SCI 204 BIOCHEMISTRY PART 3: Identification of an Unknown Organism
We’ve talked about the different ways we can attempt to identify an unknown organism, which is what is often done in real life by technicians and medical professionals in their jobs. We have looked at staining (e.g. Gram stain results, Acid-fast results), structures (capsule present? endospores present?), and growth on various types of media (differential/selective/enrichment). We’ve also studied what happens when we try to inhibit growth (changes in pH and temperature, antibiotic interference). While these methods can eliminate some organisms from contention, they are often lacking and can’t allow us to definitively identify an unknown organism.
Biochemistry utilizes different metabolic reactions and pathways to study of enzymes that catalyze these reactions and pathways. We know that many organisms share similar (or in many cases, identical) enzymes. This is because many of the enzymes are required to keep the cells alive and functioning and without these enzymes, the cells would perish.
Each organism, however, has some enzymes that might not be necessary for survival under supportive conditions, but might aid in a cell’s metabolism, and possibly survival, if conditions deviate from optimal. Each organism has a unique biochemical profile; that is, while organisms may share many of the same enzymes, no two organisms (even those in the same genus like Bacillus or Staphylococcus) have an identical enzymatic fingerprint. By examining a sufficient number of enzymes via biochemical testing, often coupled with the results of staining, growth patterns, etc., we can frequently deduce the identity of an unknown organism.
The point of these next six weeks is to study an array of different biochemical tests and learn how these tests are done and what sort of results can be obtained from a set of twelve control organisms. The last two weeks will be devoted to the examination of an unknown organism of your own, and you will attempt to apply the results collected during the studies of the control organism to determine the identity of an unknown organism.
If we were doing this in-person, we would be using the 12 organisms which were used back in October for the differential, selective and enrichment media lab. As a reminder, these are:
Bacillus cereus Neisseria sicca
Bacillus megaterium Proteus vulgaris
Bacillus subtilis Pseudomonas aeruginosa
Candida albicans Serratia marcescens
Escherichia coli Staphylococcus aureus
Micrococcus luteus Staphylococcus epidermidis
For Biochemistry parts 1 and 2, we inoculated different types of media to determine if these 12 organisms had specific enzymes. Tests were performed or observations made after the incubations. The professor collected the results submitted by students for these control organisms and will create a table with these results of biochemical testing.
If we were in-person at RCC, we would then inoculate these same types of media with unknown organisms during Part 3 of Biochemistry. Each student would get their own unknown organism, although we set this experiment up to ensure that the unknown is one of the twelve organisms that we’ve been studying. We would also inoculate the differential, selective and enrichment media. We would also perform a Gram stain.
After the incubation of the unknown, students would perform the tests and make the observations of their unknown on the different types of media. All of these different types of data can help us determine the identity of our unknown organism. Results would be recorded. Then these results can be compared to the results obtained with the control organisms, to see which of the 12 control organism best matches the student’s unknown.
SCI 204 Lab Report #2
Identification of an Unknown Organism
Let’s assume that you did the biochemical tests described in Biochemistry Parts 1 and 2. You also inoculated differential and selective media plates as we did back in October. The results you obtained follow. You need to look at the results and determine which of the twelve possible organisms you were given. Then you need to write a “lab report/homework assignment” as described after the results.
Your choices are:
Bacillus cereus
Bacillus megaterium
Bacillus subtilis
Candida albicans
Escherichia coli
Micrococcus luteus
Neisseria sicca
Proteus vulgaris
Pseudomonas aeruginosa
Serratia. marcescens
Staphylococcus aureus
Staphylococcus epidermidis
Gram stain and shape: Gelatin hydrolysis
Dextrose fermentation Lactose fermentation Maltose fermentation Mannitol fermentation Sucrose fermentation
Nitrate Reduction: Urea hydrolysis: Catalase
MacConkey agar: EMB: Mannitol salt agar
Tryptic soy agar Blood agar
This photo below is blood agar with all three types of hemolysis. Alpha is partial, Beta is compete and gamma is negative.
TSI SIM Simmon’s Citrate MR VP
Oxidase Litmus Milk
ASSIGNMENT
Write a lab report with the following sections. This assignment is due on Wednesday, December 20th at 11:55 PM.
Introduction (20 points): A brief introduction should be included. This should include some background information about metabolism and enzymes and how each microbe has a unique enzymatic ‘fingerprint’. By performing various tests to determine which enzymes are present and comparing with the enzymatic profile of controls, the identity of an unknown microbe can be determined. (Think about how this could be helpful in different situations.) Any historical information about the various tests used might be a nice addition.
Results (10 + 30 points from Required Homework #1 and #2): Include the results of your unknown on the attached chart. (e.g. You need to interpret the photos and give the result for each test.)
Analysis (35 points): Explain how you came to the identification that you did. Be sure to explain discrepant results and any other possible microbes that your unknown could be. You might need to use some additional references (Bergey’s Manual, online searches) to help settle any discrepancies.
Conclusion (20 points): Summarize your findings in a paragraph or two. Include information about the unknown organism.
References (10 points): Be sure to include full reference (MLA or APA citation style) so someone could find the information you’ve included; this would include items like the name of the article, the name(s) of the authors, journal title, volume, page numbers, year of publication. If you include at least one scientific reference you obtained by using the library’s databases, you will receive extra points. Remember, you can access the library databases from home with your RCC library bar code that you were reminded to obtain during the workshop videos. References should be dated from 2020 forward.
If you use a website as a reference, the actual page of the site used should be noted and not just the home page of the website. In other words, if the instructor types in the webpage listed, she should be taken to the actual information that was included and not just an introduction to the website. Reminder: If you do a search on the RCC library webpage, copying and pasting that link as a reference is not going to take your instructor to that page for the reference. It will take her to the library homepage.
Also, be sure to annotate the reference in the text of the report, so it’s clear to the reader from where you obtained the information.
EXAMPLE: If you say “Candida albicans can help cure cardiovascular disease,” you would want to reference this information, since it’s new information and people will want to read about this since it’ll be difficult to believe. Immediately after you make this statement, you would include the name of the author. Or, you could number your references in the list at the end and include the number of the reference which includes this information.
Your statement would look something like this ““Candida albicans can help cure cardiovascular disease,”(Olson) or ““Candida albicans can help cure cardiovascular disease,”(3), where reference #3 in your list is the paper or book by Olson. You still will have your list of references, but the reader can go look and read about this research themselves.
• You will lose all credit on the references as well as a substantial portion of credit everywhere you include lots of obviously researched information with no correct annotations. (The annotated resource has to contain the information you are mentioning.)
• If you do not italicize the genus and species names of organisms, you will lose five points automatically from the final score on this lab report.
Continue to next page for tables to fill in results for the unknown organism.
TEST Initial Appearance Final Color, Appearance or Result of Test Comments
Tryptic soy agar plate
McConkey agar plate
Blood agar plate
EMB plate
Mannitol salt agar plate
Gelatin hydrolysis
Dextrose fermentation
Lactose fermentation
Maltose fermentation
Mannitol fermentation
Sucrose fermentation
Carbohydrate ferm./gas production on TSI slant
Urease with urea agar
Nitrate reduction with nitrate broth
H2S production on TSI
H2S production on SIM
Citrate utilization with Simmon’s Citrate
Motility on SIM
Indole production on SIM
Methyl red with MR-VP
VP result with MR-VP
Catalase with tryptic soy agar
Oxidase with tryptic soy broth
Litmus Milk
Gram Stain
Shape