Micro-Unknown Research

BIOL 2420 Unknown Identification Instructions and Rubrics

General Guidelines All unknown work and inoculations will be done in lab under instructor supervision. Under no circumstance may a student interrupt another instructor or course to ask about unknowns or reports. Should this occur, 5 points will be deducted from the final report grade. Five points will be deducted for each instance an instructor must re-prepare an unknown culture due to student negligence. A student who is absent on the day unknowns are handed out will be penalized 5 points. For 24-hour and 48-hour incubations, the student is responsible for ensuring his/her test tubes and plates are placed in the appropriate locations designated for the desired incubation times. These locations will be marked or indicated on the white board, cart, or incubators. The student is also responsible for correctly labeling their media to ensure there is no confusion when returning the media.

The student should use the provided log sheet to document all activity beginning the moment unknown cultures are handed out and should continue to bring the activity sheet every class period until the identification process is complete.

The report is due at the beginning of lecture on the due date. Deductions for late reports begin 5 minutes beyond the start time of class per the classroom clock. After the first 5 minutes, an automatic 10 points deduction occurs and this is considered one day late. Late reports will be accepted up to two days beyond the due date (NOT two class periods) at a deduction of 10 points per day. Late reports will not be accepted beyond two days. Reports will be accepted early for any reason.

Lab Report Contents This is an upper-level college science course and the lab report should be written using appropriate grammar, spelling, and scientific nomenclature.

Cover Sheet- Name, class period, and unknown # Report Form - The results should be typed on the appropriate report form (Gram-negative or Gram- positive). Any test on the report form that is not performed should not be left blank. An entry of N/A should be used instead for any line that is left blank. You can find the report forms on eCampus. The report forms will be graded using the following criteria:

 Consistency - The report form should be consistent with what was tested according to the log. If a test was performed according to the log, then there should be entries for those tests on the report form. In addition, the results for tests that overlap in biochemistry should be consistent. If they are not consistent, then an error report must be included.

 Scientific nomenclature - At the end of the report form is a single line that asks for the identity of the unknown. The microbe name should be written in correct scientific nomenclature. If typed, then the name should be italicized. If handwritten, then the name should be underlined to indicate italics. Do not abbreviate.

 Observations - All observations should be recorded on the report form. This includes any color changes upon addition of reagents, or changes in media color, etc.

 Interpretations - Students should interpret the meaning of each result. For example, if the student observed a clear tube after the addition of Reagents A&B and zinc in a nitrate reduction test, then the student should interpret the results to mean that the organism is capable of undergoing anaerobic respiration using both nitrate and nitrite as alternative electron acceptors. Another example, if the organism produced an alkaline end product in a fermentation tube and the result was recorded as “K”, then the student should explain that peptones were used, pH was increased, and fermentation did not occur. Generally, interpretations are going to center around biochemical capabilities and enzymatic activities of the microbe.

 Results - Based upon the observations, the correct results should be drawn and recorded on the report form in the appropriate format. For example, when recording phenol red carbohydrate broths, the results should be recorded as AG, A, neg, or K and not “yes” or “pos.” Additionally, in

the lab manual is a set of required tests within the red box. All of these tests should be completed and recorded on the report form. All of the required follow-up tests should also be included.

Dichotomous Key - A flow chart of the unknown identification process should be constructed using word processing software. The dichotomous key will be graded using the following criteria:

 Consistency - The key should be consistent with the report form.  Dichotomy - Each test should be arranged with only two possible outcomes. Do not lump phenol

red carb broths into a set of reactions (mannitol, lactose, sucrose, glucose) or AG, A, neg, or K. Instead, separate out the reactions and use only those carbohydrates that eliminate microbes from the list. For example, separate glucose broth into acid production and gas production and use two separate lines on the key. The first line being “acid production” with results being positive or negative and the second line dealing with “gas production.”

 Progression - The progression through the key for the unknown microbe should be highlighted. Use one color for all test results that are consistent with the unknown microbe. Leave the other option colorless.

 Reduction - The student should start with all of the possible organisms, both Gram-positive and Gram-negative. Each test should reduce the number of organisms in the list. Once organisms are eliminated from the list, they should not be added back. The key should continue until there is one microbe name left in the list. If the tests were performed correctly and results interpreted accurately, this is the identity of the unknown microbe. Scientific names should be written in appropriate format and checked for autocorrect and spelling issues.

 Reagents/Stains - Any test in the key that required a reagent or stain should also be indicated and written in appropriate scientific terminology.

 Confirmation Tests - Any test that was not part of the progression should be listed as a confirmation test at the bottom in a separate box. In general, these are tests that did not reduce the microbe list but were still beneficial in the identification process. A minimum of two confirmation tests must be included.

 Format - The SmartArt function on Microsoft Word or PowerPoint is useful for the construction of the key but is not the only option and, if never used, has a learning curve that can be frustrating. Simply using arrows and text boxes is also allowed. The lists of microbes should be placed to the left or right of the appropriate test.

Identification - The identification of the unknown microbe will occur in two stages: 1.Before the student is allowed to continue with biochemical testing, the correct Gram-reaction

must be identified and verified with the instructor. The student should keep in mind that for some microbes, the Gram-reaction is difficult to interpret. If the student believes this is the case, then the correct follow-up test must be performed and the reaction verified with the instructor.

2.The correct identification of the microbe.

Putting It All Together… This is the order you should use when putting your report together. Points will be deducted if you merge any section. Do not staple or bind your report in any way. Use only a paperclip.

Cover Sheet Report Form Error Report, if required Dichotomous Key

Do’s and Don’ts  Do read ALL instructions.  Do write your unknown number on ALL materials, including activity log, report form, media, etc.  Do not staple or bind your report in any way. Use a paperclip.  Do organize your report.  Do add reagents to your dichotomous key.  Do not add back microbes in the dichotomous key.  Do outline the progression through the key.

 Do make sure there are only two possible outcomes in the dichotomous key.  Do italicize scientific names. If the results are handwritten on the report form, underline the scientific

name.  Do examine for autocorrect issues. Autocorrect will correct “pneumoniae” to “pneumonia” every single

time. You will get points deducted for this.  Do not write expected results in the report form. Write what actually happened; otherwise, this is

falsifying data.  Do not write neg/pos for any result that you did not actually get. If you did not do the experiment, write

N/A in the appropriate location on the report form.  Do not indicate that pigment was NOT produced, yet it was soluble or insoluble or had a color. If pigment

was not produced, solubility does not exist and should be an N/A. The same is true for color of pigment. If there is no pigment, there is no color. Write N/A.

 Do not write in first or second person.  Do not use contractions (i.e. didn’t, can’t, isn’t).  The use of an online rubric means deductions must be taken out manually.

Unknown Lab Report and Process Grading Rubric

Grade Possible Points

Component Information

METHODS LOG – 10 points total

10 Log The score from the separate Communication rubric. A log that is not typed will earn a grade of zero.

REPORT FORM – 40 points total

5 Consistency Consistent with log and key. Consistent within the report form results. Inclusion of an error report, when appropriate.

5 Nomenclature Correct use of scientific nomenclature when writing/typing the identity. 10 Observations Proper recording of all observations where indicated on the report form. 10 Interpretations Appropriate interpretation of observations and results.

10 Results Correct recording of the results based upon the observations. All required tests (red box) included. All appropriate follow-up tests included.

DICHOTOMOUS KEY – 40 points total 5 Consistency Consistent with the report form. 5 Dichotomy Clearly identified test with only two possible outcomes. 5 Progression Progression through the process is easily identified.

10 Reduction

A complete and comprehensive starting microbe list is provided. Each test reduces the number of microbes in the preceding list. A list of possible remaining bacteria is included after each test. Bacteria are not added back to the list in subsequent tests. The outcome is one possible microbe.

5 Reagents/Stain All reagents or stains used are listed in the key with the appropriate test.

5 Confirmation A minimum of two confirmation tests with results are included and placed at the bottom of the key. These tests should not be included within the key.

5 Format Typed using the SmartArt or arrows/text boxes Scientific names and terminology written in correct format. Spelling counts.

IDENTIFICATION – 10 points total

8 Gram Stain Correct interpretation of the Gram stain reaction as Gram-positive or Gram- negative. If Gram-variable, the correct identification of the follow-up reaction instead.

2 Identification Correct identification of the unknown bacterium.

DEDUCTIONS – negative points as indicated Absence on Handout Day (-5 pts) Interrupting another instructor or course (-5 pts/incident) Re-preparation of unknown culture due to student negligence (-5 pts/incident) Stapled or bound report (-2 pts) College-level grammar and spelling (-0.5 pt/incident) 3rd person only (-0.5 pts/incident) Correct order of sections (-2 pts) Including neg/pos for any test not performed (-2 pts/incident) Not writing N/A for any line on the report form left blank (-1 pt/incident) Late submission (10 pts/day beginning at 5 minutes into lecture time up to two days beyond) Safety (Failure to adhere to all established safety standards may earn a zero for the report) Any other penalty deemed appropriate by your instructor

100 TOTAL POINTS This is the score that will be in the gradebook.

BIOL 2420 - Fall 2017 Core Competency – Communication Isolation Log Instructions for Students

General Guidelines Students must document the day-to-day activity of the work performed during this identification process. Students should read Lab 31 Isolation and Identification of Unknown Bacteria with special attention to pages 308 and review the Isolation Log Grading Rubric at the end of these instructions for how this section will be graded. Below is a breakdown of the expected information for each of the graded log components.

1. Dates – Students should accurately record the date for each activity. 2. Times – Students should accurately record the time for each activity. 3. Reagents/Stains

a. For staining, include any smear preparation, drying, and heat fixation. Aseptic technique is implied. Be specific, but do not copy the lab manual. Summarize what YOU did for your unknown as if you were charting for a patient.

b. For all staining and biochemical testing, the reagents or stains should be correctly listed. Two examples follow:  The catalase test should indicate the substrate added, which was H2O2.  The nitrate reduction test should list the NAMES of the reagents added. Do not simply state

“Reagent A” or “Reagent B.” 4. Identification of Media – Any media that is inoculated must be correctly identified by full name

first prior to assigning an abbreviation For example, a student must write MacConkey out in its entirety and assign an abbreviation (MAC) before the student may use the abbreviation.

5. Inoculum Source – The source of inoculum should be indicated whenever a student inoculates test media or performs a staining procedure. For example, a Tryptic Soy Agar stock plate.

6. Type of Inoculation – The student must determine the type of inoculation that he/she is performing, such as “streaking for isolation” or “broth inoculation”, etc.; or briefly summarize how the inoculum was transferred if media was not the destination (ex: glass slide, oxidase paper).

7. Incubation Parameters in degrees Celsius – The temperature along with the number of hours for incubation must be accurately recorded and written for EACH inoculation. Points will be deducted if the student just copies the range that the lab manual procedures state for each test. a. Generally, cultures are incubated at 37°C or 25°C for either 24 hours or 48 hours. Since

students are not allowed to come in at 24 hours to remove their cultures, the instructor will remove the 24 hour cultures and place them in cold storage until the next day. The student MUST indicate this in their log activity as a separate entry and include that their instructor removed their cultures.

8. Observations/Disposal of Media and Stains – Students should make note of when they performed observations for every test as well as when they disposed of media or stains in biohazard. This does not include the actual observations or any results – all results get recorded on the Report Form, not in the Methods Log. For example, if the student inoculated a nitrate broth on a Tuesday, there should be a separate entry on the following Thursday for when the student added the reagents, made their observations of those results, and then disposed of the media in biohazard.

9. Grammar, Spelling, and Format – Every effort should be made to use complete sentences and correct grammar and spelling. Using a word processor’s grammar and spelling check does not always work, especially with science nomenclature. Proper format in scientific writing includes use of 3rd person and past tense for methods sections.

10. Scientific Nomenclature – Any science term should be correctly spelled. Chemical formulas and reagent names need to be written correctly. If a chemical formula must be written, such as H2O2, the “2” must be subscripted. The use of any organismal scientific name should be correctly spelled and either be formatted in italics if typed or underlined if handwritten. Please be aware that word processors will “correct” Latin names like pneumoniae to pneumonia every time. The responsibility is on the student to ensure this does not happen. Additionally, scientific names should not be abbreviated unless the full name has been written out ahead of time.

Format Your instructor may ask you to submit a typed and/or handwritten log. Please make note of his/her preference. The log should be clear, concise, and written in the 3rd person using full sentences and college-level grammar and spelling. Below are two examples of appropriate log entries:

Inoculum from tryptic soy agar (TSA) stock plate 1 was aseptically transferred to lysine iron agar (LIA) using the stab and streak method and placed into incubation at 42 C for 19 hours.

Aseptically transferred inoculum from tryptic soy agar (TSA) stock plate 1 to lysine iron agar (LIA) using the stab and streak method and placed into incubation at 42 C for 19 hours.

BIOL 2420 – Unknown Report Activity Log

Bring this with you every day! You will type up this information as the Methods Log, which will be graded using the separate Communication Rubric.

Date Activity Media/ Reagent

Results/Observations/ Comments

10/20 Received broth of unknown bacteria # ______. Streaked for isolation on ____ and ___. Incubated @ __oC for __ hrs.

Blood agar TSA

Cultural characteristic of the broth culture:

10/22

Observed the plates from _____(date). Removed one isolated colony from _________agar and made stock plate. Incubated @ ___oC for ___ hrs.

TSA Cultural characteristics of the colonies: