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Endospore Stain: An endospore stain is used to detect Bacillus and Clostridium, which produce endospores after its vegetative cycle, when the cell dehydrates to conserve its energies. It’s kind of like cell hibernation. Capsule Stain: A capsule stain allows you to see the protective layer on some microbes, such as Streptococcus pneumoniae or S. mutans. The technique used is called negative-staining because it uses stains called Indian ink and nigrosin, which makes your sample appear virtually all black. The capsules, however, are unable to absorb the stain and can be seen clearly though a bright-field microscope low in low light. Acid-Fast Stain: The acid-fast stain is used primarily to detect species of Mycobacterium, which produce diseases like tuberculosis and leprosy. The cell wall of this type of organism is layered with a waxy substance (mycolic acid) that resists most staining techniques. This method of staining produces a red-green sample that makes this microorganism easy to identify. The specific stain used in this process is called Ziehl-Neelsen (also called Ziehl’s carbolfuschin), after the two men who created this stain in the 19th century.
A WORD ON THE BUGS
Saccharomyces cerevisiae is a yeast that is commonly used in bread and beer making. Saccharomyces uvarum and Saccharomyces elipsoideus are also commonly used in the production of alcoholic beverages. Chlorella vulgaris is an alga. It is edible, and was used by the Russian space program in deep space travel simulations.
2) Hanging drop preparation.
1) Consolidate ubiquity results on the board: location, colony size, numbers, shape and color.
2) Perform the methylene blue stain on Saccharomyces cerevisiae(or ellipsoideus)
3) Prepare a hanging drop slide of and view Chlorella vulgaris in a phase contrast microscope.
4)Prepare a smear containing your own saliva (the so called ‘spit smear’) and stain with methylene blue.
5)) Look up the following properties of the Saccharomyces and Chlorella - Reproduction, shape, niche.
Answer these questions:
1) Are yeasts prokaryotes or eukaryotes? Do they have nuclei? Describe their chromosomes.
2) What type of organism (prokaryotic or eukaryotic) is Chlorella? What is its energy source? Its carbon source?
1)YPD culture of Saccharomyces cerevisiae
2)Depression slides out on counter
Exercises for the Microbiology Laboratory, Burton E. Pierce & Michael J. Leboffe, Morton Publishing Company, Colorado, © 1999, pages 34-48, and 50.
Laboratory Experiments in Microbiology, 6th Edition, Johnson & Case, Benjamin Cummings (Addison Wesley Longman, Inc.), San Francisco, © 2001, pages 17-21, 27-60 and 77-80.
Laboratory Manual and Workbook in Microbiology: Applications to Patient Care, 6th Edition, Morello, Mizer, & Wilson, McGraw-Hill Companies, Inc., Boston, © 1998, pages 31-50.
Microbiological Applications, 6th Edition, Harold J. Benson, Wm. C. Brown Publishers, Dubuque, © 1994, pages 48-65.
Microbiology Experiments: A Health Science Perspective, 3rd Edition, Kleyn & Bicknell, McGraw-Hill Companies, Inc., Boston, © 2001, pages 29-48.
Microbiology a human perspective, 3rd Edition, Nester et al McGraw Hill Companies, Inc Boston 2001 p49-50.
Day 4: Streaking for Isolation and More Staining
STREAKING FOR ISOLATION
As stated in a prior lab exercise, microbes are ubiquitous. Streaking for isolation is the process by which you separate, or isolate, individual microbes from their neighbors. This is accomplished by depositing individual cells on a nutrient agar plate, and then allowing the single cell to reproduce and grow up into colonies. A colony is simply a cluster of the same species of organism derived from the originally isolated cell. All of the cells in a colony are clones of the original cell. This procedure is also referred as “Pure Culture Technique”. Since microbes from your hands, breath, and countertop can contaminate your sample, you will want to minimize contact with the environment external to the Petri dish, i.e. don’t contaminate your plate!!
PETRI DISHES AND AGAR
The use of solid growth media is essential to the ability to generate pure cultures of bacteria and other microbes. Initially Koch used potato slices to this purpose. The wife of Koch’s lab assistant had used agar in her home food canning, and made the suggestion that it might be useful for solidifying nutrient media. Nutrient agar is a typical nutrient medium used commonly in the microbiology laboratory. Agar is a polysaccharide derived from seaweed, and is often used to solidify nutrient media. The nutrient component of nutrient agar consists of a complex mixture of proteins, lipids, and carbohydrates. Difco's Nutrient Agar contains beef extract and peptone (peptone is protein that has been digested with enzymes to produce free amino acids and/or free nitrogen) as nutrient ingredients. Petri dishes were invented by another of Koch’s laboratory assistants, Julius Petri. These two technologies led to the ability to streak for isolation, thereby separating one species of bacteria (or other microbe) from all the others. This in turn allowed investigators to begin studying and characterizing individual species of microbes.
Gram’s stain: This is a complex stain developed by Christian Gram, a Danish physician, in 1884.
1) Prepare a smear of the bacterium or yeast as described on p. 9.
2)Place slide on rack and cover smear with crystal violet. Incubate 60 seconds.
3) Rinse crystal violet off with water, and cover smear with iodine (Gram’s or Lugo’s) for 45 seconds, then rinse off with water. Iodine is a mordant that helps retain crystal violet in gram-positive cells.
4) Rinse smear with 95% ethanol just until color no longer bleeds from smear. Then rinse with water. Note: This is the tricky stage of the Gram’s stain—do not over rinse the specimen with the alcohol.
5) Cover smear with safranin for 60 seconds. Rinse with water and then blot dry with bibulous paper. Safranin is the pink counterstain. It is taken up by gram-negative cells, which are unable to retain the crystal violet during the ethanol rinse.
Staphylococcus epidermidis and Saccharomyces ellipsoideus
1) Inoculate broth cultures with same bugs.
2) Describe nutrient agars and broths.
1) In teams of two, streak for isolation--One of you use S. epidermidis, the other use Saccharomyces. Incubate at 37C for 48 hours.
2) Each person inoculate a broth culture with the same organism.
3) Gram stain the same organism.
4) Look up properties of the organisms - Gram character, shape, niche. Use Bergy’s for bacteria and other resources for the yeast.
1) Is Saccharomyces larger or smaller than the bacteria? How much so?
2) Look up components of nutrient agar and record in your lab notebook.
3) Look up properties of the microbes in Bergey’s manual of Determinative Bacteriology. Are they pathogens?
1) 48 Nutrient agar plates
2) 6 NA plate cultures of S. epidermidis and 6
Saubarad's Dextrose agar plate with Saccharomyces cerevisiae.
3) 48 nutrient broth tubes each with 5ml nutrient broth.
Exercises for the Microbiology Laboratory, Burton E. Pierce & Michael J. Leboffe, Morton Publishing Company, Colorado, © 1999, pages 51-53.
Laboratory Manual and Workbook in Microbiology: Applications to Patient Care, 6th Edition, Morello, Mizer, & Wilson, McGraw-Hill Companies, Inc., Boston, © 1998, pages 57-61.
Microbiological Applications, 6th Edition, Harold J. Benson, Wm. C. Brown Publishers, Dubuque, © 1994, pages 76-79.
Day 5: Osmosis
|Day 1: Syllabus, Safety, and Movie introduction to the microbiological laboratory||The microbiological safety and quality of food / M. Barbara Lund, Tony C. Baird-Parker, Grahame W. Gould|
|Uws laboratory Safety Guidelines||Study on Strip Pillar Safety Based on Laboratory Coal Tests|
|Syllabus bsc 105-introduction to biology||Syllabus for cs 100 – Introduction to Computers and Information Systems|
|Syllabus for meng 3354. 001 – introduction to fluid mechanics||We will meet at 3: 30 in Room 2200b symons, unless Nancy is meeting her class that day. Day when Nancy meets, we ILL meet at 3: 00. Such days are marked with an asterisk. Those responsible for presenting each topic to the group are indicated|
|The author who inspired the movie||As taken from the Internet Movie Database|