Laboratory Investigations in Microbiology
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Chapter 31: Unknown Identification

Why would you want to identify an unknown bacterium? In what circumstances does this occur in real life? If you pause for a moment to think about the many ways in which microbes impact our lives, it should become clear that there are times when it is vital that we know exactly what bacteria we are facing. For example, Medical Technologists try to identify bacteria on a daily basis - bacteria which have been collected from a site of infection. Knowing which bacteria have invaded the human body is important for two reasons: 1) Diagnosis of the disease, and 2) Selecting the appropriate treatment. However, identification of 'unknown' bacteria is not limited to medicine. Researchers are constantly seeking ways to better classify (group) bacteria together based on shared characteristics, in the hope that, through understanding one bacterium, we can learn a lot about those bacteria that are closely related. Researchers are also constantly finding completely 'new' bacteria that have never been described before. Such bacteria hold the promise of new miracle drugs, novel ways for cleaning up the environment, or modern biotechnology tools.

Identification of bacteria traditionally follows a step-by-step process of elimination. When certain characteristics are observed, we narrow down the number of possibilities. The first step is usually a Gram stain, which (if done correctly) will divide bacteria up by shape, Gram reaction, and perhaps even arrangement (strepto, diplo...). Knowing this, the scientist will carefully choose the next step in the identification process - one that is appropriate for the bacteria he/she has observed. Take a look at your identification chart for a moment, and suppose your Gram stain reveals Gram-positive cocci. Immediately your choices are narrowed down to just 11 bacteria. How would you go about distinguishing these 11 Gram-positive cocci? Perhaps a citrate test? A catalase test? A test of motility? An examination of the chart handed out to you in lab will show that a citrate test is a poor choice...all 11 bacteria react the same way, and this test does not tell you anything you did not already know! Likewise, a test of motility is a poor choice if only two bacteria show motility and the others do not. [Unless, of course, you have good reason to suspect one of these two bacteria to be your unknown, based on things such as colony color]. The catalase text, however, is an good  choice - 4 cocci have catalase and 7 do not. Once you have divided these Gram-positive cocci into these two groups, each group is examined separately to determine the next logical test to distinguish them. For the 4 catalase-negative cocci (EF, SL, SP, SV), perhaps a methyl red test would work. Look at the chart and you will notice that EF and SL are MR-positive while SP and SV are MR-negative. As a last step, one needs to now only distinguish between two bacteria! This type of strategy described here is called a dichotomous key strategy. 

Your unknown ID project will take place over the next 2 weeks. Four days are dedicated for you to work on the unknown ID, in which time you will be expected to carry out all your identification tests. If you plan ahead, you can do one or two identification tests every lab period. You may need to come in to check on your unknown outside of regular lab periods.

Your Unknown ID will begin with you receiving broths containing your unknowns - one Gram-positive and one Gram-negative. The bacteria given to you are among those listed on the handout chart. Your first objective will be to make a dichotomous key. You should make your key to fit all the bacteria on your chart. This key is your identification strategy and you will be expected to follow it.

Your next step will be to isolate pure cultures of your two unknowns, and to perform a Gram stain on your unknowns. By streaking bacteria out on an agar plate, you will accomplish two things: 1 - separate your unknowns so you can prepare pure cultures, and 2 - observe colonies for characteristics useful in identification. Your Gram stain will eliminate many of the bacteria on the chart and is an important feature of this exercise. You can repeat your Gram stain as often as you like. Once you have pure cultures of your two unknowns, select each test you wish to perform according to your dichotomous key. Every subsequent test, of course, depends on the outcome of the previous test. In the example key shown above, the urease test would only be needed if the H₂S test turned out positive! As you "order" each test, you will receive the culture media and/or reagents from me and pay $10 MicroBucks. Write your name and the name of the test you are buying on the back of the $10. Turn your inoculated media in "to be incubated" and don't forget to check on your media in a timely manner.

Materials & Methods

Materials per student

• TSB broths containing your two unknowns

• 1 TSA plate

• Unknown ID chart of bacteria  (Excel)

• Unknown ID report form (need 2 copies)

• chart of Gram-positive and Gram-negative bacteria

• chart of physical & colony characteristics

• $100 in MicroBucks

Unknown ID Instructions

1. You will receive your unknown bacteria in TSB broth cultures. Each culture contains 1 bacterial strain (1 Gram-positive, 1 Gram-negative). Please write down your unknown ID number and use this number in labeling all media.
2. The cultures are guaranteed to be uncontaminated and healthy on the day that you receive them.
3. You are responsible for obtaining and maintaining pure cultures of your unknown. Any erroneous results due to contamination are your responsibility.
4. All unknowns and inoculated media must be clearly labeled and must remain in the laboratory.
5. Instructor is responsible for providing sterile media and for incubating all inoculated media at the appropriate temperature.
6. Students are responsible for checking on their inoculated media in a timely fashion. The appropriate incubation time for tests is 24 hours except:
1. Methyl Red test: 2 - 5 days
2. Gelatin: 1 – 2 weeks
3. Citrate: up to 5 days
4. If insufficient growth is observed
7. Students are responsible for correctly inoculating and interpreting all tests.
8. You may perform as many tests as you want, but you will only be allowed one test at any one time/on any one day. Please do not get behind.
9. You will receive $100 in MicroCash to spend on tests. Each test will cost $10 except for TSA plates, TSA slants, and TSB broths (2 of each are free).
10. Slides and staining reagents are free.
11. You must have completed a dichotomous key before you may purchase any media.
12. For each unknown, record all observations on the data sheets provided, including drawings of the colony appearance, diagrams of the staining result, and comments on unusual observations.
13. Morphological characteristics:

·        Gram stain: can tell you shape and Gram type.                                   

·        Colony characteristics: shape, size, color, opacity

·        Flagella: use Motility deeps for best determination of motility

·        Endospores:  May be visible in stains. Bacteria will survive 15 minutes boiling

14. Biochemical characteristics

·        Fermentation tubes: type of product (acid, gas) and type of sugar fermented

·        SIM deeps can tell you H₂S production and Indole production.

·        MR-VP broth can be used for methyl red or Voges-Proskauer test

·        Casein, DNA, starch plates, gelatin deeps: hydrolysis reactions

·        Citrate slants, nitrate broths, urea broths

·        Catalase and Oxidase can be determined on isolated colonies on a streak plate

·        Special characteristics for each of the bacteria can be found in Bergey’s Manual. Take note of these – they may help you in your identification.

15. Note the following:

·        Some bacteria can appear as short rods or as cocci

·        +/–  indicates that the reaction is weak, slow, or variable (may be + or –);  the same applies to A/– and A(g).

16. Suggested schedule

• Day 1: Streak your unknown mix 
• Day 2: Check streak plate. Prepare pure culture slants. Gram-stain both colonies & a drop of your mix. Start on your dichotomous key.
• Day 3: Check pure culture slants. Have dichotomous key approved. Start tests.

17. Hints for testing your unknowns

• For your Gram-negative unknown, consider starting with the fermentation profile (Glucose, Lactose, Sucrose) in a mini-plate. This test saves materials and money. The profile will also break down your unknowns very well. Combine this with a citrate and SIM test and you may be done before you know it.
• A SIM test will give you 3 answers at once: H₂S, Indole, and motility. 
• SIM, citrate, and motility tests are not useful for Gram-positive bacteria.
• Colony traits can be used in your key.  But if you are unsure of a color, it is better to stay away from using this. Color production is usually vivid (purple, red, yellow, pink, golden, green).
• If you get behind, consider doing catalase or oxidase tests. Both can be done right on your streak plate and will give you immediate results for both unknowns. 
• If you notice distinct arrangements in the Gram-stain (chains, tetrads) this trait is useful, but keep in mind that cells will often clump together on a slide anyway, so they will appear clustered even if they are not normally so.
• Check your cultures for contamination!  

18. Price list

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