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




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BUGS TO BE USED


Lactobaccilus bulgaricus, Yogurt culture (various Lactobacillus species are found in yogurt, and often Streptococcus thermophilus)

THE EXERCISE


1)Streak yogurt (for isolation) onto one half of tomato juice agar plate

2)Streak (for isolation) Lactobacillus bulgaricus out onto other half.

3)Look up principles of tomato juice agar – record in notes

4)Gram stain the Yogurt - juice vs solids- and Lactobacillus

5)Look up properties of Lactobacillus – record in notes

6)View NaCl plates from last week.


QUESTIONS


1)Why is yogurt solid?

2)Why is it sour?

3)Why eat Yogurt while undergoing antibiotic therapy?

4)Why do organisms that have electron transport chains out-compete those that don’t?

5)What is meant by the phrase ‘principle of a test medium’?


FOR LABORATORY HELPERS

1)50 Tomato Juice agar plates

2)Yogurt containing live cultures from Safeway

3)Isolation Streak of Lactobacillus species onto tomato juice agar

4)750mls of NA ready to autoclave


Day 7:

BIOCHEMICAL TESTS AND SELECTIVE/DIFFERENTIAL MEDIA

BIOCHEMICAL TESTS


All microbes produce proteins called enzymes. Enzymes are biological catalysts, which means that they increase the rate of chemical reactions without being used up in the reaction. Most organisms have evolved gene sets that when expressed lead to unique combinations of biochemical pathways that allow an organism to extract energy, carbon, and other nutrients from its environment. A biochemical pathway is a series of biochemical reactions that leads to synthesis of a specific product. Often, we can devise a test that allows us to detect that specific product. These tests are termed biochemical tests. These tests are frequently formulated in a growth medium containing the chemicals so that the as the organism grows and produces the product of interest, it is released into the growth medium. You will be using a number of biochemical tests during this lab exercise. The principle of a biochemical test (or a test agar) is the simply the characteristic chemical reaction that allows you to either select or differentiate between various species of bacteria or other microbe (see below example of catalase produced by some bacteria).

CATALASE


Hydrogen peroxide is a reactive (and toxic) oxygen metabolite formed in many cells during normal metabolism. Catalase is an enzyme which breaks down potentially harmful hydrogen peroxide (H2O2) into water and oxygen. It is produced by some bacteria, and by many eukaryotic cells where it occurs in peroxisomes for in order to degrade H2O2 which forms as a by-product of oxidizing amino- and fatty acids.


CATALASE TEST PROTOCOL:

1)Place a drop of fresh hydrogen peroxide on a microscope slide

2) Using a sterile toothpick, introduce a small amount of bacteria to the hydrogen peroxide and stir.

3)Watch for bubbles of oxygen (H2O2 in the presence of catalase yields oxygen gas plus H2O), which indicates that the bacterium is producing catalase.

4)Rinse into disposal beaker with 70% ethanol


Selective and Differential Media

Read chapter 6 (Nester et al) on microbial growth. Instructor will provide definitions of Selective vs Differential media. Selective and differential media allow us to distinguish between bacterial species based upon differences in their biochemistry. For example, hemolysis on blood agar plates is caused by enzymes called hemolysins that are released by some species of bacteria.-hemolysis is complete lysis of red blood cell (RBC) allowing all light to pass where before red was reflected. Alpha hemolysis is partial breakdown of RBC, yielding a greenish zone. Staphylococcus aureus produces the alpha toxin which leads to B hemolysis, and Streptococcus pyogenes, the cause of Strep Throat, produces a similar toxin..


Mannitol Salt Agar - is an agar that can differentiate between bacterial genera on the basis of the biochemical pathways. Look up the principles of this test in the Difco manual.


MacConkeys Agar - used to characterize enteric bacteria. Look up the principles in Difco.


Bile esculin agar - Used identify Enterococcus faecalis. Look up principles in the Difco manual.
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