Microbiology (Bio 352) First Exam

This material is from chapters 1-3 in addition to labs 1 and 2.

Who was the first person to see bacterial cells with the microscope?

Leeuwenhoek

What process was studied by Redi and Spallanzani?

Spontaneous generation

1. Redi proposed that fly maggots arise from hatched eggs laid on decaying meat and does not sprout spontaneously from it.

2. Spallanzani suggested that microorganisms came from the air and would therefore grow in a brother of the cooled flask. Concluded that microbes from the air, not spontaneous generation, accounted for the presence detected in the flasks

The process of ___________ involved the inoculation of dried smallpox scabs under the skin.

Variolation/Vaccination

What is the name for the field of study established by Semmelweis and Snow in the mid-1800s?

Epidemiology- studying how diseases were transmitted and how simple measures could prevent transmission

The process of controlled heating, called ____________, was used to keep wine from spoiling.

Pasteurization

What surgical practice was established by Lister?

Antisepsis

Which one of the following statements is not part of Koch’s postulates?

1. The microorganism must be isolated from a dead animal and pure cultured.

2. The microorganism and disease can be identified from a mixed culture.

3. The pure cultured organism is inoculated into a healthy, susceptible animal.

4. The same microorganism must be present in every case of the disease.

The microorganism and disease can be identified from a mixed culture→ The identical microorganisms are isolated and recultivated from the tissue specimens of the experimental animal

Match the lab with the correct set of identified diseases:

1. Pasteur: tetanus and tuberculosis

2. Koch: anthrax and rabies

3. Koch: cholera and tuberculosis

4. Pasteur: diphtheria and typhoid fever

Koch: cholera and tuberculosis

What group of microbial agents would eventually be identified from the work of Ivanowsky and Beijerinck?

1. Viruses

2. Fungi

3. Protists

4. Bacteria

Viruses

What microbiological field was established by Winogradsky and Beijerink?

1. Virology

2. Microbial ecology

3. Bacteriology

4. Mycology

Microbial ecology

What group of microorganisms has a variety of internal cell compartments and many of which act as decomposers?

1. Bacteria

2. Viruses

3. Archaea

4. Fungi

Fungi

Which one of the following organisms was not a model organism related to the birth of molecular genetics?

1. Streptococcus

2. Penicillium

3. Escherichia

4. Neurospora

Penicillum

Which group of microbial agents is eukaryotic?

1. Bacteria

2. Viruses

3. Archaea

4. Algae

Algae

The term _______ was used to refer to antimicrobial substances naturally derived from some ________.

1. antibiotic; bacteria and fungi

2. antisepsis; other living organisms

3. antibiotic; viruses

4. fermentation; microbes

antibiotic; bacteria and fungi

Which one of the following is not considered an emerging infectious disease?

1. Polio

2. SARS

3. Lyme disease

4. AIDS

Polio

What strategy is not part of evolutionary medicine?

1. Understanding the role of the human microbiome

2. Understanding how life began on Earth

3. Reducing the emergency of infectious diesases

4. Stimulating the development of antimicrobial drugs

Understanding how life began on Earth

True or false: Leeuwenhoek believed that animalcules arose spontaneously from decaying matter.

False; Needham

True or false: Semmelweis proposed that cholera was a waterborne disease.

True

True or false: Some bacterial cells can convert nitrogen gas (N2) into ammonia (NH3).

True

True or false: Fungi are eukaryotic microorganisms, some of which are decomposers.

True

True or false: Koch proposed the germ theory.

False; Pasteur

True or false: Variolation involved inoculating individuals with smallpox scabs.

True

True or false: Most microbes today exist as independent, free-living cells.

False; biofilms

True or false: Pasteur proposed that “wine disease” was a souring of wine caused by yeast cells.

False bacterial

Describe the concept of spontaneous generation and distinguish between the experiments that supported and refuted the belief (Key concept 1.1).

• Spontaneous generation is the theory that living creatures could arise from nonliving matter and that such processes were commonplace and regular.

• Francesco Redi first tested this theory by jarring pieces of rotting meat and covering one with gauze to keep out any flying insects while the other wasn’t.

• Needham proposed that spontaneous generation resulted from a “vital force” that reorganized decaying matter into life by boiling animal broth in flasks and then kept at room temperature.

• Spallanzani challenged Needham’s experiments and suggested that microbes came from the air and would grow in the broth of the cooled flasks. Repeating Needham’s experimental design, he left some flasks open and the others were sealed. He concluded that microbes from the air accounted for the presence of microbes in the flasks.

• Pasteur hypothesized that organisms appearing in the sterile flask come from other organisms into the air. He used a bent neck flask so that dust and microbes lay trapped away from the interior. Once he snapped the neck of the flask or tilts the flask organisms appear

• Pouchet tried a similar experiment substituting the yeast based broth with boiled hay and still had growth. This was one of the main supportive studies for the belief.

• Tyndall solved the dilemma between Pouchet and Pasteur, determining that with Pouchet, boiled hay had spores that survived the singular heating process. What he did was create the process of heating, cooling, heating, cooling to germinate the spore so that it can die.

Compare Jenner’s work on smallpox and Pasteur’s studies on rabies to the concept of preventing diseases through vaccination (Key concepts 1.2 and 1.3).

• Jenner’s work with smallpox led to the first vaccination when he noticed that milkmaids (a certain demographic) occasionally contracted a mild disease of cowpox that would protect them from contracting smallpox. He took a scab of a cowpox blister from the milkmaids and infected a child with it. The child survived and when they were exposed to smallpox, failed to show any sign of smallpox.

• Pasteur’s lab also focused on preventing disease through vaccines. In 1885, he saved the life of the young boy who had been bitten by a rabid dog

Judge the importance of (a) the germ theory of disease and (b) Koch’s postulates to the identification of microbes as agents of infectious disease (Key concept 1.3).

• Germ theory of disease is the theory in which human infectious disease was due to chemical changes brought about by microorganisms infecting the body.

• Koch’s postulates:

  • The same microorganisms are present in every case of the disease

  • The microorganisms are isolated from the tissues of a dead animal, and a pure culture is prepared

  • Microorganisms from the pure culture are inoculated into a healthy, susceptible animal. The disease is reproduced

  • The identical microorganisms are isolated and recultivated from the tissue specimens of the experimental animal

• Both Germ theory of disease and Koch’s postulates were very important to label the specific microorganisms to a specific disease.

Provide evidence to support the statement: “Not all microbes cause disease; many play important roles in the environment” (Key concept 1.4).

• Out of the many microorganisms that have been discovered, only a small fraction are disease causing agents

• Sergei Winogradsky and Martinus Beijerinck discovered that some bacterial organisms convert N2 into ammonia (NH3) that plants need

  • Found that many soil microorganisms play a key role in recycling matter

  • In Plant Ecology with Professor Corbin, we had discussed the mutualism between plants and fungi that allows for easier access to nutrients (limiting root growth) in exchange for the sugars produced by the plants photosynthesis (up to about 70%).

Assess the importance of microbial ecology and microbial evolution to the current Golden Age of microbiology (Key concept 1.6).

• Microbial Ecology:

  • Discovery that microbes do not act as individuals but survive in complex communities called biofilms→ meaning that they can be difficult to treat when they cause disease.

  • Currently looking at how bacterial and archaeal species is being applied to problems that have potential to benefit the plant.

• Microbial Evolution:

  • Follow the accumulation of unpredictable, chance events that led to evolutionary novelty

    • Currently trying to slow down the evolution of antibiotic resistance, treat infectious diseases, and better understand the role of microbes in human health

As a microbial ecologist, you discover a new species of microbe. How could you determine if it has a prokaryotic or eukaryotic cell structure? Suppose that it has a eukaryotic structure. What information would be needed to determine if it is a member of the protists or fungi?

Eukaryotes would have a cell nucleus that can be seen with a light microscope. If it was a prokaryote, rRNA base sequencing would place the organism in either the domain Bacteria or Archaea. The information needed is protists are unicellular and fungi are multicellular.

You isolate and pure culture a bacterial organism from ill humans that you believe causes the disease. However, you cannot find a susceptible animal for testing that contracts the disease. What would you conclude from these observations?

Since the bacterial organism will not grow in another susceptible animal, the organism could have a special requirement for growth that is found only in humans. Thus, you couldn't use Koch's postulates and you would have to see if the bacterial organism is found in everyone who has the disease but is absent from those who don't have the disease.

Bacterial cells do not grow on bars of soap (that are very alkaline) even though the soap is wet and covered with bacterial organisms after one has washed. Explain this observation.

The bacteria can grow on soap, until it is not an antibacterial soap. But the soap(due to hydrophobicity & hydrophilicity properties together) always loosens the bacteria so that they get rinsed with water easily so with every wash bacteria will be washed . same thing occurs with hand. the soap loosen the bacteria so they get rinsed from your hands easily on washing with water. But some additives in antibacterial soaps now kill the bacteria.

Milk production typically has the bacterium Lactobacillus added to the milk before it is delivered to the market. In the milk, the organism produces lactic acid.

1. Why would this organism be assed to the milk

2. Why was this particular bacterium chosen?

It seems counterproductive to add a souring factor to a product one does not usually desire as a sour product. One reason to add acid to milk products is to change the pH to aid in digestion. The saliva in the mouth is almost neutral; the acid in the stomach is extremely acidic.

In yogurt, lactobacillus is especially helpful for those with digestive and bowel issues, as well as yeast issues, and prevents diarrhea in children. Lactobacillus is found naturally in the gut, and promotes good biotic growth, which also crowds out bad biotic growth.

The toxin associated with the foodborne disease botulism is a protein. To avoid botulism, home canners are advised to heat preserved foods to boiling for at least 12 minutes. How does the heat help to avoid the disease?

Boiling results in denaturing proteins in general- because the toxin associated with botulism is a protein it loses its tertiary structure and its effect as a result.

What is the term that describes the ability of organisms to maintain a stable internal state?

1. Metabolism

2. Homeostasis

3. Biosphere

4. Ecotype

Homeostasis

Which one of the following is not an organizational pattern common to all organisms?

1. Genetic organization

2. Protein synthesis

3. Compartmentation

4. Endomembrane system

Endomembrane system

Which one of the following is not found in bacterial cells?

1. Ribosomes

2. DNA

3. Mitochondria

4. Cytoplasm

Mitochondria

Who is considered the father of modern taxonomy?

1. Woese

2. Whittaker

3. Haeckel

4. Linnaeus

Linnaeus

___________ was first used to catalog organisms into one of three domains.

1. Photosynthesis

2. Ribosomal DNA genes

3. Nuclear DNA genes

4. Cell respiration

Ribosomal RNA genes

Several classes of organisms would be classified into one ___________.

1. order

2. genus

3. phylum

4. family

Phylum

An important automated method used in the rapid identification of a pathogen is ________.

1. rRNA gene sequencing

2. polymerase chain reaction

3. molecular taxonomy

4. biochemical tests

Biochemical tests

What metric system of length is used to measure most bacterial cells?

1. Millimeters (mm)

2. Micrometers (μm)

3. Nanometers (nm)

4. Centimeters (cm)

micrometers (μm)

Before bacterial cells are simple stained and observed with the light microscope, they must be _________.

1. smeared on a slide

2. heat fixed

3. air dried

4. All the above (A-C) are correct

All the above (A-C) are correct

If you wanted to study the surface of a bacterial cell, you would use a _________.

1. transmission electron microscope

2. light microscope with phase-contrast optics

3. scanning electron microscope

4. light microscope with dark-field optics

Scanning electron microscope

__________ The domain containing organisms whose cells have no cell nucleus or mitochondria in the cytoplasm.

Bacteria

__________A member of this group of bacteria is thought to have undergone symbiosis and evolved into the chloroplast.

Cyanobacteria

__________Type of electron microscope for which cell sectioning is not required.

Scanning

__________ The form of microscopy where only the light bouncing off the specimen is seen.

Dark-Field

__________ The organelle, absent in bacteria, that carries out the conversion of chemical energy to cellular energy in eukaryotes.

Mitochondrion

__________Domain in which fungi are protists are classified

Eukarya

__________ Staining technique that differentiates bacterial cells into two groups.

Gram

__________ This form of microscopy uses a special condenser lens to split the light beam.

Phase-Contrast

__________The staining technique employing a single cationic dye.

Simple

__________ Type of microscopy using UV light to excite a dye.

Fluorescence

Identify the cell structures:

a: Flagellum

b: Free ribosome

c: Mitochondrion

d: DNA (chromosomes

e: Cilia

f: Smooth endoplasmic reticulum

g: Rough endoplasmic reticulum

h: Cytoskeleton

i: Plasma membrane

j: Cytoplasm

k: Golgi apparatus

Identify the cell structures:

l: Ribosome

m: Cell membrane

n: Cell wall

o: DNA (chromosome)

p: Cytoplasm

Explain the assignment of organisms to one of the three domains on the “tree of life.” (Key Concept 3.2)

All of life can be divided into three domains, based on the type of cell of the organism: Bacteria: cells do not contain a nucleus. Archaea: cells do not contain a nucleus; they have a different cell wall from bacteria. Eukarya: cells do contain a nucleus.

All prokaryotes are assigned to the domain Bacteria or domain Archaea based on different DNA nucleotide base sequences coding for the small subunit ribosomal RNA. Additionally, all organisms in the domain Eukarya have rRNA small subunit genes have yet another nucleotide base sequence coding for the small subunit ribosomal RNA. Currently, other biochemical and genetic characteristics also separate organisms.

Assess the importance of magnification and resolution to microscopy. (Key Concept 3.3)

• Magnification: the increase in the apparent size of a specimen being observed

• Resolution: the lens system of the microscope allows for the observer to see a magnified object distinctly and can distinguish closely spaced objects as separate objects.

• Without both magnification and resolution, observers would not be able to observe objects through the microscope both in terms of size and clarity.

• Magnification increases the apparent size of a specimen not otherwise clearly visible to the naked eye and is generally achieved with three objective lenses: the low-power (10x), high-power (40x), and oil-immersion (100x), plus the ocular lenses that magnify the image from the objective lens by another 10x, resulting in an image appearing 100x, 400x, or 1,000x bigger, respectively. Resolution (or resolving power) allows a magnified object to be seen distinctly, and closely spaced objects can be distinguished clearly as separate objects.

Compare the uses of the transmission and scanning electron microscopes. (Key Concept 3.3)

• The transmission electron microscope uses magnets to focus a beam of elections to observe the object in question- the object is thinly cut and stained with very heavy metals to provide contrast. Normally it is the interior of microbial cells. As some electrons pass through while others are blocked by the object, an image forms on the screen below the tube.

• The scanning electron microscope also uses magnets to focus the electron beam, but covers the whole object being studied in gold. Electrons that strike a sloping surface yield fewer electrons, producing a darker contrasting spot to give the image a 3D image.

• The transition electron microscope (TEM) is used to observe internal fine details (ultrastructure) in cells and viruses. The scanning electron microscope (SEM) is used to study whole cells or the surface topology of cells

A student is performing the Gram stain technique on a mixed culture of gram-positive and gram-negative bacterial cells. In reaching for the counterstain in step 4, he inadvertently takes the methylene blue bottle and proceeds with the technique. What will be the colors of gram-positive and gram-negative bacteria at the conclusion of the technique?

• The gram-positive bacterial cells will be purple

• The gram-negative bacterial cells will be blue instead of red

Would you obtain the best resolution with a light microscope by using red light (λ = 680 nm), green light (λ = 520 nm), or blue light (λ = 500 nm)? Explain your answer.

Blue light because it has the shortest wavelength. When plugged into the resolving power equation, 500 nm gives the smallest resolvable object.

The accompanying electron micrograph shows a group of bacterial cells. The micrograph has been magnified 5,000×. Calculate in micrometers (μm) the actual length of the bacterial cells.

One cell measures about 10 mm = 10,000 µm (10,000 µm/5,000x = 2 µm)

A local newspaper once contained an article about “the famous bacteria E. Coli.” How many errors can you find in this phrase? Rewrite the phrase correctly

It should read: "The famous bacterium Escherichia coli."
Explanation: "bacterium" is singular and "bacteria" is plural; the first time the species is mentioned, Escherichia should be spelled out; coli is the specific epithet and is not capitalized; the species name should be in italics (Escherichia coli) or underlined (Escherichia coli).

Prokaryotes lack the cytoplasmic organelles commonly found in the eukaryotes. Provide a reason for this structural difference.

Since bacterial cells are small, they have a large surface-to-volume ratio, making the transport of materials a quick process. Eukaryotic cells are much larger and have a smaller surface-to-volume ratio. This indicates that transport would be much slower, and possibly lethal to the cell. The cell thus becomes more efficient through compartmentalizing functions with organelles.

A student of general biology observes a microbiology student using immersion oil and asks why the oil is used. “To increase the magnification of the microscope” is the reply. Do you agree or disagree? Wh

Disagree. Oil doesn't increase the magnification, it allows the gathering of enough light by the oil-immersion lens to resolve the specimen

How have revelations from studies on viruses and prions complicated some of the traditional views about the principles of biology?

The traditional view of biology was that a living organism was one that had homeostasis, grows, reproduces, responds to stimuli, and adapts to its environment. Viruses do not meet this definition which is why they were first thought of as poisons. It took over 30 years for scientists to agree that viruses were, in fact, living entities. It required a broadening of the definition of a living organism.

Viroids and prions are now pushing the limits of the definition of a living organism again. Viroids are simply small strands of nucleic acids; and, prions are nothing more than misfolded proteins. These two entities are even further from the traditional view of a living organism than viruses were. Viruses at least had genomes with the ability to synthesize proteins and enzymes in a host cell.

Lab exercise 2.1.1:

1. Consider plates 7 and 8:

   1. What was the purpose of incubating the unopened plates? Be specific.

   2. What is an appropriate name for these plates?

   3. If growth appears on both unopened plates, what are some likely explanations?

   4. What if growth appears on only one plate?

   5. How does growth on the unopened plates affect your interpretation of the other plates?

1. The unopened plates show sterility of the medium prior to exposure.

2. An appropriate label would be “control.”

3. If growth appears on both unopened plates, the medium used to pour those plates was probably contaminated.

4. If growth appears on only one of the plates, the stock medium may have been contaminated, but more likely, the plate was contaminated during pouring or incubation.

5. Any growth on the control plates casts doubt on the source of growth on the exposed plates. That is: is the growth a contaminant or from the sample source?

Lab exercise 2.1.2:

1. Why do you think the specific types of exposure (air, hair, tabletop, etc.) were chosen for this exercise?

These exposures were chosen because they represent major sources of environmental contamination of cultures during transfers.

Lab exercise 2.1.3:

1. Did you get different-appearing colonies on plates 2 and 3? If so, explain why.

2. What is the likely source (reservoir) of organisms that grew best at 35°C, and how do they survive at room temperature without nutrients?

1. The answer will be “Yes” or “No,” but probably some differences will be seen. One valid reason for differences is that different parts of the doorknob were sampled. Another is that the organisms on the tabletop are so sparse that two samples may not pick up all of the same types of organisms. However, the answer that we’re looking for here is that the differences could be due to different incubation temperatures. Not all organisms grow at both 25°C and 35°C. Failure to recognize this fact indicates that the student has missed an important component of the experimental design.

2. 35°C is human body temperature. Given the sites sampled (even the environmental ones), humans are the likely reservoirs. Their survival on the tabletop or other location “without nutrients” can be explained in several ways: (1) there may actually be nutrients available that we are unaware of, (2) the organism may be in a relatively inert state (stasis) in which nutrients are not required, or (3) some organisms produce highly resistant resting stages called spores.

Lab exercise 2.1.4:

1. Suppose plate 4 (cough) has no growth after incubation. It is highly unlikely the “cougher” has sterile coughs! Suggest reasons why no growth was recovered on the plate.

Some reasons would be: The student’s cough missed the plate (we’ve seen it!), they didn’t cough deeply enough, or (and this is the point of the question) one or more necessary resources for growth are not included in the recipe for nutrient agar.

Lab exercise 2.1.5:

1. The plates you are using for this lab will be autoclaved eventually to completely sterilize them. The measures taken to disinfect the tabletops (the source of the organisms on plates 2 and 3) are not as extreme. Why?

The plates (should) have a much higher cell density than any growth on the table. Since the ability to produce infection is dependent upon (among other things) the number of organisms introduced into the host, the plates present a greater threat than does the tabletop.

Lab exercise 3.1.1:

1. Why aren’t the magnifications of both ocular lenses of a binocular microscope used to calculate total magnification?

Each ocular of a binocular microscope magnifies the image coming from the objective lens, but it does not magnify the image coming from the other ocular. The image reaching the eye has only been magnified by two lenses: the objective lens and one of the oculars.

Lab exercise 3.1.2:

1. What is the total magnification for each lens setting on a microscope with a 15x ocular and 4x, 10x, 45x, and 97x objective lenses?

1. 60

2. 150

3. 675

4. 1455

Lab exercise 3.1.2:

1. Assuming that all other variables remain constant, explain why light of shorter wavelength will produce a clearer image than light of longer wavelengths.

Using the limit of resolution formula (D= λ/(NA_condenser +NA_objective), it is determined that a smaller wavelength (λ) will allow for a smaller resolution limit (D) → leading to greater clarity as a result.

Lab exercise 3.1.2:

1. Why is wavelength the main limiting factor on limit of resolution in light microscopy?

Based on the limit of resolution formula above, the smallest wavelength will allow for greater clarity as a result. Depending on the microscope, such as the phase contrast microscope, light waves that are in phase reinforces their visibility- anything that goes beyond the small wavelength will be canceled out and result in varying levels of darkness the image has in contrast to the background.

Lab exercise 3.5.1:

1. Why doesn’t a negative stain colorize the cells in the smear?

Both the stain’s chromophore and the bacterial cells have negative charges. Like charges repel, so the cells don’t get stained.

Lab exercise 3.5.2:

1. Eosin is a red stain and methylene blue is blue. What should be the result of staining a bacterial smear with a mixture of eosin and methylene blue?

Eosin is an acidic stain with a negatively charged chromophore; methylene blue is a basic stain with a positively charged chromophore. Since bacterial cells are usually negatively charged, the cells will attract the methylene blue and repel the eosin, making the cells blue and the background red.

Lab exercise 3.6.1:

Predict the effect on Gram-positive and Gram-negative cells of the following “mistakes” made when performing a Gram stain. Consider each mistake independently.

1. . Failure to add the iodine.

2. Failure to apply the decolorizer

3. Failure to apply the safranin.

4. Reversal of crystal violet and safranin stains.

1. Probable decolorization of Gram positives and no noticeable effect on Gram negatives. All cells are pink.

2. All cells will appear purple since the crystal violet stains both Gram-positive and Gram-negative cells, and the safranin is not likely to stain over the darker crystal violet.

3. Gram positives will be purple and Gram negatives will be colorless since they’ve been decolorized but not counterstained.

4. A mess. Safranin will probably be washed out during decolorization, so both Gram positives and Gram negatives will be colorized by the “counterstain” crystal violet. Even if the safranin is not completely washed away, the darker crystal violet will cover the lighter safranin.

Lab exercise 3.6.2:

Both crystal violet and safranin are basic stains and may be used to do simple stains on Gram-positive and Gram-negative cells. This being the case, explain how they end up staining Gram-positive and Gram-negative cells differently in the Gram stain.

The important differential feature is the ability of Gram-positive cells to retain the crystal violet when subjected to alcohol decolorization. This ability is enhanced with the use of the mordant iodine.

Lab exercise 3.6.3:

If you saw large, eukaryotic cells in the preparation made from your gumline, they were most likely your own epithelial cells. Are you Gram-positive or Gram-negative? (You can make a good guess about this even if you didn’t see your cells.)

Normal people are Gram negative. It is the thick peptidoglycan of Gram-positive walls that makes them able to retain the crystal violet during decolorization. In fact, most cells—prokaryotic and eukaryotic—are Gram negative.

Lab exercise 3.6.4:

One of your lab partners has followed the recommended procedure of running Gram-positive and Gram-negative control organisms on her Gram stain of an unknown species. Her choices of controls were Escherichia coli and Bacillus subtilis. She tries several times and each time concludes she is decolorizing too long because both controls have pink cells (one more than the other). What might you suggest she try and why?

The problem is most likely due to her choice of Gram-positive control. Bacillus is notorious for losing its ability to retain crystal violet during the Gram stain as it gets older (often, less than 24 hours). You should suggest using a different Gram-positive control, such as Staphylococcus epidermidis.

Lab exercise 1.5.7:

Most colonies on streak plates grow from isolated colony-forming units (CFUs). On rare occasions, however, a colony can be a mixture of two different organisms. If a culture is started from this colony (thinking it is pure), correct identification will be next to impossible because the extra organism could confound the identifying test results. How could you verify the purity of a colony? (The answers may vary depending on what experience you have had prior to performing this exercise.) If you found the colony to be a mixture of organisms, what could you do to purify it?

A Gram stain or another streak plate can be used to verify colony purity. If the colony is not pure, then it should be re-streaked (or, the streak plate used to verify purity could be used) and a colony of the “correct” organism should be picked from this second plate and used to start a pure culture.

Lab exercise 1.6.2:

What is the primary negative consequence of not spreading the inoculum evenly over the agar surface?

The cells in the inoculum may not have enough room to grow into individual colonies. This defeats the purpose (i.e., isolation) of the spread plate.

Lab exercise 1.6.3:

To get isolated colonies on a plate, only about 300 cells can be in the inoculum. What will happen if the cell density of the inoculum significantly exceeds this number?

As in question 2, the cell density may be so great that individual colonies don’t have room to develop, even if your spreading technique is okay.

Lab exercise 1.6.4:

Suppose you have two organisms in a mixture, and Organism A is 1,000 times more abundant than Organism B. Will you (without counting on good luck!) be able to isolate Organism B using the spread plate technique? Explain your answer.

It will be difficult to isolate Organism B. When Organism B is at a dilution that would produce individual colonies, it will be overgrown by the more abundant Organism A. By the time Organism A is diluted enough to produce individual colonies, Organism B is likely to be gone.

Lab exercise 3.7.2:

Are acid-fast negative cells stained by carbolfuchsin? If so, how can this be a differential stain?

What makes this a differential stain is the fact that the carbolfuchsin is easily removed from the walls of acid-fast negative cells, but it is “locked into” the mycolic acid of acid-fast positive cells. As with the Gram stain, this is a differential test not because of the stains, but rather how the different cell types respond to decolorization.

Lab exercise 3.7.3:

Why do you suppose the acid-fast stain is not as widely used as the Gram stain? When is it more useful than the Gram stain?

The Gram stain divides up the microbial world into two main groups. A Gram-stain result for an unknown organism considerably narrows the field of possible organisms it could be, regardless of the result. The vast majority of cells are acid-fast negative, so an acid-fast result for an unknown is not likely to eliminate many organisms. It is most useful when a sample is brought in from a patient with symptoms consistent with a disease caused by an acid-fast organism. (That is, when the percentages are good that an AFB—acid-fast bacillus—is present.) Then the staining result can support or eliminate AFBs as the causative agent of the condition.

Lab exercise 3.9.1:

Why does this exercise call for an older (5-day) culture of Bacillus?

Spores are produced by Bacillus when the organisms are deprived of nutrients. Older cultures are more likely to have depleted nutrients in the medium and begun sporulation.

Lab exercise 3.9.2:

Consider the possible results of an endospore stain.

1. What does a positive result for the endospore stain indicate about the organism?

2. . What does a negative result for the endospore stain indicate about the organism?

1. If you get a positive result for the endospore stain, it indicates (barring contamination) that the organism produces spores.

2. A negative result for the endospore stain might mean the organism cannot produce spores, or CAN and just ISN’T.

Lab exercise 3.9.3:

Why is it not necessary to include a negative control for this stain procedure?

Vegetative cells act as controls. They will stain red, whereas any spores present should stain green. Frankly, the hard part is getting the spores to stain, not the vegetative cells.

Lab exercise 3.9.4:

Endospores do not stain easily. Perhaps you have seen them as unstained white objects inside Bacillus species in other staining procedures. If they are visible as unstained objects in other stains, of what use is the endospore stain?

When the structure is an unstained white object, it might be a spore, or it might be a storage granule or other cellular inclusion of some sort. The spore stain technique provides good evidence that the structure stained is, in fact, a spore.

Lab exercise 3.10.2:

You are told that viewing is best done with as little illumination as possible. Why will transparent cells be easier to view with less light?

Reducing the illumination by closing the iris diaphragm cuts down on spherical aberration of the microscope and makes the image clearer. While this is a general principle, with these preparations it is even more important because no stain is used to add contrast between the cell and background.

Lab exercise 3.10.3:

Why should Brownian motion increase the longer you observe a hanging drop or wet mount preparation?

The longer the organisms stay on the microscope, the more the slide heats up due to illumination from the microscope’s lamp. This heating causes an increase in the kinetic energy of the particles responsible for Brownian motion, so there are more collisions with the cells and with greater force.

Lab exercise 5.24.1

Consider the tube stabbed with the sterile inoculating needle.

1. Is it a positive or a negative control? It is a negative control.

2. What information is provided by the sterile-stabbed tube?

1. It is a negative control.

2. It shows what a stab looks like in the absence of microorganismal growth. In most cases, this is unnecessary because if the organism grows and is nonmotile, the stab line will be the red color of reduced TTC. We included it only to provide one more opportunity to practice stabbing in the low agar concentration medium.

Lab exercise 5.24.2:

Why is it important to carefully insert and remove the needle along the same stab line?

Growth will occur anywhere that the inoculating needle contacts the medium. Since spreading from the stab line indicates motility, poor stabbing technique will result in growth in many parts of the medium and perhaps give the impression of motility. A single stab line is much easier to interpret.

1. Describe the major scientific contribution(s) of the following individuals:

a)    Francesco Redi and Lazzaro Spallanzani

b)    Antony Van Leeuwenhoek

c)     Ignaz Semmelweis

d)    John Snow

e)    Joseph Lister

a)    Francesco Redi and Lazzaro Spallanzani: Redi found that if flies were prevented from landing on meat, it did not produce maggots. Spallanzani boiled yeast broth and left one flask open and one flask closed; again showcasing that organisms appear in open environments but not in closed ones.

b)    Antony Van Leeuwenhoek: Suggested that maggots arose from eggs in the decaying material but not the material itself

c)     Ignaz Semmelweis: Published data on source of perpetual fever/blood poisoning of women in childbirth

d)    John Snow: determined the cause of cholera transmission in London

e)    Joseph Lister: developed the practice of antisepsis healed wounds without infection after surgery

Louis Pasteur – describe Pasteur’s swan-neck flask experiment and the significance of his observations. Explain why John Tyndall’s findings were at odds with those of Pasteur.  Compare and contrast vegetative cell vs. endospore.

Pasteur believed that the air was filled with microbes. With the swan neck flask, he was able to visualize that when the flask remains upright, no microbial growth occurs; if the flask is tipped, microorganisms trapped in the neck reach the sterile liquid and grow. The media used is why Pouchet finding were at odds- he used hay which doesn’t kill any of the spores that may lay on it whereas Pasteur used a yeast based broth. When both broths boil, Pasteur was the only one that removed possible microorganisms whereas Pouchet endospore filled hay, did not denature. It wasn’t until Tyndall came and solved the discrepancies between the two researchers. He developed the process of rapid heating, then cooling, heating, then cooling to germinate the spores that may lay dormant and denature them for the study.

Summarize the doctrine of spontaneous generation and explain why refuting it was essential to the development of the germ theory of disease.

Spontaneous generation is the theory that living organisms develop from nonliving matter. It was instrumental to refute it because it allowed for new medicinal and culinary developments to form.

1. Redi proposed that fly maggots arise from hatched eggs laid on decaying meat and does not sprout spontaneously from it.

2. Spallanzani suggested that microorganisms came from the air and would therefore grow in a brother of the cooled flask. Concluded that microbes from the air, not spontaneous generation, accounted for the presence detected in the flasks

3. Pasteur demonstrated that microorganisms appear and grow not by spontaneous generation but because of the spores both in the air and on food

Pasteur also discovered the processes of fermentation and pasteurization.  Define the terms fermentation and pasteurization and give some examples/applications.

Fermentation: the process in which sugars are transformed into a new product through chemical reactions carried out by microorganisms (any alcohol, kimichi, etc.)

Pasteurization: heating liquids at a high temperature for a short period of time to kill harmful bacteria (milk, beer, etc.)

Robert Koch – describe Koch’s postulates and explain the need for pure cultures to satisfy the postulates.