MICROBIOLOGY 101 LABORATORY MANUAL

EXERCISE #4: CONCLUSION OF SIMPLE STAINING, & STREAKING; NEGATIVE STAIN & CHARACTERIZATION OF COLONIES

NAME, ID #:_______________________________________________.

TA Name:__________________________

REVISED: 08/10/99

INTRODUCTION TO EXERCISE 4

The SIMPLE staining procedure performed in the previous exercise can result in DISTORTION of the fine structure of bacteria due to the heating & the chemicals in the stains. The NEGATIVE staining procedure is less distorting to cells. Stains like CONGO RED and NIGROSIN are negatively charged (-) and are repelled by the negative change (-) of the bacteria. This PREVENTS them from entering the cell and staining the cytoplasm. This technique produces a STAINED BACKGROUND which surrounds the UNSTAINED MICROBE. The term "NEGATIVE" has two connotations here that cause some confusion:

• (1) the strains are negatively charged, hence the "negative" aspect OR
• (2) because they are not taken up by the cytoplasm of the cell it is NOT STAINED, but the background is, thus negative also refers to this non-staining of the cell.

PURPOSE OF LABORATORY:

1. To learn what a negative stain is.
2. To learn how to carry out a negative stain.
3. To learn how to observe & describe microbial colonies & to use their colonial form for diagnostic purposes.
4. Grading of your first streak plate.

RELATIONSHIP TO LECTURE MATERIAL

• NetText101/102: CHAP. III , Bacterial architecture.

GENERAL INSTRUCTIONS AND MATERIALS:

1. Fixed and/or stained smears from Exercise #3.
2. Stains, negative: Nigrosin.
3. Stains, positive: Crystal violet and safranin.
4. Streak plates from previous experiment.
5. Cultures of Bacillus subtilis or other bacteria for negative staining.

STAINING PROCEDURES

Figure 1. Negative stain. As seen in the Atlas in the negative stain the bacteria should colorless (transparent). A procedure similar to this will be used in Exercise #7 to visualize the bacterial capsule and in this procedure the bacteria will be colored red and the background blue.

NEGATIVE STAIN

1. Read pg. 31 & section 3 of A Photographic Atlas for the Microbiology Laboratory and compare your results with the figure of a negative and capsule stain.
2. CLEAN SLIDES as demonstrated using Bon Ami and dry with a lint-free cloth/paper.
3. Place a small drop of the NEGATIVE STAIN approximately 1 cm from one end of a slide.
4. ASEPTICALLY procure a loop-full of bacterial sample and mix it with the negative stain on a slide.
5. While holding a clean slide at about a 20^o angle (Fig. 1), touch the edge to the drop so that it spreads across the edge. Then spread the suspension across the long axis of the slide while lifting the spreading-slide to "feather" the stain as demonstrated by your TA. Heat the end of the spreading-slide to sterilize it (NOT TOO HOT).
6. Air dry the slide, but DO NOT HEAT-FIX it.
7. Examine the dried slides under the microscope as before (e.g. 10X first etc.). Especially look at the transition area between the "thick 'n thin" areas of the stain (Fig. 1). Draw representative negative stained bacteria. Try to relate them to ones you have previously seen. Have the TA verify that what you're seeing is really bacteria and see if your TA agrees with your identification. Compare your results with those in the Atlas fig. 3.4.
8. Draw the results in the circles below.

CONCLUSION OF STREAKING EXPERIMENT FROM EXERCISE #3

1. Collect your streak plate from the previous experiment.
2. Examine it and assess how well you have succeeded in isolating clones. Compare your plate with the Atlas figures on pg. 2-4, 9 and give yourself a grade from A to F, if the TA has not done so (no points). For a look at some other streaking and colony characteristics (takes a while to load) click here for colonies and stains of Gram negative bacteria; click here for colonies and stains of Gram positive bacteria. If you have received a C or lower be sure and talk to your TA about how to improve your streaking technique.
3. Based on Figure 1 below, draw the two different colonies on the two plates in the circles below and describe the characteristics of each: the shape; the diameter (in millimeters); the elevation; the margin; the color; any other significant feature. Include such terms as "wet", "dry", "wrinkled" etc. in you description. Ask your lab partner to describe your selected colonies and vice versa. Discuss any different perspectives & come to a consensus. Use the dissecting scope to view the colonies if you're having trouble deciding.
   • Touch each of the colonies with a sterile loop and gently pull away to see if the colony is "sticky"; describe the "texture" of the colony.
   • Look at your Rodac plates and draw and describe 2 different colonies you see on them. Use your neighbor's plates if nothing grew on yours or if all the colonies looked alike.
4. Save your Bacillus streak plates in your drawer for Exercise #6. Place them in a zip-locked bag to prevent drying out.

Figure 2. Some Typical Colony types (Not All). Visit this site for pictures of typical colony types.

Figure 3. Flares at the edge of a spreading bacterial colony. This gives rise to irregular or undulate or filiform or lobate colony depending on the bacterium.

SIMPLE STAINS RESULTS

NEGATIVE STAIN RESULTS

STREAK PLATE COLONIES

RODAC COLONIES

SAMPLE QUESTIONS: You should be able to answer these questions at the conclusion of this laboratory.

• What charge is the typical bacterium?
• How does streaking a plate result in the obtaining of a pure culture?
• How does heating distort the microbes form?
• Describe how the negative stain works and why do the cells appear the way they do?

Copyright © Dr. R. E. Hurlbert, 1999.
This material may be used for educational purposes only and may not be duplicated for commercial purposes.
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