Review for Practical Exam #1

I. Lab 1/ lecture 1: Microscopy and the Ubiquity of Microorganisms.

A. Microorganisms are ubiquitous, but they do not generate spontaneously. It is the ubiquity of microorganisms that makes it so important to use sterile technique when working in the laboratory with sterile media or pure cultures.
B. Know the parts of a bright-field microscope and the function of each. Be able to identify the parts if they are pointed out on a microscope.
Although we work only with the bright-field microscope, it's important to remember that there are other types of microscopy.
Which type of microscope is commonly used to view living cells as it converts slight differences in refractive index and cell density into easily detected variations in light intensity?
C. Understand the difference between magnification and resolution.

1. Magnification is the process of enlarging the size of an object as an optical image. Be able to calculate total magnification = the product of the magnifying power of the oculars and the objectives.
2. Resolution is the ability to distinguish objects that are close together. The numerical aperture and the wavelength of light used are the factors that affect microscope resolution. How do these factors affect the working distance? How does immersion oil affect the resolution?

D. In a microscope that is parfocal, the image should remain in focus when the objective lens is changed.
E. Be able to determine the approximate size of an object if you are told the diameter of the field of view. Understand the inverse relationship between the power of magnification and the diameter of the field.

II. Lab 2 / lecture 2: Simple Stains: Direct and Negative

A. Know the purpose of fixing and staining a sample. Understand the difference between a fixed sample and a wet mount.
B. Understand the difference between basic (also called direct, cationic or positive) and acidic (also called indirect, anionic or negative) stains.
C. In simple staining, a single dye is used and all organisms take on the same color when stained with this dye. Simple stains can either be acidic or basic. Be able to tell the difference between these two types of simple stains if viewing a sample under the microscope.
D. Know that a simple stain is generally used to determine shape [e.g. spheres (coccus) and rods (bacilli)] and arrangement [e.g. strepto - chains of cells, staphylo - irregular clusters of cells, diplo - two cells together, tetrad - four cells together, sarcina - eight cells together].
E. You may be given a slide and a microscope and be asked to focus the microscope in order to determine the shape of the bacteria.

III. Lab 3/ lecture 3 and lab 4/ lecture 4: Differential Stains

A.In differential staining, bacteria are divided into separate groups based on their staining properties. How is this different from simple stains?

1. Gram stain

a. Know the difference between a Gram-positive cell wall and a Gram-negative cell wall. Don’t forget to note the presence of LPS in Gram-negative cells and teichoic acid in Gram-positive cells. Be sure that you understand that the Gram reaction of a cell has nothing to do with the charge that it carries on its surface. All cells, Gram-negative and Gram-positive, have a negative surface charge.
b. Know all the steps in the Gram stain procedure (Come In And Stain). The differential step is the decolorization with ethanol. This step is also the most error prone step.
c. Be able to identify Gram-positive or Gram-negative cells on a Gram stained slide.

2. Acid-fast stain

a. Used to detect cells capable of retaining a primary stain when treated with acid alcohol (these cells are termed acid-fast). Acid-fast cells generally contain a great deal of mycolic acid. a waxy hydroxy-lipid that resists staining.
b. Be able to identify an acid-fast / nonacid-fast cell if viewing a slide that has been stained using the acid-fast stain. This will require some familiarity with the staining procedure.
c. Realize that this is an important technique for identifying bacteria in the genus Mycobacterium, in specific the pathogens Mycobacterium leprae and Mycobacterium tuberculosis.

3. Endospore stain

a. Know what an endospore is and why it forms. Bacteria of several genera form endospores two of which are Bacillus and Clostridium.(e.g. the pathogen that we are all too familiar with : Bacillus anthracis) What are some of the pathogenic species of Clostridium?
b. Be familiar with the structure of an endospore and the steps in spore formation. Also realize that a spore can transform back into an active, vegetative cell. What two things must occur before the spore will develop into a viable cell?
c. The endospore staining procedure must be fairly extreme because in addition to being resistant to environmental stresses, it is also resistant to staining. The primary stain, malachite green must be driven into the spore with steam heating. Why is the counterstain necessary in the endospore staining procedure?
d. Be able to recognize an endospore on a very clear stain.

4. Capsule stain

a. Bacterial capsules are composed of polysaccharides or polypeptides forming a well organized layer of material lying outside the cell.
b. Know the functions of a capsule.
c. If viewing capsule stained cells under a microscope, be able to recognize cells that have a capsule. This will require some knowledge of the capsule staining procedure.

B. If given background (e.g. shape, Gram-positive or negative etc..) of an unknown bacterial isolate, be able to use the dichotomous key to determine the possible genus of the unknown.

IV. Lab 5 / lecture 5 : Cultivation of Bacteria I

A. All organisms require the major elements. The CHNOPS acronym will help you remember some of these elements but don't forget that potassium, magnesium, iron and calcium are also major elements. Some elements are required in only trace amounts, these include manganese, molybdenum, zinc, cobalt, nickel and copper. Why are all of the major elements and micronutrients important in the cell. Organisms that can't synthesize their growth factors from the major elements, must have these growth factors provided in the medium.
B. Know the definitions of photoautotroph, chemoautotroph, photoheterotroph and chemoheterotroph.
C . A defined medium has a known (defined composition) whereas an undefined medium is prepared with complex natural extracts and digests of plants, yeasts and beef; its exact composition is unknown. A defined medium is often used to determine an organism’s exact growth requirements. If an organism is incapable of growth on a particular defined medium, it means that this unknown requires growth factors that are not provided by that medium. Be able to pick out an organism for which this is true by comparing its growth an a defined and an undefined agar.
D . A selective medium suppresses the growth of certain bacteria and thus selects for those bacteria whose growth is not suppressed. (e.g. EMB selects for the growth of Gram-negative bacteria by suppressing the growth of Gram-positives. The Eosin Y and Methylene Blue dyes are the selective ingredients in EMB). Two bacteria, one labeled A and the other labeled B, are streaked on and EMB plate. The bacterium labeled A grows well whereas the bacterium labeled B does not grow at all, which one, A or B, is Gram-positive?
E . A differential medium differentiates between different types of bacteria growing on the same medium. This is different than selection because the different types of bacteria all grow (no growth is suppressed) but there is a difference in colony characteristics. The different appearance often tells us that the bacteria are utilizing the medium differently. (e.g. EMB differentiates between bacteria that can ferment lactose (colony is purple and often has a metallic sheen) and those that cannot.) Be able to recognize a lactose fermenting bacteria on an EMB plate.
F . Quantitation of bacteria can be either direct or indirect. What's the difference? Understand that the standard plate count is a direct method for the enumeration of bacteria. What advantages does it have over the other direct method?

1. When we count the number of colonies on a plate, we are determining the number of cells that were plated on the plate BECAUSE 1 COLONY COMES FROM ONE CELL THAT DIVIDES EXPONENTIALLY.
2. Be able to determine the original titer of a sample if given the final plate (with between 30 and 300 colonies) and the dilution scheme. (*Don’t forget the extra practice dilution problems on the web page.)

V. Lab 6 / lecture 6: Cultivation of Bacteria II

A. A pour plate, a spread plate and a streak plate are all methods used to derive pure cultures. A pure culture is a culture that is derived from 1 bacterial cell so it contains only 1 species. Since 1 colony comes from 1 cell that divides exponentially it represents a pure culture (see above). However, if the colony itself is different colors, different consistencies or has an irregular shape then it is probably not a pure colony (it did not originate from a single cell but instead from two or more cells that divided exponentially very close to one another on the agar surface.)
You will need to be able to streak a T-streak plate - demonstrate good technique and get an isolated colony.
B. Environmental Influences

1. Temperature - all organisms have a temperature range over which they can grow. Within this range there are 3 cardinal temperatures: the minimum, the maximum and the optimum. What happens to cells at temperatures below their minimum or above their maximum. *Note - the damage caused by temperatures above the maximum is irreversible.

a. Most bacteria are mesophiles, they grow optimally at temperatures between 20 and 45oC. Most pathogens are mesophiles. Some organisms are thermophiles (heat-lovers) (55-65oC) and some are even hyperthermophiles (70-110�C). Other organisms prefer cold temperatures (psycrophiles (cold-lovers) (< 15oC) and psychrotrophs (prefer temps around 20-30�C but can grow at temperatures as low as 0�C). Psychrotrophs are commonly responsible for the spoilage of refrigerated foods.

b. If viewing a plate with an organism grown at several different temperatures, be able to identify the organism’s optimal growth temperature and into which category (listed above) the organism belongs.

2. pH: Most bacteria grow optimally at pH 5.5-8.0 (neutrophiles). However, some are acid-lovers (acidophiles) pH = 0.1 to 5.5 and some are alkalophiles pH 8.5 - 11.5.

3. Osmotic pressure and water availability -

a. Know the difference between a hypotonic and a hypertonic environment. Most bacteria, fungi and algae have a rigid cell wall that allows them to tolerate and even enjoy a slightly hypotonic environment. Some microorganisms will even synthesize solutes within their cytoplasm in order to maintain a slightly higher cellular solute concentration and keep their membrane forced against their cell wall. Most microorganisms are less appreciative of a hypertonic environment as it commonly results in plasmolysis. *It's important to understand that the higher the concentration of dissolved solutes in the environment, the less water that is available ---> It's not that there's less water there, it's just tied up in interactions with solute molecules. (Think about drinking sea water and the Rhyme of the Ancient Mariner).
b. There are some prokaryotes that can maintain the availability of water even in a hypertonic environment. They do this by increasing the solute concentration within the cell. These prokaryotes are called osmotolerant.
c. Bacteria that require high salt concentrations for growth are called halophiles.
d. Be able to pick out an osmotolerant or halophilic organism by comparing a set of plates containing different solute concentrations.

4. O2 concentration

a. Aerobes require free O2 for growth, whereas anaerobes are killed by or cannot grow in the presence of O2. Some bacteria are aerotolerant anaerobes, they can survive in the presence of O2 but do not use it in their metabolism.
b. A facultative anaerobe does not require O2 for growth, but it grows better when it’s present. Microaerophiles require a low concentration of O2 (5 -10%).
c. Be able to determine whether an organism is an aerobe, anaerobe etc... by looking at the growth pattern in a BHI agar deep.

5. Be familiar with the environmental requirements of the organisms that we worked with in class. (e.g. review pg. 62 and 63 of your notebook).

VI. Lab 7 / Lecture 7: Bioluminescence and pipetmen

A. Be familiar with the biochemistry of the bioluminescence reaction (e.g. understand what reactants are needed (e.g. O2) and that an enzyme (Luciferase) acts as a catalyst.)
B. Luminescence costs Vibrio fischeri a great deal of energy, but allows for the mutually beneficial symbiotic relationship between Euprymna scolopes and Vibrio fischeri. What benefits do each of the organisms get from this relationship?
C. What are the current ideas as to how the relationship between the juvenile squid and the bacteria is established?
D. V. fischeri bacteria only bioluminesce at high concentrations. It is only at these high concentrations that the bacteria can communicate with one another using quorum sensing. Describe the quorum sensing process.
E . Be able to determine what volume a pipetman will dispense by viewing the volume indicator.
F . Remember that a pipetman should not be used to dispense volumes outside of its intended range. (Practice: Which pipetman would be used to dispense 28 microliters?)
G . If asked to pipet a particular volume, know which pipetman to use and be familiar with proper pipetting technique.

VII. Lab 8/ Lecture 8: Control of Microbial growth I

A. UV irradiation

1. UV is a high energy (low wavelength) form of electromagnetic radiation that induces the formation of thymine dimers. These dimers inhibit the accurate replication and transcription of the DNA genome.
2. Some bacteria have evolved mechanisms to repair thymine dimers (photoreactivation and excision repair). What is the difference between these two mechanisms?
3. Be able to identify areas on a plate that have been exposed to UV irradiation. If a colony forms on a region of plate that was exposed to UV, this cell was able to repair the thymine dimers using one of the above repair mechanisms.
4. Understand the limitations of UV irradiation.

B. Antibiotics

1. Substances produced by one living thing to inhibit the growth of another. Realize that this definition is used rather loosely as some antibiotics are either partially or completely synthetic.
2. Study page 81 of the notebook. Be familiar with the antibiotics that we used in class, their sites of action and why they were effective or ineffective on the organisms that we used. Remember that Staphylococcus aureus was originally very sensitive to penicillin but with increased antibiotic resistance, most strains of this organism have become resistant. Focus your study on the antibiotics that were also discussed in lab lecture 8: Naladixic acid, sulfa drugs, polymyxin, penicillin/s, tetracycline, streptomycin, gentamicin and erythromycin.
3. If presented with a Kirby Bauer assay plate, be able to measure the zone of inhibition and use a provided chart to determine if the organism is sensitive, resistant or intermediate.
4. Remember: Antibiotics do not induce mutations, but they can be used to select for them. If a bacterial cell is resistant to a particular antibiotic and it is plated on that antibiotic with a bunch of other bacterial cells that are not resistant, it will have a selective advantage. We demonstrated this in lab by selecting for E.coli cells that were resistant to the antibiotic streptomycin (inhibits protein synthesis by binding to the small ribosomal subunit). This is a spontaneous mutation! What is the other way that a mutation can occur??

C. Bacteriostatic = stops growth, bactericidal = kills and prevents regrowth.
D. Selective toxicity is the ability of a substance to kill one specific organism but not another.

VIII. Lab 9/ Lecture 9: Control of Microbial Growth II

A. Sterilization is the removal of all living cells as well as spores and viroids whereas sanitization means to reduce the microbial populations to levels considered safe by public health standards.
B. Know the difference between an antiseptic and a disinfectant. Be able to recognize a disinfectant in a group of antiseptics and vise-versa.
C. If a disinfectant or antiseptic is strong enough to kill than it may advertise that it is -cidal against what ever type of organism it can kill (e.g. bactericidal, fungicidal etc.)
D. Both phenolic compounds and detergents disrupt microbial membranes and denature proteins. Alcohols also denature proteins and they actually extract membrane lipids. The halogens and hydrogen peroxide oxidatively damage cell constituents.

IX. Lab 10/ lecture 10: Transformation

A. The three mechanisms of bacterial recombination are transformation, transduction and conjugation. Be familiar with the latter 2 but know the details of transformation.
B. In lab 10, we transformed E.coli cells with the plasmid called pGLO.

1. pGLO contains a gene encoding for the beta-lactamase enzyme. Thus, bacteria that are successfully transformed with the pGLO plasmid will produce beta-lactamase and this enzyme will allow them to be resistant to antibiotics containing a beta-lactam ring, such as ampicillin. (If a cell takes up the plasmid (is transformed), it will be resistant to ampicillin).
2. pGLO also contains a gene encoding for the Green Fluorescent protein (GFP). Expression of this protein is under control of the arabinose operon. When arabinose (a sugar) is present, it binds to the DNA binding protein called the Regulatory C protein (AraC). AraC then changes its conformation and allows RNA polymerase to bind and transcribe the GFP gene. (If a cell takes up the plasmid (is transformed) and arabinose is present, the cell will produce GFP and fluoresce under UV light.)
3. Know the purpose of the two negative control plates that were carried through this experiment. Be able to determine which plate has colonies that will fluoresce based on the composition of the plate. Be able to determine which plate shows successful transformants based on its composition.

X. Lab 11/ lecture 11: Bacteriophage

A. A bacteriophage is a virus that uses a bacterium as its host cell.
B. Know the five steps of the bacteriophage replication cycle (lytic) and know all the parts of a T4 bacteriophage.
C. There are two types of bacteriophage: Virulent (multiplies rapidly in host cells and then destroys them by lysis) and Temperate (integrates into the host cell’s DNA = a prophage (the host cells are called lysogenic bacteria), can later take the lytic pathway.
D. Bacteriophage are very small and thus can pass through a filter with pores small enough to exclude bacteria.
E. If virulent bacteriophage are spotted onto a lawn of sensitive bacteria, then they will infect and lyse the host cells. This will create a zone on the plate where no viable bacteria reside. This zone is called a plaque. Be able to recognize a plaque on a bacterial lawn.

XI. Lab / Lecture 12: Titration of Bacteriophage

A. A plaque is a clear zone in the bacterial lawn where a phage has infected and lysed the bacteria.
B. Since one plaque originates from a single bacteriophage, a phage suspension can be diluted, plated in the presence of bacteria, plaques can be counted and the original phage concentration (titer) can be determined. (*Note- This is the same as what was done in lab 5 to determine the concentration of bacteria in an original culture. Both a colony and a plaque originate from a single parent.)

XII. Lab / Lecture 13: Fungus

A. Fungi are eukaryotic chemoorganoheterotrophs that secure their nutrients mostly from decaying organic material (saprophytes).
B. Fungi include both yeasts and molds. Be familiar with the general morphology of these two types of fungi. Recognize that there are two types of hyphae and that individual hyphae can intertwine to form mycelium.
C. Yeast generally reproduce asexually by budding. Molds can reproduce either sexually or asexually. Be familiar with the types of spores involved in asexual reproduction.
D. Be familiar with the six subdivisions of fungi that we discussed: Chytridiomycota, Ascomycota, Basidiomycota, Zygomycota, Glomeromycota and Microsporidia. Focus your study on the three most discussed divisions: Ascomycota, Basidiomycota and Zygomycota
E. Although there are many pathogenic fungi (e.g. the causative agents of Athlete’s foot and ergot), it is also important to remember that without these efficient decomposers the entire ecosystem would collapse.
F. Fungi are important economically, not only as a food source, but also as a source of antibiotics and other medicinal agents.
G. Don’t forget about mycorrhizae, ergot and black truffles.

XIII. General:

A. Aseptic technique - you will need to be competent with aseptic pipetting.
B. Volume conversion (practice: 0.31 mL equals how many microliters? Which pipetman would you use to measure this volume?)
C. Organisms to know (these are included on the microorganism search on the web page): For this exam all you need to know about these microorganisms is what we have learned about them*. To assist in this endeavor, there is a list of organisms after every lab in which they were either introduced or further discussed.

Escherichia coli
Saccharomyces cerevisiae
Bacillus subtilis
Mycobacterium smegmatis (also M. leprae and M. tuberculosis)
Staphylococcus epidermidis
Staphylococcus aureus
Klebsiella pneumoniae
Pseudomonas aeruginosa
Pseudomonas fluorescens
Bacillus stearothermophilis
Campylobacter jejuni
Clostridium sordellii (also C. botulinum and C. tetani)
Vibrio fischeri
Micrococcus luteus
Halobacterium salinarium

*Be familiar with the characteristics of these organisms that were discussed in lab (e.g. Campylobacter jejuni is a microaerophile that is responsible for many cases of food poisoning). Also realize that the names must be either in italics or underlined.

Rachel Watson, M.S.
AG 5010
766-3524
Cell: 307-314-9636
rwatson@uwyo.edu