LAB 4 Bacterial Staining Techniques III I. Differential Stains (Structural): Endospore and Capsule Stains II. Morphological Unknown I. DIFFERENTIAL STAINS (STRUCTURAL) A. Endospore Stain B. Capsule Stain A. Endospore stain The most important endospore-forming bacteria are members of the genera Bacillus and Clostridium, both of which are Gram-positive rods. An endospore is a dense, multilayered structure that contains the genetic material of the bacterial cell. Endospores are formed within a vegetative bacterial cell when the environmental conditions no longer support cell growth. As the vegetative cell dies, the endospore is released into the environment where it can survive indefinitely in the presence of many environmental stresses, such as dessication, extremes in temperature, radiation, and lack of nutrients. When more favorable conditions arise the endospore germinates, again forming a viable vegetative cell. The presence of endospores in a bacterial culture can be detected by staining with malachite green. Because the endospore coat is so tough, steam is used to enable dye penetration. After washing, only the endospores will retain the primary stain Malachite green. Safranin is then used as a counterstain for vegetative cells. The endospore stain is a differential stain because it differentiates spore-formers from non spore-formers. Note: Formation of an endospore. The spore stains green and the vegetative cells stain red to orange.

Germination

Vegetative Cell

Spore

Vegetative Cycle Sporulation PROCEDURE: (EACH STUDENT) 1. Prepare a thick smear of Bacillus subtilis. 2. Air dry the smear and heat fix it. 3. Cover the smear with the primary stain Malachite green. Place a paper towel on top of the dye and steam the slide for 5 min. 4. After the slide is cool, rinse both the top & bottom well with H2O. 5. Counterstain with Safranin for 30 seconds. 6. Rinse both the top and the bottom of the slide well with water. Clean the bottom of the slide with a paper towel. 7. Allow the preparation to air dry and observe the stain using the 10X and 40X objective as well as the oil immersion lens. Draw a typical microscopic field and record in the Results Section for Lab 4. 31

B. Capsule Stain Many bacteria are surrounded by a slimy layer called a capsule that usually consists of a highly hydrated layer of polysaccharide or in a few cases polypeptide. The capsule can have a number of different functions, helping bacteria escape phagocytic white blood cells, protecting against bacteriophage infection, dehydration or facilitating the adherence of bacteria to surfaces. Staining of the capsule requires that the cells NOT be heat-fixed, since any exposure to heat destroys the capsule. PROCEDURE: (EACH STUDENT) 1. Make a very heavy smear from the milk culture of Klebsiella pneumoniae. Use 4-5 loopfuls. Do not use a water drop on the slide. 2. Air dry thoroughly – DO NOT HEAT FIX! 3. Flood the slide with 1% Crystal Violet. Allow the stain to sit for 1 minute. 4. Rinse very gently with water. 5. Flood the slide with 20% CuSO4 (copper (II) sulfate) and allow this reagent to sit on the slide for 20 seconds. 6. Rinse very gently with water. 7. Air dry; Do Not Blot.

Examine the stain using the 10X, 40X and oil immersion lenses. Note: Blobs of the milk will stain blue to purple. Cells will appear as small dark brown rods with a light clear to bluish halo surrounding them. Draw a typical microscopic field. Record in the Results section of Lab 4. B. Flagella Stain Motile bacteria are characterized by propeller-like structures called flagella. These structures vary in distribution but allow equipped bacteria to move quickly, albeit via a convoluted path, through aqueous, sometimes even highly viscous environments. The flagella stain is the only stain that we will not perform in lab, however, please take time to observe this stain in the demo microscope at the back of the room and draw this stain in the Results section of Lab 4. II. 1. 2. 3. 4.

Morphological Unknown: continue your investigation of your morphological unknown. Collect your unknown from the side bench. Perform an endospore stain as described in the above section. Record your observations. Based on the data you have acquired on your morphological unknown, identify the organism using the dichotomous key provided on the course web page. Please verify the identity of your unknown with either the TA or instructor. Be sure that you are provided with a species name in addition to your determined genus name. This will enable you to further investigate the organism before writing the unknown press release (see results section at the end of this lab). 32

NOTES

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LAB 4 RESULTS I. DIFFERENTIAL STAINS (STRUCTURAL) A. Endospore Stain B. Capsule Stain Bacillus subtilis

C. Flagella Stain

Klebsiella pneumoniae

QUESTIONS: 1. What is the primary stain used in the endospore stain?________________________________ 2. What special procedure is used in the endospore stain? _______________________________ 3. What is the function of an endospore? ___________________________________________ _____________________________________________________________________________ 4. List two genera of bacteria that produce endospores. _________________________________ 5. Which dye is used in the capsule stain? Is it acidic or basic? __________________________ 6. Capsules are composed of what types of molecules? _________________________________ 7. What is the relationship between the presence of a capsule and bacterial pathogenicity? _____ _____________________________________________________________________________ _____________________________________________________________________________ 8. In the capsule stain, why is the bacterial smear not heat fixed? _________________________ _____________________________________________________________________________

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RESULTS: Morphological Unknown 1. Number of Unknown: _______________ 2. Colony color and morphology (as viewed on the agar slant):____________________________________ 3. Staining Characteristics: Direct Stain

Gram Stain

Color: _________________

Color: ___________________

Shape: _________________

Shape: ___________________

Arrangement: _________________

Arrangement: ___________________ Gram Reaction (G+ or G-): _________________

Acid-fast Stain

Endospore Stain

Color: _________________

Color: ___________________

Shape: _________________

Shape: ___________________

Arrangement: _________________

Arrangement: ___________________

Acid-fast (yes/no): __________________

Endospore (yes/no): ____________________

4. Use the key to determine which organism you might have_______________________________. Although you will only be able to determine a possible genus, please verify this with either the instructor or TA. At this point, you will be given both a genus and species name to enable you to perform some additional research on the bacterium. UNKNOWN PRESS RELEASE ASSIGNMENT DESCRIPTION (5 POINTS) Write a press release about the microorganism identified above. The circumstances surrounding the article can be fictional, however, any information about the unknown itself must be factual. This press release is an informal report but must include all of the data collected and reported above, including the unknown #: Colony color / morphology and staining characteristics 3 points _________ (e.g. cell shape, arrangement, Gram reaction etc…) Correct identification (Both genus and species as given by TA) 1 point _________ Creativity / quality of the composition will also be considered 1 point _________ *Note - Information obtained from outside sources must be cited. PLEASE include in-text citations where pertinent. Two points will automatically be deducted for failure to cite sources. Examples of previous press releases have been posted on the course web page. These examples were chosen for content and creativity. They are not necessarily perfect examples but they are well-written and received high scores. 35

QUESTIONS: 1. If you did not observe endospores in your unknown, how confident are you that it is not a spore-former? Explain. ___________________________________________________________________ __________________________________________________________________________ __________________________________________________________________________ 2. Which bacteria produce water-soluble pigments (hint – the dichotomous key will help in answering this question)? _________________________________________________________________ __________________________________________________________________________ 3. List two bacteria that produce colored colonies: ____________________________________ __________________________________________________________________________ __________________________________________________________________________ 4. Indicate the bacteria that typically grow in grape-like clusters, in long chains, and in packets of 4 or 8. __________________________________________________________________________ __________________________________________________________________________ 5. What other information about the unknown would make you more confident in your identification? __________________________________________________________________________ __________________________________________________________________________ __________________________________________________________________________

Organisms introduced in this lab: Bacillus subtilis Clostridium botulinum Clostridium tetani Clostridium perfringens Klebsiella pneumoniae

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