Biol 251 lab 3
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Why is the media in the test tubes referred to as differential media?
Because it is showing different characteristics of different types of bacteria. For example; Biochemical characteristics and motility (movement)
What is a bacterial exoenzyme and what is the function? Why are they important for bacteria?
- Allow the bacteria to breakdown large molecules into smaller ones.
- The smaller molecules then, can enter the bacteria cell and be used as nutrient
What is the evidence that your bacteria did or did not have the citrase or urease enzymes?
if each or both produced ammonia, they tasted positive for citraze and/or urease enzymes.
Why are indicators used in the differential media tubes? Name the indicators in the urea agar tube
and the SCA tube.
- To show existence of chemical reactions by changing their color.
- urea: phenol red
- SCA: bromthylmol
Write the chemical reaction for indole production.
Tryptophanase released by the bacteria →hydrolyze amino acid tryptophan→produce indole & pyruvate
Explain the different types of metabolic processes that can be observed and read from the triple
sugar iron agar media.
- Fermentation of any of the the three sugars, glucose, lactose, and fructose by the bacteria.
- Peptoniziation: Usage of peptones (amino acids in the media) as an additional food source by the bacteria.
- Production of H2S
Describe 3 properties of Enterobacteriaceae. Why is it important to identify them correctly?
- Normal gastrointestinal microbiota
- Identification of these bacteria is an important feature of clinical microbiology because some of them can cause diseases in humans and animals.
What are Indicators?
A chemical that change the appearance (color) of the media in response to the products of biochemical characteristics of the bacteria
What the SIM Media was used for in lab 3?
Test for motility and production of H2S
Why is rapid identification important in a clinical setting?
So we can begin treatment for infection early.
Are the rapid identification (Chromagar & biochemical agars) tests definitive?
What is the purpose of the smear preparation?
To obtain accurate staining results
What is the purpose of heat fixation of a smear?
to stick the bacteria to the slide so it will not be washed off during staining
Discuss three errors that may occur when preparing the smear.
- Obtain too much culture will cause cells to pile on top of each other
- Use too much saline will increase drying time
- Use saline with liquid culture. Only broth is used with liquid culture. Adding saline will result in too little cells in the smear.
List the Gram stain reagents and the purpose of each in the Gram stain procedure.
- Crystal violet; stain peptidoglican of gram (+) and gram (-) purple
- Gram's iodine; use as mordant. cause Crystal violet to bind to gram(+) cell wall.
- Gram's Alcohol; decolorizing gram(-) and lock in iodine and crystal violet in gram(+)
- safranin; stains gram(-) pink-red
- Purified water; use to wash each reagent between each step.
Explain how bacterial cell wall structures account for the two different Gram reactions.
- Crystal violate absorbed in the peptidoglican layer (cell wall) of gram(+) & gram(-) dye them purple.
- Gram's iodine prevent crystal violet to be absorbed in the cell membrane.
- Gram's alcohol dissolve and can remove the crystal violet/iodine but also shrinks the pores of the peptidoglican. because gram(+) cell wall is thicker than gram(-) cell wall some of the crystal violet/iodine got locked in deep in gram(+) cell wall while the gram(-) turn colorless.
- Safranin stains gram(-) pink. because the dark purple color of gram(+) is overpower the pink color of safranin, gram(+) stays purple.
Explain why the Gram stain is a differential stain.
because it allow us to tell the difference between two types of bacteria Gram(+) & Gram(-)
Explain why the decolorization step is the most critical step in the procedure.
Because if we do it too long, it might remove the stain from gram(+) and if we not do it long enough gram(-) will stay purple
Why is the Gram stain an important first step in identification of an unknown bacterium?
It will help to chose the type of antibiotic and allow chemotherapy begin quickly.
What clinical information does a Gram stain provide?
Identification of unknown bacteria in relatively short time
Why does culture age affect the results of a Gram stain?
- old cultures may yield in false positive/negative results.
Discuss two causes each for false positive and false negative stain reactions
Preforming the decolorizing step incorrectly or using too thick or old culture may result in false negative or positive results
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