Day 1: Syllabus, Safety, and Movie introduction to the microbiological laboratory




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OSMOSIS IN MICROBIOLOGY


Diffusion is the chemical process by which molecules move from a high concentration to a low concentration. Osmosis is the diffusion of water across a semi-permeable membrane. The cell membrane is a biological example of a semi-permeable membrane through which water readily diffuses. The term osmotic pressure refers to the force exerted on the membrane when concentration of solute on inside of cell is different than that on the outside of cell. When discussing the biological membrane and its environment, here are three types of tonicity: isotonic, hypotonic, and hypertonic. Isotonic means that the concentration of solute is the same inside the cell as it is outside the cell. Hypertonic means that the concentration of solute is higher outside the cell, and hypotonic means that the solute concentration outside cell is lower.


Many microbes can live in either hypotonic or hypertonic environments, such as freshwater or saltwater lakes. Many microbes even use high concentrations of amino acids within the cell to maintain osmotic equilibrium, and some, like the freshwater microbe Paramecium, actively pump water out of the cell (Paramecium utilizes a contractile vacuole to accomplish this). However, if the osmotic pressure is greater on the outside than on the inside, the cell will eventually dehydrate due to loss of water molecules and it will not be able to grow.


Osmotic pressure can cause bacteriostasis, which means cessation of microbial growth. Once the bacteriostat is eliminated, the microbes can resume growth. Knowledge of osmotic pressure is used to help preserve our food. By having high concentrations of salt and/or sugar, microbes lose their ability to grow within that environment; therefore, foods that are packed with preservatives are in essence just another form of antimicrobials. However, certain bacteria, molds, and yeasts can grow within a high-salt or high-sugar environment; these spoil food more often than bacteria.


THE BUGS

Micrococcus luteus, Staphylococcus epidermidis, and Escherichia coli


DEMONSTRATION

Show them how to streak the bugs (not for isolation!)





THE EXERCISE


1)In groups of two, label plate and streak Esherchia coli, S. epidermidis, and Micrococcus luteus onto each of the salt plates provided (0%, 2%, 4%, 8% NaCl). Place upside down in 37C incubator for 48 hours.

2) Look up properties (as for other laboratories)of the new bugs in Bergey’s manual of Determinative Bacteriology.


3)View streak from last week


4)Gram stain M. luteus and E. coli (one partner stain one , the other partner the other).

QUESTIONS

1)What physically happens to a cell in hypertonic conditions? What happens to a cell in hypotonic conditions?

2)Look up properties of the bugs – staining characteristics, shape, size, medical significance.

3)What is salt? What other chemicals influence osmotic pressure?

4)What variables will influence the growth on these plates?


HELPERS

1)24 plates of each: 0% NaCl, 2%NaCl, 4% NaCl, 8%NaCl (percent by weight)

2)6 nutrient agar plates of Staphylococcus epidermidis, 6 with E. coli, and 6 of Saubaraud’s dextrose agar for Micrococcus luteus.


REFERENCES

Microbiology: An Introduction, 6th Edition, Tortora, Funke, & Case, Benjamin Cummings (Addison Wesley Longman, Inc.), San Francisco, © 1998. pages 88, 92-93, 157-158, 181, 188, and 190.


Microbiology: A Human Perspective, 2nd Edition, Nester, Roberts, Pearsall, Anderson, & Nester, McGraw-Hill Companies, Inc., Boston, © 1998, pages 94-95.


Day 6: Lactobacillus and Tomato juice agar

Lactobacillus species are common inhabitants of mouth, intestine, and vagina. They also contaminate milk and are often responsible for souring. They are commonly used in production of yogurt, certain cheeses, kefir, sauerkraut, and pickles. Yogurt manufacturers often employ Streptococcus thermophilus as well. Streptococcus and Lactobacillus species contain no cytochrome systems, hence derive all of their energy from fermentative pathways that produce acids as end products. Lactic acid is a common end acid produced by Lactobacillus species. Lactic acid is sour to the taste. It inhibits the growth of competing microbes, allowing the relatively inefficient (they don’t have electron transport systems and organisms that do have electron transport outgrow those that don’t) lactobacillus to grow.


Demonstration

Explain the autoclave an its workings


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