Lab Midterm (labs 1-7) Flashcards
Define transmission electron microscopy (TEM)
Electrons pass through the cell, so you can see detailed images of its inside
Define scanning electron microscopy (SEM)
Electrons bounce off the cell, so you can capture detailed images of its surface
Define ocular lens
Binocular lenses to which you place your eyes for viewing the specimen. These lenses have a set magnification of 10x
Define objective lenses
Three or four lenses mounted on a turret that provide the major magnification power of the microscope. These lenses are rotated into place immediately above the specimen. Typical magnification powers usually include 4x, 10x, 40x, and 100x. In concert with the ocular lenses, these objective lenses yield total magnification powers of 40x, 100x, 400x, and 1000x
Define revolving turret
A mechanism allowing movement of the different objectives into place above the specimen
Define stage
Flat plate on which the specimen is placed. Light passes from the condenser through a hole in the plate and then through the specimen on a microscope slide.
Define slide clamp
Holds the glass slide with the specimen in the correct place for observation, allowing the entire slide to be moved by the slide manipulator
Define slide manipulator
Through the use of a system of gears, the manipulator moves the slide clamp holding the slide. This allows for smooth movement of the slide, allowing precise location of different parts of the slide
Define condenser
A series of lenses that focuses light traveling from the light source on the specimen. Depending on the type of microscope, condensers can provide multiple settings. Settings often seen include bright field (used most often), dark field (providing excellent illumination of the edges), phase contrast (enhancing poorly contrasting objects), and many other options
Define iris diaphragm
Allows for the reduction of or expansion of the amount of light passing from the condenser to the specimen
Define coarse focus adjuster
Allows for focusing the image on lower power objective lenses (4x or 10x lenses). Often used to quickly scan specimens and focus in on interesting items on the slide
Define fine focus adjuster
Allows for focusing the image on higher power objective lenses (40x or 100x lenses). Since the working distance of these higher power lenses is relatively small, the fine focus is the only focus that should be used with these; use of coarse focus on the higher power lenses could lead to lens damage
Define rheostat
Power control that increases or decreases the voltage applied to the light source, increasing or decreasing the brightness. Generally ranging from 0 to 10. Properly stored microscopes should always have their rheostats adjusted to 0 to minimize possible bulb filament breakage if the power is switched on with too high an initial voltage.
Define power switch
Controls the application of electrical power to the rheostat.
Define light source
Generally a tungsten filament bulb
Define magnification
The relative enlargement of the image. Total magnifications are calculated by multiplying the magnification of each lens the light passes through after passing through the specimen. For the microscopes in this lab total magnification is calculated by multiplying the power of the objective and ocular lenses
Define resolution
The ability of a microscope to enable visualization of two closely spaced points on a specimen. In practice, known as the resolving power of a microscope, the lower the resolving power the better the resolution
Define resolving power
Defined as the smallest distance between two points where those two points are clearly visible. Mathematically related as follows: RP = wavelength / (NAobj. + NAcond.), where NA is the numerical aperture of either the objective or condenser lens systems. Numerical aperture = n (Sin0), where n is the lens refractive index and 0 is the angle from the center of the beam of light as it passe through the specimen to the outer edge of the objective lens.
Define refraction
The bending of light
Define refractive index
Different substances that will transmit light have different refractive indices. Air has a refractive index of 1.0, glass is about 1.5, and immersion oil is very close to 1.5
Define immersion oil
A highly purified oil used to fill the space between the specimen and the objective and condenser lenses. Usually, for the sake of keeping the microscope clean, the oil is not added to the bottom of the slide. When added the oil displaces the air, reducing refraction of light passing through the specimen. This decreases the resolving power of the lens system, increasing the microscope’s resolution. We only use this on 1000x
Define focal length
The specific working distance of the lens system when the specimen is in focus to the observer
Define parfocal
A geometric property of a lens system and microscope allowing for nearly focused images when switching from a focused lower objective magnification to a higher power lens (which should be in focus with minimal movement of the fine focus).
Define working distance
The space between a focused objective lens and the surface of the slide. For lenses on typical microbiology laboratory microscopes the working distances are as follows: 4x, about 2cm; 10x, about .5cm; 40x, about .4mm; and 100x, about .1mm
All culture media must provide what components?
1) Major elemetns (C, H, O, N, P, S)
2) Minor elements (Fe, Ca, K, Mg, etc)
3) Trace needs (e.g., vitamins)
4) An energy source
5) Buffering capacity
6) Special needs (e.g. unique terminal electron acceptors or donors, unique temperatures or other physical parameters)
What are the two types of bacterial media?
Complex or defined
Define broth
A liquid based medium provided in either tube or flask. Broth tubes are filled after thorough mixing. These broth tubes must be sterilized to ensure having sterile media for your cultures
Define agar
An extract of seaweed that can be used to make a solid, gel-like product after heat sterilization. The spaces within the agar matrix will be filled with whatever aqueous phase was mixed with the agar initially. Thus, beef-based, soy-based, and any number of aqueous additions can be made.
Define petri dish
A shallow dish with a loose fitting lid designed to hold a relatively small volume of molten agar, which then solidifies, giving a sold flat surface to grow cultures. They can be made out of glass or plastic; most are plastic and come pre-sterilized
Define agar slant
Agar-based media poured into a test tube prior to heat sterilization; upon removal from the autoclave, these tubes are placed on a slant and allowed to cool where they solidify, leaving a solid slanted surface in the tube. Media produced in slants is much less susceptible to drying than in agar petri dishes.
Define complex medium
The major constituents of this medium are provided by extracting components of plants or animals; the most common being soybean or beef based, because they provide a complex mixture of carbohydrates, proteins, lipids, nucleic acids, and other elements not easily defined. In general complex media are used to grow the largest number of different types of bacteria.
What were the two types of media used in lab 2?
Tryptic soy broth (TSB) and tryptic soy agar (TSA)
Define an autoclave
A device used to sterilize equipment/ supplies by subjecting them to high pressure saturated steam at 121C for 15-20 minutes
What do you use with a magnetic stirrer?
A hot plate
Describe what was done with the TSB media in lab 2
1) Measured out 9 grams of TSB powder and combined with water in a flask to produce 300ml of media
2) Transferred 10ml of the media to each of the 30 tubes
3) Capped the tubes, and removed one tube and labelled it as “nonsterile”
4) Tubes were then put in the autoclave
Describe what was done with the TSA in lab 2
1) Measured out 9 grams of TSB powder and mixed with 4.5 grams of agar as well as water to make 300ml of molten agar; a hot plate and magnetic stirrer were used to ensure it was mixed well.
2) Transferred 12ml of the solution into 25 tubes
3) Capped the tubes and labelled them as TSA slants
4) Autoclave
Describe how autoclaves work
They work because moist heat kills due to:
1) Increased motion of molecules
2) Cleavage of H bonds in/between proteins
3) Water molecules in steam become more energized
4) More penetration (into liquids/ surfaces)
Describe what was learned in lab 2 overall
The preparation of bacterial culture-based media
What are the 3 main reasons aseptic technique is important?
1) Safety: When working with cultures to minimize potential contamination of you and your neighbor
2) Sterility: When working with sterile substances to avoid contamination
3) Purity: When working with pure cultures
How do you aseptically pour tryptic soy agar (TSA) plates?
1) Flask closed; open and pass over the flame
2) Pour into petri dish
3) Recap petri dish and pass the flask over the flame again
4) Recap the flask
* All of this must be done within ~1.5 feet of a flame
How do you aseptically transfer a bacterial pure culture?
1) Sterilize the loop with a flame
2) Pass the lid of the culture’s tube over the flame
3) Open tube, put loop in the tube and collect bacteria
4) Close bacteria tube
5) Open the sterilized petri dish or tube and deposit bacteria with the loop
6) Re-sterilize loop
* All must be done within ~1.5 feet of a flame
When working with ‘dry’ bacteria (i.e. in a petri dish of agar), why is it important to slowly put the loop into the flame slowly when re-sterilizing?
So you don’t accidently allow the bacteria to go airborne
Define colonial morphology
The outwardly visible structure, color, and odor of colonies of a pure culture of bacteria, used to characterize that culture
Define streak plate isolation
A technique involving the use of sterile nutrient agar Petri dishes and an inoculation loop to physically spread large numbers of bacterial cells over the surface of the agar until individual cells end up well separated from each other; individual colonies may grow from separated cells.
Define pour plate isolation
The use of dilution of liquid bacterial samples in sterile isotonic diluents that leads to dramatically reduced numbers of cells per unit of volume in the tubes; requires multiple dilutions. The bacteria are then added to molten agar
Define spread plate isolation
The use of dilution of liquid bacterial samples in sterile isotonic diluents that leads to dramatically reduced numbers of cells per unit of volume in the tubes; requires multiple dilutions. The bacteria are then spread over an agar Petri dish.
Who created serial dilutions in broth?
Pasteur
Who created diluted cultures on agar (streak plate isolation)?
Koch
Define streak plate isolation
The careful dilution of cultures on agar
What was accomplished during lab 4?
We streaked 4 Petri dishes; 3 of the 4 dishes had different bacteria (Bacillus subtilis (Bs), Staphylococcus coli (Sepi), and Escherichia coli (Ec)) and one dish had a mixed culture of all 3.
We used 3 types of streaking techniques: continuous, quadrant, and t-streak
What were the 3 types of streaking techniques used?
Continuous, quadrant, and t-streak
Describe how to do continuous streaking
Deposit the loop into a dot at the top of the plate, then drag some of that bacteria across the entire Petri dish, making squiggly lines with the loop
Describe how to do quadrant streaking
1) Divide the plate into 4 even sections and label them
2) Apply a loop of bacteria and spread across the entire first quadrant
3) Sterilize loop and drag the loop through quadrant 1 into quadrant 2, making a zig-zag pattern
4) Sterilize loop and drag the loop through quadrant 2 from bottom to the top, then drag the loop through quadrant 3 making zig-zag pattern
5) Sterilize loop and drag the loop through quadrant 3 from bottom to the top, then drag the loop through quadrant 4 making zig-zag pattern
Describe how to do a T-streak
1) Divide the plate into 2 large sections and one small section at the top; label the small section as 1 and the others as 2 and 3
2) Apply a loop of bacteria and spread across the entire first quadrant
3) Sterilize loop and drag the loop through half of quadrant 1 into quadrant 2, making a zig-zag pattern
4) Sterilize loop and drag the loop through quadrant 2 from bottom to the top, then drag the loop through quadrant 3 making zig-zag pattern
List the 3 steps of a simple stain
1) Select a dye and add to a heat-fixed sample, leave on for one minute
2) Rise slide with water
3) Blot dry
How should you observe a simple or Gram stain?
Under 100x magnification with oil
List the 4 steps of transferring a culture to a slide
1) Put a loopful of water onto the slide
2) Aseptically collect a small amount of bacteria from a slant, then deposit and spread into the water on the slide using circular motions
3) Allow to air dry
4) Heat fix by passing the slide through a flame 4 times
List the 5 steps of performing a Gram stain
1) Primary Stain: Crystal violet for 1 minute; rinse with water
2) Mordant: Iodine for 1 minute; rinse with water
3) Decolorize: holding slide at a tilt for 4 seconds (until liquid is colorless); rinse with water immediately
4) Counterstain: Safranin for 1 minute; rinse with water
5) Blot slide with KimWipe
What color is gram negative bacteria on a gram stain?
Pink or red
What color is gram positive bacteria on a gram stain?
Purple
Give an example of a gram negative bacteria
E. Coli
What is the primary stain of gram stains?
Crystal violet
What is the primary stain step of acid fast stains?
Carbol fuchsin flood smear (paper towel)
What is the primary stain step of endospore staining?
Malachite green flood smear (paper towel)
What is the mordant step of a gram stain?
Iodine
What is the mordant step of an acid fast stain?
Heat carbol fuchsin to 90-95 degrees C (steaming) for 3.5 minutes, then rinse with water
What is the mordant step of an endospore stain?
Heat malachite green to 90-95 degrees C (steaming), rinse with water
What is the decolorization step of a gram stain?
95% alcohol
What is the decolorization step of an acid fast stain?
Add alcohol dropwise for 20-30 seconds, rinse with water
What is the decolorization step of an endospore stain?
There isn’t one
What is the counterstain step of a gram stain?
Safranin
What is the counterstain step of an acid fast stain?
Methylene blue for 1 minute
What is the counterstain step of an endospore stain?
Safranin for 1 minute, rinse with water
What are the steps of an endospore?
1) Primary stain: Malachite green flood smear (paper towel)
2) Mordant: Heat at 90-95C (steaming), keeping paper towel wet, for 3.5 minutes. Rinse with water.
3) Counterstain: Safranin for 1 minute, rinse with water
What are the steps to an acid fast stain?
1) Primary stain: Carbol fuschin (paper towel) flood smear
2) Mordant: Heat to 90-95C for 3.5 minutes, rinse with water
3) Decolorization: Add alcohol dropwise 20-30 seconds, rinse with water
4) Counterstain: Methylene blue for 1 minute
What are the acid fast colors?
Acid fast positive = purple/ red
Acid fast negative = blue
How do you make bacteria make spores?
Leave them in an incubator for a week or more
Would you ever acid-fast stain a species that gram-stains well?
No
How do you calculate stepwise dilution factor? Give an example
Volume added divided by the volume of dilution (volume + volume added)
-Ex: 1: 1ml + 00ml > 1:100, then 1/100 = 0.01, which = 10^-2
1) How do you calculate total dilution factor? Give an example
2) What is the typical initial total dilution factor of the culture?
1) Stepwise dilution factor + previous total dilution factor
- Ex: 10^-2 x 10^-4 = 10^-6 (adding not multiplying)
2) Typically 10^0 for the culture
In the 1890s, we learned the causative agent of what two things? What was the causative agent? Why wasn’t this very beneficial at first?
1) Typhoid fever and cholera
2) Causative agent was mammalian feces
3) Monitoring those agents was time consuming
What technique was created to monitor for potentially contaminated water?
Monitoring fecal contamination with an indicator species called coliforms
What do coliforms do?
1) Indicate fecal contamination
2) Lactose fermentation (usually within 24 hours)
Define lactose fermentation
The production of acid + gas
Historically, the issue of enumerating bacteria arose due to what?
Public health concerns for clean, uncontaminated water, which led to attempts to monitor water quality
How is water quality counted?
Using MPN tests with phenol red lactose fermentation
What are other ways to count bacteria? (3)
1) Serial dilutions
2) Viable plate counts
3) Hemocytometer (graph thing)
When does phenol red turn yellow?
At pH greater than or equal to 6
What does an MPN test do, and what does MPN stand for?
Tests water quality; stands for ‘most probable number’
What are the 3 parts of an MPN test?
1) Presumptive: data generated
2) Confirmed: data verified
3) Complete: published and acted upon (recommendations, policy, etc)
What is the formula to calculate the number of bacteria in the original culture (cells/mL)?
Number of colonies divided by total dilution factor
True or false: The number of colonies on a plate is the minimum number of cells originally put on that plate
True
The statistically valid range of colony counts depends on what?
The size of the plate
What range is considered a reliable colony count?
30-300
Define a batch culture
The making of a culture in which bacteria are initially provided with a finite amount of nutrients and space, and nothing is ever added to the culture. A closed system.
Define a continuous culture
-An open-system culture that utilizes a chemostat and probe to monitor and adjust pH, oxygen, and temperature.
-It has 3 openings; one to allow atmosphere to enter, one to allow for ‘refreshing’ , and one to allow for collection.
What did we do during the bacterial growth lab? (lab 7)
We measured the optical density of our samples at 3 different times using a spectrophotometer, and made and plated serial dilutions from that sample at 3 different times
List and describe the 4 phases that happen in a batch culture (closed system)
1) Lag: No growth, but genes are being turned on/off based on available nutrients.
2) Log: Rapid growth, use of nutrients, and waste generation
3) Stationary: Cell division happens at the same rate as cell death, can last days
4) Death: Nutrients are depleted, toxic wastes accumulate, cell division is less than cell death
What is the X axis of the graph of closed-culture growth? What is the Y axis?
1) X-axis: Time
2) Y-axis: Number of bacteria
What were the serial dilutions we made during the bacterial growth lab? (lab 7)
1) .1ml into 9.9ml
2) 1ml into 9ml
3) 1ml into 9ml
What does a spectrophotometer measure?
The amount of light that passes through the medium
What type of flask makes using a spectrophotometer easier? What makes it easier?
A Nephlo flask, which has a cuvette built into its side
What is 0.01 equal to in scientific notation? What about as a fraction?
10^-2; 1/100
What is 0.1 equal to in scientific notation? What about as a fraction?
10^-1; 1/10
How do you calculate MPN?
After you’ve completed the experiment and you have one group of 5 10ml tubes, one group of 5 1ml tubes, and one group of 5 .1ml tubes, count how many out of each group of 5 is yellow (positive), and use the chart
List the steps to an acid fast stain
1) Primary stain: carbol fuchsin flood smear (paper towel)
2) Mordant: Heat to 90-95⁰ (steaming) for 3-5 minutes; rinse with water
3) Decolorization: Acid alcohol drop wise 20-30 seconds; rinse with water
4) Counterstain: Methylene blue for 1 minute; rinse with water. Blot and view
List the steps for an endospore stain
1) Primary stain: Malachite green flood smear (paper towel)
2) Mordant: Heat to 90-95⁰ (steaming) for 3-5 minutes; rinse with water
3) Counterstain: Safranin for 1 minute, rinse with water. Blot to view.
Describe the differences between simple stains and differential stains. Give 3 examples of differential stains
1) Simple stains only provide simple information about the cells on the slide (cellular morphology and arrangement of cells)
2) Differnetial stains provide data that can be used to differentiate bacteria into at least 2 groups.
3) Gram stain, Acid-fast stain, Endospore stain
Give examples of colony form, margin, and elevation
1) Form: Circular, irregular, rhizoid, filamentous
2) Margin: Entire, undulate, lobate, filiform, or curled.
3) Elevation: Flat, raised, convex, pulvinate, or umbonate
