Gram stain of S.aureus (http://textbookofbacteriology.net/staph.html) 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 has been relevant to your group discussion so far? We have found some information from the book written by Minor and Marth (T. E. Minor and E. H. Marth, 1976. Staphylococci and their significance in food. Elsevier Scientific Publishing Company, Amsterdam). From reviewing this book, we know that staphylococci are spherically-shaped and Gram-positive bacteria as shown in picture above. Moreover, they can grow in the presence of high salt concentration (5-15% sodium chloride). This information helps us in planning our isolation for staphylococci from other microorganisms. They are strongly catalase-positive and they are facultative anaerobes which mean that their metabolism is both respiratory and fermentative (E. W. Koneman, 1992. Color Atlas and Textbook of Diagnostic Microbiology (4th edition). J. B. Lippincott Company, Philadelphia). From all of this information, we have a rough idea on how to isolate our desired microorganism. In addition, we also have individually gathered many resources. One of which is written by Sleigh and Timbury (J. D. Sleigh and M. C. Timbury, 1990. Medical Bacteriology (3rd edition). Longman Group Limited, Edinburg). This book provides us a brief outline of information about the characteristics of staphylococci, which allows us to choose the environments from which to collect our samples and furthermore design our plan to isolate and test our pure sample. There are some articles mentioned about the identification of the different species of Staphylococcus, including analysis of biochemical tests and schemes for isolation (Geary C. Stevens M. Sneath PH. and Mitchell CJ,1989. Construction of a database to identify Staphylococcus species. Journal of Clinical Pathology. 42(3):289-94, and Use of enzyme tests in characterization and identification of aerobic and facultatively anaerobic Gram-positive cocci, 1998. Clinical Microbiological Reviews, 11(2):318-40). Although at this stage we are concentrating on isolating the genus Staphylococcus, this research will be valuable if we decide to progress to isolating individual species. Furthermore, information about the normal flora of the human body is obtained from a book written by Prescott (L. M. Prescott, J. P. Harley, and D. A. Klein, 2005. Microbiology (6th edition). McGraw-Hill Education, Boston).

Catalase positive shown on S. aureus

What will be your group's initial approach in this investigation? What are your individual and collective reflections regarding this approach at this point in time? Our group's initial approach to the investigation is to: 1. Select the type of bacteria to be identified. 2. Research the living conditions of the bacteria. 3. Identify several environments in which the bacteria is commonly found. 4. Attain information on the life cycle, nutritional requirements, growth rate and morphology of the bacteria. 5. Select an appropriate collection technique for staphylococci. 6. Research the type of media and the conditions necessary for cultivating the bacteria in a laboratory setting. 7. Select and demonstrate the correct techniques and tests for isolating the bacteria in the laboratory according to its metabolic and physiological attributes. 8. Research the techniques to attain a pure culture of the bacteria from the sample after identifying it from other organisms present in the sample.

Our group's isolation plan 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. Our first step is to collect the samples. Each of these will be grown in both salt agar and a nutrient broth. As a group we will perform Gram stain tests on selected colonies, aiming for a positive result. The colonies that display this characteristic will be examined microscopically to determine their microbial morphology, and those that display a cocci shape will be isolated and subcultured. A pure culture of the bacteria will be isolated from this and a catalase test performed. Positive-catalase colonies will be separated and grown on an Oxidation Fermentation (O/F) media. From this particular test we can determine that our isolated bacterial colonies are faculative anaerobes and thus present the required characteristics of staphylococci. Timeline: 1. Collect the samples - week 4. 2. Grow the culture on nutrient broth and salt agar media - week 4-5. 3. Perform Gram stain and morphology test for each type of colonies - week 6. 4. Sub culture for Gram-positive, cocci colonies - week 6. 5. Catalase test - week 7. 6. O/F test for catalase positive culture - week 8. 7. Transfer the culture that produces the yellow colour through the whole column of the O/F media onto the nutrient medium and allow time to grow - week 8. 8. The pure culture of staphylococci will be obtained - week 9. 9. If we have more time, we will get down to the species level of Staphylococcus - from week 10 onwards.

Mannitol salt agar plate using for differentiate between species of Staphylococcus a) non-ferment mannitol b) ferment mannitol

As a group, develop a set of criteria for the expectations for each member of your team during this research project. How will you ensure that this contract will be honoured? Group Contract As a group we discussed the relevance of the contract and concluded that the agreement should include the following points: Each group member is to actively contribute to discussions, both at meetings and through WebCT. All students are to come prepared to each laboratory and tutorial session with any work or equipment required for the group assignment. All ideas are to be posted on WebCT and treated fairly by all members of the group. The Contract shall be honoured using continual evaluation on WebCT.

What is the aim of your investigation? The aim of our investigation is to isolate the staphylococci from a selection of six different source sites. Our group will approach the experiments by initially researching the unique characteristics of staphylococci, developing selection criteria for their isolation and then utilising this plan to isolate and culture a pure sample of our particular genus from the six difeerent sources. If time permits our group also hopes to retest our pure cultures and hopefully isolate particular species from staphylococci.

What other questions or concerns does your group have at this time? At this time our group is happy with the method and timeline established and has no further questions or concerns about the methodology of isolation and identification. However we still have some concerns regarding some factors of our isolation that are beyond our control. One such concern is that many of the samples that we have obtained, such as the food samples, have had prolonged human exposure. Therefore, there is a possibility that a variety of microorganisms inhabit that sample. This will make it slightly more difficult to identify the targeted microorganism as microbial growth is rapid and extensive. Furthermore, the selected microorganism may become "out competed" in the sample and exist in minute numbers.

What will you do next to address these questions/concerns? As previously mentioned, our group members are satisfied with the progress and methodology of the isolation and identification. However, one minute concern of vast microbial inhabitance of our cultures does exist. Nevertheless, we have attempted to minimise this concern as much as possible. Such measures include taking food samples that are rich in salt to further the possibility of the presence of the selected microorganism and minimise the occurance of other microorganisms. It must be noted that this concern is only a minute concern and appropriate techniques and tests have been proposed for the cultivation and identification of Staphylococcus alone. If time permits the identification process will be extended to the species level.

From where will you 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? From joint collaboration our group has decided to select the samples from the following six sources - human skin, nasal passage, food, soil, lift buttons and bathroom taps. Staphylococci bacteria thrive in many areas due to their low requirements for survival. They grow in temperatures ranging between 10-42 degrees with an optimum temperature of 37 degrees. Staphylococci are facultative anaerobes, which means that they are able to continue respiration in both oxygenated and deoxygenated environments (E. W. Koneman, 1992. Color Atlas and Textbook Diagnostic Microbiolgy (4th edition). J.B. Lippincott Company, Philadelphia). The above sources were selected not only because of their ease of isolation of the organism but also because it was important to have a diverse range of sources, to hopefully isolate different strains of the bacteria and to prove the abundant existence of staphylococci in the environment. Skin, nasal passages, food and soil were selected because of their moist and rich nutrient environment. Lift buttons and bathroom taps were selected as sources due to the large amount of human contact they receive throughout the day. Nasal passages, bathroom taps, lift buttons and skin samples will be sampled using saline swabs. Food and soil samples can be collected without the use of swabs. It is crucial in all cases that correct asceptic technique is maintained. All swabs and samples taken will be collected and transported in sterile collection jars. The environmental niche the group has chosen is evidently in touch with the atmosphere and constantly in contact with humans. Frequently new microorganisms will be introduced in the samples allowing a variety of microorganisms to be found. Access to the atmosphere introduces both aerobic and some kinds of anaerobic bacteria. It is also enriched with carbon dioxide, the primary carbon source for autotrophic organisms. Our samples also have energy source from light for phototrophic microorganisms. Due to the constant human activity that occurs around the sample areas, the variety of microorganisms may be transferred around. As a result we are looking at all sorts of microorganisms, although some will be more dominant in these conditions. Although we are trying to isolate staphylococci, there must be some other microorganisms present. Skin contact always occurs on the bathroom taps, lift buttons, food and soil, making it possible to find some microorganisms beside those of the genus Staphylococcus.Other microorganisms that might be found in our samples may include diphtheroids (including Propionibacterium acnes), streptococci, Bacillus spp., Malassezia furfur, Candida spp., and Mycobacterium spp. (L. M. Prescott, J. P. Harley, and D. A. Klein, 2005. Microbiology (6th edition). McGraw-Hill Education, Boston). The reason is because these microorganisms are normally found on human skin. Escherichia coli may present in the samples from the bathroom taps, as well as legionella, which is often found in the environment where there is a water system. Causes of meningitis, Neisseria meningitidis and Streptococcus pneumoniae, and other normal microbiota of the nose such as Viridans streptococci and Haemophilus spp. can exist in the samples from the nose (G. W. Tannock, 1995. Normal microflora :an introduction to microbes inhabiting the human body. Chapman and Hall, London). We may also find some agrobacterium in soil as they are often found around plant roots. As for the food sample, there may be Clostridium botulinum and Escherichia coli.