Lab Exam 1 Flashcards
Aspergillus Conidiophores
Penicillium
Colony Morphology includes:
- ________ (name 3 subcategories)
- ________ (name 5 subcategories)
- ________ (name 5 subcategories)
- ________ (name 3 sub categories)
- ________ (name 4 subcategories)
- Shape
- Circular
- Irregular
- Punctiform (tiny)
- Margin
- Entire (smooth, mo irregularities)
- Undulate (wavy)
- Lobate (lobed)
- Filamentous
- Rhizoid (branched like roots)
- Elevations
- Flat
- Raised
- Convex
- Pulvinate (very convex)
- Umbonate (raised in the center)
- Texture
- Moist
- Mucoid
- Dry
- Pigment production
- Opaque
- Transluscent
- Shiny
- Dull)
With regards to bacterial growth, what does the term pure colony or colony-forming unit mean?
Pure colony: Microbial culture containing only one species
Colony-Forming Unit: Cell or groups of cells that produces a colony when transferred to a plated media
What are the important precautions taken to avoid contamination?
- Do not perform transfers over books, study materials, etc
- Be organized with your materials and be sure they are not set up in ways that might result in contamination
- Plase all media tubes i the test tube rack when not in use whether they are sterile or not
- Take your time
- Never hold tube culture by it’s cap
- Try not to breathe over cultures
- Minimize talking with peers to avoid contamination
In which hand should the tube cap be held while transferring cultures from one medium to another?
In your dominant hand, same as the loop.
Why should Petri dishes be incubated in the upside down position?
Moisture can build up on the lid if the plated culture is set with the culture on the bottom part. To avoid this you place the dish upside down.
Why is the mouth of the test-tube passed over the flame after opening and before closing the tube cap?
To sanitize the tube and the air around it.
Why is the inoculating loop flamed before and after use?
To sanitize and avoid contamination.
What is the pH of nutrient agar?
6.6-7.0 at 25º C
What are the ingredients of a nutrient agar and a nutrient broth?
Nutrient agar: 3.0 g beef extract, 5.0g peptone, 15.0g agar, 1.0L distilled or deionized water
Nutrient broth: 3.0 g beef extract, 5.0g peptone, 1.0L distilled or deionized water
What is the limit of resolution and how do you calculate it?
Limit of resolution: The closest two points can be together for the microscope lens to make them appear separate; two points closer than the limit of resolution will blur together
(Note: the resolution improves as the limit of resolution is made smaller)
Formula:
D (rtransmitted in the image): the minimum point at which two points can be resolved (best is usually 2 µm in electron microscope) this is the value you are normally trying to calculate
λ: (lambda) measurement of wavelengths in nm, normally provided in the problem
Nacondenser: the numerical aperture of the condenser
Naobjective: the numerical aperture of the selected objective lens (this is not the same value as the magnification, it will look like this: 10x/1.30 - use the 1.30 value)
How do you calculate total magnification?
Objective lens magnification x Ocular lens magnification
Example:
Low power objective lens x Ocular lens
10 x 10 = 100x magnification
What is the magnification of the Ocular lens?
10x
What are the magnifications of the three objective lenses?
10x, 20x, and 40x
Objective lens
The microscopic
Examlpes of simple stains
Methylene blue
Safranin
Crystal Violet
Why does a basic stain color the cells?
Because the positive charge of the auxochrome of a basic stain allows its attachment to the negative charge of most bacterial cell surfaces, thus the cell becomes colored.
Steps of Gram Staining procedure.
What is the other name for the reagant?
- Heat fixed emulsion
- Cover smear with crystal violet stain for 1 min, rinse with distilled water
- Cover smear with iodine stain for 1 min, rinse with distilled water
- Decolorize smear with ethanol/acetone for about 10 seconds, rinse with distilled water
- Counterstain smear with safranin for 1 min, rinse with distilled water
- Pat dry and view under microscope
Other name: iodine?? (need to update)
What is the function of each substance used for gram staining?
Crystal violet: primary stain for both gram negative and gram positive bacteria
Iodine: acts as mordant for keeping crystal violet dye in gram positive bacteria
Ethanol/Aceton: to decolorize gram negative bacteria
Safranin: to re-stain the gram negative cells for visual comparison
Identify Gram positive vs Gram negative bacteria under a microscope
Question 1 from 3.6
Predict the effect of the following mistakes made when performing Gram stain.
a. failure to add the iodine
b. failure to apply the decolorizer
c. failure to apply the safranin
d. reversal of crystal violet and safranin stains
a. the crystal purple die will not fix with the material of the cell and the dye could leave
b. the dye will not be removed from the gram neg cells and both neg and pos will appear purple
c. you will not be able to see the gram neg cells as they will have no dye
d. both gram pos and gram neg cells will be purple and indistinguishable
Endospore Staining
A differential stain used to visualize bacterial endospores
What are the steps and stains used in the Endospore staining process
- Start with heat-fixed emulsion.
- Cover smear with a strip of bibulous paper, aple malachite green stain and steam for 5 minutes
- Rinse slide with water
- Counterstain with safranin for 1 min
- Gently blot dry
Malachite green is forced into the spre while it is being steamed, breaking down the keratin of the endospore. **Malachite green is water-soluble and has low affinity for cellular material, so vegetative cells and spore mother cells can be decolorized with water and counterstained with safranin.
The safranin counterstain is to stain bacterial cells so there is contrast between themselves and the endospores under the microscope
What stain can stain vegetative cells?
Safranin
What is the purpose of steaming the slide during the staining process?
To expedite the ability of the malachite green to enter the endospores.
Identify vegetative cells and endospores on a microscopic slide.
Blue arrows indicate vegetative cells/spore mother cells (pink)
Black arrow indicates endospores (blue)
Question 1 from 3-9 Lab (Endospores)
Why does this exercise call for an older (5-day) culture of Bacillus?
So the bacterial cells are well into the stabilization phase and have plenty of endospores created for us to stain and view.
Be able to determine motility results by looking at a stab culture in semi-solid agar (Example of non-motile bacteria - Micrococcus luteus; Example of highly motile bacteria - Proteus mirabilis)
What is the agar concentration in a typical nutrient agar medium and in a semi-solid medium?
Typical nutrient agar medium: 1.5%
Semi-solid medium: 0.4% (just enough to maintain fom while allowing movement)
Acid-fast staining
A differential stain used to identify acid-fast organisms such as members of the genus Mycobacterium . Acid-fast organisms are characterized by wax-like, nearly impermeable cell walls; they contain mycolic acid and large amounts of fatty acids, waxes, and complex lipids.
Steps of Acid-fast staining
- Obtain 3 motility stabs. Label two tubes, each with the name of the organism, your name, adn the date. Label the third tube “control.”
- Stab-inoculate two tubes with the test organisms. Do not inoculate the contro.
- Incubate the tubes aerobically at 35 degrees celsius for 24-48 hours
- Examen growth patterns
What is the use of carbolfuschin in acid-fast staining?
When carbolfuschin is used as the primary stain because it is lipid-soluble and penetrates the waxy cell wall. It also stains acid-fast negative cells. Staining of acid-fast cells by carbolfushin i further enhances by steam heating the preparation to melt the wax and allw the stain to move into the cell
Recognize acid-fast and non-acid-fast bacteria.
Which stain stains acid-fast bacteria?
Which stain stains non acid-fast bacteria?
Carbolfuschin stains acid-fast bacteria
Methylene blue stains non acid-fast bacteria
Why is myobacterium acid fast?
Because it has wax-like, nearly impermeable cell walls; they contain mycolic acid and large amounts of fatty acids, waxes, and complex lipids.
How does heating the bacterial smear during a ZN stain promote entry of carbolfuschim into the acid-fast cell wall?
The steam melts the waxy wall allowing the carolfuschin to enter the cell and stain it.
Are acid-fast negative cells stained by carbolfuschin? If so, how can this be a differential stain?
Yes, it can stain acid-fast negative cells which can be used to identify a genus and then help start determining the species.
Why do you suppose the acid-fast stain is not as wifely used as the Gram stain? When is it more useful than the gram stain?
Because it is not always necessary to break down a thick, waxy outer membrane. some gram neg bacteria have more porous membranes;
Aerobic bacterium from a facultative anaerobe by looking at the thiglycollate test results.
What is the purpose of adding thioglycollate to the medium?
It is a liquid medium designed to promote growth of a wife variety of fastidious microorganisms. It can be used to grow microves representing all levels of oxygen tolerance; however, it generall is associated with the cultivation of anaerobic and microaerophilic bacteria.
What are the ingredients of FTM and what is the use of each?
Yeast extract - provides nutrients
Pancreatic digest of casein - provide nutrients
Dextrose - notmentioned
Sodium chloride - notmentioned
Sodium thioglycollate - reduce oygen to water
L-cystine - reduce oxygen to water
Agar - slow oxygen diffusion and stabilize growth
Resazurin - indicator
Distilled or deionized water - notmentioned
What is the indicator used int hemedium?
Resazurin
How are bacteria classified based on the oxygen requirements?
Microaerophiles: survive only in environments containing lower than atmospheric level sof oxygen
Aerotolerant: microorganisms that don’t require oxygen and are not adversely affected by it
Facultative anaerobes: grow in the presence or absence of xygen. when xygen is available, they respire aerobically.
Obligate anaerobes: organisms for which even small amounts of oxygen are lethal, and therefore will be seen only in the lower regions of the medium
Obligate anaerobes: organiss that require oxygen for respiration, grow at the top where xygen is most plentiful
Nostoc
Rhizopus Nigricans
Saccharomyces
