MICR 211, Lab Quiz 2
What stain penetrates the endospore and how?
Malachite green w/ heat.
What type of staining involves mixing cells with the stain before they are smeared on the slide?
Negative and capsular.
What is a palisade arrangement?
Parallel arrangement of rod-shaped cells.
What 3 factors can affect the outcome of gram staining?
1. Age of cultures. 2. Smear thickness. 3. Decolorization.
What are the 3 goals in preparing a smear?
1. Cause cells to adhere to slide. 2. To ensure shrinkage of cells doesn't occur during staining. 3. To prepare thin smears.
How can capsular staining be done?
1. Place drop of india ink or nigrosin near end of slide. 2. Disperse organisms into drop of nigrosin or india ink. 3. Move spreader slide toward drop of suspension. 4. Push spreader slide to the left. 5. Allow to dry. 6. Stain w/ methylene blue or safranin. 7. Allow to dry.
How do you do spore staining by the Schaeffer-Fulton method?
1. Saturate paper toweling w/ malachite green. 2. Steam. 3. Remove paper toweling and rinse with water. 4. Counterstain w/ safranin. 5. Rinse w/ water. 6. Blot dry.
How do you do acid-fast staining by the Ziehl-Neelson method?
1. Stain w/ carbolfuchsin. 2. Heat. 3. Wash w/ water. 4. Decolorize w/ acid-alcohol. 5. Rinse w/ water. 6. Counterstain w/ methylene blue. 7. rinse w/ water. 8. Blot dry.
How do you destroy endospores?
Autoclave for 15-20 minutes in 121 degrees Celsius steam.
When essential nutrients are exhausted, which 2 genera of bacteria produce endospores?
Bacillus and Clostridia.
Why aren't anionic chromophores attracted to bacteria?
Bacteria are negative.
Why are cationic chromophores attracted to bacteria?
Bacteria are negatively charged.
What are endospores?
Bacterial structures that allow them to survive conditions not favorable for growth.
What is the primary stain in gram staining?
Crystal violet.
How does the age of the culture affect the outcome of gram staining?
Cultures older than 16-18 hours can convert gram-positive bacteria into gram-variable or gram-negative bacteria.
Which type of microscopy produces an image most similar to the one achieved by negative staining?
Darkfield microscopy.
What is the most critical step in Gram staining and why?
Decolorization, because the cells become differentiated after this point. Gram-positive bacteria remain purple and gram-negative bacteria become colorless.
How might fluorescence microscopy be used to view the bacterial capsule?
Fluorescent dyes adhere to antibodies on the capsule which can be viewed by fluorescence microscopy.
What is another word for capsule?
Glycocalyx.
Why is negative staining also useful for determining cell dimensions?
Heat fixation is not performed, so so no shrinkage of cells occurs.
What is acting as the mordant in acid-fast staining and how?
Heat, which makes the stain complex more permeable to the mycolic acid and cell wall lipids.
Why can't staining of the bacterial capsule be accomplished by ordinary staining procedures?
Heat-fixation shrinks or destroys the capsule.
How do you prevent air contamination during agar plate inoculation?
Hold lid over top of agar plate and inoculate near open flame.
What are 2 examples of negative stains?
India ink and nigrosin.
What is pleomorphism?
Irregularity of form.
How does a counterstain differ from a primary stain?
It is a different color.
How does the endospore trait enhance Bacillus anthracis's capabilities as a biological weapon?
It protects it from harm or death that vegetative cells would normally experience.
Are acidic dyes positive or negative?
Negative.
What type of staining is useful for viewing spirochetes?
Negative.
How is heat in the Ziehl-Neelson method unsafe?
Phenol can vaporize when heated and give rise to noxious fumes.
What is the color of Bacillus megaterium after mordant?
Purple.
What is the color of Pseudomonas aeruginosa after primary staining?
Purple.
What is the color of Staphylococcus aureus after decolorization?
Purple.
What are metachromatic granules?
Reddish-purple granules w/in C. diphtheriae when stained w/ methylene blue.
What type of staining uses water to remove excess stain?
Simple.
How does Streptococcus pneumonia's capsule protect it?
The capsule prevents prevents phagocytosis by white blood cells.
How should you incubate agar plates and why?
Upside-down, to prevent condensation from dripping on to the agar plate and spreading the inoculated microbes.
What is simple staining?
Use of a single stain to color cells.
Disinfectants are only effective against which organisms?
Vegetative cells and viruses.
What are 3 characteristics of endospores?
1. Dehydrated. 2. Not actively metabolizing. 3. Resistant to harm and death.
How do you do a gram staining?
1. Heat-fix cells. 2. Stain w/ crystal violet. 3. Wash w/ water. 4. Gram's iodine. 5. Wash w/ ethyl alcohol. 6. Stain w/ safranin. 7. Wash w/ water. 8. Blot dry.
What are 3 common dyes for simple staining?
1. Methylene blue 2. Basic fuchsin 3. Crystal violet
How do prepare a smear from liquid media?
1. Place 2 loopfuls of liquid containing organisms on the slide. 2. Disperse organisms over entire area of slide. 3. Allow smear to dry. 4. Pass slide through flame several times.
How do you prepare a smear from solid media?
1. Place 2 loopfuls of water on slide. 2. Disperse organisms in water. 3. Allow smear to dry. 4. Pass slide through flame several times.
How can negative staining be done?
1. Place drop of india ink or nigrosin near end of slide. 2. Disperse organisms into drop of nigrosin or india ink. 3. Move spreader slide toward drop of suspension. 4. Push spreader slide to the left. 5. Allow to dry.
What characteristics of Corynebacterium diphtheriae are present from simple staining?
1. pleomorphism 2. metachromatic granules 3. palisade arrangement
Why is it important to limit the quantity of cells in smear preparation?
A high quantity of cells obscures details about individual cells and can entrap stain, preventing it from being washed or destained.
Why aren't endospores easily penetrated by stains?
Because of their resistant properties.
Why are endospores so resistant?
Because of their: 1. Exosporium. 2. Low water content.
Where should agar plates be labeled?
Bottom side.
How are acid-fast bacteria made more permeable to staining in acid-fast staining?
By heat and the phenol present in the carbolfuchsin.
What type of staining is useful for viewing the glycocalyx of certain bacterial species?
Capsular.
The common dyes for simple staining have color-bearing ions called?
Chromophores
What is the color of Moraxella (Branhamella) catarrhalis after decolorization?
Colorless.
What is the color of Staphylococcus aureus before primary staining?
Colorless.
What color are bacterial endospores after Gram stain is performed and what does this tell you about its physical properties?
Colorless. Endospores are resistant to staining.
What is mycolic acid?
Complex lipid composed of fatty acids and fatty alcohols.
What type of stains are used in a simple stain procedure?
Crystal violet and methylene blue.
Why are encapsulated strains of Streptococcus pneumoniae more likely to cause disease than non-encapsulated strains?
Encapsulated strains are protected from phagocytosis by white blood cells.
Disinfectants aren't effective against which type of organism?
Endospores.
What is the decolorizer in gram staining?
Ethyl alcohol.
Compare and contrast the cell walls of gram-positive bacteria and gram-negative bacteria?
Gram positive bacteria have a cell membrane surrounded by a thick peptidoglycan layer. Gram negative bacteria have a cell membrane surrounded by periplasmic space, a thin peptidoglycan layer, and an additional outer membrane.
Which of the 3 differential stains should be used first to identify unknown bacterium?
Gram staining, because all bacteria can be differentiated by gram staining.
What is the mordant in gram staining and what does it do?
Gram's iodine. It forms an insoluble complex w/ crystal violet in gram positive bacteria.
How is gram staining a differential stain?
Gram-positive bacteria become purple and gram-negative bacteria become pink.
How is Mycobacterium grouped in terms of Gram classification?
Gram-positive.
How can decolorization affect the outcome of gram staining?
If destaining reagent is over applied, the dye-mordant complex can be removed from gram-positive cells.
What type of stains are used in a negative stain procedure?
India ink and nigrosin.
Which genera of bacteria have a high lipid content?
Mycobacterium and Nocardia.
The acid-fast stain is important in identifying what 2 species of bacteria?
Mycobacterium tuberculosis and Mycobacterium leprae.
How does the presence of mycolic acid affect staining properties?
Mycolic acid prevents stains routinely used in Microbiology.
What makes Mycobacterium resistant to staining?
Mycolic acid.
What type of staining uses a stain that does not penetrate cells, but is used to color the background?
Negative and capsular.
Why don't negative stains penetrate bacterial cells?
Negative stains are acidic and therefore, have negatively charged chromophores. Bacterial cells are also negatively charged.
What type of staining should not involve heat fixation?
Negative.
What disinfectant is used in the lab?
Phenolic disinfectant.
Besides polysaccharides, a capsule can also consist of?
Polypeptides.
What do bacterial capsules consist of?
Polysaccharides, and sometimes polypeptides with unique amino acids.
What are most capsules usually composed of?
Polysaccharides.
Are basic dyes positive or negative?
Positive.
Are chromophores positively charged or negatively charged?
Positively charged.
What is the counterstain in gram staining?
Safranin.
What types of staining uses a basic dye to stain bacterial cells?
Simple and capsular.
What problems can arise when the slide is heated in flame?
Slide can shatter.
Why is negative staining useful for observing spirochetes?
Spirochetes are thin and do not stain with positive stains.
How does the acid-fast nature of Mycobacterium contribute to its virulence?
The mycolic acids protects it from phagocytosis in the host.
How do the cell structure of gram-positive and gram-negative bacteria contribute to their different staining properties?
The thick peptidoglycan layer of gram-positive bacteria retain the primary stain better than the thin layer of gram-negative bacteria.
How does the color of gram-positive bacteria change throughout the gram staining procedure?
They are colorless after heat fixation, but purple after crystal violet primary staining.
How does the color of gram-negative bacteria change throughout the gram staining procedure?
They are colorless after heat fixation, purple after crystal violet primary staining, colorless after ethyl alcohol decolorization, and pink after safranin counterstaining.
How does smear thickness affect the outcome of gram staining?
Thick smears can entrap the primary stain that cannot be removed by alcohol or acetone.
What is the purpose of heat fixation in smear preparation?
To adhere the cells to the slide.
Give 3 reasons why aseptic technique is essential when handling microbial cultures in the lab?
To prevent contamination of: 1. the handler or others 2. culture materials 3. lab area
Give 2 examples of how the Bunsen burner is used during tube culture inoculation.
To sterilize inoculating loop and tube.
Why are negative stains useful?
To study the morphology and external structures of bacterial cells.
How does smear preparation differ for liquid medium and solid medium?
Water is required for solid medium, but isn't required for liquid medium.
What makes a bacteria acid-fast?
When acid-alcohol cannot remove the primary stain.
How can negative staining demonstrate structures such as capsules?
When combined with positive staining, the capsule can be seen as a halo surrounding the positively stained cell against a dark background.
When do endospores germinate to form a new vegetative cells?
When nutrients become available.
Is the capsule also a means for bacteria to attach to solid surfaces?
Yes.
A wet smear must first be __________ before heat fixation.
air-dried
