LAB Microbiology Q&A

Why do we maintain an aseptic environment?

1) Create a safe work environment

2) Minimizes the risk of sample contamination

3) Ensures you maximize the number of microbes you are working with

4) Prevents contamination of yourself and the environment

5) Keeps the environment completely sterile

1) Create a safe work environment

2) Minimizes the risk of sample contamination

4) Prevents contamination of yourself and the environment

Why does working close to the bunsen burner flame or an incinerator help to prevent contamination?

1) Hot air kills all the microbes in the air around your work area

2) Hot air rises and carries the microbes in the air away from your work area

Hot air rises and carries the microbes in the air away from your work area

When is it important to use aseptic technique?

1) When transferring cultures

2) When obtaining a new isolate

3) When we don't want to contaminate ourselves or others with the microbes

4) When working in a shared lab space

1) When transferring cultures

2) When obtaining a new isolate

3) When we don't want to contaminate ourselves or others with the microbes

4) When working in a shared lab space

To what depth is the loop inserted?

1) Until the handle just enters the orange portion

2) As far back into the incinerator as possible

3) Just until the loop enters the orange portion

Until the handle just enters the orange portion

How long does the loop have to remain in the incinerator?

1) A full 10 seconds

2) 2-3 seconds but can leave laying in the incinerator longer while preparing other supplies

3) No set time just wait until the loop portion glows red

4) 2-3 seconds if you have not touched anything else

1) A full 10 seconds

Why does an open tube have a greater chance of contamination?

1) Microbes floating in the air will fall into the tube

2) Microbes from your hands will get transferred to the tube

3) While speaking aerosols from your mouth may enter the open tube

Microbes floating in the air will fall into the tube

When do we "flame" or heat the tube?

1) Right after removing the cap and after insertion of the loop

2) Before the cap is removed and right after the cap is replaced

3) Only right before the loop is inserted

4) Only right after the cap is removed

Right after removing the cap and after insertion of the loop

When transferring from one tube to another which actions should you NOT do?

1) Work close to the incinerator or bunsen burner

2) Put caps on the table

3) Keep tubes closed as much as possible

4) Take caps off while in the rack

5) Hold tube in front of the incinerator before and after transfer

6) Hold caps with your little finger and keep the cap pointing down

2) Put caps on the table

4) Take caps off while in the rack

Which statements are true about how staining techniques help you in the visualization of microorganisms. Write T for True and F for false

Increases the magnification of the organism

Increase the contrast

Increases the resolving power

Increases the magnification of the organism - F

Increase the contrast - T

Increases the resolving power - F

When preparing your smear you forgot to pass the slide over the bunsen burner or flame. Why is this a problem when you move on to staining?

1) Cells will be distorted and morphology can not be correctly visualized

2) Cells will not stick to the slide and will wash off with the staining steps

3) Stain will not be able to enter the cell during the staining process since protein were not denatured

Cells will not stick to the slide and will wash off with the staining steps

When using a basic stain the background will be ___________ and the cells will be ___________.

clear, colored

clear, clear

colored, colored

colored, clear

clear, colored

In a simple stain how many dyes are used?

One

Two

Three

Four

One

In the 1st type of simple stain just described why does the stain adheres to the cells?

Cells are negatively charged; stain is basic

Cells are negatively charged; stain is acidic

Cells are positively charged; stain is acidic

Cells are positively charged; stain is basic

Cells are negatively charged; stain is basic

When using a acidic stain the background will be ___________ and the cells will be ___________.

clear, colored

colored, clear

clear, clear

colored, colored

colored, clear

When performing an acidic stain as just described if you rinse off the stain what will you see when the smear is viewed under the microscope?

Clear cells and a pale grey background

No cells or stain

Clear cells and dark grey background

No cells or stain

Which statements below are true about the second simple stain discussed. Write T for True and F for false.

Increases Contrast

Requires excess stain to be removed

Multiple stains need to be applied

Requires a small drop of stain

Increases Contrast T

Requires excess stain to be removed F

Multiple stains need to be applied F

Requires a small drop of stain T

Microscopes are used to see objects that are __________.

Too large to be seen with the unaided eye

Too dense to be seen with the unaided eye

Too dilute to see with the unaided eye

Too small to be seen with the unaided eye

Too small to be seen with the unaided eye

The course focus is use to make___________ adjustments, while the fine focus is used to make __________ adjustments to the height of the slide.

small, large

higher, lower

Large, small

lower, higher

Large, small

If you use a 40x objective what is the total magnification?

40x

400x

4000x

400x

Define resolving power

1) The minimum distance between two objects that they can still be observed as one object

2) The minimum distance between two objects that they can still be observed as two separate objects

3) The maximum distance between two objects that they can still be observed as one object

4) The maximum distance between two objects that they can still be observed as two separate objects

The minimum distance between two objects that they can still be observed as two separate objects

You are viewing a specimen in focus at 10x and when you change to the 40x objective the specimen is now slightly blurry. What should you do to ensure you could see your specimen clearly with the new objective?

1) Adjust the stage adjustment knob until the specimen is in the middle of the field of view.

2) Adjust the fine focus until the specimen is sharp and clear.

3) Adjust the course focus until the specimen is sharp and clear.

4) Adjust the stage adjustment knob until the specimen is on the edge of the field of view.

Adjust the fine focus until the specimen is sharp and clear.

You are viewing a specimen in focus at 4x and when you change to the 10x objective you can no longer see it in your field of view. What should you have done before changing objective lens to ensure you could see your specimen?

1) Adjust the fine focus until the specimen is sharp and clear.

2) Adjust the course focus until the specimen is sharp and clear.

3) Adjust the stage adjustment knob until the specimen is on the edge of the field of view.

4) Adjust the stage adjustment knob until the specimen is in the middle of the field of view.

Adjust the stage adjustment knob until the specimen is in the middle of the field of view.

The working distance for the 10x objective will be __________ than the working distance with the 4x objective.

Larger

Smaller

the same

smaller

When do you use immersion oil and what is its purpose?

1) With the 100x objective; focuses light onto the slide

2) With the 40x and 100x objective; minimizes refraction of light away from lenses

3) On the 100x objective; minimizes refraction of light away from the lens

4) With the 40x and 100x objective; focuses light onto the slide

On the 100x objective; minimizes refraction of light away from the lens

On a light microscope, what do you adjust to properly focus the light onto the specimen?

ocular lens

fine focus

condensor lens

objective lens

condensor lens

The magnification of the ocular lens is?

40X

20X

10X

100X

10X

Why is it important to prepare a thin smear instead of a thick one?

1) morphology of the cells can be observed

2) Stain evenly penetrates the smear

3) Even distribution of the cells

4) morphology of the colonies can be observed

5) Even distribution of colonies

morphology of the cells can be observed

Stain evenly penetrates the smear

Even distribution of the cells

A negative stain has chromogen that is ______ charged. This means the stain will be ______ from the cell wall.

1:negatively

2:repelled from

While preparing your smear you did not let the slide dry before heat fixing. As a result when viewed under the microscope after completing staining what would you expect to see.

1) Cells did not absorb the stain due to the excess water

2) Cells would be distorted due to the excess liquid on the slide

3) Cells would look normal as this is the correct procedure

4) Cells will have all washed off since they did not adhere to the slide

2) Cells would be distorted due to the excess liquid on the slide

Negative staining can be useful for accurately determining the dimension of a cell.

True

False

True

What is colored by the negative stain?

Background surrounding the cell

The cell

Background surrounding the cell

You should wash your hands before leaving the lab.

True

False

True

Any type of shoes can be worn in the lab.

True

False

False

Biological waste must be discarded in a bio-hazard bin.

True

False

True

To get an organism to grow successfully in the lab we have to take into account the original environment. If I want to grow an organism originally from the ocean what condition might be most important.

Salinity

temperature

oxygen

pH

salinity

If you want to maximize the different types of unknown organisms that would grow from your sample what medium would you choose?

Rich medium

minimal medium

rich medium

If you want to see if an organism is able to metabolize a specific carbon source what type of medium would you use?

Complex medium

Defined medium

Defined medium

Which of these is NOT a reason why the plates and tubes are labeled as instructed.

Ensure sample label always remains with the sample

Makes it easier to view the sample after incubation

Requires your lab notebook to look up the sample ID number

Requires your lab notebook to look up the sample ID number

When labeling the plate shown on the right what was done incorrectly by the student?

Label was not on the lid

Label was across the middle of the plate

Label was on the bottom

Label was across the middle of the plate

If multiple colony morphologies are observed from the sample it suggests?

The sample has only one type of organism present

The sample has more than one type of organism present

The sample has more than one type of organism present

You can observe individual colonies in a broth.

True

False

False

Which statements below apply to differential stains and not to simple stains?

1. Only uses one stain

2. Uses multiple stains

3. Stains all organisms the same

4. Allows you to determine specific characteristics

5. Always requires the slide to be heat fixed

1, 3, 5

1, 3, 4

2, 3, 4

2, 4, 5

2. Uses multiple stains

4. Allows you to determine specific characteristics

5. Always requires the slide to be heat fixed

When a microorganism is viewed using TEM or SEM you can closely observe the surface. Why does a Gram negative organism appear rough on the surface?

The presence of lipopolysaccharides

The thinness of the peptidoglycan layer

The thickness of the peptidoglycan layer

presence of cytoplasmic membrane

The presence of lipopolysaccharides

What is the purpose of the alcohol?

counterstain

mordant

primary stain

decolorize

decolorize

A good control for a Gram stain would consist of:

A mixture of known gram positive and gram negative organisms that have different cell shapes

A known gram negative organism

A known gram positive organism

A mixture of known gram positive and gram negative organisms that have the same cell shape

A mixture of known gram positive and gram negative organisms that have different cell shapes

In the lab how do we get the culture to start forming spores?

Exposure to UV light

Exposure to chemicals

Exposure to low-nutrient conditions

Exposure to low-nutrient conditions

If the staining procedure is done correctly at the end the procedure the vegetative cells should be _________, the endospores will be _________, and the background will be __________.

green, red, clear

red, green, clear

clear, green, red

red, clear, green

green, clear, red

red, green, clear

What would see if after you added malachite green you failed to use moist heat during the staining procedure but completed everything else correctly?

red spores, clear or no vegetative cells

clear or no spores, red vegetative cells

nothing all clear

green spores, red vegetative cells

green spores, clear or no vegetative cells

clear or no spores, red vegetative cells

What would you see if you forgot to add safranin in the staining procedure but performed all other steps correctly?

red spores, clear or no vegetative cells

nothing all clear

red spores, green vegetative cells

clear or no spores, green vegetative cells

green spores, clear or no vegetative cells

green spores, clear or no vegetative cells

What type of staining procedure is the Endospore stain? Why is the technique useful?

Basic stain, increases contrast between cells and background

Simple stain, allows you to see cells structures

Differential stain, able to determine presence of specific cell structures

Acidic stain, able to see cell size and shape clearly

Differential stain, able to determine presence of specific cell structures

What would you see if you forgot to place the slide into the incubator when performing the endospore stain but completed everything else correctly when viewed under the microscope?

Red vegetative cells and red spores

No cells, everything is clear

Green vegetative cells and red spores

Red vegetative cells and clear spores inside sporulating cells

Green vegetative cells and clear spores

Red vegetative cells and clear spores inside sporulating cells

In the endospore stain what is the purpose of the safranin? What is the purpose of the malachite green?

Primary stain that stains the endospores red; counterstains the vegetative cells green

Basic stain that stains everything purple; primary stain that stains the endospores green

Counterstain for the vegetative cells; stains the endospores

Mordant that traps the stain in the endospores, counterstain for vegetative cells

Counterstain for the vegetative cells; stains the endospores

What type of staining procedure is the Gram stain?

Basic stain, increases contrast

Differential stain, able to determine presence of cell structures

Simple stain, allows you to see cells structures

Acidic stain, able to see cell shape clearly

Differential stain, able to determine presence of cell structures

What aspect of the microorganism determines the color of the cells at the end of the Gram Stain procedure.

Number of cell membranes

Cell shape

Thickness of the peptidoglycan layer

Thickness of the LPS

Presence of endospores

Thickness of the peptidoglycan layer

Match the Reagents with their purpose in the Gram Staining procedure.

Alcohol

Safranin

Crystal Violet

Iodine

Counterstain

Primary Stain

Decolorizer

Mordant

Alcohol- Decolorizer

Safranin- Counterstain

Crystal Violet- Primary Stain

Iodine - Mordant

Determine which statements are true or false when describing a pure culture. Write only T or F for credit not the full word. (Worth 4 Interactions)

Consists of only one strain of bacteria

A collection of bacteria of the same genus and multiple species

An isolated colony on a plate

A sample that originated from one person

Consists of only one strain of bacteria - True

A collection of bacteria of the same genus and multiple species - False

An isolated colony on a plate - True

A sample that originated from one person - False

How do we dilute the culture on the plate?

Drag in a single direction from high to low concentration and sterilize the loop between quadrants

Sterilize the loop after the original sample is picked up and then only drag from high to low concentration

Drag in a single direction from high to low concentration

Sterilize the loop between quadrants

Drag in a single direction from high to low concentration and sterilize the loop between quadrants

Which of the following is NOT true?

Streak plates can be used to:

1) Estimate if the sample is a pure culture

2) Isolate organisms from a mixture

3) Observe individual cell morphology

4) Observe individual colony morphology

3) Observe individual cell morphology

Why is it recommended that you label on the edge of the plate in the 1st quadrant?

Label obscures colony morphology

Label does not obscure colony morphology

There will be lots of isolated colonies in that quadrant

To be consistent

Label does not obscure colony morphology

Why does the loop go back and forth in the 1st quadrant?

Dilute the culture in the quadrant

Evenly spread the culture

To cover the label completely

Evenly spread the culture

When performing the quadrant steak after the first quadrant you should _________.

pick up more cells for each remaining quadrant

go back and forth in each remaining quadrant to evenly distribute the sample

overlap with the two previous quadrants you streaked

flame/incinerate the loop between each remaining quadrant

flame/incinerate the loop between each remaining quadrant

For each of these methods the end goal is to _________ the original sample until we can see _________.

concentrate; isolated colonies

dilute; isolated colonies

concentrate; lawn of growth

dilute; lawn of growth

dilute; isolated colonies

If you pick an isolated colony from this plate and restreak it what should you observe on the new plate?

only colonies that match the individual colony used to inoculate the plate

may only have two of the morphologies observed on the original plate

All three different morphologies again isolated on the plate

only colonies that match the individual colony used to inoculate the plate

Loops should be held in the Bacti-Cinerator for 30 seconds to ensure sterilization.

True

False

False

Which of the following may indicate that you have correctly transferred an organism from a pure broth culture to a slant?

The slant has colonies and growth of only one color and morphology

After gram staining a smear was prepared from the slant, all of the cells have a similar color and morphology under the microscope

The slant has colonies of three different colors

The slant appears turbid throughout

The slant has colonies and growth of only one color and morphology

After gram staining a smear was prepared from the slant, all of the cells have a similar color and morphology under the microscope

How is air contamination prevented when an inoculating loop is used to introduce or remove organisms from an agar plate?

the plate lid is flamed before insertion of the loop

the loop is flamed just before placing the organisms on the plate surface

the plate lid is flamed before incubation

the plate lid is kept closed or lifted only slightly when inoculating

the plate lid is kept closed or lifted only slightly when inoculating

When a liquid culture contains microorganisms the microbes are always visible to the unaided eye.

True

False

False

Why is the mouth of a culture tube passed through the Bunsen burned or in front of an incinerator after removing and before replacing the cap?

to prevent the entry of ambient air, which might introduce contaminants

to heat up the glass to get the culture to temperature for growth

to make sure the cap will be easily replaced onto the tube

to sterilize the tube

to prevent the entry of ambient air, which might introduce contaminants

Indicate the statements that correctly describe microbial colonies. (Select all that apply)

Microbial colonies are readily observed in broth medium

A microbial colony is a visible accumulation of microbial cells on the surface of an agar plate or slant

Microbial colonies can be observed intact with a microscope at 1000x magnification

A microbial colony is often visualized as an opaque spot on the surface of an agar slant or plate

A microbial colony is a visible accumulation of microbial cells on the surface of an agar plate or slant

A microbial colony is often visualized as an opaque spot on the surface of an agar slant or plate

Select the sites where microorganisms are likely to be found. (Select all that apply)

Laboratory bench surface

hands

surface of sterile agar inside Petri Dish plate

computer keyboard

air

inside tube of sterile broth

Laboratory bench surface

hands

computer keyboard

air

Microorganisms are found in nearly every environment and are said to be

omnipresent

contaminated

ubiquitous

omniscient

ubiquitous

Which term refers to the introduction of unwanted microorganisms?

contamination

inoculation

ubiquitous

sterilization

contamination

You have cultured several different objects and surfaces. Which result on an agar plate indicates more diversity obtained from the sample site?

the plate with colonies of many different morphologies

the plate with the largest colonies

the plate with the greatest number of colonies

the plate with colonies of many different morphologies

In the semi-log graph of CFU/ml vs. time which axis is log scale?

x--axis, time

X-axis, CFU/ml

Y-axis, time

Y-axis, CFU/ml

Y-axis, CFU/ml

During which phase of growth do we calculate the generation time?

Lag phase

Log phase

Stationary phase

Death or decline phase

Log phase

Generation time is the same for an organism no matter the growth conditions.

True

False

false

When you do direct counts with a Petroff- Hausser Counter you count both live and dead cells.

True

False

true

With viable or spread plate counting there are some disadvantages. Of the options below which one is NOT a disadvantage of the technique.

Requires cells to be stained to see viability

Requires incubation so is slower

Requires lots of supplies

Requires knowing the basic growth conditions of the organism

Requires cells to be stained to see viability

Why does less light reach the sensor if cells are present in the sample?

cells absorb the light

medium absorbs the light

cells scatter the light

medium scatters the light

cells scatter the light

If you only want to measure the number of viable cells which technique or techniques can be used?

Turbidity

Petroff Hausser Chamber

Spread Plates

Turbidity and Spread Plates

Turbidity and Petroff Hausser Chamber

Petroff Hausser Chamber and Spread Plates

Spread Plates

What pipet should I use to most accurately measure 150uL of sample?

P20

P100

P200

P1000

P200

Pipets have two stop points when depressing the plugger. When used correctly the first stop point is used to ___________ and the second stop point is used to __________.

fully expel the sample volume after the transfer, accurately measure the sample volume before aspiration

accurately measure the sample, volume before aspiration; fully expel the sample volume after transfer

partially expel the sample volume after the transfer, accurately measure the sample volume before aspiration

accurately measure the sample volume before aspiration, partially expel the sample volume after transfer

accurately measure the sample, volume before aspiration; fully expel the sample volume after transfer

What is the liquid in the blank? What is the purpose of the blank?

water, remove any effects of the liquid so only the cell absorbance is measured

Saline, remove any effects of the liquid so only the cell absorbance is measured

growth medium, remove any effect of the liquid so only the cell absorbance is measured

water, remove any absorbance due to the liquid

saline, remove any absorbance due to the liquid

growth medium, remove any absorbance due to the liquid

growth medium, remove any absorbance due to the liquid

In the streak plate method, what is the best way to ensure that the loop is cool after it has been sterilized?

touch the loop with your finger to determine the temperature

touch the loop to an uninoculated portion of the plate

touch the loop to an inoculated portion of the plate

touch the loop to an uninoculated portion of the plate

Please match the type of culture to the statement that most accurately describes it.

The type of culture most frequently used in the laboratory which contains only a single species of microorganism

A second level culture where an isolated colony from one culture is taken and then transferred into a new medium

A culture which contains two or more species

A culture which contains one or more unwanted and often unidentified microbes.

Contaminated

Subculture

Pure culture

Mixed

The type of culture most frequently used in the laboratory which contains only a single species of microorganism

Pure Culture

A second level culture where an isolated colony from one culture is taken and then transferred into a new medium

Subculture

A culture which contains two or more species

Mixed Culture

A culture which contains one or more unwanted and often unidentified microbes.

Contaminated culture

When completing a quadrant streak, when do you sterilize the loop? (Select all that apply.)

before you pick up a loop of the organism from the original culture

before you streak quadrant one

before you streak quadrants two, three, and sometimes four

before you put the loop away

before you pick up a loop of the organism from the original culture

before you streak quadrants two, three, and sometimes four

before you put the loop away

A ___ is a mound of cells on a solid medium that represents the progeny from one original bacterial cell.

colony

Please select all of the statements which are true regarding isolated colonies.

An isolated colony is a visible mass that results from the multiplication of a single cell or occasionally a few cells located very closely

The cells within a single colony are within the same species

Isolated colonies form in liquid nutrient media

Isolated colonies can only be obtained via the streak plate method

Isolated colonies form on solid nutrient media

An isolated colony is a visible mass that results from the multiplication of a single cell or occasionally a few cells located very closely

The cells within a single colony are within the same species

Isolated colonies form on solid nutrient media

A quadrant streak can be used for which purpose or purposes below?

(Select all that apply.)

to obtain isolated colonies

to observe the morphology of colonies

to see the shape of individual cells

to estimate the purity of a culture

to determine CFU/mL

to obtain isolated colonies

to observe the morphology of colonies

to estimate the purity of a culture

Match the terms to the statements that most accurately describe them.

Process of purposely adding a microbe to a growth material.

The material which provides the nutrients for growth.

To cultivate (verb) for the observable growth in or on lab media (noun).

Medium

Inoculation

Culture

Process of purposely adding a microbe to a growth material. Inoculation

The material which provides the nutrients for growth. Medium

To cultivate (verb) for the observable growth in or on lab media (noun). Culture

The optimal temperature for growth is closer to the ________ temperature because enzymatic reactions _________ to reach their optimal rate.

minimum; speed up

minimum; slow down

maximum; speed up

maximum; slow down

maximum; speed up

In a closed system the pH is maintained by incorporating a(n) _______ into the medium.

carbohydrate

buffer

base

acid

buffer

Will bacteria isolated from a freshwater pond grow equally well in the ocean?

Yes

No

No

What parameters are required based on the organisms' classification as an extreme alkaliphile and halophile.

Acidic pH, Low salinity

Neutral pH, Low salinity

Basic pH, Low salinity

Acidic pH, High salinity

Neutral pH, High Salinity

Basic pH, High salinity

Basic pH, High salinity

Organisms that require lower than atmopsheric levels of oxygen are classified as?

Microaerophile

Obligate Anaerobe

Facultative Anaerobe

Aerotolerant

Obligate aerobe

Microaerophile

Organisms that cannot live with oxygen are classified as?

Facultative Anaerobe

Microaerophile

Obligate aerobe

Obligate Anaerobe

Aerotolerant

Obligate Anaerobe

Which conditions indicate that the environment and media are free of oxygen?

Resazurin is pink, Brewers Agar is pink, Methylene blue strip is white

Resazurin is clear, Brewers Agar is pink, Methylene blue strip is blue

Resazurin is clear, Brewers Agar is clear, Methylene blue strip is blue

Resazurin is pink, Brewers Agar is clear, Methylene blue strip is blue

Resazurin is clear, Brewers Agar is clear, Methylene blue strip is white

Resazurin is pink, Brewers Agar is pink, Methylene blue strip is blue

Resazurin is clear, Brewers Agar is clear, Methylene blue strip is white

Match the special techniques to experimental set up.

A. Anaerobic Jar

B. Anaerobic Culture Bottle

C. Anaerobic Chamber

An ___ works well when you want to take multiple samples over a period of time.

An ____ is best for working with very oxygen sensitive samples.

An ______ works well for samples that are not as oxygen sensitive and only require a single time point.

An Anaerobic Culture Bottle works well when you want to take multiple samples over a period of time.

An Anaerobic Chamber is best for working with very oxygen sensitive samples.

An Anaerobic Jar works well for samples that are not as oxygen sensitive and only require a single time point.

What is released by the gaspak to create an anoxic environment?

Nitrogen

Carbon Dioxide

Hydrogen Peroxide

Oxygen

Hydrogen

Water

Hydrogen

Match the type of medium(numbered) to the correct to the correct goal of the experiment. .

1. Complex

2. Selective

3. Differential

4. Selective and Differential

Determine if an isolated organism has a specific characteristic but growth is not dependent on that trait

From a mixed sample only grow organisms with a specific characteristic

Maximize the number of organisms grown from an environmental sample

From an environmental sample grow only specific types of organisms and determine if they have an additional characteristic

Determine if an isolated organism has a specific characteristic but growth is not dependent on that trait - Differential

From a mixed sample only grow organisms with a specific characteristic - Selective

Maximize the number of organisms grown from an environmental sample- Complex

From an environmental sample grow only specific types of organisms and determine if they have an additional characteristic - Selective and Differential