Streak Plate And Viable Cell Count

Streak Plate And Viable Cell Count Aim and introduction should display insight into what the streak plate and viable cell count method are employed to achieve. They should also introduce MacConkey agar and how its selective and differential properties allow the characteristics of the test organism to be determined. Escherichia coli (E.coli) The aim of this experiment is to allow a certain bacterium to divide and multiply enabling us to view the bacteria in a single cell structure. E.coli is one bacterium that is good for such an experiment. E.coli can be said to be both bad and harmless, some E.coli bacteria have are highly toxic and can harm humans and animals. However, the majority of E.coli strains are relatively harmless with low toxicity. These harmless strains of E.coli are found naturally occurring in the human body, especially in areas such as the human intestines. Some E.coli can even benefit their hosts; they do this by producing specific vitamins. It is for reasons like the ones mentioned why E.coli is an appropriate bacteria to use for this experiment. Another reason is that E.coli bacterial cells have an average bacterial size of 2um; this can be seen under a light microscope. Other bacteria however may be even smaller and may require a larger microscope for viewing or even an electron microscope. Also the incubation period for E.coli to multiply and grow rapidly isnt very long and temperatures arent too high or too low. E.coli can be incubated overnight at 37oC and then stored at 4oC until its requirement. The technique use to manipulate and isolate the E. coli bacteria is known as the streak plate procedure. The technique was developed to allow bacteria to multiply and produce many colonies, during the incubation period, depending on the amount of bacteria present. Each colony will contain millions of bacteria cells derived from a single parent cell. (Talk in more detail about this procedure). We will be using this technique to allow the E. coli to multiply and divide splitting itself into colonies. The viable cell count, also known as viability count, is a method used to determine the number of living cells within a suspension, in this case E. coli. To obtain an understanding of how much E. coli cells are present in a sample this method must be put into action. (Expand) The MacConkey agar is specifically designed to allow gram negative bacteria to grow; its a recipe of many substances such as bile salts, sodium chloride proteose and many more. One of the properties of the MacConkey agar is its selective isolation and identification of bacteria; it is a medium that allows us to distinguish gram-negative bacteria. E. coli is a rod shaped gram negative bacteria, so using the MacConkey agar plate to multiply it would be appropriate, the agar will also cause the E. coli to change colour from pink to red, and this is an indication of gram negative bacteria present. A Nutrient agar is a growth medium used for the cultivation of bacteria, this specific agar remains solid even at high temperatures. The gram-stain technique was developed for viewing cells clearly under a microscope and to enable us to establish their structures. It is a very simple procedure of just adding 4 different substances accordingly, however one of these substances is toxic to humans therefore the procedure must be carried out in a fume hood. The gram stain method was introduced by It is important for scientists and medics to know the structure and function and identity of bacteria and viruses, it is for reasons like this why such experiments are carried out. Without such procedures so many bacteria and viruses wouldnt be known and could spread and become out of control. Methods: Explain why each procedure was done highlight key points state any deviation from protocol document any errors or difficulties you had with the technique Discuss the importance of aseptic technique and what steps could be taken to prevent contamination during manipulation of bacteria All methods were doing under aseptic conditions; the reason for this is to prevent contamination of the bacteria during its manipulation. Many errors could arise if aseptic conditions arent used, eventually resulting is wrote results. Streaking bacteria on MacConkey agar method: Prior to the experiment, an E. coli sample was made and given to during the practical. Risk assessment: Materials used: 10ml liquid culture of E. coli the bacteria sample to be use in this practical Sterile plastic loops used for transferring E.coli bacteria from one place to another uncontaminated. MacConkey agar plate used to allow E.coli bacteria to grow as it provides energy recourses and support Sharps bin for loops etc. these are used to keep the laboratory area as uncontaminated as possible and to make sure bacteria doesnt spread Marker pens and labels to label the plate Step 1: using a sterile plastic loop I touched a given sample of E. coli and streaked an inoculum onto a MacConkey agar plate in a specific pattern (see..). This plastic loop is then disposed of into the sharps bin. Step 2: using another sterile plastic loop, I created a run of parallel lines from the edge of the initial streaks Step 3: step 2 was repeated 2 more times with a new sterile loop used. Step 4: a final streak was made, creating a simple streak from the previous streaks into the centre of the plate. The picture below illustrates this. The MacConkey plate was then given to the technicians to incubate. It was important to dispose- on the plastic looks by placing them into the sharps bin because they are contaminated and if they touch any other surface it can lead to the spread of bacteria resulting in major contamination. Throughout this procedure plastic gloves and a lab-coat were worn, also to prevent contamination. Viable cell counts: Risk assessment Materials used: P1000 and P100 pipettes and tips used to transfer certain amounts of PBS etc. Three Nutrient agar plates 10ml sterile PBS buffer, used to maintain the pH Sterile plastic spreaders to spread the E.coli on the Nutrient agar plates Eight sterile bijoux bottles for dilutions To start the dilution, using a pipette I transferred 900 ul of diluents (PBS) in eight different sterile bijoux bottles. The PBS (phosphate buffered solution) solution is a commonly used buffer to maintain a pH; it is used in this practical because of its ability to aid biological research. After the PBS was placed into the labelled bottles, using a new sterile tip for the pipette I transferred 0.1ml of E.coli liquid culture sample (neat) into the first bottle (10-1). For the dilution to continue a new pipette tip was placed and 0.1ml of the 10-1 diluted E.coli was transferred to the 10-2 bottle, this process continued up till bottle 10-8. By doing so the E.coli will become more and more dilute within the different solutions, because less E.coli is being added each time. 10-5, 10-6 and 10-7 samples were then spread onto three different Nutrient agar plates using different sterile plastic spreaders so contamination wouldnt occur. This was done by placing 0.1ml of each dilution onto th e centre of the agar plate and then spreading it over with a sterile spreader. The agar plates were labelled and given to the technician for incubation. Gram Stain of bacteria from an isolated colony Risk assessment In order to stain the bacteria I selected an appropriate colony to stain, the colony must appear to be uncontaminated and its appearance must obviously look grown.. After this procedure is complete, the bacterial cells will be visible under a microscope. Materials needed: Bunsen burner used to heat-fix bacteria onto microscope slide Saline (PBS) emulsifier Light microscope to view bacterial cells Lens tissue to clean the lens Immersion oil for light microscope lens to allow better view at 100x magnification Stains for Gram stain method Before the bacteria can be modified to be viewed under the microscope clearly, the microscope glass slide must be cleaned to prevent contamination. After doing so a drop of sterile saline was placed onto the centre of the slide, the saline drop was placed because it can emulsify any bacterial colony that will be placed on top. To move some of the bacteria off the agar plate onto the slide, a sterile loop was used I touched the bacterial colony on the agar plate with the top of the loop and then spread the bacteria into the saline drop making it thin. Due to the moisture of the liquid I let the slide dry then used a Bunsen-burner to heat-fix the bacteria onto the slide by passing it through a few times then allowing it to cool. Heat-fixing was done so that during the staining the bacteria or wouldnt move or fall off. Once that was complete the slide was moved to a laboratory fume hood where the staining can take place, the follow 4 stage method was used: at first the bacteria sample on the slide was soaked in crystal violet for 30 seconds, after so it was rinsed with distilled water and drained. The second part is to soak the bacteria with gram iodine (mordant) for another 30 seconds then rinse with distilled water and drain it. Gram iodine is a toxic substance; it is for this particular reason why this part of the practical was carried out in a laboratory fume hood. Acetone decolouriser was then added for 10 second and the bacteria was again rinsed with distilled water and drained. The final part is to add Safranin, a counter stain, to the bacteria. It was placed on the bacteria for 30seconds and then the bacteria was further rinsed with distilled water, drained, blotted and allowed to dry. Substance Duration Further action Crystal violet (primary stain) 30 seconds Rinse with water drain Gram Iodine (Mordant) 30 seconds Rinse with water drain Acetone/alcohol (Decolouriser) 5-10 seconds Rinse with water drain Safranin (Counter stain) 30 seconds Rinse with water, drain, blot dry Stain was carried out in a laboratory fume hood due to the toxic gram iodine substance used. The transparent plastic shield of the fume hood was lowered so that only my hands were inside dealing with the chemical and biological substances. Gloves were worn during this procedure so that no stain would come into contact with the skin. When the slide was rinsed with water, it was rinsed gently with distilled water so that the bacteria are not shifted. After the staining was completed the sample can now be viewed under a light microscope and compared to other bacterial samples. The slide is placed on the stage with a drop of oil for immersion, the microscope is focused on 100x and the bacteria is viewed. Results: Should describe your findings in : prose/text, diagrams, tables and graphs which includes a description of growth characteristics and how successful your aseptic technique was MacConkey agar plate results: During the experiment there were no results to be noted as it was too early for anything to occur. After the agar plate containing the E.coli was incubated at 37oC and then stored at 4oC, its appearance was as expected. Colonies were separated, and as the streaks moved on less E.coli was present. The colonies were well distinctive and were round in their shape. The sample initially given was simply liquid, the result showed significant growth of this E.coli liquid into 3D structures. This indicates the growth of the bacteria in a fine way; the 3D structures appeared in a yellowing solid colour. Because the practical was conducted in aseptic techniques no contamination occurred. Aseptic techniques were successful in allowing me to produce accurate results. Viable cell counts My results: The colonies that appeared on the nutrient plate had a badge colour; visually they all appeared relatively same sized and volume. 10-5 10-6 10-7 10-8 TMTC 46 1 For the 10-7 the calculation for the number of bacteria in 1ml of the original culture is: (1107/0.1) x (X/1) [cross multiply] 0.1X = 1 x(1107) [divide by 0.1] Therefore X = 1.0108 For the 10-6 the calculation for the number of bacteria in 1ml of the original culture is: (46106/0.1) x (X/1) [cross multiply] 0.1X = 1 x(46106) [divide by 0.1] Therefore X = 4.6108 The number of bacteria present in 1ml of the 10-5 culture cannot be calculated as there was no value noted (TMTC). X= number of bacteria. The number of bacteria present in 1ml of 10-6 dilution is 4.6108 and in the 10-7 dilution culture is 1.0108. Class results: Pair number 10-5 10-6 10-7 10-8 1 TMTC 46 1 2 95 9 0 3 TMTC 52 9 4 TMTC 23 1 5 34 2 6 84 11 4 7 TMTC 24 18 8 2 2 26 9 102 6 0 10 19 3 3 11 TMTC 140 15 12 TMTC 57 9 13 61 12 0 14 8 6 1 15 195 51 4 16 55 27 3 17 TMTC 94 11 18 TMTC TMTC TMTC 19 TMTC TMTC TMTC 20 TMTC 113 18 Total 266 985 177 28 Average 66.5 57.9 9.8 7 Should describe your findings in : prose/text, diagrams, tables and graphs which includes a description of growth characteristics and how successful your aseptic technique was To determine the amount of bacteria within a culture a simple calculation must be done using my personal results for this experiment. There was no value for the 10-5 so this cannot be done. The result for 10-6 was 46, 46 x 10 = 460ml. To estimate the amount of E.coli present this is further multiplied by 106, therefore 460 x 106 = For the 10-7 result Æ’Â   7 x 10 = 10 Æ’Â   10 x 107 = However, I have selected some reasonable results from the table to calculate an average. Gram-stain of bacteria from an isolated colony: (view method number 3) Gram stains help us distinguish between microbial organisms, for example gram negative bacteria and gram positive bacteria. This method was developed to know the identity of bacteria present. (See procedure for The Gram Stain in the methods section). During step 5 of the Gram Strain Method above the following results were made when applying the four different substances: Substance Colour after stain Crystal violet (primary stain) Purple Gram Iodine (Mordant) Purple Acetone/alcohol (Decolouriser) Transparent (dye was washed off) Safranin (Counter stain) Reddish-pink The appearance of the E.coli bacteria under a microscope with 100x magnification was quite clear; it had a rod-like structure with a reddish-pink colour. The rods were all more or less the same size, however some were packed together and others were on their own. Discussion: Were the results the expected? Did the methods adopted achieve their aim? How the experiments could be improved. Include background information, critical evaluation of results Throughout all the experiments and procedures a lab-coat and gloves were worn to avoid skin contact with bacteria and harmful substances. Overall the aims were accomplished and the results were as predicted. MacConkey agar The colonies were expected to be in such a form, indicating that it was E.coli present and that it has rapidly multiplied into individual colonies. This further suggests that when E.coli is present under conditions where it could multiply, it multiplies by forming a round colony and expanding from there. However, some of the colonies were stuck together making it difficult to count the number of them present. What this means is that the growth of the bacteria was a success and the method adopted was accurate. The reason why some colonies were packed together may be the result of pressing too hard on the agar while streaking, with more streaking practice more accurate results can be obtained with colonies being on their own. The methods adopted for this practical achieved what was aimed for. After the incubation of the MacConkey agar plate the plate was stored for a week at a temperature of 4oC, this may have changed the appearance in colour and in shape of the formed colonies. Contam ination of the agar plate may have even occurred. An improvement to the experiment is to note down results straight after incubation is finished. Gram Stain results After analysing the microscope slide which contains the Gram Stained E.coli under the microscope its features were obvious. There were many average sized rods with a reddish-pink colour, some of these rods were packed together whist others were separated. Comparing this with another prepared sample of B.subtilis, the B.subtilis was a purple colour and has a longer and curved shape, like fine threads. However some again were packed together and others separated. The purple colour of the B.subtilis indicates that it is gram positive, and the pink colour of E.coli indicates its gram negative. When the Gram Stain method was applied to the B.subtilis it obviously stayed purple though out, with E.coli it will decolourise once the decolouriser is added. The gram stain method is highly effective and efficient when dealing with different bacteria; it helps identify them to a great extent. B.subtilis remains purple throughout the Gram Stain procedure, this itself can be an indication that it is a Gram positive bacteria. Bacterial cells have different types of cell walls, the gram negative and gram positive terms describe the nature of their structural differences. One of the important differences is that Gram positive bacteria have no outer membrane whereas Gram negative bacteria do, the purpose of this outer layer is to cover the peptidoglycan layer. When staining occurs the outer membrane of a gram positive bacterial cell wall becomes permanently stained as the strain can easily penetrate the thick peptidoglycan layer, so that if a decolouriser or distilled water is added the colour will remain purple. In the case of the gram negative bacterial cell wall the stain gets attached to the far outer membrane layer (lipopolysaccharide and protein), this layer decreases the penetration depth of the strain on the peptidoglycan, so the stain can be decolourised or removed. The diagrams below illustrate this. Gram positive: Æ’Â   Æ’Â   Æ’Â   Primary stain Mordant Decolourisation Counter-stain Note: colour remains the same throughout addition. Gram negative: Primary stain Mordant Decolourisation Counter-stain Note: colour changes Æ’Â   Æ’Â   Æ’Â   The aim of the Gram Stain method was confirm that the bacteria that was initially being dealt with was E.coli, after tests and results it confirmed that it was so the results were as expected and predicted. The methods used for this procedure were successful at achieving good results, however some can be altered. For example, the E.coli used for this experiment was used from experiment number one, not that that is a problem but when the E.coli was incubated over night and it had successfully multiplied it was stored at 4oC for quite a while (this experiment was carried out 1 week after the first one). This possible may have altered the activity of the E.coli and also its appearance. Many resources state that gram negative bacteria should have a pink colour after the counter-stain has been added and rinsed off. In this case the E.coli bacteria in this experiment had quite a dark pink colour which was really close to the colour red, this appearance of colour was visual both with the naked eye and under the microscope as individual bacterial cells. Viable cell counts As I predicted, the more dilute (10-8) solution will have less E.coli bacteria growing on its surface. As there were 20 different pairs doing the practical, and the dilutions were all done 20 times by different people, there is plenty room for error from contamination of inaccurate measurements. The 10-5 agar plates had many E.coli bacterial colonies growing on it, according to the results there was far too many bacteria that it was too many to count (TMTC). Gradually as the dilution increased the bacteria became less, 10-6 dilution had numbers ranging from 6-140. Obviously with such great difference within what is meant to be the same dilution there was some error/contamination present. The most obvious ones which had error are pair numbers 18 and 19 there was TMTC throughout (10-6, 10-7 and 10-8). What would be expected is that fewer bacteria should be present in the 10-8.