What is the aim of your investigation? The aim of our investigation involves the process of isolation and identification of a bacterium genus called Proteus. The samples will be obtained from soil, creek water and mud, as well as meat. These samples reflecting the environmental niche for Proteus will be cultured using various laboratory techniques and suitable microbiological media, and tested with an appropriate selection of biochemical tests.

What will be your group's initial approach in this investigation? The most logical way to plan our investigation was to research Proteus individually, and share our findings both on the Discussion forum and in class. By now, we have understood the fundamental morphology and physiology that enables us to effectively formulate a sound experimental plan. From the various literature searched, we have found that Proteus can be isolated from a variety of environments, such as soil, polluted water, and animal intestines. It is from these suggested environments that we plan to individually obtain our samples, on which we will conduct the following outlined procedures as a way to increase the chance of isolating Proteus. We have decided to place our samples of soil, and mud from a creek into a nutrient broth, to promote the growth of Proteus from a low number to a higher number. If the concentration of bacteria is too high, judging from the cloudiness of the broth after incubation, we may have to perform serial dilutions. It is from there that we inoculate the bacteria colonies from the broth on to a general nutrient agar. This step allows colonies to be formed. Once the colonies are formed, gram stains and the oxidase test are then conducted to confirm the gram-negative and oxidase-negative characteristics of Enterobacteriaceae. This biochemical test will be carried out relatively early so we can narrow-down from a large number of bacterium colonies to a manageable number. The colonies that satisfy the criteria are inoculated individually onto a selective and differential medium - McConkey agar. The reason for the use of MacConkey agar is, firstly, the bile salt constituent selectively inhibits microbes that are not suited to grow in intestinal environments, therefore, favouring the growth of Proteus as it is a part of the intestinal flora. Secondly, it is due to its ability to differentiate the non lactose-fermenting nature of Proteus from the lactose fermenting organisms that makes MacConkey agar an extremely useful instrument in our investigation. The agar plate will be incubated in an anaerobic jar, as Proteus is an anaerobic organism. Once a colourless culture on the MacConkey agar is obtained, we have agreed to use the following biochemical tests to confirm the identity of our pure cultures: The Potassium Cyanine (KCN) test - this is used to substantiate the oxidase test conducted in the previous step, due to the fact that cytochrome oxidase systems are absent in Enterobacteriaceae and thus, the resistance of KCN in Proteus allows it to grow while inhibiting others that possess cytochrome c. Methyl Red - we know Proteus carries out fermentation of sugars to mixed-acids, the Methyl red indicator allows us to detect the acidic end-products produced by Proteus. Motility test - due to the fact that Proteus possesses peritrichous flagella, motility test allows us to demonstrate whether our culture is motile in the medium, and the movement in the medium causes a uniform red colour throughout the medium that will assist our identification. Urease test - A unique characteristic of Proteus is its ability to hydrolyse urea by generating enzyme urease. The ammonia produced as a by-product creates an alkaline environment optimal to its growth, in relation to the environment that it is found - animal intestines. In addition, the ammonia is utilised for respiration. Phenylalanine deaminase - Proteus species are able to deaminate phenylalanine by flavprotein-linked amino acid oxidase, to form phenylpyruvate. This test identifies the presence of this compound by reacting it with the ferric chloride solution to form phenylpyruvic acid, which gives a bright green colour. Our initial approach outlined here, including the appropriate media and biochemical tests, incorporated with careful aseptic techniques, will help us in many ways in isolating and identifying our microorganism. This preliminary plan provides an overview, but more importantly, an understanding of the unique characteristics of Proteus and how we can relate these characteristics to help us to choose the correct methodology in this investigation. The attached Flowchart summarises the whole initial approach of our group.

Figure 2. A Flowchart of the Isolation and Identification of Proteus

What resources/references have you found so far that might aid you in your investigation? What question(s) do you have about the work described in this resource material? What information in these resources have been relevant to your group discussion so far? Reference 1: O'Hara, C.M., Brenner, F.W., & Miller, J.M., 2000, 'Classification, Identification, and Clinical Significance of Proteus, Providencia, and Morganella', Clinical Microbiology Reviews, vol. 13, no. 4, pp. 534-546. This article became the basis of our investigation, as it provided important and in-depth information on the morphological, physiological and chemical characteristics of Proteus, such as the ability to form swarming colonies, and the non-lactose fermenting nature of Proteus. The reference also included an extensive list of suitable biochemical tests relevant to four species of Proteus, such as the Methyl Red test, Phenylalanine deaminase test and the Motility tests. Although some tests are utilised as a means to differentiate between species of Proteus, a large range of tests are suitable for the identification of at least four known species. In addition to this, the knowledge that some Proteus species are susceptible to antimicrobial agents may be helpful later on should antibiotic testing be taken into account in our investigation. The technical details of this reference were difficult to grasp. For example, the short description of typing systems in the article was brief and unclear. While some members of the group concerned the process of DNA hybridisation, some have difficulty in understanding some of the technical identification systems employed to classify Proteus, such as the Becton Dickinson Microbiology Systems. Overall, this informative reference is vital to the current, and future stages of our investigation as we gain an understanding of the critical tests that will enable us to biochemically identify Proteus from all other bacteria. Reference 2: Rozalski, A., Sidorczyk, Z., Kotelko, K., 'Potential Virulence Factors of Proteus bacilli', Microbiology and Molecular Biology Reviews, vol. 61, no. 1, pp. 65-89. This journal article emphasises on the pathogenicity of Proteus. We are not particularly concerned about the pathogenicity, but are more interested in the characteristics of Proteus. Some questions that arose when analysing this article include: Can Proteus be found in all areas of ANY soil and water? What other biochemical tests can be used to identify Proteus' presence (other than the Urease test)?- tests mentioned in the article were based on Proteus' ability to infect, rather than non-pathogenic Proteus (these were only relevant to an extent). How can non-pathogenic Proteus take advantage of the type of cell wall/surface it has? It mentions in the article that pathogenic Proteus has a cell surface that is crucial to their virulence and ability to infect the host i.e. Urinary tract. This article does provide useful information on the characteristics of Proteus. This includes its swarming abilities and the reason behind this swarming phenomenon (Figure 3). Understanding the swarming characteristic is essential, as it is an important identification that is unique to Proteus species. The article also listed some of the biochemical tests for Proteus, and although not as extensive and thorough as Reference 1, it provided interesting comparison to the biochemical tests listed in Microbiological Method manual for Proteus vulgaris. The structure of Proteus is also relevant to the group discussion. The article states that flagella are present on Proteus. Therefore it has a swimming behaviour also known as 'swimmer cells'. When placed on a solid medium, Proteus morphs into 'swarmer cells', thus having the swarming ability and also supports an increase in the number of flagella. The explanation given assists us to understand the evolutionary perspective for the presence of peritrichous flagella in Proteus. The environment which Proteus lives in is also revealed in the article - polluted water, soil and manure. This has been a long discussion among the group, as to where students should obtain their samples. The article was able to tie our understanding of Proteus, in relation to the environment. For example; we knew that Proteus are both anaerobic and facultative anaerobic, hence, this unique adaptation possibly increased their etiological role in infections. This has also led to the discussion of the depth required to obtain our soil samples from. Overall, the article as a whole is too detailed, however, some parts of this article encouraged critical thinking and appreciation for the microbial roles within our group.

Figure 1. Proteus vulgaris, a gram-negative bacterium is demonstrated by the red/pink colouration from the gram stain. Its bacillus (rod) shape is revealed along with its peritrichous flagella. Reference: Fankhauser, D.B., 2005, 'Proteus vulgaris flagella', David B. Fankhauser, Ph.D., viewed 6 April 2005, <http://biology.clc.uc.edu/fankhauser/Labs/Microbiology/ Prepared_Slides/Proteus_vulgaris_flagella_P7311251.JPG>.

Briefly outline a plan and predicted timeline of your group's approach and process for your isolation and identification. Identify any steps or topics you are unsure of, and how you might seek guidance in clarifying these areas of uncertainty. Week 1 - Obtain scientific facts on Proteus Week 2 - Further research on Proteus and general discussion on isolation procedures Week 3 - Relate Proteus characteristics to the media and biochemical tests used Week 4 - Development of E poster v1 Week 5 - Hand in E poster v1. Methods of isolation for the whole group clearly defined, and in detail. 12 samples are brought in to the laboratory for isolation. Inoculate samples to nutrient broths and allowed to incubate Week 6 - Determine whether the bacterial concentration in samples requires serial dilutions. Inoculate nutrient agars with samples from nutrient broths. Incubation of nutrient agar plates commences Week 7 - A list of biochemical tests used for the identification of Proteus confirmed. Modification of the isolation methodologies if required. Apply gram stain and the oxidase test to colonies of bacterium. Inoculate gram-negative, oxidase-negative colonies to MacConkey agar. Allow colony to grow to obtain a pure culture. Week 8 - Select non-lactose fermenting colonies from the MacConkey agar and transferring them to nutrient agar. Incubate the nutrient agars in an anaerobic jar to inhibit the growth of obligatory aerobes. This allow obligatory anaerobic and facultative anaerobic organisms to grow so that enough pure cultures are obtained for the purpose of biochemical tests. Week 9 - Development of E poster v2. Biochemical tests commence ( The KCN test, the Methyl red test and the Motility test). Select appropriate colonies to culture if they are required for further testing. Week 10 - Hand in E poster v2. Biochemical test continues (The urease and the phenylalanine deaminase test) Week 11-12 - Biochemical test continues if required. Development of the final E poster Week 13 - Finalise results of the investigation. Final E poster development Week 14 - Submit final E poster and individual paper We hope that we will be able to formulate our methods early on for the isolation and the growth of Proteus. At this point in time, we have an abundance of information that is very useful. However, in order to maximise its usefulness to our task, we need to relate the specific characteristics of Proteus to its growth in the laboratory. And how well we are going to perform this is uncertain, and this is a crucial step as it will ultimately reflect the successfulness of growing a pure culture. We are unsure of how the latter stages of the investigation will unfold. We need to work as a team and follow the timeline to make certain that we will finish the project within the given timeframe. We are expecting obstacles and challenges ahead, and we also realised that sometimes not everything will go according to plan. We can seek help from our tutor, Sophie, who has been extremely helpful and supportive in many aspects of MICR2201. She may be able to provide useful advice throughout this investigation and provide insights as to how we can improve, modify and refine our methodologies at various stages of the task. We can also gain an understanding of our problems from journals, where comparisons can be made between how we conduct our investigation, and how microbiologists did their research.

From where will collect your samples for isolation? How will you collect your samples? What are the characteristics of your organism upon which you have based these decisions? What other organisms might be present in the environmental niche you have chosen? Our group is collecting our samples from a variety of locations, including soil, mud from a creek, and meat. Soil (9 samples): Group members that selected to collect their samples from soil, will collect soil from their backyard. When collecting, we try to remove plant materials and root mass by digging to the desired depth of about 10 cm below ground. We extract the soil and place it in sterile containers provided. We have chosen soil based on the characteristics that Proteus species are: Not strictly anaerobes, but are also facultative anaerobes, in which soil provide some oxygen to low oxygen level suitable for Proteus growth and development; Chemoorganotrophs, where Proteus extracts energy and electrons from the organic molecules that soil provides, particular in soil profile O. Most abundant in soil. In addition, soil supports many microbial lives, primarily due to the rich nutrients that it contains. Soil is an environmental niche suitable for many organisms, including many bacteria, such as the Enterobacteriaceae (Escherichia coli being one of the most prominent), photosynthetic microbes such as cyanobacteria and purple non-sulfur bacteria. Other organisms frequently found are actinomycetes, algae, fungi, protozoa, and other organisms such as earthworms, nematodes, and arthropods. Creek water and mud (2 samples): Collect creek water and extract mud from the bottom of the creek and deposit the sample in to a sterile container. We have chosen creek water and mud based on the characteristics that Proteus species are: Facultative anaerobes, in which water and mud provide limited oxygen supply suitable for Proteus growth and development; Chemoorganotrophs, where Proteus extracts energy and electrons from the organic molecules that mud provides. Mud is an environmental niche suitable for many organisms as demonstrated in the Winogradsky column experiment. This includes many bacteria, such as green and purple sulphur bacteria, clostridium, photosynthetic bacteria such as cyanobacteria and non-sulfur purple bacteria. Other organisms present are green algae. Meat (1 sample) We have chosen meat based on the characteristics that Proteus species are: Anaerobes, in which oxygen is limited inside an animal host, such as the intestines Chemoorganotrophs, where Proteus extracts energy and electrons from the organic molecules that host provides. A part of the natural flora of animal intestines Other genera of bacteria are present in animal internals such as E. coli, clostridium, and many bacteria displaying symbiotic relationships with the host. The bacteria found in our samples share many characteristics with Proteus. Clostridium, for example is obligatory anaerobic, which shares the anaerobic characteristic with Proteus. E. coil found in soil, animal intestines and polluted water shares the gram-negative characteristic with Proteus. It is also a gram-negative rod shaped bacteria. E. coli can be facultative anaerobic like Proteus as well. Following the safety precautions is very important when collecting our samples of soil and mud. Due to the fact that many pathogenic bacteria may be present on site, for example, harmful strands of E. coli from animal faecal material may be embedded in the soil, garden gloves must be worn when extracting substances from the ground or creek. Broken glasses and other sharp objects are other major dangerous hazards in which we need to be aware of.

What other questions or concerns does your group have at this time? 1. We are unsure of whether all of the biochemical tests relevant to Proteus identification are available to us in the lab. 2. We are concerned whether there is a "right" type of soil that allows us to find relatively high concentration of Proteus species. 3. How can we collaborate as a group, when everyone is doing their own isolation and identification? 4. Will we be able to stick to our predicted timeline of isolation and identification? If not, how might this impact on our experimental results? 5. How can we design effective control throughout our investigation?

What will you do next to address these questions/concerns? "We are unsure of whether all of the biochemical tests relevant to Proteus identification are available to us in the lab." In cases where certain tests are unavailable, the way we address this particular concern is to use a different approach in the laboratory, such as utilising other appropriate substitutable tests to identify Proteus, rather than following a strict and inflexible guideline. There are over 10 biochemical tests that are appropriate for the identification of Proteus, including four important tests that we would like to use, as they give us the best chance of identifying the most species of Proteus, as opposed to only one or two in some tests. In cases where we do become restricted to few tests, the fact that working as a team means that we can replicate each other's results by repeating the available tests a number of times to obtain clear and consistent results for each test. "We are concerned whether there is a "right" type of soil that allows us to find a relatively high concentration of Proteus species." Our samples are diverse, not just soil samples. This includes samples of meat and mud from a creek. Also, this is one of the reasons why each member is going to obtain samples from different areas, in an attempt to increase our chances of finding Proteus. If our soil samples contain relatively low numbers of Proteus, we must be extremely thorough in growing and locating bacterium colonies, to make certain that we do not miss our microbe. "How can we collaborate as a group, when everyone is doing their own isolation and identification?" We need to utilise the time in our face-to-face tutorial meeting extremely carefully. We have planned the topics of our tutorial specifically dedicated to the development and progress of our investigation. We are committed to continue our great work as a team by actively posting our current findings, ideas and reflections on the Discussion forum. Our weekly results may be summarised and shared in tutorial the week after, or on the forum. We could also arrange extra meetings on campus to further discuss our progress as a group. "Will we be able to stick to our predicted timeline of isolation and identification? If not, how might this impact on our experimental results?" We hope that as a group, we can be efficient in the laboratory, but more importantly, take great care along every step of the way to avoid time-wasting. We need to have a time management plan, much like the planning of the tutorial topics, to tackle time concerns like this. If we do not follow our timeline, we are risking of falling behind. This will impact on our experimental results, as well as the development of future E posters enormously. However, we could set aside some of our free time to come to the laboratory, with permission from Sophie, to catch up if necessary. This question also illustrates the significance of our tutorial session, as it is required to convey the objectives and steps of the investigation in the laboratory for that particular week. Mind maps and flowcharts are a useful tool in helping us achieve this. "How can we design effective control throughout our investigation?" A controlled experiment is required in our investigation, so that new and meaningful results are drawn from our investigation. A controlled experiment involves every factor in the experiment kept constant except one - the factor being examined, or the experimental variable. Therefore, a controlled experiment requires us to test one particular factor at a time. For example, the first step of our investigation is to grow bacteria from our sample in a nutrient broth. A control is one that only contains the nutrient broth, without adding the variable, that is, the sample. The second trial will consist of both the broth and the sample. While everything is kept the same, including the temperature, the incubation time and the amount and type of nutrient broth, the results obtained would be meaningful as these are the results of the variable being tested. In simpler terms, any growth in the nutrient broth that contains the sample must have stemmed from the sample itself, provided that no growth is evident in the control. We must conduct controlled experiments throughout our investigation, from the initial step of culturing bacteria to the very last step of identifying bacteria with biochemical tests. These detailed protocols will be planned prior to each section of our investigation so that valid, reliable results and accurate scientific methods are demonstrated.

Figure 3. The Swarming Phenomenon of Proteus within the 16-hour period Reference: Williams, F. D., 'Nature of Swarming Phenomenon in Proteus', Annual Review of Microbiology, vol 32, pp 101-122.