2051 MBIO Midterm Practice 1

Beef extract in Nutrient Agar makes NA a ___________________________ medium.

complex

What is the difference between TSA and TSB?

TSA stands for tryptic soy agar, TSB stands for tryptic soy broth
TSA=solid medium made from agar
TSB=liquid medium (broth)

Identify each of the following as selective or differential:
a. an agar plate that only allows the growth of Gram bacteria.

b. an agar deep that allows you to see if bacteria produces an enzyme.

c. an agar deep that only allows the growth of bacteria capable of using citrate as a carbon source.

d. a flask of broth that contains no nitrogen, allowing only the growth of bacteria that can use nitrogen from the air.

e. an agar plate that turns color if the bacteria can produce acidic fermentation products.

f. a tube of broth with a Durham tube to collect gas if it's produced by bacteria growing in the tube.

a. selective
b. differential
c. selective
d. selective
e. differential
f. differential

What sealed device is used to sterilize the growth media used in this lab?

autoclave

An autoclave heats to __________ °C at _____ psi for at least 15 minutes.

121°C at 15 psi

When would the following sterilization techniques be used?
a. dry-heat sterilization

b. filter sterilization

c. cold sterilization

d. UV radiation sterilization

e. ionizing radiation sterilization

a) glassware
b) heat-sensitive gases or liquids
c) heat-sensitive objects (ex: plastic Petri dishes)
d) exposed surface
e) food industry/medical field to decontaminate food/medical tools

Name five categories of colony morphology properties which can be used to describe the growth of a microorganism.

whole colony shape, margin shape, elevation, optical properties, surface characteristics

What can influence the colony morphology properties of a microorganism?

The type and amount of nutrients present in the medium will influence the size, shape, and even the color of a colony.

Describe the growth medium used for this experiment 1 (cultivating microorganism from the environment). Would a broth culture work just as well?

TSA (trypticase soy agar) is a solid medium in a Petri dish. Using a broth culture would make it difficult to determine colony morphology because the microorganisms are suspended in the liquid. We would be unable to see colony formation.

Some areas sampled had more microorganisms capable of growing on TSA than other areas. Give a couple of explanations as to why this was observed.

The type and amount of nutrients present in the medium will influence the size, shape, and even the color of a colony. The nutrients available in the medium may not have been suitable for growth.

Most of the areas sampled that had a high average number of colony morphologies were high traffic areas that would regularly come into contact with these microbes. The area with the lowest averages are areas that may be cleaned more regularly or do not come into contact with as many microbes as high traffic areas.

Do microorganisms live in all the areas sampled or might some just be transiently there? List the areas sampled where microorganisms are most likely to be residents. Consider the areas where transient microbes were found. How did the microbes get to these areas?

Areas with resident microbes could be mouth/nose, fingers, and hair. These areas can also contain transient microbes transferred onto these areas through daily activities. Areas with transient microbes can also be floor, sink, clothes, air, and dirt.

Where did the microbes come from that landed on the agar surface of the plate left open for 30 minutes?

Airborne particles containing microbes could have landed onto the afar plate and stuck to agar. For example, sneezing, coughing, breathing, etc. could be common methods of transport for these airborne microbes.

Which of the sites tested are sources of contamination when working with cultures at your lab bench? Suggest a way to prevent contamination from each of these sites.

Wearing gloves and washing your hands can prevent contamination from your fingers, which is the most likely method of contamination. Keeping the Petri dish closed can minimize exposure from airborne particles. If you're sick, covering up your mouth and nose will prevent contamination from coughing and sneezing.

What was the purpose of experiment 1?

It helped demonstrate the presence of possible contaminants in the lab and the necessity of using proper aseptic technique to prevent pure bacterial culture from becoming contaminated with undesired organisms from the environment.

What are the different types of whole colony shape?

round, irregular, rhizoid

What are the different types of margin shape?

smooth (entire), lobate, filamentous

What are the different types of elevation?

convex, umbonate (button-like), flat

What are the different types of optical properties?

opaque, translucent

What are the different types of surface characteristics?

shiny vs dull

True or False: The majority of microorganisms are pathogenic.

false

a bacterium, virus, or other microorganism that can cause disease

pathogen

an infection acquired in a healthcare setting

nosocomial infection

Why is handwashing important?

Handwashing physically removes microorganisms and is very important in preventing the spread of disease by reducing the possibility of transmitting an infectious dose.

the number of microogranisms or viruses sufficient to establish an infection

infectious dose

microorganisms that live in or on the body, and do not produce disease under normal conditions

normal flora

How can normal flora become harmful?

An organism harmless on skin may cause disease if introduced into the bloodstream or tissues

nonresident microbes you encounter, most are unable to multiply, reside on or in you and get removed with washing or other daily behavior

transient flora

Why is it difficult for transient flora to multiply and survive on your skin?

The pH of the skin is usually acidic, ranging from 4 to 6.low water content and frequently dry

How is normal flora able to survive on our dry skin?

The normal skin flora is associated with the apocrine and sebaceous glands in the skin, which provide not only moisture but also nutrients for microbial growth

Since most of the normal flora is not harmful, why must it be removed in a surgical scrub?

They may become opportunistic pathogens if the patient is exposed to your normal flora during surgery. If it enters the bloodstreams, it may causes disease.

Why would liquid soap be used in a surgical scrub instead of bar soap?

Even after use, bar soap may retain the bacteria and can be passed onto the next user. Liquid soap does not collect contaminants as easily as bar soap.

Why could there be more bacteria present after handwashing?

Handwashing removes the dead skin from the top layer and expose the normal flora.

What is meant by the term normal flora?

Normal flora are microbes that naturally live on the body but do not produce disease unless exposed

a population of cells which arise from a single cell growing on a solid medium

colony

True or False: Cells in a single colony are genetically different

False, they should all be genetically identical

Why is a streak used to obtain a pure culture rather than a broth?

Unlike examining growth on a streak plate, it is impossible to assess if that broth culture is a pure or mixed culture just by viewing the tube. An isolated colony on a streak plate is an example of a pure culture

If you only see one type of colony morphology on the plate, is your culture a pure culture?

Not necessarily, many organisms have similar colony morphologies, so colony morphologies alone is not sufficient to determine whether you have a pure or mixed culture

What is another way of determining the purity of a broth culture?

staining the sample can reveal the different cellular morphologies (shape and/or arrangement) of the microbes present while a differential stain like a Gram stain can reveal structural differences helping you to determine the purity of a culture

What is subculturing?

transferring culture to a new growth medium

What is an addition benefit of subculturing?

This second plating will also allow any hidden cells of a different species to be diluted when streaking the plate for isolation

Why must the inoculating loop be flamed between each quadrant? Flaming is what type of sterilization method?

So you do not introduce contaminants from an unsterile loop
dry-heat sterilization

What is the difference between a mixed culture and a pure culture?

Pure culture--a culture containing a single organism
Mixed culture--a culture that contains more than one type of organism

What is meant by the term colony? Is a colony a pure culture?

A colony is a population of cells that arise from a single cell, making them all genetically identical

After examining your streak plate, you notice that three colony types are present while the TA says only two organisms were in the mixed broth. What might have caused this?

Contamination from the air or your hands may have landed on the streak plate.

Why is it necessary to isolate individual colonies from mixed broth?

You can't tell the difference between cells just by looking at the broth mixture. By creating a streak plate, you create a pure culture of a single organism, all genetically identical.

Explain why a mixed broth culture may appear pure on the simple stain, but mixed when different colonies were observed on the streak plate.

Cellular morphology may appear the same and indistinguishable. However, colony morphology may make them distinguishable. For example, one may be shiny and the other is dull.

In nature, how do microbial communities exist?

biofilms

What type of interfaces do microbial communities have?

water-air or water-solid

Why do microorganisms produce polysaccharides?

Polysaccharides help trap nutrients and aid in the attachment of the microorganisms to the surface. It also provides protection for the inhabitants.

How can biofilms be a problem?

They are difficult to treat because antibiotics and immune cells cannot penetrate the layers of a biofilm. Companies also spend millions of dollars trying to control biofilm growth in pipes, on boats, and on oil rigs.

How do biofilms develop?

Attachment: adhesion of a few cells to a surface
Colonization: growth, polysaccharide production, cell communication
Development: continued growth

How do cells in biofilm communities communicate with each other?

quorum sensing

What compound is produced in quorum sensing?

homoserine lactone

What is the purpose of homoserine lactone?

When it has accumulated to a certain level, it causes effects in neighboring cells--to recruit other microbial cells to join the biofilm or to induce all members of a biofilm to produce an enzyme or virulence factor at the same time

________ microscopes allow light rays to pass directly to the eye without being deflected by an intervening plate in the condenser.

brightfield

Can you view live, motile organisms with brightfield?

Not really, because there is little contrast between unstained cells and their background so you must stain and heat fix them in order to see them, which means you kill those cells.

What is a wet mount?

live cells suspended in a droplet of solution covered with a coverslip for viewing under the microscope

light microscopes that are fitted with a condenser that allows light to pass through the specimen from the side only

darkfield microscopy

What do images produced by darkfield microscopy look like?

Images appear light on a dark background without staining

condenser and objectives used that take advantage of and amplify these slight differences in refractive index so that objects appear with significant contrast to their surroundings

phase-contrast microscopy

What is the advantage of using phase-contrast microscopy over brightfield?

Brightfield microscopy does not allow us to view live motile microbes under the microscope because they are usually stained and heat fixed, which kills live cells. However, we still cannot view them unstained because there is very little contrast between the cell and its surroundings.

What do phase contrast and darkfield microscopes have that brightfield scopes do not?

They have plates or filters in the condensers and objectives that increases the contrast between cells and their surroundings without staining to allow for viewing of live cells

What is the function of flagella?

locomotion

What is the eukaryotic flagellum made of?

microtubules (made of the protein tubulin)

What causes the eukaryotic flagellum to move back and forth?

dynein motor proteins

What anchors the flagella in the cell envelope?

a complex of proteins called the basal body/motor

What connects the basal body to extracellular component of the flagellum?

hook

What connects the basal body to extracellular component of the flagellum?

hook

What is the current model of movement of bacterial flagella?

by a "rotary engine" in which a proton gradient, or sometimes a Na+ gradient, provides the energy to rotate the flagellum

What generates the force to spin the motor proteins?

When protons from the periplasm flow back into the cell through Mot proteins, which are located adjacent to the motor

What does it mean when you have a polar flagella?

One or more flagella are attached at the "ends" of the cells

What are the different forms of polar flagella attachment?

monotrichous
amphitrichous
lophotrichous

a single flagellum attached at one end

monotrichous

a "tuft" of flagella are attached at one or both ends

lophotrichous

one flagella at each end of the cell

amphitrichous

multiple flagella around the cell

peritrichous

Can you see flagella under a light microscope?

No, you must use special flagella stains to coat the flagella sufficiently to be seen with a light microscope

What is a motility test medium?

a low concentration agar medium used to determine whether microorganisms are capable of motility

What is the agar concentration of motility deeps?

0.4%

How should the motility test medium be stored?

It should be kept in the fridge until immediately before it is inoculated due to the low agar concentration.

What is the purpose of tetrazolium salt (TTC) being in the medium?

provided in growth medium such as motility medium as an alternative electron acceptor

What does TTC look like in its oxidized form?

colorless and soluble

What does TTC look like in its reduced form?

red and insoluble

What does the motility test look like if the bacteria is motile?

the red color will radiate red in all directions away from the original stab line

What does the motility test look like if the bacteria is not motile?

You will see a red line only where you stabbed the deep

How are we able to see larger live motile microbes?

hanging drop method

What is a hanging drop method?

the suspension of cell culture from a coverslip over a depression slide to allow freedom of movement for motile cells to be viewed under the microscope

the random movement of particles suspended in a fluid

Brownian method

What is the opposite of Brownian motion and why do microbes do this?

directed motion, they dart from place to place trying to find nutrients and light (if photosynthetic)

Explain the "rotary engine" model for how a bacterial flagellum rotates.

Protons or Na+ gradient provides energy for flagellar rotation. Protons flow back into the cell via Mot proteins, which are adjacent to the motor and generate the force needed to spin the motor proteins

How do the two flagella of the eukaryotic green algae Chlamydomonas reinhardtii move? Could you see the flagella when viewing the live Chlamydomonas cells?

The flagella whip back and forth to move cell.
yes.

Why does motility test medium have a low agar concentration?

to allow motile bacteria to move

Explain the color change that occur in the motility test medium after inoculation (TTC).

Tetrazolium salt (TTC) is an electron acceptor in the medium. When oxidized, TTC is colorless and soluble but in reduced form, it is red and insoluble. The red color from TTC follows the organism's path if motile.

Describe and explain the appearance of a positive and negative motility medium test result.

If red radiates multidirectionally, it indicates a positive motility test. If red only appears in one direction, it indicates a negative motility test.

What are the primary stain, mordant, decolorizer, and counterstain used when doing the Gram stain?

primary stain: crystal violet
mordant: Gram's iodine
decolorizer: ethanol
counterstain: safranin
*REMEMBER P.I.E.S.*

What is the function of the mordant? If the mordant was not added when performing the stain, what would then be the Gram reaction of all the cells? Why?

binding to the crystal violet forming a complex not easily removed from Gram-positive cells
Red b/c the crystal violet molecules wouldn't form a complex (that remains in thick peptidoglycan layer of Gram-positive bacteria) and would be washed away.

What would be the appearance of all cells if the decolorizer were not added? What would be the result if the smear were over-decolorized?

Purple.
Red.

Why is a counterstain added in a Gram stain? What cells does the counterstain stain? Are these cells Gram-positive or Gram-negative?

-added to the smear to stain the Gram-negative cells red
-gram negative

A Gram stain was performed on Wednesday using Staphylococcus epidermidis from a streak plate done on Monday. The cells appeared Gram-negative. It is known that Staphylococcus epidermidis is a Gram-positive bacterium and no steps were omitted in the stain. What is the most obvious explanation for this incorrect Gram reaction?

If bacteria stained was more than 24 hours old, the gram positive cells may have lost the ability to retain primary stain

What structural component of bacteria determines their Gram reaction? How is this structure different in Gram-positive and Gram-negative bacteria?

-Gram- positive cell walls have many layers of peptidoglycan which make the structure rigid. In addition to the thick peptidoglycan layer, most Gram-positive bacteria contain teichoic acids.
-In contrast, Gram-negative bacterial cell walls consist of few layers (thin) of peptidoglycan surrounded by an outer membrane.