MICROBIOLOGY 101 LABORATORY MANUAL

EXERCISE #6: EXAMINATION OF #2 STREAK PLATES, GRAM STAIN #2 AND SPORE STAIN

NAME, ID #:_______________________________________________

NAME of TA: ______________________________

REVISED: 08/02/99

INTRODUCTION

SPORES

Bacterial SPORES (or ENDOSPORES) are the toughest forms of life known. They are so resistant to destruction that some scientists have proposed that life arrived on earth when bacterial spores drifting through space fell to earth. Only G+ cells form spores, specifically members of the genera Bacillus and Clostridium. Spores are formed by bacteria to survive during PERIODS OF DEPRIVATION, such as the loss of a food or water supply. When a spore-forming-bacterium (SFB) senses that tough times are coming a series of COMPLEX EVENTS are triggered that lead to the formation of a spore. Basically a spore is a structure that contains the ABSOLUTE MINIMUM of genetic information and associated materials required to produce the vegetative form once times become good again. The genetic material (DNA) and other indispensable substances are packaged into a dry, heavily shielded spore that is able to resist high temperature, drying, UV light, deleterious chemicals and other harmful conditions. All metabolic activity ceases and spores appear "DEAD" or in deep HIBERNATION (stasis). We do not know how long this resting state can last, but viable spores have been found in sealed tombs that are 1,000s of years old. Recently, Jurassic spores that are approximately 65 million years old were reported to have been germinated after recovery from insects in amber. When spores sense that the environment is suitable for growth the spore is triggered to GERMINATE. Germination is the process whereby spores change (DIFFERENTIATE) into VEGETATIVE CELLS that grow, reproduce and metabolize. One spore turns into ONE vegetative cell. The vegetative cell has NONE of the resistant properties of the spore; that is, it is easily killed by heat, UV light and drying.


Figure 1. of spore from NetText. The spore is shown containing the genome (DNA) and surrounded by a thick spore coat. The spore is shown inside of the vegetative cell, however, with time the vegetative cell parts are lost and only the spore remains.

Because the spore is protected by a thick, tough covering, it is difficult to stain. To force the stain into the spore body we use the thermal energy present in stream. We then counter-stain any vegetative cell components that are attached to the spore with a dye of a contrasting color. Therefore a spore stain contains spores alone, spores inside vegetative cells and vegetative cells without spores formed in them.

 

 

PURPOSE OF LABORATORY:

  1. To learn how to perform and interpret a spore stain.
  2. To improve your gram stain technique and data interpretation.

RELATIONSHIP TO LECTURE MATERIAL

GENERAL INSTRUCTIONS:

  1. Place drawings and notes on the back of the manual sheets as before.
  2. Examine streak plates from exercise #5 and compare with the colony pattern shown in the Atlas, pg. 1.4. Give yourself a grade on your streaking technique. Then, before you leave class, take to plates to the TA for their grade.

PROCEDURES:

REPEAT OF GRAM STAIN

  1. Divide two slides into 3 sections each.
  2. Using the bacterial cultures provided (control G+ and G- and unknown), place smears on one of the slides AS BEFORE. Use the second slide to prepare smears of the 2 colonies from Exercise #5 streak plates. Gram stain the smears on both slides.
  3. Determine the gram-staining characteristics of the unknown mixture and of the 2 streak plate colonies; i. e., is the unknown a pure culture and if so what is its gram characteristic. If it is a mixed culture describe the gram reactions of the various bacteria that are present and make a guess as to what they are.
  4. Compare your results with those on pg. 32-33 of A Photographic Atlas for the Microbiology Laboratory. Give yourself a grade on your stains. Verify your conclusions with your instructor if your can.

SPORE STAIN

Figure 2. Illustration of spore staining protocol and example of various types of spore arrangements.

  1. Read pg. 32-33 in A Photographic Atlas for the Microbiology Laboratory.
  2. Prepare two smears from a colony of the spore-formers Bacillus subtilis from your Exercise #3 streak plates that you've kept in your drawer and a colony from your (or someone else's) Rodac plate that you think might be a spore-former.
  3. Cut a piece of blotting paper the size of the smears, but small enough so it doesn't hang over the edges of the slide.
  4. Place the slide over the boiling streamer and lay the paper on top of the smears.
  5. Flood the paper with the malachite green stain (CARCINOGEN) and time for 3 min. Keep the PAPER WET with the stain if it begins to dry.
  6. After 3 min. remove the slide and lift off the paper with your loop and discard.
  7. Gently wash the slide with water.
  8. Flood the slide with the counter-stain safranin to stain the vegetative cells.
  9. After one minute, wash off the safranin, blot the slide dry and examine under the microscope.
  10. Draw and identify the spores and the vegetative cells in the circles below. Describe the characteristics (location, form, size etc.) of the spores compared with the picture of the various types of spores shown above and in the NetText (Chap 3).
  11. Click here to see a spore done by medical students; how does yours compare? Compare your spore stain with these: 1, 2, 3

EXAMINATION OF STREAK PLATES FROM EXERCISE 5

  1. Examine your streak plates. The TA will have given you a grade.
  2. If you receive a C- or lower, you must repeat the streaking, but first the TA will discuss with you how to improve your technique.

For Gram stain use split circle.

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

TEST OF YOUR KNOWLEDGE OF THE SPORE STAIN

Click here to take the test.

Copyright Dr. Ronald E. Hurlbert, 1998.
This material may be used for educational purposes only and may not be duplicated for commercial purposes.
SCIENCE HALL, ROOM 440CA
PHONE: 509-335-5108
FAX: 509-335-1907
E-mail address: hurlbert@wsu.edu
Phone: 509-335-5108