Biol 251 - Microbiology Lab

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Introduction

Bacteria are usually transparent. Without staining most bacteria are impossible to see with brightfield microscopy. Your text has a good general overview of principles of staining. Be sure to read it before proceeding. You are required to master the most common and essential staining techniques. You have several weeks in lab to learn and practice your staining. Before you proceed to the more complicated and time consuming techniques, you need to master making a smear. Since you can’t view your smear directly, you will do a simple stain on your smear to check your technique. You need to successfully do a smear from both a broth tube and a plate culture since you will be working with both during the rest of the semester. Smear preparation is a case of less is better! You can proceed to the Gram stain after your smear and simple stain have been approved by your IA or the instructor.

Bacteria are initially characterized by their reaction to the Gram stain - either gram positive, gram negative, or gram variable. You will use this stain throughout lab. You will also learn some specialized stains that elucidate the unique characteristics of some bacteria. The negative stain yields accurate information on the size and shape of bacteria. Because there is no heat fixing in this technique, the bacteria undergo the least amount of morphological changes. The capsule stain is a modified negative stain that allows you to see the slimy capsules that surround and protect a variety of bacteria. Bacteria that form endospores such as Clostridium and Bacillus can be distinguished with the endospore stain. The shape and position of the endospore is a useful diagnostic tool. The presence of Mycobacterium, the bacteria responsible for tuberculosis and leprosy, can be determined with the acid-fast stain. The characteristics these specialized stains target are involved in the organism’s pathogenesity or its ability to resist antibiotics.

Assignment
Check your syllabus to see which of the stains you are required to complete. It is a good idea to keep your completed and signed off slides in the box provided until the end of lab. This portion of the lab can be frustrating for the student. It is hard to get a good stain. It is even harder to get your instructor’s or IA attention to view your slide! Please remember there are 24 of you and two of them! If your slide does not get checked off in that lab period, carefully blot off the oil with a Kimwipe and save in your drawer until the next lab period. Be sure to label every slide with some identifying code that you can refer back to. There usually isn’t enough room to record all the pertinent information directly on the slide.

Staining is a messy procedure. Look at the tops of the lab benches! Hopefully your lab coats will protect your clothes. These labs would be good ones to dress “down” in.

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