cappuccino and Sherman, 2001) ~ 15 ~
2.5. Catalase test Gram stain analysis under a microscope allowed the identification of several Gram positive cocci. All species of Staphylococcus are known to be gram positive cocci therefore the catalase test is an excellent way of distinguishing between them. The three major types are Staphylococcus aureus, Staphylococcus epidermidis and Staphylococcus saprophyticus. The catalase test is able to determine whether these microorganisms produce the enzyme catalase which leads to the breakdown hydrogen peroxide to water and oxygen (Chelikani et al, 2004). 2 H2O2 2 H2O + O2 To begin with, 200µl of 3% H2O2 is pipetted onto a sterile glass slide. A sterile loop was then used to delicately touch and extract a miniscule amount of culture and the visible mass of cells are then mixed in the drop of H2O2. The formation of bubbles indicates a positive test while no bubbles, due to no oxygen production, indicate a negative test result. The catalase test therefore allowed a distinction to be made between Staphylococcus, which is catalase positive and other gram positive cocci, such as Streptococcus. 2.6. Dryspot Staphytect Plus The Dryspot Staphytect Plus (Oxoid ) test and is a latex slide agglutination test, similar to a coagulase test (Essers and Radebold, 1980). It enables the differentiation of Staphylococcus aureus from other species. Principally, it is a test that works by the detection of clumping factor and Protein A that Staphylococcus aureus contain. Those Staphylococci that do not possess these properties give a negative result. ~ 16 ~
Dryspot Staphytect Plus uses blue latex particles coated with both fibrinogen and rabbit IgG. The fibrinogen will react with clumping factor, while Protein A is said to be found on the cell surface of almost all of the known human strains of Staphylococcus aureus, it also has the capability to join the Fc portion of immunoglobulin G (IgG) (Taussig, 1984). In order to undertake the test, cultures of suspected Staphylococcus aureus are mixed onto the reaction cards provided that already contain the reagents dried onto them. Upon mixing, after a short period of time rapid agglutination will occur between, firstly, fibrinogen and clumping factor and then IgG and Protein A if Staphylococcus aureus is indeed present, providing a positive result. If neither clumping factor nor Protein A are present then agglutination will therefore not occur. In such cases, Staphylococcus epidermidis is shown to give this negative result. 2.7. Mannitol salt agar Further culture media can be made in order to more accurately differentiate between Staphylococcus aureus and other species of Staphylococci. Mannitol salt agar (Oxoid ) was made up from dissolving 55.5g of dehydrated medium into 500ml of distilled water. The unprepared solution was then autoclaved and left to cool in a water bath at 50oC, much like the technique used when preparing nutrient agar. The prepared medium is then poured, to make up approximately twenty petri dishes and left to set overnight. Mannitol salt agar (Oxoid ) is a selective medium for the isolation of presumptive Staphylococci. The medium contains a high concentration of salt which inhibits the growth of most bacteria. Coagulase-positive staphylococci that are presumed to be Staphylococcus aureus produce yellow colonies which are surrounded by yellow halo zones whilst ~ 17 ~
staphylococci such as Staphylococcus epidermidis produce colonies with a pink colour while the medium also gains a pink colour after initially being red. 2.8. Bacillus cereus selective agar Bacillus Cereus selective agar (Oxoid ) was made up by dissolving 21.5g of dehydrated media in 500ml of distilled water. It is then sterilized by autoclaving as normal, however, once allowed to cool in the water bath to 50oC, Egg Yolk Emulsion (Oxoid ) and 1 vial of Polymyxin B Supplement (Oxoid ) are added and mixed thoroughly. Then only are the plates poured. The agar is a selective and differential medium that relies upon the failure of Bacillus cereus to utilise mannitol and the ability of most strains to produce phospholipase C. In the typical formula for the agar, a peptone level of 0.1% and the addition of sodium pyruvate improve the precipitation of the egg yolk and improve the production of spores. The primary diagnostic features of the medium are the colonial appearance, precipitation of hydrolysed lecithin and the failure of Bacillus cereus to utilise mannitol. As a result it is possible to differentiate from the accompanying mannitol-positive microorganisms which are identified by a change in colour of the indicator phenol red to yellow. Bacillus cereus produces lecithinase, which degrade the lecithin that is contained within the egg-yolk suspension. The hydrolysed lecithin gives a white precipitate around the Bacillus cereus colonies. The colonies of Bacillus cereus can also be seen on the agar to be a rough, dry and pink to purple color surrounded by a ring of white precipitate. Colonies surrounded by a yellow, pink or a clear zone are not Bacillus cereus. In addition, Bacilllus subtilis is ~ 18 ~
known to form colonies of yellow/pink while forming a precipitate. The formation of the precipitate is due to an egg-yolk reaction. 2.9. Spore stain (Schaeffer-Fulton) This technique is designed to isolate endospores by staining any present endospores green, and any other bacterial bodies red. Since Mycobacterium are not known to produce endospores one can expect to see visible red cells present (Cappuccino and Sherman, 2001). The staining procedure uses involves the use of a primary stain, decolourising agent and counterstain. The primary stain used is malachite green and due to the spore's impervious coat heat is additionally required to allow for better penetration. The decolourising agent used is water. However, if the spores accept the malachite and have been stained green previously, then they cannot be decolourised. Finally, the counterstain used is safranin which is absorbed by vegetative cells which then appear red. The spores retain the green of the primary stain and a distinction can be made between the two colours using a light microscope and oil immersion (Cappuccino and Sherman, 2001). 2.10. Acid-Fast stain (Ziehl-Neelson) The genus of Mycobacterium are resistant to gram staining procedures due to the presence of a thick, waxy wall. This lipoidal wall makes the penetration by certain stains extremely difficult. The organisms are therefore called acid-fast as once a stain has penetrated the wall it is difficult to readily remove, even while using large amounts of acid alcohol as a decolourising agent (Cappuccino and Sherman, 2001). ~ 19 ~
The acid-fast procedure involves preparing a heat fixed slide much like when undergoing a gram stain of the unknown culture. The primary stain of carbol fuchsin is then applied over heat while ensuring when doing so that the stain does not evaporate. The carbol fuchsin penetrates the lipoidal wall as it is soluble in the materials found in the wall, this penetration is further enhanced by the use of heat and the red phenolic stain is retained (Cappuccino and Sherman, 2001). Acid alcohol is then added as a decolourising agent once the smear has been permitted to cool. This is important as the waxy cells can be allowed to harden. On application of the acid alcohol, cells that are acid fast will retain their colour and be resistant to decolourisation since the primary stain is more soluble in the cellular wall waxes than in the decolourising agent. Non-acid fast cells will lose their colour and thereby appear colourless (Cappuccino and Sherman, 2001). The counterstain used is methylene blue. As previously mentioned, prior to the application of acid alcohol, non-acid fast cells will lose their colour and thereby appear colourless. They may now however absorb the counterstain and take on its blue colour, while acid fast cells, such as those of Mycobacterium retain the red colour of the initial carbol fuchsin primary stain (Cappuccino and Sherman, 2001). ~ 20 ~