Final Unknown Identification 4 (This Is Your Final Lab Report)

BI 234 Unknown Identification 2014

Final Unknown Identification 4 (this is Your Final Lab Report)

Introduction During the course of the term, you will perform several assays that will be used to identify the unknown organism assigned to you. You are expected to identify at least the Genus of your organism, using the provided chart and results of your assays. It is most important that your conclusion is consistent with the observations and conclusions of your tests. Some things to keep in mind as you are trying to identify your unknown and prepare your assignment:  No one test may give you a definitive identification.  Draw logical and rational conclusions based on your data.  Your hypothesis as to the ID of your unknown may change as more tests are conducted – remember, this is how science works!  Let your test results tell you what the organism is – do not try to make your results and data fit your expectations.  You are not limited to the unknown ID chart as your only resource. You may want to consult other sources of information in your process: o Bergey’s Manual of Determinative Bacteriology (in library) o Bergey’s Manual of Systematic Bacteriology (in library) o Photographic Atlas o Your textbook o Online Textbook of Bacteriology - http://www.textbookofbacteriology.net/

What you will turn in for the Lab Report: Observation and Conclusions. Final Observations and Conclusion - is in the lab report that is due on the final due date. For this portion of the assignment, you are asked to identify your organism (at least to Genus level) based on your accumulated data from all your tests. Simply add this as a paragraph or two to your discussion in the Lab Report (see below). You may find example lab reports (good and bad) on my main lab web page. Discussion This is due on the final due date. The discussion should address all tests that were conducted. Briefly discuss the tests that are listed in the unknown ID chart. Your discussion should include ALL tests used in the unknown exercise. As you discuss the assays, indicate how the tests were used – e.g. , used to eliminate possibilities, confirmed other tests, were not used to due to absence of data in chart, were not used due to problems with assay. Do not reiterate your dichotomous key in your discussion. It should be between 1-1.5 pages. Dichotomous Key This page is due on the final due date. You will construct a dichotomous key that should use the fewest number of tests possible to positively identify your unknown. Your key should repeat your discussion. The structure and requirements of the key are outlined in the next section.

An Unknown Summary Template (see link on main web page)

1 BI 234 Unknown Identification 2014

An unknown summary template that has all the tests you did and your results as +, -, ?, or whatever it says on the line to record the test as. Use my unknown summary template (see link on main lab page) or yours that has the same order of tests.

An Exclusion Worksheet (see link on main web page)

In the exclusion worksheet you must exclude all families of bacteria except one, the one family your unknown belongs to. For full credit, state at least one reason for excluding each family and if you exclude a family you must exclude all members of the family (not just some). If you give more than one reason, each must exclude all members of the family, so it is best to only give one reason. The first family has been filled in for you as an example.

How to construct a dichotomous key Dichotomous keys are routinely used in the identification of many different kinds of organisms. The principle behind a dichotomous key is that at each step (test) there are only two outcomes or possibilities: yes or no, + or -, coccus or bacillus, etc. For this exercise, you will prepare a flow-chart type dichotomous key. At each step through the key, those organisms that do not fit your data will be eliminated, and the key will lead to your final conclusion. Requirements for your key:  Names of Families and Genera must be enclosed in circles  You must start with the complete list of families as given in the unknown chart

 Names of all tests used in the key must be enclosed in squares. Possible outcomes must use arrows and indicate test outcomes (yes/no, +/-, etc.)  Fewest number of tests possible must be used (not all tests will be used in the key)

 Final step - use double exclamation marks (!!) to indicate your final conclusion

On the following pages are examples of a discussion and the type of dichotomous key you must construct.

Please note – the discussion and key were constructed using an unknown ID chart different from the one given to you. You will need to list and use all the correct possibilities from the current unknown ID chart. Do not use the list and dichotomous key provided here!

Example Discussion

The simple stain and Gram stain both indicated that our organism was bacillus in shape. We eliminated the families with coccus shape at this point. The Gram stain produced pink cells, so we concluded that our organism was Gram-negative. The endospore stain did not reveal the presence of any endospores, which was expected, as Gram-negative cells do not produce endospores. At this stage of testing, we concluded that our organism most likely belongs to either the Family Alcaliginaceae or Family Enterobacteriaceae.

There was no growth on MSA, which indicated that our organism was not a halophile. Growth on MAC, gooey KOH and lack of growth on PEA all confirm that our organism is Gram-negative. The oxidase test indicated that cytochrome oxidase was absent, and the catalase test indicated that our organism produces the enzyme catalase. These tests indicate that our organism is most likely not a member of the Alcaliginaceae, but is more likely from the Family Enterobacteriaceae.

Growth in thioglycollate was consistent with a facultative anaerobe, which agrees with expected results for Enterobacteriaceae. Results from the SIM tests indicate that our organism is motile, does not reduce sulfur, and does not produce indole. Based on these tests, we could eliminate Citrobacter freundii and Escherichia coli. There was no zone of clearing on the starch plate, and the nutrient gelatin agar was liquefied. These results indicated that our organism was most likely Erwinia amylovora, Serratia liquefasciens or Serratia marcescens.

Methyl red turned yellow when added to MR-VP broth, so our organism did not use a mixed acid fermentation. This eliminated Serratia liquefasciens as a possibility. The Voges-Proskauer test turned red after 40 minutes, so this is consistent with both Erwinia amylovora and Serratia marcescens. Two of our Simmons citrate tubes had no growth and remained green, and two turned blue. Blue would agree with our hypothesis, while no growth is inconsistent with our other findings. We think that maybe we made a mistake, such as the needle being too hot when we streaked our slants on the two tubes w/o growth. Fortunately, the phenol red carbohydrate broths could be used to differentiate between our two remaining possibilities, Erwinia amylovora and Serratia marcescens.

Phenol red glucose broth was yellow with bubbles, this eliminated Serratia marcescens as a possibility, and is consistent with Erwinia amylovora. Phenol red lactose broth was red with no bubble. My phenol red sucrose remained red with no bubble, while the other three had yellow tubes with no bubble. We think this was a problem with inoculation again. Three mannitol broths were red with no bubble, indicating no fermentation occurred. One of the mannitol broths was yellow and had a bubble. This test was discounted as acid and gas production was not consistent with our other findings, and may have been due to contamination with one of our controls.

Based on these fermentation results, when the problematic tubes are removed, our assays, as a whole, are most consistent with the conclusion that Erwinia amylovora was our unknown. This was confirmed when our research showed that Serratia marcescens produces pigmented colonies and ours were not pigmented.

Example Dichotomous Key

Note – this key was constructed using an unknown ID chart different from the one given to you. You will need to list and use the correct possibilities from the current chart. Do not use this key! Lactobacillaceae Micrococcaceae Streptococcaceae Enterobacteriaceae Bacillaceae Neisseraceae Corynebacteriaceae Pseudomonadaceae

Gram Stain Gram + Gram -

Enterobacteriaceae Lactobacillaceae Neisseraceae Micrococcaceae Pseudomonadaceae Streptococcaceae Bacillaceae Corynebacteriaceae

Obligate aerobe NO YES (From here on family Enterobacteriaceae Enterobacteriaceae Neisseraceae Pseudomonadaceae members are only considered) Gelatin Hydrolysis NO YES

Erwinia amylovora Citrobacter braakii S. marcescens C. freundii Enterobacter aerogenes E. cloacae Ferments Mannitol E. sakazakii Escherichia coli Serratia fonticola YES NO S. marcescens

!! Erwinia amylovora !!