MCBL 121L: Exam 1

Diopter ring

A ________ _______ on a microscope allows you to adjust the focus of the left ocular lens independently of the right lens.

Condenser

A ______________ on a microscope will help to concentrate light on the specimen.

Counterstain

A _________________ is the application of second stain with a contrasting colour to sample for microscopy. In the case of Gram stain, this would be safranin, which binds to cell membranes.

Decimal point

A line on a micropipette should be considered like a __________ _________. E.g. 0|05

Axenic

A pure culture is also known as _______. It's relatively easy to determine the cell concentrations of microorganisms growing in these. But what about for a mixed culture? Combine selective media and differential media!

Optical density

After putting a tube in the spectrophotometer when quantifying bacterial concentration through turbidity, it takes a second or two for the ____________ ___________ value to stabilize. Once it does, record the value. If you wait longer, it will decrease as the bacteria settle to the bottom of the tube.

10-15

Allowing a 10-ul of bacterial solution on a glass slide to air dry takes about __-___ minutes.

Heat, malachite green

Although endospores are not effectively stained by standard methods, they can be stained by using a combination of ______ and _____________ __________ dye.

Oxford

An __________ pipette has its own tip ejector button apart from a plunger button and volume adjustment knob and volume adjustment lock.

Eppendorf

An ________________ micropipette has only one button and 3 stops for each function. It has a volume indicator, volume adjustment lock, pipette shaft for the disposable tip, a plunger button and volume adjustor knob & tip ejector button.

Coat, cortex, core

An endospore has a _______, which is outer and protein rich, providing much of its chemical and enzymatic resistance capabilities. Within that, its _______ has specialized peptidoglycan that contributes to its heat resistance capabilities. The _____ contains DNA, ribosomes, and small acid soluble proteins (SASPs) that protect DNA from UV light and DNA damaging chemicals.

Stressor

As endospores are survival structures, they are typically formed when the bacteria are exposed to an environmental ___________. In this case, that will be low nutrient levels. When bacteria are grown on laboratory media, in the beginning, the nutrient levels are plentiful. But after a day or 2 of cell division, some of the key nutrients are depleted. For our exercise, we examine bacteria that have been grown on agar media for several days, and therefore should have produced endospores.

DD-transpeptidase

Bacteria constantly remodel their peptidoglycan cell walls, simultaneously building and breaking down portions of the cell wall as they grow and divide. Beta-lactam antibiotics inhibit the formation of peptidoglycan cross-links in the bacterial cell wall, allowing them to continue to break down their own cell walls but not rebuild them. Penicillin acts when the four-membered Beta-lactam ring of penicillin binds to the bacterial cell-wall building enzyme called __-___________________.

Low

Before plugging the electrical cord into the socket, turn the rheostat knob for the light down (front), by lining up the two dots. Anytime you turn on the light it is better for the life of the bulb to start in this [high or low?] light position.

Endospores

Certain bacteria can survive extreme environmental conditions. _________________ from Bacillus stearothermophilus, the standard indicator bacterium used by the canning industry, can withstand boiling temperatures for 1 week!

800000000

Direct Microscopic Count Method The cell concentration of a water sample if 4 of the medium sized squares have 34, 26, 39, and 39 cells, when the 10^(-2) dilution was analyzed, based on the volume of a medium sized square being 4.0 x 10^(-6) ml, is _________________. Do not use scientific notation for your answer.

Malachite green, steam, safranin, KimWipe

Endospore Staining Procedure: 1. Place a few drops of __________ ________ on the cells and _______ the slide for 5 minutes (do NOT allow the stain to go dry). 2. Hold the slide at a 45° angle and gently rinse excess dye from both sides of the slide with water. 3. Apply 1-2 drops of _____________ and wait 1 minute. 4. Hold the slide at a 45° angle and gently rinse excess dye with water. 5. Blot the slides dry with ________ and observe the bacteria with the microscope.

Firmicutes

Endospores are survival structures formed by bacteria in the phylum ______________ such as Bacillus and Clostridium.

Stained

For [stained or unstained?] microorganisms, the iris diaphragm should be wide open and the light regulated with the power source. For the other, reducing the diaphragm opening while increasing the light intensity with the power source produces greater contrast.

Compound

For a ____________ microscope, two lenses produce the magnification. Total magnification = objective magnification x ocular magnification.

Resolution

For a microscope, ________________ is the minimum distance between distinguishable objects in an image. Its limit depends on the wavelength of light that passes through the lens system and the numerical aperture, which describes the light gathering ability of the system. Some light is lost between the illumination system and the lens system due to refraction.

Chloroform

For our test panel in lab 2 - antibiotic producers, ______________ was used to kill the antibiotic producing bacteria in the middle of the TSA plate.

Direct microscopic count

For the ________ ______________ _________ method, to calculate cell concentration, divide the number of cells by the volume of the square(s). Don't forget to consider the

30, 300

For viable plate count method, statistical confidence is increased with more colonies as long as they're not so large they're running together on the agar media. In practice, the optimal range is between ___ and _____ colonies per plate. If too many to distinguish individual colonies, enter data as "lawn" or "TMTC."

Standard curve

How do we obtain cell concentration values from turbidity measurements? We use a __________ ________, which allows determination of unknown values based on their relationship to known values.

Dye

If Gram-negative bacteria are heated too much in the heat-fixing step, they can retain _____ and appear to be Gram-positive.

Inhibition

If an antibiotic from an antibiotic-producing soil organism inhibits the growth of a specific bacterium like E. coli, an ____________ zone will be produced around this colony. This zone will have no growth because the colony of bacteria in the middle has secreted an antibiotic that kills/inhibits E. coli.

Lens

If oil is not cleaned from a microscope, it will seep inside the ______ and make it impossible to focus.

Glycerol

If you need to store your bacterium for several months, rather than using an slant, you would need to make a frozen ___________ stock.

More

In general, older cultures will have [more or less?] endospores than newer ones.

Hemocytometer

In the Direct Microscopic Count method, cell concentration is determined by counting the number of cells in a specific volume of solution. In this exercise, we'll accomplish this by using a ___________________. (Careful: these and their special cover slips are breakable and very expensive). The chamber is ruled with a grid of known dimensions and a depression well with a known depth. The volume of liquid retained over the grid and between the cover slip is a fixed distance (0.1 mm) so that cell counts can be converted to concentrations (cells/ml).

Endospores, vegetative

In the Endospore stain, ______________ stain green and _______________ cells stain red.

Crystal violet, iodine, alcohol

In the Gram stain, __________ __________ and ___________ are applied to bacteria cells, producing a purple colored complex inside them. In the next step, _____________ is applied to the cells. This removes the purple complex from the Gram-negative cells, but not from the Gram-positive cells. This is caused by the dehydration (and therefore closing) of the pores in the peptidoglycan rich cell wall of Gram-positive bacteria, resulting in the "purple" complex becoming trapped in the cell. Finally, a red colored dye called ______________ is applied to the cells. The result of this entire process is that Gram-positive bacteria are stained purple and Gram-negative bacteria are stained red.

Antibiotic

In the picture provided, the E. coli colony surrounded by an inhibition zone is considered an ______________ producer.

30

It is important to spread the solution within about [how many?] seconds of adding it to the media. Otherwise, the solution can diffuse into the agar, leading to an uneven distribution of the solution across the surface of the agar.

Artificial

Lab 1: Fundamental Techniques Studying microorganisms frequently involves growing them on ____________ laboratory media. When performing such experiments, it is important that organisms from the outside environment do not contaminate these cultures. To accomplish this, all of the solutions and instruments used in these experiments must be sterile (or free of other living organisms).

Sterile

Lab 1: Fundamental Techniques Studying microorganisms frequently involves growing them on artificial laboratory media. When performing such experiments, it is important that organisms from the outside environment do not contaminate these cultures. To accomplish this, all of the solutions and instruments used in these experiments must be ________ (or free of other living organisms).

Bench

Lab 1: Fundamental Techniques Studying microorganisms frequently involves growing them on artificial laboratory media. When performing such experiments, it is important that organisms from the outside environment do not contaminate these cultures. To accomplish this, all of the solutions and instruments used in these experiments must be sterile (or free of other living organisms). In addition, the likelihood of contaminating the cultures can also be decreased by performing the experiments on a sterile laboratory _______.

Pipettes

Lab 1: Fundamental Techniques ______________ are instruments used to measure or transfer specific volumes of solution from one location to another. Knowing how to properly use one is a skill needed for most microbiological experiments. They are delicate and must never be used outside of their volume ranges.

Force

Lab 1: Fundamental Techniques Never f_______ anything on the pipette.

Phylogeny

Molecular _____________ is the use of molecules to classify organisms and examine their evolutionary relationships. This is particularly useful for microorganisms, as the other characteristics (shape, etc) usually do not provide meaningful information. Compare nucleotides in a DNA sequence for "relatedness"!

Cross-kingdom

Neocallimastigomycota is a fungal phylum describing anaerobic gut fungi living in the GI tracts of ruminant animals like cows. They have an extraordinary ability to degrade plant biomass. There is evidence of ____-______________ horizontal gene transfer across bacteria, plants and animals to help them break down complicated proteins and carbohydrates from the grasses that cows eat.

10X

On a microscope, always use the ___ objective to initially focus the specimen: then proceed to higher objectives.

Iris diaphragm

On a microscope, an _____ ______________ controls the amount of light passing through the specimen.

14

Solution Preparation The volume needed from a 20% solution of crystal violet to make a 1400 ml, 0.2% solution is ____ ml.

Solution

Spread plating: Uneven distribution of cells can be caused by improper "spreading" technique or by allowing the ____________ to diffuse into the media before it is spread. In the figure provided, the spread plating procedure was not properly performed, as the colonies are not evenly distributed across the entire surface of the agar media. Make even contact with the agar, but do not press too hard.

Square, volume, dilution factor

Steps for Calculations Using the Direct Microscopic Count Method: Determine the average number of cells per ___________. Then, concentration will be that number divided by the ______________ of the uniformly sized portion. FINALLY, take that concentration and multiply it by the __________ ____________.

T-streak

Streak-plating is also known as a _-_______.

Cell spreader

The "hockey stick" looking pieces of plastic are known as a _____ ____________ and used in conjunction with a turntable for spread plating.

Power

The 10X objective is considered the lowest ________ objective and that's what the nosepiece of your microscope should be set to when putting it away. Also, the power cords should be wrapped vertically around the microscope, the binoculars should be cleaned, the stage needs to be completely lowered, the light needs to be turned off, and the metal holding the slides should be returned to neutral.

Differential

The Gram stain, made by Christian Gram in 1884, is the most commonly used _________________ stain in bacteriology.

Great Plate Count

The ________ ______ __________ anomaly refers to the fact most microbes don't grow on laboratory media.

Revolution

The molecular ___________________ happened because the ability to sequence DNA completely reshaped our ability to view microorganisms. "The cell is basically an historical document and gaining the capacity to read it (by the sequencing of genes) cannot but drastically alter the way we look at all of biology" - Carl Woese, 1987 Woese recognized potential of ribosomal RNA subunits. Multiple subunits with different rates of evolution (SSU, LSU). Use of ribosomal DNA identified domains of life. Gave us a culture-independent means of describing microbes in any sample.

Interpupillary

The oculars (eyepieces) on a binocular microscope should be adjusted to your __________________ distance.

0.5-10, 2-20, 10-100, 50-200, 100-1000

The pipette types and volume limits are as follows: P-10 is for ___-____ ul P-20 is for ___-____ ul P-100 is for ____-_____ ul P-200 is for __-_____ ul P-1000 is for ____-_______ ul

P-10, P-20, P-100, P-200, P-1000

The pipette types and volume limits are as follows: ____ is for 0.5 - 10 ul ____ is for 2-20 ul ______ is for 10-100 ul ______ is for 50-200 ul _______ is for 100-1000 ul

Colonies

The purpose of the streak plating procedure is to create individual ____________ of bacteria, which are derived from single cells. When you pick up bacteria with your inoculating loop, there is a very large number of bacteria on the end of the loop. By rubbing (or "streaking") the loop across the surface of the agar media, the number of bacteria on the loop will be gradually reduced until individual cells are deposited far apart from each other on the media along the "streaks." These individual cells can then replicate, forming thousands of copies of themselves.

Grams

To measure how many _______ something is, use the equation: (Molarity) * (Volume) * (Formula weight) = X

False

True or false: Black Eppendorf Pipettes also have a volume adjustment lock.

True

True or false: During the Gram stain, after applying crystal violet and before applying iodine, you must wait ~30-40 seconds and gently rinse off excess dye with water and holding the slide at a 45° angle.

False

True or false: For a successful transfer of bacteria in a culture, the bacteria MUST be visible on the loop.

True

True or false: If a pipette tip does not eject, you can gently twist it off with your fingers and place it into the tip waste container.

True

True or false: When disinfecting your lab bench, throw the paper towel away in the biohazard trash container.

Dilution

Turbidity method: If an optical density value is above 1.0, repeat the process using the next ____________ until you obtain a value between 0.1 and 1 (the most accurate range for spectrophotometers).

Immersion oil

Using ______________ _____ for a microscope reduces refraction, therefore increasing resolution.

12400000, 15600000

Viable Plate Count Method The concentration of bacteria in CFU/ml of a water sample given that the colony counts on duplicate plates was 124 and 156 based on 0.01 ml portions plated from the 10^(-3) dilution is ________ CFU/ml and __________ CFU/ml respectively. Do not use scientific notation for your answers.

2

We typically use [how many?] significant figures when reporting our results from calculations in labs.

300

When a solution contains fewer than approximately [how many?] bacteria, each cell will have enough room to divide and form an isolated colony when performing spread plating.

Lock

When changing the volume setting on a pipette, make sure the volume adjustment _____ is not engaged.

Cells/ml

When determining cell quantity using a hemocytometer, your calculation is in ____/___, NOT CFU/ml.

Heating

When performing an aseptic transfer of bacteria, _____________ the end of a slant tube will cause the air around the tube to rise, reducing the probability that organisms in the air will fall into the tube and contaminate the culture.

10

When performing aseptic technique, you need to wait [how many?] seconds after heating your loop before collecting your bacteria, or else you can kill all your bacteria. Another trip is to cool the loop by touching it to sterile agar media.

Damaging, thick, overlapping

When streak plating, ______________ the agar, creating a _________ line, or __________________ the zig-zags will interfere with the results.

Contaminants

When transferring liquid solution to the surface of an agar plate before spread plating, holding the Petri dish lid over the plate reduces the possibility of airborne ___________________ landing on the agar media.

43.83

You work in a Lab Prep Facility and you need to make 1.5 liters of a 0.5 M solution of NaCl. You will need [how many?] grams of NaCl. Note: Formula weight of NaCl = 58.44 grams/mol

260

_____ nm is the optimal wavelength DNA absorbs at.

Soil

_______ is one of the most bacteria-rich sources on Earth.

Binding matrix

__________ _________, a solution containing silica with a high salt concentration, is used to purify DNA. DNA (not other cell components) binds to silica when salt concentration is high. It can then be removed from this by adding low salt solution.

Spread plating

__________ ____________ is when a sample of bacteria are smoothed out over the top of a solidified plate of agar. Uses the entire Petri dish instead of quadrants. Like with streaking, this can be used to obtain pure cultures of bacteria and to isolate colonies. In addition to other techniques, it can also be used to determine the concentration of bacteria in a culture.

Aseptic

___________ technique can also be used to transfer specific volumes of solutions from one location to another without introducing contaminants, such as bacteria from the environment. In the example pictured, a large-volume sterile pipette is used to transfer the solution.

Antibiotics

____________ are drugs that kill or inhibit the growth of bacteria. In medicine, they're used to treat bacteria infections. Some infectious bacteria have developed resistance to these, so it's important to discover new ones that can treat the resistant bacterial infections.

MacConkey

______________ agar is considered selective. Its components are meant to mimic gut conditions. The bile salts in this medium inhibit many non-enteric bacteria like Bacillus.

Penicillium rubens

_______________ ______________ is the commercial source of penicillin. This is the most famous antibiotic-producing fungus.

Refraction

_______________ is the change in direction of a propagating wave of light when passing from one medium to another. The more light that is lost due to the this, the lower the resolution.

Metagenomics

____________________ is the study of genomes recovered from the environment using a culture-independent approach.