Micro Lab Exam 2

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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

Whee should a label be written on an agar plate?

bottom of the plate

How should agar plates be incubated? Why?

upside down to prevent condensation on the agar surface

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

An inoculating loop or needle is sterilized in a flame

until it is ENTIRELY bright red

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.

Why is dilution a necessary part of pure culture preparation?

in order to get isolated colonies

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 basic dye is utilized to stain bacterial cells. (simple, negative, capsule)

simple

A stain that does not penetrate cells is used to color the background. (simple, negative, capsule)

negative

Useful for visualizing spirochaetes. (simple, negative, capsule)

simple

Useful for visualizing the glycocalyx of certain bacterial species. (simple, negative, capsule)

capsule

Cells are mixed with a stain before they are smeared on the slide. (simple, negative, capsule)

negative & simple

Heat fixation of the slide is not recommended. (simple, negative, capsule)

negative & simple

Water is used to remove excess stain from the slide. (simple, negative, capsule)

negative & simple

For the simple stain procedure, one can use

crystal violet AND methylene blue

For the negative stain procedure, one can use

india ink AND nigrosin

Before heat fixation, a wet smear of bacterial cells on a slide must first be

air-dried

Bacterial capsules can consist of

polysaccharides AND polypeptides with unique amino acids

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.

What is the function of a mordant?

adherence

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.

Staphylococcus aureas before primary stain is added. (pink, purple, no color)

no color

Pseudomonas aeruginosa after primary stain is added. (pink, purple, no color)

pink

Bacillus megaterium after the mordant is added. (pink, purple, no color)

no color

Staphlococcus aureas cells after the decolorizer is used. (pink, purple, no color)

pink and purple

Moraxella (Branhamella) catarrhalis after the decolorizer is used. (pink, purple, no color)

no color

Bacillus megaterium after the counterstain is added. (pink, purple, no color)

no color

Pseudomonas aeruginosa after the counterstain is added. (pink, purple, no color)

purple

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)

What are the colors for the cell type in gram stain?

gram (+)=purple; gram (-)=pink

What are the colors for the cell type in spore stain?

sporangium=green; vegetative=pink

What are the colors for the cell type in acid-fast stain?

acid-fast=red; non-acid-fast=blue

Bacterial cell wall is composed of

peptidoglycan

The exosporium, or endospore coat, is composed of

proteins

Endospores are produced by bacteria in the genus

Bacillus AND Clostridium

Acid-fast staining is useful for identifying the causative agent of

leprosy AND tuberculosis

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