Lab Quiz 1 Flashcards
Where is the stage, nose piece, course focus, eye piece, and fine focus
3-1 Microscopy
Stage- Where slide sits
Nose piece - Rotating lenses
Course focus - Moves stage up and down quickly
Eye piece - Adjustable to eye width
Fine focus - Smaller stage movements to get clear focus
What are the four names of the lenses?
3-1 Microscopy
Scanning
Low power
High power
Oil immersion lens
What is the ocular magnification, object magnification, and total magnification
3-1 Microscopy
- Ocular magnification is 10x for the microscope
- Object magnification can be adjusted to 4x,10x, 40x, and 100x
- Total mag. = ocular x object. therefore 40x, 100x, 400x, 1000x
What is oil immersion?
3-1 Microscopy
- Oil has similar refraction to glass and helps focus light through lens
- Must only be used on 100x and NOT 40x
What is aseptic technique? What is innoculation?
1-3 Aseptic Technique
- Aseptic technique is when bacteria is transfered without contamination
- Innoculation is the introducing of microorganisms to the environment
Outline the steps involved in a slant culture.
1-3 Aseptic Technique
- Sterilize innoculation loop with burner
- Flame the tube’s lip and when loop cools pass wire over growth
- Flame tube and streak sterile agar surface in a zig zag pattern
- Flame tube and innoculation loop
What is a chromagen and what are it’s two components?
3-5 Simple Stain
- Colored molecule to increase visibility on slide
- Auxochrome is the charged portion allowing binding
- Chromophore is colored portion
What are four examples of Basic stains? What is their charge?
3-5 Simple Stain
- Methylene Blue
- Crystal violet
- Safranin (red-pink)
- Malachite green (teal)
- (+)
What is the purpose of a streak plate?
1-4 Streak Plate
- Identify what bacteria are in a sample
- Allows us to isolate colonies of bacteria
What is the purpose of a negative stain?
3-6 Negative Stain
(-) charge repels (-) of cell resulting in outlining the morphology, arrangement, and cell size
Why is the negative stain medically important?
3-6 Negative Stain
Some bacteria can only be seen with negative stain (spirochetes)
What is the charge of Negative dyes? Give 4 examples.
3-6 Negative Stain
- Negative charge to repel from cell’s charge
- Congo Red
- Nigrosin (black)
- India Ink
- Eosin
What is the purpose of a capsule stain?
3-9 Capsule Stain
- Identify capsule producing cells and non encapsulated cells
What are stains? What do they consist of?
3-5 Simple Stain
- Stains are solutions that help make transparent cytoplasm and organelles visible
- Solutions consisting of a solvent and colored molecule
What is a chromophore? An auxochrome? What kind of charge does the auxochrome have in basic stains?
3-5 Simple Stain
- Chromophore - Portion of chromagen that gives color
- Auxochrome - Charged portion of chromagen
- Basic charges (+) are attracted to cell surface (-)
Why are most bacteria most easily stained by basic stains (hint: think about the charges)?
3-5 Simple Stain
- Most cells have a negative charge. Thus a basic charge (+) is attracted to the charge of bacteria
Name 3 common basic stains.
3-5 Simple Stain
Methylene Blue
Crystal Violet
Safranin
List 3 reasons for heat-fixing bacterial smears.
3-5 Simple Stain
- Kills bacteria
- Causes adhesion to slide
- Coagulates cytoplasm proteins increasing visibility
Why is it usually necessary to stain bacterial cells in order to see them?
3-5 Simple Stain
- Bacterial cells have transparent cytoplasms and by staining you can see cytoplasm as well as organelles
In the medical laboratory, bacterial characteristics such as morphology and arrangement are usually determined by a _____________ stain.
3-5 Simple Stain
Gram
What is wrong with the heat-fixing technique illustrated on pg 186?
3-5 Simple Stain
Allow the flame to completely pass through the flame and cool off on the other side. do not swipe it through the fire quickly.
Why do we use a needle when making a smear from slant culture instead of using a loop?
3-5 Simple Stain
Allows for more precise culture collection and avoids getting too much sample.
How does the chromogen in a negative stain differ from the chromogen in the simple stain?
3-6 Negative Stain
Negative stain has (-) charge that creates an outline to be able to see morphology, arrangement, and cell size. Chromagen in a simple stain has a positive charge that attaches to bacteria and stains them.
The chromogen in a negative stain carries a ___________ charge. Is the negative stain, acidic or basic?
3-6 Negative Stain
- Negative
- Negative stain is acidic
Why do the bacterial cells remain unstained in a negative stain?
3-6 Negative Stain
- The similar charges makes the stain repel from the cell
Two stains often used in negative staining are ________________ and ____________________.
3-6 Negative Stain
- Congo Red
- Nigrosin
Negative stains are used to determine ____________________ and _______________________ in bacteria that are:
3-6 Negative Stain
- Morphology
- Arrangement and cell size
- Fragile and heat sensitive
A spirochete that is usually stained by a negative stain is ___________________________. This organism causes the disease __________________________. Another spirochete is ______________________________, which causes the disease _________________________.
3-6 Negative Stain
- Treponema pallidum
- Syphilis (STI)
- Borrelia burgdorferi
- Lyme disease
Why are negative stains used when determining the accurate size of a micro-organism is crucial?
3-6 Negative Stain
Heat fixing can result in microorganisms shrinking. Negative staining does not
In a good negative stain, what should the background of the slide look like?
3-6 Negative Stain
Uniformly dark
Simple stains stain the __________________ and not the _________________; negative stains stain the ______________________ and not the ________________________.
3-6 Negative Stain
- Microorganism
- Background
- Background
- Microorganism
What is wrong with the amount of acidic dye that the pg 192 recommends that you use? How much dye should you use?
3-6 Negative Stain
A whole drop was used. A half drop can be used by touching the dropper to the slide.
Differential stains allow a microbiologist to:
3-7 Gram Stain
Differentiate gram (+) and gram (-) bacteria
Which are used more frequently, differential stains or simple stains?
3-7 Gram Stain
Differential Stains are used 95% of the time
The most commonly used differential stain in bacteriology is the _______________ stain.
3-7 Gram Stain
Gram
Besides the gram stain, other differential stains test for the presence of _____________, _____________, _________________, or __________________ in bacteria.
3-7 Gram Stain
- Acid-fastness
- Capsule
- Spores
- Flagella
In the gram stain a _____________________ step occurs between the application of two ___________ stains.
3-7 Gram Stain
- Decolorization
- Basic
The primary stain in the gram stain is _____________________. What do bacterial cells look like after application of this primary stain?
3-7 Gram Stain
- Crystal violet
- Purple
Iodine acts as a ___________________ in the gram stain. What is the purpose of adding iodine?
3-7 Gram Stain
- Mordant
- Binds negative charge to positive charge of crystal violet allowing more purple to stay on the cells
The most critical step in the gram stain is the ______________________ step.
3-7 Gram Stain
Decolorization
The decolorizing solution we use in the gram stain is _______________________________.
3-7 Gram Stain
Acetone Alcohol
In the last step in the gram stain, gram negative cells are colored by safranin, which is a ____________________________.
3-7 Gram Stain
Red (basic) counterstain
The ability to resist or not resist decolorization in the gram stain is based on:
3-7 Gram Stain
Composition of the cell wall in Gram positive and Gram negative cells
What do gram negative and gram positive cells look like before crystal violet is added in the gram stain?
3-7 Gram Stain
They are transparent
What do gram negative and gram positive cells look like after the mordant is added in the gram stain?
3-7 Gram Stain
- Gram positive cells will look purple
- Gram negative cells will be red
- Over exposure results in redish gram positive cells
- Underexposure results in purple gram negative cells
-Red bc iodine as safranin hasn’t been added yet
What do gram negative cells look like after the decolorization step in the gram stain? Why?
3-7 Gram Stain
They will appear transleucent because both the crystal violet and iodine will have been washed off.
What do gram positive cells look like after the decolorization step in the gram stain? Why?
3-7 Gram Stain
They will look purple because the crystal violet and iodine would have stained the cells and remained attached after the wash.
What will happen to gram negative cells if you don’t add enough alcohol in the gram stain?
3-7 Gram Stain
They will maintain a red hue from the iodine
What will happen to gram positive cells if you add too much alcohol in the gram stain?
3-7 Gram Stain
The gram positive cells will become more red in addition to purple giving poor visibility
Why is it best to use cultures that are 24 hours old or less for gram staining?
3-7 Gram Stain
Bacillus and Staphylococcus can lose the crystal violet-iodine complex in more than 24 hours
Name 3 Gram negative bacterial genera and 2 Gram positive bacterial genera other than the genera used in this exp.
3-7 Gram Stain
Gram Negative:
- Eschericha coli
- Citrobacter freundii
- Klebsiella pneumoniae
Gram Positive:
- Staphylococcus aureus
- Streptococcus pyogenes
What do the gram negative cells look like after successful completion of the gram stain? Gram positive cells?
3-7 Gram Stain
- Gram positive cells will be purple
- Gram negative cells will be pink
Why are you asked to gram stain a mixture of gram positive and gram negative bacteria on the same slide?
3-7 Gram Stain
To create clear contrast between gram positive and gram negative bacteria
Why do you need a counterstain in the gram stain?
3-7 Gram Stain
- Crystal violet attaches to both gram positive and negative cells
- Mordant fixes crystal violet onto gram positive cells
- Acetone alcohol Washes the crystal violet off gram negative cells
- Saffranin is then used to stain gram negative cells to provide visual contrast as the gram positive cells remain purple while the gram positive cells become pink
Why do gram-negative bacteria stain differently than gram-positive bacteria in the gram stain? Be specific!
3-7 Gram Stain
Gram Positive:
- Thick Peptidoglycan layer
- Crystal violet is retained by PG layer with the help of mordant even after acetone alcohol wash
Gram Negative:
- Thin Peptidoglycan layer
- Lipid A dense layer
- Crystal violet is washed out by acteone alcohol due to thinner PG and Lipid A layer
- Safranin is then used to stain the transparent gram negative cells
What is the Optic lense? What is the objective lense?
3-1 Microscope
Optic Lens - Where your eyes go
Objective lens - Lenses on the nose piece
What are teichoic acids?
3-7 Gram Stain
- Unique to gram positive bacteria
- Teichoic acids are supporting structures embedded in the PG layer of gram positive bacteria
What is the charge of an auxochrome in a simple stain? What about a negative?
3-6 Negative Stain
- In a simple stain an auxochrome would be basic and therefore have a positive charge
- In a negative, an auxochrome would be acidic and therefore have a negative charge
Which is easier to get antibiotics for, gram positive or negative bacteria?
3-7 Gram Stain
- Harder for gram negative as meds struggle to pass lipid A layer
- Most medicine is water based and cannot cross the lipid layer
