Your LAB UNKNOWN Start Guide 8/22/22

GETTING STARTED ON YOUR UNKNOWN BACTERIA!


MICROBIOLOGY INDEX (install to check Microbes and the diseases they cause): http://www.medmaster.net/atlasofmicrobiol.html
Here is more help for your Unknown identification (NEW CHARTS/KEYS): http://www.uiweb.uidaho.edu/micro_biology/250/IDFlowcharts.pdf


How to start your Unknown Experiment!

The test cards you made are most though only SOME of the tests needed to identify your unknown.  You must PLAN your Unknown Identification Process.  To begin your Unknown follow the directions listed below.  Everything begins with your UNKNOWN ISOLATION LAB.  Part of the isolation is the "Pouring an Agar Plate," "Dots-R-Us" Lab followed by the Purity Lab and the Stock/Working Log Rotation Lab.

Remember that discovering your unknown bacteria requires your careful application of the following: (1) proper ISOLATION from the mixture with S. aureus (2) maintaining your STOCK & WORKING slants as a proper isolate and in LOG GROWTH PHASE (no more than 10 days to 2 weeks old) - you cycle a new slant into the incubator overnight and then move it to refrigerate between tests (this is called Maintaining Log growth by 12 hr incubation refrigeration and subculture at 10-14 days) (3) proper INOCULATION of the test media (4) proper INCUBATION (5) proper EVALUATION.  Please be cautious and re-test your WORKING and STOCK PURE CULTURE TUBES on a regular basis by Gram staining.

MY ISOLATION PLAN OVERVIEW (You should summarize mine in your own words to understand it): 

 (1) This is the Streak Plate Isolation Lab 1.  Obtain your "mixed" culture (S. aureus + "X").  Plan your Isolation (1st use Quadrant Streak Plate Isolation technique).  Using the maximum in Aseptic Technique (sterile technique), pour your agar plate and cool it.  Streak the cooled plate for Isolation using the 4 Parallel Stipe/Quadrant Streak method listed below (your plate should NOT be open more than a total of 10 sec. during the streaking process).  Incubate the plate up-side-down overnight at 37 C.

(2) This is the "Dots-R-Us" Lab 2.  Observe the plate and number (on the bottom) each completely round small separate colony. Using a heated cooled sterile loop, pick up 1/2 of each numbered colony and make a Gram Stain of the material (use little or no water in making this smear as it is such a small amount of sample).  Observe the microbe under 100X.  If it is grape-like clusters of round Gram-positive cocci, put an "X" through that colony number and move on to the next colony to test.  If the microbe is some OTHER shape than gram-positive cocci (diplococci are another shape CAUTION!), then take the other 1/2 of that colony with your heated and cooled sterile loop and "zigzag" it on a new sterile Nutrient Agar Slant.  Incubate overnight at 37 C. Make sure the new slant is incubated in a RED cup that says 24hrs ONLY INCUBATION as it can be "over" incubated and not function correctly for Unknown Testing (see INVOLUTION Chapter 6 - Maintaining Log Growth for testing).  Move the 24hr incubated slant to refrigeration until you have time to test for contamination and purity of isolation.

(3) This is the "Purity Lab" Lab 3.  Remove the slant from refrigeration. CHECK the "Zigzag" growth area on the slant in 3 regions by Gram stain (use a normal smear preparation with a drop of water) - you are checking your slant for proper isolation of 1 bacteria.  If all the microbes are the same size and shape and gram stain and are NOT Gram-positive cocci in clusters, then you have isolated your UNKNOWN MICROBE.  Make a second slant from the first slant and test it by gram stain overnight.  Label the first slant WORKING UNKNOWN and the second STOCK UNKNOWN.  Keep both in the refrigerator until in use.  Use the Working Stock to do the other 2 major stains and to inoculate your Unknown Tests.   

 (4) This is the Working/Stock Rotation Log Growth Lab  4. Use your original pure WORKING slant for 10 days then discard it and move the STOCK to WORKING and make a new STOCK (artificially maintaining LOG GROWTH by refrigeration and slant rotation/subculture).  This is the Working/Stock Rotation Log Growth Lab.  Using a "FRESH" working slant that has been purity checked at least once, perform the Unknown Tests beginning with the Optimum Growth Procedure (Lab 5) , Colony Morphology and Pigmentation Procedures followed by all the Stains.

AFTER the collection of your data (recorded in your Lab Record Book), you must utilize either the dichotomous keys PROVIDED BY YOUR INSTRUCTOR and/or tables of similar organisms located in the reference texts.  Weight for the reliability of each piece of data must be assessed and judgment "calls" will be required. 

Please check your GENUS and species...  with your instructor BEFORE beginning to write your APA-style Formal Unknown Report...

UNKNOWN EXPERIMENT FLOW CHART


UNKNOWN EXPERIMENT FLOW CHART: (READ THIS or you won't know where to begin your testing!)  This is a summary of the entire Unknowns project which leads to the required typed formal Unknown Report due for grading on the last day of the semester.

(1) obtain a melted TSA deep and aceptically pour your agar plate, leave the lid "cracked" for it to cool ----> (2) obtain your unknown, label your plate around the edge on the bottom with your last name, class and date... record your unknown number in your LRB, heat your loop completely red hot and cool it, dip it 1X into your unknown slant (NEVER DIP AGAIN) and streak 4 stripes across the agar, heat the loop, cool the loop and cross the last line and make 4 more streaks... do this 4 times as demonstrated in class----> (3) tape your plate shut and incubate it up-side-down in MY INCUBATOR overnight ---->(4) Obtain your plate from the Refrigerated bin... dry it off, turn it up-side-down and look for individual separate "DOTS."  Number the dots.  Using a heated cooled loop (or needle if your dots are very small)... take 1/2 the dot and put it on a slide for a gram stain. If the Gram Stain shows Gram-positive cocci in grape-like clusters then "X" out that numbered DOT and move to the next one.  If the Gram Stain on your dot is NOT Gram-positive cocci in grapelike clusters (diplococci?) then go back and with your heated cooled loop, pick up the remainder of the dot and zigzag it on a new TSA slant ----> (5) Incubate this new WORKING SLANT overnight in MY INCUBATOR-----> (6) check the slant in 3 regions by Gram Stain.  If they are all the same shape and stain then confirm that with the instructor ----> (7) Make a new TSA zigzag slant and incubate it overnight using your first slant, check it in 3 regions the next day and make sure it is the same as the first slant... label it STOCK----> (8) Put the STOCK slant in the REFRIGERATE Basket with the WORKING slant each day... Use the STOCK when the Working is 2 weeks old (discard the Working or use it for the Oxidase or Catalase Tests) and make a new STOCK----> (9) Do an Acid Fast and Endospore Stain on the WORKING Stock, confirm your results with the instructor----> Do the Opt Temp. Experiment (use that temperature for all other testing)-----> (10) Inoculate and incubate the remaining Unknown Tests... record your information on a blank Library Assignment Worksheet and put it in the back of your LRB where the Lab Data is to be recorded...
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ALSO See ISOLATION (Streak Plate - or "POOP" spreading dilution method)

On day one you will be given a small numbered test tube containing a small amount of liquid.  Inside this liquid are TWO (2) microbes.  One is a KNOWN or Staphylococcus aureus which is a Gram Positive grape-like cluster of cocci.  The second microbe is your UNKNOWN.  Your job during the rest of your Microbiology laboratory time is to independently work to apply yourself in order to isolate a pure culture of your Unknown, maintain your unknown as a pure culture in Log Growth, and perform all the available tests on the microbe and then to determine the Scientific Name of your unknown microbe.  Finally, you are to compose a publishable paper about your Unknown.

Step 1:  Isolation (see Unknown Testing):

The most useful of the 3 unknown isolation procedures is called Agar Plate Streaking for Isolation.   The Quadrant Streak is the most likely to produce acceptable results quickly and is recommended for your use.

To accomplish this procedure see the Unknown Isolation Test - Streak Plate Method.... Should you NOT be able to complete this successfully (after at least 20-30 colony tests) you may try the POUR PLATE METHOD of Isolation (with the permission of the instructor).
Step 2: Maintaining LOG GROWTH and using the Stock and Working Cultures to perform Unknown Testing... (See Isolation - Streak Plate Method)

IMPORTANCE OF THE LIBRARY BONUS ASSIGNMENT - knowing HOW to determine your Genus and species... AND knowing how to take a "READING" and put it into a table.

Step 3: Matching your results to a dichotomous key for common bacterial Genera

    See Genus keys on the LIBRARY ASSIGNMENT OR UNKNOWNS Button pages

STEP 4: Determination of your species.... use the OTHER tests and these plus your reference texts (Bergey's Determinative 6, 7, 8, and 9th Editions, MacFaddin 2nd and 3rd Editions) and READ each microbial species' description - make a chart of the species and results and select the one species that most matches the tests you did and TRUST... REMEMBER - YOU WERE NOT GIVEN A HUMAN PATHOGEN AS YOUR UNKNOWN!  For the purposes of the 50 pt Bonus for the Unknown Flashcards, I believe there should be approximately 30 procedure cards and about 26 experiment cards (if you include one for each stain and one for each method of making a smear and one for each "section" of the Unknown Report)...

DETERMINING YOUR UNKNOWN FLOW CHARTS & KEYS!

Use the following dichotomous keys to determine your Unknown identity with the observations and data collected from your Unknown Procedures and Experiments.  Remember, often you may find the GENUS on a key but have to go to the 8th or 9th or Systematic "list of species" under that GENUS to use your data to determine your species.  PLEASE HAVE BOTH YOUR GENUS and species "signature/stamped" and confirmed by your instructor!

 Click here for an additional set of good dichotomous keys to unknown bacteria!

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