BIOL 2051 Lab Midterm Questions

What chemical is responsible for the acid-fast property of Mycobacterium species?

mycolic acids

What was the appearance of Mycobacterium smegmatis on the Myco plate? Why?

The Mycobacterium smegmatis on the Myco plate had compact, wrinkled colonies. This looks this way because of the hydrophobic nature of the cell wall.

Why was heat used with the stain?

Initially, the organism is heat fixed to the slide to prevent the organism from being washed off during subsequent steps. Later in the procedure, the slide with the heat fixed organism is steamed to make the cell wall a little more penetrable, allowing the stain to enter the cell wall.

What are the primary stain, decolorizer, and counter stain used when performing an acid-fast stain?

The primary stain is the carbol fuchsin. The decolorizer is the acid-alcohol. The counterstain is methylene blue.

What important genus of bacteria is this stain used to identify?

Mycobacterium

Are acid-fast bacteria Gram (+) or Gram (−)? Explain.

Positive.

What are two diseases caused by members of the genus Mycobacterium?

M. Tuberculosis and Hansen's disease are caused by members of Mycobacterium.

Why is this stain so useful for clinical diagnosis?

This stain is useful because it can quickly identify the harmful microorganism which allows for a quick diagnosis and treatment for the patient with the harmful organism.

Aseptic technique is performed to prevent ____.

contamination

What is the purpose of gently shaking a broth culture prior to using it?

To make sure everything is mixed and not settled on the bottom.

When is flaming the loop required in aseptic technique?

Before being used to transfer bacterial culture and after the transfer.

In what way should you hold the culture tube or flask to prevent contamination from contacting the media?

It should be held at an angle of about 45 degrees.

How is the lid to a tube handled during aseptic technique?

It is taken off with the hand holding the inoculation loop, with the fingers not holding the loop.

When is the mouth of the tube flamed?

Before and after obtaining the microorganisms with the loop.

Is the culture tube recapped before or after placing the loopful of microorganisms on the slide? Explain why the timing would matter.

Before so contaminants from the air do not enter the tube.

What types of microorganisms were found in your biofilm? (bacteria, algae, protozoa?)

spirostomum , chlamydomonas (protozoa)

How does your biofilm appear different under phase contrast relative to the Gram stain?

Under phase contrast, there is a significant contrast, while gram stain shows the shape of the cell in color.

Is there a correlation between the type of environment and the type of microorganism found in the biofilm? (For example, do you see Cyanobacteria and algae in samples that were exposed to the sun for most of the day?)

Yes, due to the lake environment, one would expect aqueous organisms, which protozoa are.

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

Phase contrast has condensers and objectives that make objects appear with significant contrast allowing for movement to be seen. Brightfield only shows the image with little contrast, making it very difficult to see movement.

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

They have condensers and objectives that give the ability to view live organisms and their motility by showing them with contrast to their surroundings without heat fixation and staining. Brightfield scopes don't have the ability to view live organisms.

What is the main component of the capsule? What are other components?

Layers of polysaccharide or polypeptide contains material that surround the cell wall of the bacteria.

Why was the smear not heat fixed?

If the smear is heat fixed, the capsule will be destroyed.

What are four possible functions of the capsule?

Four functions are: prevent dessication of the cell, attach certain pathogenic microorganisms to host cells, resist phagocytosis, enhance the organism's ability to cause disease

Describe the appearance of the capsule-forming bacteria on the surface of the agar. Why might this indicate that it possesses a capsule?

Gram-negative, oval shaped stained cell with a clear white "halo" ring around it

Why is it important to stain the background with this capsule stain?

The capsules themselves will remain unstained and appear as a clear area surrounding the cell and the cells will be stained red/brown.

A spore-forming, capsule-forming bacterium is grown on a medium rich in glucose and other required nutrients. Would you expect to see spores with the endospore stain? Explain. Would you expect to see capsules when performing a capsule stain? Explain.

No, because endospores are formed in unfavorable conditions. Yes, because there is an abundance (excess) of nutrients.

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

1. whole colony shape

2. margin shape

3. elevation

4. optical properties

5. surface characteristics

6. pigementation

What can influence the colony morphology properties of a microorganism?

the media used

Describe the growth medium used for this experiment. 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 morphology.

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 color of the 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.

What are two genera that commonly form endospores? What is the Gram reaction and morphology of these bacteria?

-Bacillus and Clostridium

-gram positive rods

Are endospores reproductive structures? Why or why not?

No, they are only produced under stressful conditions. An endospore is a specialized DORMANT structure. Will form within a genetically capable cell when essential nutrients are depleted or when water Is unavailable.

Describe the process of sporulation. When will sporulation occur?

Occurs when a vegetative cell (reproductive) produces endospore (dormant); when nutrients or water becomes depleted and the environmental conditions are harsh.

What component of an endospore protects against harsh chemicals? What component protects the DNA from damage?

The spore coat is responsible for the resistance of the endospore to harsh chemicals; Endospores also contain high levels of calcium complexed with the dipicolinic acid. This complex may play a role in protecting the spore DNA from damage.

What are three common diseases associated with Gram-positive spore forming rods? How does the presence of endospores in these bacteria contribute to the capacity to cause disease?

Botulism, anthrax, and tetanus; endospores allow them to survive during harsh conditions.

What is the primary stain, decolorizer, and counterstain used in the staining of endospores?

-primary stain: malachite green

-decolorizer: water

-counterstain: safranin

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

-Primary stain - Crystal violet

-Mordant - Gram's iodine

-Decolorizer - alcohol

-Counterstain - Safranin

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?

A mordant binds to the crystal violet forming a complex not so easily removed from the Gram positive cells. If the mordant was not added, the cells would be gram negative, bc there would be no dye from the primary stain.

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 (gram +); red (gram -)

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

It is added to stain the gram negative cells red; 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?

It was either over-decolorized with ethanol or the stain was left on for so long that it lost its retention and appeared gram negative when stained with the safranin.

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

color; red for negative and purple for positive

Which soap tested was the most effective in reducing the number of bacteria present? The least effective?

-most effective: softsoap antibacterial liquid soap

-least effective: softsoap NON antibacterial liquid soap

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

They could be harmful if introduced into the bloodstream or tissue; to prevent nosocomial or hospital-acquired infections.

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

Liquid soap would lessen the probability of cross contamination between the uses of bar soap. Also liquid soap dries more quickly without residue and is more effective.

Why could there be more bacteria present after hand washing?

Washing skin with soap and water sloughs off the dead outermost layers of the skin and may expose the normal flora.

What is meant by the term normal flora?

Normal flora are microbes that establish themselves on or in the body, but do not produce disease under normal conditions.

Beef extract in Nutrient Agar makes NA a ___ medium.

complex

What is the difference between TSA and TSB?

TSA (Tryptic Soy Agar) is a type of solid growth medium, usually containing agar.

TSM (Trypic Soy Broth) is a type of liquid growth medium.

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, 15 psi

When would the following sterilization techniques be used?

a. dry heat sterilization

when flaming inoculating loops and inoculating needles

When would the following sterilization techniques be used?

b. filter sterilization

to physically remove bacteria and other contaminants from heat-sensitive liquids or gases

When would the following sterilization techniques be used?

c. cold sterilization

to sterilize heat sensitive objects like petri dishes, paper, leather, wood, metal and rubber productsit is also used by NASA to sterilize interplanetary spacecraft

When would the following sterilization techniques be used?

d. UV radiation sterilization

for exposed surfaces

When would the following sterilization techniques be used?

e. ionizing radiation sterilization

used in the food industry and for sterilization and decontamination of medical supplies

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

A proton gradient (Na+, K+, and Rb+ can also be used) provides energy to rate the flagellum. The force to spin the motor proteins can be obtained when protons from the periplasm flow back into the cell through Mot proteins, which are located adjacent to the motor.

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

They sway back and forth to move the cell. Yes, you could faintly see the flagella.

Monotrichous flagella

single flagellum at one end of the cell

Lophotrichous flagella

cluster of flagella at one or both ends

Amphitrichous flagella

one flagella at each end

Peritrichous flagella

flagella that cover the surface of a cell

Why does motility test medium have a low agar concentration?

to allow for movement of motile bacteria

Explain the color change that occurs in the motility test medium after inoculation.

TTC, tetrazolium salt, is in the medium. It's an electron acceptor. Oxidized = colorless and soluble, reduced = colorful and insoluble.

If the bacteria are motile, then the red color will radiate in all directions away from the original stab line. The entire deep may even turn red. If the bacteria are not motile then you will see a red line only where you stabbed the deep.

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

-Red radiating multidirectional (very pronounced) is POSITIVE.

-Red in only the direction of insertion = NEGATIVE, meaning the microbe didn't move beyond where it was placed.

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

Flaming the inoculating loop between each quadrant preserves the dilution you're establishing. If you don't flame it, you risk carrying over more bacteria and invalidating the streaking procedure Flaming is aseptic technique...

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

-mixed culture = multiple species

-pure culture = 1 species

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

A colony is a grouping of genetically identical bacteria from a single cell on an agar plate. An ISOLATED colony is a pure culture.

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?

The third colony could be a transient microflora in the air that infected the plate when you were performing the streak technique Note: this is technically contamination, but "contamination" is not suitable answer for midterm.

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

Most lab tests for identifying bacteria require pure cultures. Clinical specimens usually contain a mix of different species. The medical technologist has to separate the different species before any other tests can be done. She separates them by doing a streak dilution, after the streak dilution plate has incubated, the technologist chooses bacteria from individual colonies. A colony consists of bacteria all descended from the same founder. Thus, it is a pure culture.

Consider the results of your simple stain and the results of your first streak plate. Do these observations agree?

The simple stain was all dyed the same whereas the first streak plate had both gram positive and gram negative bacteria present (done with differential stain so that pink and purple cells present).

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

A simple stain reveals CELLULAR morphologies but covers all the cells the same whereas different colonies could be observed on the streak plate differentiating between species because although the species may have the same cellular morphology they could form very different COLONY morphologies.

What is the purpose of performing a streak plate?

to obtain a pure culture and determine how many organisms are present

How many times do you dip into the original culture for a streak plate? For creating a lawn?

-streak plate = only once for all 4 quadrants

-lawn = twice for each half of the dish

How does a lawn differ from a streak plate?

A streak plate is a dilution technique to obtain a pure culture where as a lawn is to obtain confluent grown to test environmental stresses.

In replica plating, what is touched to colonies on the agar surface of the master plate to transfer the colonies to a new plate?

the velvet side of a sterile velveteen square cloth

In replica plating, explain why the master plate and new plate are marked with a line on the bottom of the plate to correspond with the white line on the locking ring of the replica-plating tool.

The new plate acts as the circumference reference point where as the master plate is placed where the locking mark of the replica plate tool is.

Which plating procedure requires the melted, tempered agar to be added to the dilution of microbial culture? What would happen if the agar was not tempered?

pour plate technique; tempering the plate allows the agar to cool

Would you use the spread plate or pour plate method if culturing an obligate aerobe? Explain.

Spread plate because colonies will only grow on the surface. if a pour plate was used then the colonies would be trapped inside the agar.

Compare and contrast the appearance of a spread plate and a pour plate after incubation.

In a spread plate, colonies will only grown on top of the agar surface where as a pour plate the colonies will grow on top and also within.

Compare and contrast the procedure for a direct stain and an indirect stain.

Direct stain = after heat fixing, place the prepared smear on a staining rack over the water through and flood with any one of the available dyes. then rinse the slide gently with water and blot with bibulous paper

Indirect stain = after air drying of the smear in the primary stain, place the smear on a staining rack over the water and flood with acid-alcohol for a couple seconds until the sear turns blue. do not rinse with water or blot. allow to air dry

Look at the genus names of the organisms stained. Do some of them indicate what the cell morphology and cell grouping should be? Which ones? What should the morphology and cell grouping be?

Streptococcus are chained round cells where as Staphylococcus are round, grape like clusters. Bacillus are rod shaped and Spirosoma are twisted that resemble corkscrews.

List the cell shapes and give a brief description of each. What are the two most common cell shapes?

The two most common are cocci, which is round shaped, and bacilli, which is rod shaped. Both share prefixes that represent the shape of cells.

Explain why staphylobacilli do not exist.

Because bacilli only divide along their axis so they will never form a cluster of rods

How does smear preparation for a direct stain differ if the culture is in a broth versus on an agar plate?

When obtaining a culture from an agar dish, you have to put roughly two drops of water on the slide first, but if the culture is in a broth you do not have to put water first.

What instrument is used to obtain culture for a smear from a broth or solid medium?

inoculating loop

The slide warmer does two things in preparing a smear. What are they? Do you do this if preparing a smear for an indirect stain?

Heat fixation kills the cells, destroying autolytic enzymes, and helps the cells adhere to the slide. It is not required for an indirect stain.

On the lab midterm exam, no slide warmer will be available. How will you dry and heat fix your smear?

Leave the slide on the bench for several minutes and heat fix the slide over the burner to ensure proper procedure. Only swipe the slide over the burner... do not leave directly over the flame.

What three terms describe the microscopes used in lab? What is the meaning of each?

-brightfield = used to observe microorganisms

-compound = because it consists of a series of ocular and objective lenses

-binocular = each eyes there is an ocular containing a lense

If you are using the 10× objective, what is the total magnification?

100x

If you are using the 100× objective, what is the total magnification?

1000x

If two cells are less than 0.2 microns apart, can you see them as two independent cells with a microscope that has a 0.2 micron maximum resolution?

No, bc the limit is 0.2 microns.

How does immersion oil help the resolution?

The use of oil reduces the amount of light lost and increases resolution.

Eyepieces

A complex piece, located at the top of the microscope. Consists of two or more internal lens and usually has a magnification of 10x. Most modern microscopes have two ocular (binocular) lens.

Observation tube

Tube in which image passes through

Revolving nosepiece

This is the part that holds two or more objective lenses and can be rotated to easily change