Provide three reasons why the use of aseptic technique is essential when handling microbial cultures in the laboratory.
1) no contaminants introduced into the culture, 2) does not contaminate the handler, 3) no contaminates remain after
Provide two examples of how the Bunsen burner is used during inoculation of a tube culture.
1) sterilized inoculating loop, 2) sterilizes mouth of tube
How is air contamination prevented when an inoculating loop is used to introduce or take a bacterial sample to/from an agar plate?
it is sterilized over the Bunsen burner
Against which organisms are disinfectants effective? Against which type of organism may not be effective? What disinfectant(s) is used in your laboratory?
Disinfectants are effective against vegetative cells and viruses, but not endospores. Lysol is used as a disinfectant in the lab.
A disinfectant is used on your work surface
before the beginning of lab procedures, after all work is complete, and after any spill of live organisms.
To retrieve a sample from a culture tube with an inoculating loop, the cap of the tube is
removed with the fingers of the loop hand and placed in the fingers of the tube hand
In regards to bacterial growth on solid media, define the term "colony".
the billions of cells that originate from one parent cell
What colony characterisitics can be used for differentiation of bacterial species? As an example, compare the properties of colonies of Serratia marcescens and Micrococcus luteus on your streak plate.
Color and shape are used for differentiation . Serratia marcescens is a gram (-) rod while the Micrococcus luteus is a gram (+) coccus.
What advantage(s) does the streak-plate method have over the pour-plate method?
The streak-plate method is more economical in materials and time.
What advantage(s) does the pour plate method have over the streak-plate method?
The pour-plate method requires less skill.
Why is the loop flamed before it is placed in a culture tube? Why is it flamed after completing the inoculation?
To sterilize the loop of any microbes on it.
Before inoculating and pouring molten nutrient agar into a plate, why must the agar first be cooled to 50° C?
In order to avoid condensation of moisture on the cover
Provide two reasons why plates should be inverted during incubation.
1) prevent condensation, 2) prevent contamination
A stain that does not penetrate cells is used to color the background. (simple, negative, capsule)
Useful for visualizing the glycocalyx of certain bacterial species. (simple, negative, capsule)
Cells are mixed with a stain before they are smeared on the slide. (simple, negative, capsule)
negative & simple
Water is used to remove excess stain from the slide. (simple, negative, capsule)
negative & simple
Which of the three differential stains would likely be the first used when identifying an unknown bacterium? Explain.
Gram stain because structures within cells display dissimilar staining reactions that can be distinguished by the use of different dyes.
For differential staining, how does a counterstain differ from a primary stain?
A primary stain uses crystal violet that is the initial stain for both gram (+) and gram (-) cells while a counterstain uses safranin that stains the colorless (gram (-) cells.
How do a gram (+) and gram (-) bacteria differ in cellular structure? How does this contribute to their differential staining properties?
Gram (-) cells have a greater lipid content in their cell walls than gram (+) cells. Lipids are more soluble in alcohol and acetone that causes more rapid decolorization of gram (-) cells.
Which is the most critical step in the Gram-stain procedure? Why? If this procedure is done incorrectly, how might that affect the final results?
Destain is the most critical step. If done incorrectly, it could cause a gram (+) read gram (-)
How does culture age affect the results of a Gram-stain?
Gram (+) cultures can convert to gram (-) over time giving false readings
How does culture age affect the results of a spore stain?
Malachite green is used to stain endospore cultures older than 36 hours while nigrosin is used 24-36 hours
Why must smear thickness be considered before performing a Gram-stain?
To better able see the morphology and characteristics of the cell
What color are bacterial endospores after a Gram-stain is performed? What does this tell you about the physical properties of endospores?
Bacterial endospores will be either green (sporangium) or pink (vegetative)
Bacillus anthracis, the causative agent of anthrax, is an endospore-former. Why does this trait enhance its capabilities as a biological weapon?
Because endospores are the most biologically resistant structure known to man
What makes Myobacterium particularly resistant to staining? How are the bacteria in this genus grouped in terms of Gram classification?
the myocolic acid in the cell wall make them resistant to sharing. Grouped as acid-fast
How do you think the acid-fast nature of Myobacterium contributes to its virulance?
It provides a coating that prevents an immune response from mounting and rather than causing an acute disease state, immediately progresses to a chronic disease state.
Moraxella (Branhamella) catarrhalis after the decolorizer is used. (pink, purple, no color)
What are chemicals used in gram-stain technique?
crystal violet (primary), iodine (mordant), 95% alcohol (decolorizer), safranin (counterstain)
What are the chemicals used in spore stain technique?
Malachite green (primary stain), heat (mordant), none (decolorizer), safranin (counterstain)
What are the chemicals used in acid-fast stain technique?
carbolfuchsin (primary), heat (mordant), acid-alcohol (decolorizer), methylene blue (counterstain)