EXERCISE 20

LABORATORY UNKNOWN

I. INTRODUCTION

The main test of one's understanding of the laboratory techniques used to identify microorganisms is whether one can use them correctly in a clinical setting. To that end, you will be given a liquid culture that represents a patient specimen. Your task is to identify all bacterial species present in the specimen, using the techniques learned in the previous lab sessions. Your unknowns have been selected from the bacterial species you have already studied. Don't panic; follow the procedural steps outlined for you below, in sequence.

II. LAB WORK

FIRST LAB SESSION: Materials supplied: (work individually)

• 1 tube, numbered, containing 1 or more organisms
• Blood Agar Plates (BAP)
• Chocolate Agar Plates (CAP)
• Gram stain reagents

Procedure:

1. Mix the patient's specimen well and using a sterile loop, streak one BAP and one CAP with your unknown.

   This is the most important step in this exercise, because if there are more than one species in your unknown, but only one grows out because of faulty technique, your total possible grade will be reduced proportionally. In past years, only one species was present in the unknown broth. Do not assume that to be the case this year! Label the plates with your name and unknown number, and incubate both plates in a candle jar at 37°C until the next lab session.

2. Prepare a Gram stain of your patient's specimen (optional).
   1. Mix the patient's specimen well before sampling for the stain.
   2. Use several loopfuls of the specimen for the smear, but remember to flame the loop before re-entering the tube (the glass slide is not sterile and you could introduce contaminants).
   3. Be careful during the staining procedure that you don't use too much Acetone-Alcohol; everything may turn out Gram negative when it really isn't.
   4. You might not get much information from the Gram stain prepared from the patient's specimen; bacterial numbers are low and often hard to find on the slide, so don't despair if you fail to find anything. Also, the bacteria in the broth are probably in late log phase and can easily become permeable to the decolorizer; do not bet the farm on the Gram stain prepared from the broth culture!

SECOND LAB SESSION:

Materials supplied: (work individually)

Blood Agar Plates		Esculin Agar / 6.5% NaCl Broth
A and P discs		Coagulase tubes
Entric media tube sets		MacConkey agar plates
Gram stain reagents		Carbohydrate utilization tubes
Tinsdale agar plates		Germ-tube tests
Oxidase reagent		Hydrogen Peroxide (H2O2)

Procedure:

1. Examine your BAP and CAP carefully, and concentrating on isolated colonies, determine whether you have a pure or mixed culture. If you have more than one unknown species, confirm this with the Lab Instructor.

   Observe for similarity of color, texture, and hemolysis pattern. Remember, differences in size may be the result of crowding, not that there are different species.

2. Record your observations on the Report Sheet. Be complete.
3. Next, prepare a Gram stain of each unknown species you observe. You must wait for these results before proceeding to the next step. Remember to make your bacterial smear broad and thin so it will air dry quickly. Do not boil your slides - your Gram stain will be wrong and you will get the wrong identification.
4. Record your results on the Report Sheet, and on the basis of your Gram stain, refer to the appropriate exercise as indicated by the table and choose the correct course of action.
5. Take your unknown plates to the instructor and explain what you have found out so far, and what secondary media you need and why.
6. Remember to check with the instructor before inoculating your secondary media.

THIRD LAB SESSION:

Materials supplied: (work individually)

• Gram reagents
• Various test reagents

Procedure:

1. Retrieve your unknown test materials from the back bench.
2. Read the individual tests and interpret the results using the appropriate lab exercise as a resource.
3. Review all of your results and check to confirm the rationality of those results. When you are confident that your final answer is consistent with all of the information you have gathered, fill out the Lab Report Sheet and turn it in.
4. Discard all materials from this and previous labs, either on your lab bench or in your lab drawer.
5. Clean the oil from the microscope oil objective before returning it to its cabinet.