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SECTION C Answers to Questions on Laboratory Reports

Exercise 1 Brightfield Microscopy A. Short Answer Questions 1. One hand should be under the base of the microscope to support its weight and one hand should be on the arm for balance. 2. The limit of resolution of the unaided human eye is 0.2 mm. For the typical light microscope, the limit is 0.2 m. 3. a. The condenser height and diaphragm can be adjusted. b. Illumination of the specimen is increased when the condenser is raised and the diaphragm is opened. 4. Unlike the voltage control, condenser adjustments will increase illumination without affecting the bulb life. 5. The maximum resolution with the oil immersion lens is achieved by using a layer of oil, using a blue filter over the light source, raising the condenser to its highest point, and opening the condenser diaphragm. 6. The oil immersion lens has the smallest working distance and one runs the risk of striking the slide with the lens when trying to achieve focus. Starting with the low power lens, which has a larger working distance, and progressing up to the oil immersion lens is advised. 7. Oil is used with the oil immersion lens because the small working distance does not allow enough light to enter the lens. The oil, which has the same refractive index as glass, directs more light into the lens. 8. As the power of the objective lens increases, the working distance decreases. B. Matching Questions 1. Oil immersion 2. High-dry 3. Low power 4. Oil immersion 5. Low power 6. Condenser 7. Ocular 8. Ocular 9. Condenser C. True-False Questions 1. False 2. True 3. False 4. False 5. True D. Multiple Choice Questions 1. e 2. e 3. c 4. d 5. a 6. d Exercise 2 Darkfield Microscopy

A. Short Answer Questions 1. Darkfield microscopy is preferred for live unstained specimens or thin cells like spirochetes that are difficult to resolve by staining and brightfield microscopy. 2. Reflection of oblique rays off of objects passes through the lens system. 3. A cardioid condenser allows more light to pass through the lens system and allows use of more powerful objectives. 4. A simple star diaphragm can be made cutting various sizes of round disks of opaque paper and cementing them to transparent celluloid disks that fit in the slot. Exercise 3 Phase-Contrast Microscopy A. Short Answer Questions 1. Staining kills live cells and does not allow observation of movement. 2. Direct rays are produced when light passes straight through a transparent medium without changing amplitude or phase. Diffracted rays are produced when light is bent during retardation by the medium due to density differences and are phase-shifted wavelength. 3. Coincidence results when direct and diffracted waves are brought into phase with one another, where the amplitude is the sum of the two waves, and creates a brighter image. Interference occurs when two waves of equal amplitude are in reverse phase and cancel each other to produce a dark image. Coincidence and interference created greater contrast in specimen being viewed. 4. Bright-phase microscopy produces a brighter image (amplitude summation) with a dark background while dark-phase microscopy produces a dark image (amplitude interference) and a lighter background. 5. Centering telescope or Optovar. B. Multiple Choice Questions 1. d 2. d 3. a 4. d Exercise 4 (CV only) Fluorescence Microscopy A. Short Answer Questions 1. Both are types of photoluminescence. Fluorescence occurs when the excited molecule returns to ground state in less than 104 seconds. Phosphorescence occurs when an excited molecule returns to ground state in more than 104 seconds and has a longer half-life. 2. Shorter wavelength of light equals higher energy. 3. Quenching occurs when fluorescence of a molecule diminishes during prolonged exposure to UV light. 4. Direct exposure to light from mercury vapor arc lamps can damage eyes and the pressurized gas creates the potential to explode. 5. The warm-up period is typically a minimum of 30 minutes where the illumination of the lamp increases to its optimum. 6. A darkfiled condenser provides the best contrast and it deflects the majority of UV rays, which protects the viewers eyes. 7. Low-fluorescing immersion oil should be used and can also be placed between the condenser and the slide. Ordinary immersion oil should be avoided. B. Multiple Choice Questions 1. a 2. a 3. e 4. a

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Exercise 5 (SV 4) Microscopic Measurements A. Short Answer Questions 1. An ocular micrometer has lines separated by an arbitrary distance. A stage micrometer has lines separated by 0.01 mm and is used to calibrate the ocular micrometer. 2. If 2 stage divisions = 0.02 mm = 13 ocular divisions, then 16 ocular divisions = 0.02 mm / 13 x 16 = 0.025 mm. 3. Calibration is necessary for each objective because of the differences in magnification (distances between lines of stage micrometer changes). Exercise 6 (SV 5) Protozoa, Algae, and Cyanobacteria A. Results 1. Sketches of protozoa 2. Sketches of algae 3. Sketches of cyanobacteria B. Short Answer Questions 1. Algae and protozoa are classified in the domain Eukarya. Cyanobateria are classified in the domain Prokarya. 2. a. Algae and plants are both photosynthetic and have cell walls composed of cellulose. b. Algae, however, can be single-celled and motile. 3. Protozoa utilize flagella, cilia, or pseudopodia (amoeboid movement) for motility. Flagella and cilia are fibrous, extracellular, protein appendages that wave. Flagella are longer and singular whereas cilia are shorter and more numerous. Pseudopodia are cytoplasmic projections that enable a crawling motion. 4. a. Cyanobacteria b. Cyanobacteria are prokaryotic cells that, unlike eukaryotic algae, lack a nucleus and chloroplasts, although they do contain chlorophyll. 5. Overgrowths or blooms of microscopic algae cause Red Tides and the cellular pigments are responsible for the oceans taking on a red color. 6. Malaria is caused by the genus Plasmodium, a Protista found in the group Apicomplexa. C. Fill-in-the-Blanks Questions 1. Protozoa = nucleus, flagella, cilia, pseudopodia Algae = nucleus, flagella, photosynthetic pigment(s), chloroplasts, cell wall Cyanobacteria = photosynthetic pigment(s), cell wall 2. Amoeboid cells = pseudopodia Flagellates = flagella Ciliates = Cilia Diatoms = gliding 3. Chloroplastida = chlorophyll a & b, flagella, cell wall, chloroplasts, starch Chrysophyceae = chlorophyll a & c, fucoxanthin, cell wall, chloroplasts, oils, leucosin Euglenozoa = chlorophyll a & b, flagella, chloroplasts paramylon Phaeophyceae = chlorophyll a & c, fucoxanthin, cell wall, chloroplasts, laminarin, mannitol Bacillariophyta = chlorophyll a & c, fucoxanthin, cell wall, chloroplasts, laminarian, oils Dinoflagellates = chlorophyll a & c, fucoxanthin, cell wall, chloroplasts, laminarian, starch, oil, fat Cyanobacteria = chlorophyll a, c-phycocyanin, c-phycoerythrin, cell wall Exercise 7 (SV 6)

Ubiquity of Bacteria A. Results 1. Colony counts 2. Variable answer 3. Variable answer 4. Variable answers B. Short Answer Questions 1. Bacterial colonies are generally smooth and small as compared to fungal colonies, which are large and fuzzy. 2. Since each colony is produced from a single cell, the number of colonies indicates the number of cells originally present or level of contamination. Colony size reflects growth rate or motility. 3. Bacteria, such as the staphylococci and the diphtheroids, are part of the normal skin flora. Molds, however, are likely transient contaminants picked up from the environment. 4. Microbial levels on skin are best controlled by hand washing, on surfaces in the environment with use of disinfectants like bleach, and in the air by HEPA filtration systems. 5. a. Bacteria are smaller, about 0.5 to 10 m in diameter. b. Bacterial DNA is not enclosed in a nucleus but rather is organized in the cytoplasm. c. Bacteria have 70S ribosomes. d. Bacteria have a cell wall composed of peptidoglycan. e. Bacteria lack mitochondria and chloroplasts but can carry out respiration and photosynthesis. f. Bacteria may have flagella that are simpler in structure but may be more numerous. Exercise 8 (SV 7) The Fungi: Yeasts and Molds A. Results 1. Yeast drawings and descriptions 2. Mold drawings and descriptions B. Short Answer Questions 1. Eukaryotic, heterotrophic, lack tissue differentiation, cells walls of chitin or other polysaccharide, propagate by spores. 2. Modern schemes of classification use genetic analysis to determine relatedness between species while traditional schemes rely on morphological characteristics and reproductive mechanisms to determine relatedness. Traditional methods are used in this exercise because examination of morphological characteristics is easily accomplished in the laboratory. 3. Important phenotypic characteristics used to identify fungi include colony appearance, types of hyphae, sexual spores, and asexual spores. 4. Molds are filamentous fungi that produce hyphae while yeasts are fungi that lack hyphae. Yeasts may form reproductive buds called pseudohyphae. 5. Hyphae are microscopic filaments produced by molds and a macroscopic mass of hyphae is called a mycelium. 6. Fungal hyphae are coenocytic if they are unbroken by crosswalls, or septa. These fungi are also termed nonseptate. 7. Asexual spores of fungi include sporangiospores as well as several types of conidia, such as phialospores, blastospores, arthrospores and chlamydospores. 8. Sexual spores of fungi include zygospores, ascospores and basidiospores. 9. Sporangiospores are asexual spores produced within a thin-walled sac called a sporangium. Sporangiospores may or may not be motile. Conidia are asexual non-motile spores that form on specialized hyphae called conidiophores. 10. Macroscopically, bacterial colonies are smoother and shinier (wet looking) than fungal colonies, which generally appear dry and cottony. Microscopically, fungal cells are far larger than bacteria, with a visible nucleus and vacuoles. Morphologically, molds may display hyphae, sporangia and spores while yeast may show budding and pseudohyphae. Bacterial cells appear far more uniform,

with no specialized structures. Exercise 9 (SV 8) Aseptic Technique A. Results 1. Variable answer. 2. Success is presence of growth. 3. Failure is no growth. B. Short Answer Questions 1. When handling microbial cultures, aseptic technique limits contamination of yourself and your workspace with the microbes in the cultures and it limits contamination of your cultures with unwanted environmental microbes. 2. The flame from a Bunsen burner is used to sterilize transfer instruments (e.g. inoculating loop) and is used to flame the opening of the tube after the cap is removed and before the cap is replaced. 3. Since the opening of a plate is not readily flamed, one should hold the lid over the top of the open plate when inoculating so that air contamination is limited. Working near a flame is also useful. 4. Labels should be written on the bottom of the agar plate. 5. Agar plates should be incubated in an inverted position to prevent condensation on the agar surface that could spread the inoculated organisms. 6. Disinfectants, such as bleach and alcohol, are generally useful against vegetative cells and viruses but may not completely eradicate bacterial endospores. C. Multiple Choice Questions 1. e 2. b 3. c Exercise 10 (SV 9) Pure Culture Techniques A. Results 1. Streak plate evaluation 2. Pour plate evaluation 3. Subculture evaluation 4. Variable answer 5. Staining and microscopy or a streak plate can determine purity of a slant culture. B. Short Answer Questions 1. A colony is a visible microbial growth on a solid medium that originated from a single parent and through cell division produced a multitude of identical daughter cells. 2. Useful colony characteristics for differentiation of bacterial species include size, color, shape, texture, opacity, and odor. For example, Serratia marcescens colonies are red, irregular in shape, and have a strong odor. Micrococcus luteus colonies, on the other hand, are yellow, opaque, round, and dome-shaped. 3. When working with culture that may contain millions of cells, dilution on to a solid medium is essential for separating the cell with enough space so that they grow into isolated colonies. 4. The streak plate method does not require any additional media for dilution and only requires one plate for inoculation. 5. The pour plate method requires less skill, has optimization built in, and will more likely produce the desired result. 6. The loop is flamed before entering a culture tube to ensure that no contaminating microbes are introduced in to the culture. The loop is flamed afterward so that no culture microorganisms are introduced into the working environment. 7. Molten agar must be cooled to 50C so that microbes added to the medium will not be killed by

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excessive heat, but it must not be cooled too much because it will solidify before the cells can be dispersed and poured. Also if too hot when poured, excess moisture will form on the lid of the plate. Plates are inverted during incubation so that moisture does not accumulate on the lid and drop on to the agar surface. This will cause the organisms to spread and negate the dilution effect. Also agar plates tend to dehydrate faster in the upright position.

Exercises 1114 (SV 1013) Smear Preparation, Simple Staining, Negative Staining, Capsular Staining A. Results 1. Pleomorphism, metachromatic granules, palisades arrangement (drawing) 2. Oral streptococci are ovoid cell is pairs and chains, yeast cells are larger ovals and may have buds, and spirochaetes are thin spirals with many turns. (drawing) 3. Since the positive stain colors the cell and the negative stain stains the background and neither stain adheres to the capsule, then the contrasting absence of stain around the cell represents the extent of the capsule. (drawing) B. Short Answer Questions 1. Cells from a solid medium are placed in a drop of liquid on the slide and then smeared. This is not necessary for a loop of cells from a liquid culture. 2. If too many cell are used to create a smear, it will be difficult to distinguish individual cell shapes and arrangements from large clumps of cells. Also staining and destaining techniques do not work well on clumped and layered cells in a smear. 3. Heat fixation of smears kills the bacterial cells and causes them to adhere to the glass so that they do not get washed off during staining. Overheating can damage and dehydrate the cells causing them to distort in shape. Also the glass can crack or shatter if overheated. 4. Basic dyes, which carry positive charge (e.g. methylene blue), will adhere to negatively charged cell surface structures (e.g. phospholipids) because of charge attraction. Acidic dyes will not adhere. 5. Darkfield microscopy of unstained cells creates and image most similar to negative staining. 6. Fluorescent dyes linked to antibodies specific to the bacterial capsule can be used to tag and visualize the capsule by fluorescence microscopy. 7. Encapsulated strains of S. pneumoniae are protected against phagocytosis in the host and are more likely to cause infection than unencapsulated strains. C. Matching Questions 1. Simple, capsule 2. Negative, capsule 3. Negative 4. Capsule 5. Negative, capsule 6. Negative (gentle heat for capsule) 7. Simple, capsule D. Multiple Choice Questions 1. e 2. d 3. a 4. d Exercises 1517 (CV 1416) Gram Staining, Spore Staining: Two Methods, Acid-Fast Staining: Ziehl-Neelsen Method A. Results 1. S. aureus should be purple (Gram-positive) cocci in singles, pairs, short chains, and clusters. P.

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aeruginosa should be pink (Gram-negative) bacilli in singles in pairs. Spores should be green and vegetative cells should be pink. Acid-fast cells should be red bacilli and the non-acid-fast cells should be blue cocci.

B. Short Answer Questions 1. Almost all bacteria can be differentiated by Gram stain into the two groups whereas only a very small percentage of bacterial species are either spore formers or acid-fast. 2. A mordant causes the primary stain to adhere better or be taken up by the cell so that it not removed during the decolorizing step. 3. The counterstain must be a different color than the primary stain to aid in differentiation. 4. A Gram-positive cell, which has a thick cell wall, retains the crystal violet-iodine complex better in the presence of the decolorizer as compared to Gram-negative cell, which has a thin cell wall. 5. The decolorizer step is the most critical because it is the step in which the cells become differentiated (Gram-positive cells are purple and Gram-negative cells are colorless). If too much decolorizer is used, Gram-positive cells will lose the primary stain and be counterstained pink. If too little decolorizer is used, Gram-negative cells will not lose the primary stain and will remain purple after counterstaining. 6. Old cultures of Gram-positive cells may not retain stain as well as younger cultures and could give false negative results, i.e. pink cells. 7. Old cultures of spore formers like Bacillus are ideal because under the conditions of nutrient depletion, sporulation is more likely to occur. 8. Smears that are too thick will not stain properly because the stains may not penetrate lower layers and thick clumps of cells may not be easily decolorized. 9. Basic dyes do not penetrate spores so Gram staining will result in colorless spores. This indicates that spores are very resistant structures. 10. Bacillus anthracis spores are ideal for biological weaponry because they are easily produced, can be dispersed in the air, and are environmentally stable. 11. Mycobacterium has a peptidoglycan layer filled with mycolic acids that make the cell wall waxy and impenetrable to stains. They are classified with Gram-positive cells because of cell wall thickness and genetic similarities. 12. The waxy cell wall of Mycobacterium protects the bacterium against phagocytosis and some antibiotics while in the host (e.g. lungs for TB) so the pathogen has greater opportunity to cause disease. C. Matching Questions 1. No color 2. Purple 3. Purple 4. Purple 5. No color 6. Purple 7. Pink D. Fill-in-the-Blanks Questions 1. Gram (crystal violet, iodine, alcohol-acetone, safranin, purple, pink) 2. Spore (malachite green, heat, water, safranin, green, pink) 3. Acid-fast (carbolfuchsin, heat, acid-alcohol, methylene blue, red, blue) E. Multiple Choice Questions 1. a 2. c 3. d 4. d Exercise18 (SV 17)

Motility Determination A. Results 1. Proteus is motile. 2. Drawing. Varible. B. Short Answer Questions 1. A bacterial flagellum is a fibrous appendage composed of protein subunits and anchored in the membrane. Flagella rotate counterclockwise to propel the cell forward and clockwise to tumble and change direction. 2. Peritrichous flagella, which are numerous arrangements across the entire cell surface, are characteristic of highly motile bacteria, such as Proteus. Confirm the flagellar arrangement with a flagella stain. 3. a. Rapid swimming and directional change indicate true motility. b. Jiggling motions without vectorial movement indicate Brownian movement. c. Sweeping motion indicates water currents. 4. Hanging drop slides are more resistant to evaporation, although wet mount slides work better with phase contrast microscopy. 5. A concentration of 0.4% agar is used to make semi-solid media, which when stabbed, allow motile bacteria to swim but hold non-motile in place. Typical solid medium contains 1.5% agar, which when stabbed, would not allow movement of motile bacteria. 6. Exposure of individuals to pathogens is more likely when making wet slides as opposed to stabbing a semisolid medium where the bacteria are contained. 7. SIM is a semisolid medium that will not only show motility (M) of P. vulgaris, but will also show sulfide production (S), which is visualized as a black precipitate that forms in the medium when H2S complexes with iron salts. Exercise 19 (SV 18) Culture Media Preparation A. Short Answer Questions 1. The exact composition of defined media is known whereas it is not known for complex media. Complex media are typically made for rich extracts of meat or plants. Defined media are made by individually measuring out all components. 2. Basic nutritional requirements in all culture media include a carbon source, an energy source, nitrogen, minerals, vitamins, growth factors, and water. 3. Blood 4. Since powdered media lack water, no growth of microbes can occur. After water is added to prepare the media, it must be sterilized to prevent microbial growth. 5. a. Sterilization is the complete removal or destruction of all microbes including bacterial endospores. b. Media are sterilized at 121C with 15 psi of steam for at least 15 minutes. c. Heat-sensitive media additives (e.g. antibiotics or milk) cannot be sterilized by autoclaving. d. Filter sterilization can be used for liquid components that cannot be autoclaved. 6. a. A selective medium allows one type of microbe to grow while another is inhibited. MSA contains 7.5% NaCl, which inhibits growth of most bacteria other than the staphylococci. b. A differential medium displays differences (usually color) between two types of microbes that grow on the medium. MSA contains a pH indicator, phenol red, that changes from pink to yellow if acid is produced, which indicates that fermentation of mannitol has occurred. c. MSA is useful for the isolation of staphylococci and the differentiation of S. aureus (mannitol fermenter) from most other staphylococci (non-fermenters). 7. a. Agar is a complex polysaccharide isolated from seaweed. b. Agar melts at 100C but does not solidify until it reaches 45C, which will not kill microbial cells so inoculation of molten media (e.g. pour plates) is possible. Agar is also relatively inert and is not degraded by most microbes. c. A semisolid medium has an agar concentration of 0.4%, which is in contrast to conventional

solid media that has a concentration of 1.5%. Semisolid media can be used for motility studies because they allow movement. B. Multiple Choice Questions 1. b 2. c 3. a 4. d Exercise 20 (CV only) Preparation of Stock Cultures A. Results 1. Drawings 2. Drawings 3. Drawings B. Short Answer Questions 1. Aseptic technique prevents contamination of stock cultures. 2. A working stock culture is useful because during multiple rounds of culture it may become contaminated, so the clean reserve stock will still be available to make a new working stock. 3. Glycerol is added to stocks to prevent water crystallization during freezing, which would damage the cells. 4. Lyophilization removes water from the cell during freezing; therefore physiological or mutational changes are unlikely to occur over time. These stocks can be kept for longer periods of time and be revitalized with use of sterile water and plating on sterile media. 5. B. megaterium is viable when left at room temperatures because it is an endospore former. Exercise 21 (SV 19) Cultivation of Anaerobes A. Results 1. Tube inoculations 2. Plate inoculations E. coli: facultative anaerobe E. faecalis: aerotolerant organism S. aureus: facultative anaerobe B. subtilis: aerobe C. sporogenes: aerotolerant anaerobe C. beijerinckii: obligate anaerobe 3. Spore drawings B. Short Answer Questions 1. Oxygen acts as a terminal electron acceptor during respiration. 2. In the absence of oxygen, anaerobic respiration or fermentation occurs. 3. Obligate anaerobes lack superoxide dismutase, which converts toxic superoxide to a harmless compound, and catalase, which converts toxic peroxide to oxygen. 4. Microaerophiles require low amounts of oxygen for metabolism although higher concentrations are lethal. Aerotolerant organisms can grow in oxygen but do not use it for metabolism and are thus indifferent to its presence. 5. Reazurin is an indicator for the presence of oxygen. 6. Brewers anaerobic agar in petri plates provides growth of organisms in an oxygen-rich environment on the surface of the medium; therefore the GasPak anaerobic jar is necessary to remove the oxygen for anaerobic growth. Anaerobes grow in fluid thiglycollate medium because in the depths of the tube the conditions are anaerobic; therefore the GasPak is unnecessary.

Exercise 22 (SV 20) Enumeration of Bacteria: The Standard Plate Count A. Results 1. Quantitative Plating Method 2. Optical Density Enumeration B. Short Answer Questions 1. Not all colonies are formed from individual cells but rather arrangements of cells (e.g. chains or clusters), so cfus are not necessarily equivalent to number of cells. 2. Plate 0.1 ml of the culture to inoculate the plate for a 1:10 dilution and plate 0.1 ml of a 1:10 culture dilution to for a 1:100 dilution. 3. A plate count is a viable cell count whereas turbidimetry only provides information on cell density, dead or alive. Exercise 23 (SV 21) Slime Mold Culture A. Results 1. Observations 2. Observations B. Short Answer Questions 1. The classification of slime molds has been difficult because they share characteristics of both the fungi and protozoa. 2. a. The plasmodium is the assimilative stage. b. A sclerotium forms from the plasmodium when the moisture and temperature conditions become less than ideal. c. Sporangia are fruiting structures that form under conditions similar to that of sclerotium. d. Flagellated swarm cells form when environmental conditions improve. e. A zygote form from swarm cells that acts as isogametes, which unite and eventually give rise to another plasmodium. Exercise 24 (SV 22) Slide Culture: Molds There is no laboratory report for this exercise. Exercise 25 (SV 23) Determination of a Bacteriophage Titer A. Results 1. Varibale 2. Variable 3. Variable B. Short Answer Questions 1. Phage attach to carbohydrate groups, e.g. LPS on gram-negative cells. 2. Viruses must invade a host cell in order to replicate. 3. Lysogenic phage can lead to altered host cell genetics. 4. Lysogenic conversion is the insertion of the phage genome into the host cell chromosome. 5. Flies are coprophagous (dung-eating) insects and feces have high levels of intestinal bacteria, e.g. E. coli. 6. Plaques and colonies are used for the enumeration of phage and bacteria, respectively. Plaques, however, are the absence of cells (i.e. lysis), whereas colonies are masses of cells (i.e. growth).

Exercise 26 (CV only) Burst Size Determination: A One-Step Growth Curve A. Results 1. Plaque counts 2. Dilution interpretation a. 5 x 108 b. 1 x 109 c. 50 d. 2 x 108 e. 2 x 107 f. 1:50 g. 5 x 106 B. Short Answer Questions 1. Burst size is the average number of mature phage particles released by the lysis of a single cell. 2. Multiplicity of infection (m.o.i.) 3. Phage adsorption was stopped by dilution. 4. E. coli without phage was plated as a negative control to show that there was no endogenous phage contamination that might skew results. Exercise 27 (SV 24) Isolation of Phage from Flies A. Results 1. Plaque Size Increase 2. Observations B. Short Answer Questions 1. Flies deposit their eggs in fecal material, which is heavily populated by E. coli. The young larvae feed, grow, pupate, and emerge as adult flies that carry E. coli as well as the parasitic phages in their guts. 2. Raw sewage 3. a. The capsid is the coating the surrounds and protects the nucleic acid whereas the sheath is a hollow tube connected to the capsid and is used for injection of nucleic acid into the host cell. b. Lysis is the break down of the host cell to release new phage particles. Lysogeny is the insertion of phage DNA into the host cell chromosome. c. Virulent phages cause lysis whereas temperate phages cause lysogeny. 4. Lysozyme Exercise 28 (SV 25) Phage Typing A. Results 1. Variable 2. Variable B. Short Answer Questions 1. Phage tail fibers and host cell carbohydrates determine specificity. 2. Phage typing can identify a single bacterial strain as the cause of an outbreak. 3. Lysogenic phage can alter the genetics of a bacterial cell by insertion of phage DNA into the host chromosome. This inserted DNA may carry a gene for producing virulence factors, such as toxins. Bacterial DNA also may be packaged into phage particles and transduce a new host. This DNA may carry a gene for resistance to antibiotics, which may easily spread to pathogens and make them more difficult to fight.

Exercise 29 (SV 26) Temperature: Effects on Growth A. Results 1. Observations a. Drawings b. 25C c. The enzyme that produces the pigment, prodigiosin, is inhibited at higher temperatures. 2. Tabulations a. Data chart b. Graph c. S. marcescens should grow best at 25C and E. coli at 37C. d. More precise results could be acquired by incubating the bacteria at a greater number of different temperatures. B. Short Answer Questions 1. Optimal growth temperatures a. Hyperthermophile > 80C b. Thermophile 50C to 80C c. Mesophile 20C to 50C d. Psychrophile 5C to 20C 2. Psychrophiles grow best at low temperatures whereas psychrotrophs are mesophiles that are capable of growth at lower (e.g. refrigerator) temperatures. 3. Refrigeration is used to control spoilage microbes in food but psychrotrophs, such as Pseudomonas, are still capable of growth at these temperatures. 4. Most human pathogens grow best at 37C, which is body temperature. 5. Temperature affects metabolism by influencing enzymes, cell membranes, and ribosomes. Exercise 30 (SV 27) Temperature: Lethal Effects A. Results 1. Tabulation 2. Tabulation B. Short Answers 1. The control plate with bacteria not exposed to high temperatures is used for comparison and to ensure that the bacteria are capable of growth. 2. If the thermometer was in a culture tube it would interfere with the subsequent transfers of culture to plates across the time intervals. 3. B. megaterium is a mesophile and in its vegetative state it would be killed at high temperatures. The endospores it produces, however, are extremely heat resistant (thermoduric) and allow the bacteria to survive until plating and incubation at an optimal temperature. 4. Endospores are more resistant to heat because they have a: a. tough spore coat b. low moisture content c. large amount of calcium and dipicolinic acid 5. Diseases caused by spore-forming bacteria include: a. Anthrax b. Tetanus c. Botulism d. Gas gangrene 6. Heating conditions used to destroy endospores include: a. Dry heat, 170C, 2 hours b. Steam 121C at 15psi, 15 minutes c. Flame inoculating tool, 1900C, until glowing red, 10-15 seconds.

Exercise 31 (SV 28) pH and Microbial Growth A. Results 1. Tabulation 2. Growth curves 3. S. cerevisiae 4. A. faecalis 5. E. coli B. Short Answers 1. Extremes of pH may affect the solubility of molecules and cause proteins to denature and lose enzymatic activity. 2. Microbes can be classified as acidophiles, neutrophiles, and alkalinophiles. 3. Sugar fermentation often results in the production of acids, which would lower the pH of the culture medium. Urea hydrolysis results in ammonia production, which would result in a rise in pH of the medium. Exercise 32 (SV 29) Water Activity and Osmotic Pressure A. Results 1. Tabulation 2. a. E. coli b. S. aureus c. H. salinarum 3. Obligate halophile B. Short Answer Questions 1. Bacteria have cell walls that resist osmotic pressure that causes cells to swell. Animal cells do not have cell walls and will burst under hypotonic conditions. 2. Hypertonic environments cause water to diffuse out of a cell and cause the cytoplasm to shrink away from the cell wall (plasmolysis) and damage the metabolic machinery of the cells. 3. Staphylococci are halotolerant and can exist on the skin, which is hypertonic because of salts contained in perspiration. 4. Halophiles require high salt concentrations for growth. Osmophiles, in contrast, grow in environments where sugar concentrations are excessive, so they are more likely to cause spoilage of jam and jellies that have high sugar concentrations. Exercise 33 (SV 30) Ultraviolet Light: Lethal Effects A. Results 1. Tabulation 2. Variable 3. The index card blocks the UV rays and provides direct comparison to the side that is exposed. 4. The ability of plastic, even clear, to block UV rays is demonstrated. B. Short Answer Questions 1. UV radiation causes the formation of thymine dimmers in DNA that result in replication errors during reproduction. These error cause gene defects, which ultimately lead to cell death. 2. Endospores are non-dividing cells and are not subject to the same effects as reproducing vegetative cells. 3. UV radiation is most germicidal at 260 nm because that is the wavelength at which DNA maximally absorbs UV light.

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Cell protection against UV damage include the repair mechanisms, such as the SOS system, which enzymatically removes dimmers and inserts in their place new pyrimidine molecules. UV radiation can cause damage to skin cells and can cause skin cancers. It also can cause cataracts and eye injury. In this experiment, goggles are worn to protect the eyes and one should avoid looking directly at the UV light source. In everyday life, wearing long clothing, a hat, and/or sunscreen will protect against the damaging UV rays of the sun. A B. megaterium culture in stationary phase will contain more endospores as nutrients become limited and therefore would demonstrate the best survival following exposure to UV radiation.

Exercise 31 (SV only) Oligodynamic Action A. Tabulation of Results B. Questions 1. Answer will depend on metals used. 2. Answer will depend on metals used. 3. Answer will depend on metals used. 4. Heavy metals combine with SH, sulfhydryl groups in proteins, thus disrupting protein structure. This would be especially damaging to enyzmes. Exercise 34 (CV only) The Effects of Lysozyme on Bacterial Cells A. Results 1. Tabulation 2. E. coli, a gram-negative bacterium, should be more resistant to lysozyme than S. aureus, a grampositive bacterium, because the gram negative has an outer membrane that limits access of the lysozyme to the peptidoglycan layer. 3. No lysozyme was added to one quadrant to serve as a negative control and show that the bacteria are growing normally. B. Short Answer Questions 1. Peptidoglycan comprises the cell wall of bacteria and protects against osmolysis. 2. Lysozyme degrades the beta (14) bond between the sugar molecules in peptidoglycan, which weakens the cell wall. 3. Lysozyme provides protection against bacteria in eggs, in eyes (tears), on skin (perspiration), and tissues (tissue fluids). 4. Gram-negative bacteria, such as Haemophilus, may cause eye infections because they are less sensitive to the effects of lysozyme found in tears. Exercise 35 (SV 32) Evaluation of Alcohol: Its Effectiveness as an Antiseptic C. Results 1. Tabulation 2. Alcohol should reduce the level of most skin contaminants. 3. Swabbing should be more effective than dipping, because in addition to the killing action of alcohol, swabbing physically also removes microbes. 4. Gram-positive micrococci are the most common bacteria found on skin. Some may reside in pores or crevices and may not have been exposed to the alcohol treatment. D. Short Answer Questions 1. Alcohol is useful for preparing the skin before insertion of a needle or catheter. In sanitizing gel, it is useful for general antisepsis of hands. 2. For antisepsis of skin, alcohol has advantages over soap because it is a killing agent, does not

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require water, and does not need to be wiped off because it readily evaporates. A sterile environment is not created by alcohol treatment of skin because bacteria hide in the pores, cracks, and crevices of skin and may be protected from exposure.

Exercise 36 (SV 33) Antimicrobic Sensitivity Testing: The Kirby-Bauer Method A. Results 1. Tabulation 2. Answers vary according to antimicrobials used. B. Short Answer Questions 1. a. Antibiotics are natural compounds produced by microbes whereas antimicrobial drugs are synthesized in a lab. b. Broad-spectrum antibiotics, e.g. ciprofloxacin, are effective against a wide range of grampositive and gram-negative bacteria whereas narrow-spectrum antibiotics may only work against a small class of bacteria, e.g. penicillin against gram-positive bacteria. 2. The size of the zone of inhibition for a particular antibiotic is influenced by diffusibilty of the agent, size of the inoculum, type of medium, and resistance mechanisms of the test organism. 3. Although the diameter of the zones of inhibition for two antibiotics is identical, resistance determination is determined by a cutoff number (see Table 36.1), which varies between antibiotics. 4. Gram-negative bacteria have an outer membrane that limits penetration of many antibiotics that attack cytoplasmic targets. 5. Gram-positive bacteria have thick cell walls that maintain cell stability even when the plasma membrane is disrupted by polymyxin B. 6. Even when dealing with an organism with intermediate or moderate resistance to an antibiotic, increasing the dosage of the antibiotic may effectively counteract the resistance and successfully treat the infection. Exercise 37 (SV 34) Evaluation of Antiseptics: The Filter Paper Disk Method A. Results 1. Tabulation 2. Variable 3. Variable 4. Pseudomonas is very resistant to most disinfectants. B. Short Answer Questions 1. Antiseptics, such as alcohol or iodine, are used on living tissues (e.g. skin). Disinfectants, such as bleach, are used on inert surfaces (e.g. tabletops). Most antiseptics (e.g. alcohol, iodine, peroxide) can be used as disinfectants, but most disinfectants are not recommended for use on skin (e.g. bleach, Lysol, ammonia). 2. The size of the zone of inhibition is influenced by the molecular weight of the agent and its rate of diffusion in the agar. 3. Gram-negative bacteria have outer membranes that may limit penetration of chemicals that harm cytoplasmic targets. Exercise 38 (SV 35) Effectiveness of Hand Scrubbing A. Results 1. Tabulation 2. Graph 3. Handscrubbing will not completely free hands of microorganisms. Scrubbing may uncover larger numbers of resident microflora. The first 69 minutes of scrubbing removes the majority of microbes, so excessively long scrubbing times are not particularly useful. 4. A higher count may be due to uncovering resident microflora.

B. Short Answer Questions 1. Diphtheroids, staphylococci, and yeasts. 2. Normal skin flora is beneficial to the host because they compete against pathogens for space and nutrients. 3. S. aureus 4. Normal flora is always found on skin. Transient flora consists of temporary contaminants of skin typically picked up through contact with contaminated items. 5. Gloves can become punctured or contain unseen defects, so it is important that surgeons continue to scrub in prior to surgery. Exercise 39 (SV 36) Morphological Study of Unknown Bacterium No laboratory report. Information recorded on descriptive chart. Exercise 40 (SV 37) Cultural Characteristics No laboratory report. Information recorded on descriptive chart. Exercises 4143 (SV 3840) Physiological Characteristics: Oxidation and Fermentation Tests, Hydrolytic and Degradative Tests, Multiple Test Media A. Results Results placed in the descriptive chart. B. Short Answer Questions 1. Morphological and cultural characterizations are important to help with preliminary classification and help one decide which tests are appropriate for further identification, rather than to run every test available. 2. Genes are recipes for making cellular proteins, including the enzymes that regulate the cells metabolism. Therefore, species with similar enzymatic capabilities will likely have similar genetic makeup. 3. Bacterial exoenzymes are used for the extracellular degradation of substances so that the simpler compounds can be taken into the cell as nutrient or energy sources. 4. a. Anabolism is the energy-driven synthesis of complex molecules from simpler ones. Catabolism is the energy-releasing degradation of complex molecules into simpler ones. b. Fermentation is the metabolism of sugars in the absence of oxygen whereas respiration is a process that does utilize oxygen. 5. Both are gram-positive cocci; however, staphylococci are catalase positive and streptococci are catalase negative. 6. a. Acids and alkalis are detected with use of pH indicators, which are colored dyes that change color in response to pH changes. b. Acid end products are a result of various sugar fermentations and fat hydrolysis. c. Alkaline end products are a result of urea hydrolysis, citrate utilization, and proteolysis (e.g. litmus milk and Kliglers iron agar). 7. a. Carbon dioxide and hydrogen gas may be produced by sugar fermentation. b. Gas can be detected in a Durham tube as a trapped air bubble or as an air pocket in a stabbed solid medium (e.g. Kliglers iron agar). c. The catalase test yields oxygen gas as a positive result. 8. Hydrolysis of casein in milk is readily visualized as the opaque milk agar shows clear zones of hydrolysis. Because starch is not readily visible in starch agar, iodine is added to form complexes with starch and reveal the clear zones of hydrolysis. Detection of fat hydrolysis is performed indirectly. Instead of visualizing the absence of the substrate (fat) of hydrolysis, spirit blue agar contains a pH indicator that reveals the release of fatty acids, the byproduct of fat hydrolysis.

9. 10.

11.

12.

13.

Proteolytic digestion is demonstrated in the litmus milk reactions and the alkaline reaction in Kliglers iron agar. a. A positive triglyceride hydrolysis test is expected for P. acnes because it feeds on sebum (oil) in clogged sebaceous glands. b. A positive urea hydrolysis test would be expected for H. pylori because one of the byproducts of this reaction, ammonia, will neutralize harmful stomach acid and allow the bacterium to survive in this hostile environment and cause infection. a. Both include a test for hydrogen sulfide production. b. Both media contain cysteine, an organic sulfur-containing substrate, and ferrous salts, which complex with hydrogen sulfide to produce a black precipitate for identification. c. SIM is a semi-solid medium that is stabbed to determine motility. Kliglers iron agar is a solid medium that is stabbed to create an anaerobic environment to test for glucose fermentation (acid and/or gas production). Differentiation of bacteria based upon physiological tests can be difficult because many tests (numerous inoculations and media preps) need to be conducted in order to find the few differences that closely related organisms might have between them. Further identification of microbes entails genetic, antimicrobial resistance, and immunological tests.

C. Matching Questions 1. Media 2,3-butanediol fermentation (b) carbohydrate fermentation (a, b, c) casein hydrolysis (f) citrate utilization (e) hydrogen sulfide production (a, d) mixed-acid fermentation (b) triglyceride hydrolysis (g) tryptophan degradation (d, h) 2. Reagents 2,3-butanediol fermentation (a, i) catalase test (e) mixed-acid fermentation (g) nitrate reduction (b, j) oxidase test (h) phenylalanine deamination (c) starch hydrolysis (d) tryptophan degradation (f) 3. Enzymes casein hydrolysis (d) gelatin liquefaction (d) hydrogen sulfide production (b) indole (e) starch hydrolysis (a) triglyceride hydrolysis (c) urea hydrolysis (f) 4. Products catalase (e) phenylalannine deamination (f) triglyceride hydrolysis (c) tryptophan degradation (d) urea hydrolysis (b) Voges-Proskauer test (a) Exercise 44 (SV 41)

Use of Bergeys Manual No laboratory report. Exercise 45 (SV 42) Enterobacteriaceae Identification: The API 20E System A. Results 1. Tabulation 2. Profile 3. Identity of unknown 4. Tabulation B. Short Answer Questions 1. The advantages are ease of use, a single inoculation, self-containment, numerous tests, little media preparation, rapid results, reliability, uniformity, and simple interpretation. The disadvantages are having to confirm questionable test results and multitest systems are designed for a particular medically important group of pathogens and are not available for a wide variety of microbes. 2. The oxidase test is done to confirm that the unknown is a member of the family Enterobacteriaceae, which test negative for oxidase. 3. API 20E vs. Enterotube II a. More time is necessary for preparation of the API 20E strip but incubation periods and results interpretation times are similar. b. A saline suspension of cells is prepared for the API 20E system. No specimen preparation is needed for the Enterotube II system. c. The API 20E system includes 23 biochemical tests whereas the Enterotube I system only contains 15. d. Anaerobic conditions are achieved with an overlay of mineral oil on certain chambers. This is not necessary in the Enterotube II system where chambers are sealed with wax. e. Interpretation of results for both systems procede by tabulating test results and constructing a numerical profile (7 digits for API 20E and 5 digits for Enterotube II) that is identified in an interpretation guide. 4. Contaminated or mixed cultures as well as non-enterobacteria (e.g. oxi/ferm bacteria) may provide nonsense profiles that cannot be found. Exercise 46 (SV 43) Enterobacteriaceae Identification: The Enterotube II System A. Results 1. Tabulation 2. Identification 3. Interpretation Guide 4. Tabulation B. Short Answer Questions 1. The advantages are ease of use, a single inoculation, self-containment, numerous tests, little media preparation, rapid results, reliability, uniformity, and simple interpretation. The disadvantages are having to confirm questionable test results and multitest systems are designed for a particular medically important group of pathogens and are not available for a wide variety of microbes. 2. The oxidase test is done to confirm that the unknown is a member of the family Enterobacteriaceae, which test negative for oxidase. 3. API 20E vs. Enterotube II a. More time is necessary for preparation of the API 20E strip but incubation periods and results interpretation times are similar. b. A saline suspension of cells is prepared for the API 20E system. No specimen preparation is needed for the Enterotube II system.

4. 5.

6. 7.

The API 20E system includes 23 biochemical tests whereas the Enterotube I system only contains 15. d. Anaerobic conditions are achieved with an overlay of mineral oil on certain chambers. This is not necessary in the Enterotube II system where chambers are sealed with wax. e. Interpretation of results for both systems procede by tabulating test results and constructing a numerical profile (7 digits for API 20E and 5 digits for Enterotube II) that is identified in an interpretation guide. All enterobacteria ferment glucose and produce acid (2) and sometimes gas (3). A 0 as the first number of the code indicates non-enterobacteria, i.e. not a glucose fermentor. Gram-negative bacilli that do not ferment glucose include Pseudomonas, Plesiomonas, Vibrio, Achromobacter, Moraxella, Pasteurella, Aeromonas, Alcaligenes, and Bordatella. These bacteria are likely oxidase positive and should be identified with the Oxi/Ferm Tube II system (see Exercsie 47). The VP test is utilized when the 5-digit code provides more than one species and the interpretation guide indicates that the VP test can differentiate between the choices. Contaminated or mixed cultures as well as non-enterobacteria (e.g. oxi/ferm bacteria) may provide nonsense profiles that cannot be found.

c.

Exercise 47 (SV 44) O/F Gram-Negative Rods Identification: The Oxi/Ferm Tube II System A. Results 1. Tabulation 2. Tabulation B. Short Answer Questions 1. The advantages are ease of use, a single inoculation, self-containment, numerous tests, little media preparation, rapid results, reliability, uniformity, and simple interpretation. The disadvantages are having to confirm questionable test results and multitest systems are designed for a particular medically important group of pathogens and are not available for a wide variety of microbes. 2. If the gram-negative bacillus is oxidase positive or not a glucose fermentor, it is not a member of the Enterobacteriaceae. 3. Contaminated or mixed cultures as well as non-oxi/ferm bacteria (e.g. enterobacteria) may provide nonsense profiles that cannot be found. Exercise 48 (SV 45) Staphylococcus Identification: The API Staph System A. Results 1. Tabulation 2. Profile 3. Final determination B. Short Answer Questions 1. The advantages are ease of use, a single inoculation, self-containment, numerous tests, little media preparation, rapid results, reliability, uniformity, and simple interpretation. The disadvantages include having to confirm questionable test results and multitest systems are designed for a particular medically important group of pathogens and are not available for a wide variety of microbes. 2. To confirm that bacteria are staphylococci, a Gram stain (gram-positive cocci in clusters) and a catalase test (catalase positive) should be performed. 3. The coagulase test confirms the identity of a staphylococcal isolate as S. aureus, which is coagulase positive. 4. Contaminated or mixed cultures as well as non-staphylococci (e.g. streptococci) may provide nonsense profiles that cannot be found.

Exercise 49 (CV only) Isolation of an Antibiotic Producer: The Actinomyces A. Results 1. Leathery appearance 2. Variable 3. Halo of no growth around actinomyces colony (zone of inhibition) 4. Variable 5. Variable B. Short Answer Questions 1. Microbes produce antibiotics to help them compete against the numerous other microbes in an environment such as soil. 2. Some microbes (e.g. fungi) produce antibiotics (e.g. protein synthesis inhibitors) against a different class of microbes (e.g. bacteria), therefore, the antibiotic attacks the proper target (e.g. bacterial 70S ribosome) but does not affect the homologous host enzyme (e.g. fungal 80S ribosome). Some microbes also have developed resistance mechanisms to protect themselves against their own antibiotic compounds. 3. Fungi, such as Penicillium (penicillin) and Cephalosporium (cephalosporins), and bacteria, such as Streptomyces (streptomycin) and Bacillus (bacitracin), produce antibiotic compounds. 4. S. aureus is typically resistant to numerous antibiotics and would therefore not be a good choice as a test organism. Exercise 50 (CV only) Nitrogen Cycle: Ammonification A. Results 1. Tabulation B. Short Answer Questions 1. Nitrogen, which is contained in amino acids for cellular proteins and nucleotides for genetic material, is essential for cell growth and reproduction. 2. a. Ammonification is the deamination of amino acids, nucleic acids, and uric acid to produce ammonia. b. Nitrification is the oxidation of ammonia to produce nitrites and nitrates. c. Denitrificaiton is the reduction of nitrate in several steps to produce nitrogen gas. d. Nitrogen fixation is the conversion of nitrogen gas into ammonia. 3. Amino acids from proteins, nucleotide bases from nucleic acids, and uric acid from animal wastes 4. Bacillus, Pseudomonas, and many other decomposing bacteria Exercise 51 (CV only) Symbiotic Nitrogen Fixation: Rhizobium A. Results 1. Bacteroids should be pleomorphic club-shaped cells (see Figure 51.1). 2. Pleomorphism is a variation in cell shape. Variable. B. Short Answer Questions 1. Symbiosis is a nutritional relationship that benefits both parties. Rhizobium forms nodules on the roots of legumes. The plant supplies carbohydrates for the bacteria to make ATP and reducing power to fix nitrogen and the bacteria fix nitrogen gas to produce ammonia, which is used by the plant for biosynthetic purposes. 2. Specificity is defined by binding proteins of the bacteria (e.g. ricadhesin) that attach to calcium complexes on the root hair. Carbohydrate-binding lectins may also play a role. 3. Leghemoglobin, a pink biosynthetic product of both bacteria and plant, is an oxygen buffer that protects oxygen-sensitive nitrogenase but provides enough oxygen for bacterial metabolism.

Exercise 52 (CV only) Free-living Nitrogen Fixation: Azotobacter A. Results 1. Descriptions B. Short Answer Questions 1. Oxygen causes irreversible denaturation of nitrogenase, which is unusual because Azotobacter is a strict aerobe and requires oxygen for metabolism. 2. To protect the oxygen-sensitive nitrogenase, there are three levels of protection. Respiratory protection involves the high utilization of oxygen by Azotobacter that limits stray oxygen from contacting nitrogenase. Conformation protection involves a protein that combines with nitrogenase to protect it from oxygen. Slime layer production limits entry of oxygen into the cell. 3. Nitrogen salts would allow any bacteria that can utilize the primary energy source to grow since a nitrogen source is provided. Without nitrogen, only bacteria that can fix nitrogen will grow. 4. Once the aerobe Azotobacter utilizes most of the available oxygen, conditions are created that are ideal for growth of the free-living, nitrogen-fixing, obligate anaerobe, Clostridium. Exercise 53 (CV only) Denitrification: Paracoccus denitrificans A. Results 15. Variable descriptions B. Short Answer Questions 1. Denitrification of the nitrate ion yields nitrogen gas. 2. Denitrification occurs under anaerobic conditions with organic acids, such as succinate, as a carbon source. 3. Denitrifying bacteria are costly to farmers because they deplete soils of ammonium nitrate from fertilizers. 4. Denitrifying bacteria are essential to life on Earth because they return nitrogen to the atmosphere so that it does not become depleted. 5. Denitirifcation benefits the organism because it serves as a terminal electron acceptor for respiration in the absence of oxygen. Exercise 54 (CV only) The Winogradsky Column Lab reports follow Exercises 55 & 56. Exercise 55 (CV only) Purple Nonsulfur Photosynthetic Bacteria A. Results 13. Variable observations B. Short Answer Questions 1. Anoxygenic photosynthetic bacteria do not produce oxygen from splitting water during photosynthesis. Reducing power comes from hydrogen, reduced forms of sulfur, or organic acids. They grow in anaerobic environments. 2. Cellulomonas degrades cellulose in the column producing glucose that can be used by itself and other organisms for fermentation, which produces organic acids in the column. Clostridium ferments monosaccharides producing organic acids such as lactate and acetate. Desulfovibrio uses sulfate as a terminal electron acceptor in anaerobic sulfate respiration to produce hydrogen sulfide.

3. 4.

The Green Bacteria and some Purple Non-Sulfur Bacteria can use the latter as a source of reducing power in anoxygenic photosynthesis. Chromatium stores elemental sulfur granules in side the cell. Chlorobium stores elemental sulfur outside the cell. Algae: oxygenic photosynthesis; 6CO2 + 12 H2O > C6H12O6 + 6H2O + 6O2 Chromatium and Chlorobium: anoxygenic photosynthesis; CO2 + H2S > C6H12O6 + SO42

C. Tabulation of Results D. Conclusions Exercise 56 (CV only) Sulfate-Reducing Bacteria: Desulfovibrio A. Microscopic Observations B. Short Answer Questions 1. Reduction of sulfate by Desulfovibrio yields metal sulfides, which are black in color, and hydrogen sulfide gas, which imparts a rotten egg smell. 2. Sulfate is more plentiful in marine environments. 3. By doing two enrichments, one can achieve almost pure cultures of Desulfovibrio. The isolates can be checked for purity by streaking onto fresh water enrichment medium and incubating under anaerobic conditions. Exercise 57 (CV only) Bacterial Commensalism Results and questions located at the end of Exercise 59. A. Results 1. Tabulation B. Short Answer Questions 1. S. aureus is a facultative anaerobe and can grow readily in the nutrient broth tube. C. sporogenes is an obligate anaerobe and does not grow well in nutrient broth except at the very bottom of the tube where oxygen is limited. 2. S. aureus utilizes the oxygen in the nutrient broth and created anaerobic condition, which are ideal for the growth of C. sporogenes. The beneficiary of this commensal relationship, C. sporogenes, offers no benefit in return to S. aureus. Exercise 58 (CV only) Bacterial Synergism Results and questions located at the end of Exercise 59. A. Results 1. Tabulation B. Short Answer Questions 1. Gas production is checked individually for organisms to see which organisms are capable of this production on their own. If two non-gas producers are combined and produce gas, then synergism is evident. E. coli should produce gas on its own for both sugars. 2. Acid production is a measure of fermentation since not all microbes produce gas. If a combination of microbes produces acid but no gas, then no synergism has taken place. 3. a. P. vulgaris cannot ferment lactose by itself but will produce gas by the fermentation of glucose. S. aureus can ferment lactose to produce acid but not gas. Therefore, S. aureus will

breakdown lactose into glucose and galactose and P. vulgaris will produce gas during glucose fermentation. b. Requires results. Exercise 59 (CV only) Microbial Antagonism A. Short Answer Questions 1. Requires results 2. Requires results 3. Gram-negative bacteria often do not allow penetration of antibiotics, such as penicillin, through the outer membrane. 4. Microbial antagonism provides a competitive advantage for the antagonistic compound producer. 5. a. Foods that are produced by microbial fermentation are protected from spoilage by the organic acids produced. The acidic pH prevents growth of other microbes. b. Bacterial infections are often treated with antibiotic compounds that are isolated from antagonistic microbes. 6. Some microbes (e.g. fungi) produce antibiotics (e.g. protein synthesis inhibitors) against a different class of microbes (e.g. bacteria), therefore, the antibiotic attacks the proper target (e.g. bacterial 70S ribosome) but does not affect the homologous host enzyme (e.g. fungal 80S ribosome). Some microbes also have developed resistance mechanisms to protect themselves against their own antibiotic compounds. Exercise 60 (SV 46) Bacterial Counts of Foods A. Results 1. Tabulation B. Short Answer Questions 1. Most likely ground meat because it is fresh and has not been frozen, which may damage some cells as water crystals form. 2. Most likely dried fruit because of the lack of water available to contaminating microbes. 3. a. During the slaughter of animals, the digestive tract may be opened and release coliform bacteria on to the meat and other surface the meat contacts. Likewise, fecal contamination is prevalent in slaughterhouses. b. Numerous bacteria of animal intestinal origin are human pathogens, such as Salmonella, Shigella, and E. coli O157:H7. c. Steaks have bacterial contamination on their surfaces. These bacteria are readily heat-killed early during cooking. Hamburgers, however, are made of ground beef, where bacteria from the surface of the meat have been mixed into the center of the patty. These bacteria are only heat-killed when the center of the patty reaches a certain temperature. d. To prevent foodborne illness, raw meat should be refrigerated until it is ready to be cooked, cutting boards and utensils should not be shared between food items, countertops should be disinfected and hands should be washed after handling raw meat, and the meat should be cooked until the center reaches the proper temperature. 4. Frozen foods should not be thawed over long periods of time at room temperature or in warm water because these temperatures allow growth of spoilage microbes as well as pathogens. Thawing overnight in the refrigerator or rapidly in the microwave oven is recommended. One should also be aware of contaminated liquids or juices from the foods being released as thawing proceeds and being transferred to surfaces and utensils. 5. Some microbes, including spoilage microbes such as Pseudomonas and foodborne pathogens such as Listeria, are able to grow and reproduce at refrigerator temperatures. 6. Dried fruits do not have enough water content to support most types of microbial growth. They also have a high sugar content (hypertonic environment) that also suppresses growth.

Exercise 61 (SV 47) Bacteriological Examination of Water: Qualitative Tests A. Results 1. Tabulation 2. Tabulation 3. Tabulation B. Short Answer Questions 1. No, a positive presumptive test indicates that the water might be unsafe to drink but further testing is warranted. 2. A false positive presumptive test may be due to two or more non-coliform bacteria working synergistically to produce sufficient gas in the tube. 3. a. Easy to identify b. Present only in sewage, not in soil c. Lives slightly longer than pathogens in water 4. Typhoid fever, bacillary dysentery, and cholera 5. Amoebiasis caused by E. histolytica, cryptosporidiosis caused by C. parvum, and giardiasis caused by G. lamblia 6. Since pathogens are so hard to find in water, it would be like looking for the needle in a haystack. 7. Coliform bacteria are gram-negative, facultative anaerobic, non-endospore forming bacilli that ferment lactose to produce acid and gas. 8. a. Detect gas production from lactose fermentation b. Inhibition of gram-positive growth and lactose fermentors will produce nucleated colonies (black centers) and may have a metallic sheen c. To make a slide for Gram staining to confirm morphology (gram-negative bacilli) 9. The IMViC tests (indole, methyl red, Voges-Proskauer, citrate) are used to confirm the identity of the isolates as coliforms. These tests will differentiate E. coli, which is associated with sewage, from E. aerogenes, which is not always associated with sewage. Exercise 62 (SV 48) The Membrane Filter Method A. Results 1. Tabulation B. Short Answer Questions 1. The membrane filter method takes less time and only one type of medium is needed for identification of coliforms. 2. The amount of water filtered depends on the water quality. No less than 50 ml should be used. Low turbidity water with few bacteria may require more than 200 ml to be filtered. In order to get an accurate count (neither too few nor too many colonies) on the test medium, the correct volume of water should be filtered. 3. Viruses are smaller than the pore size of the filter and will pass through. A filter with a smaller pore size can be used to achieve sterilization.

Exercise 63 (SV 49) Reductase Test A. Results B. Short Answer Questions 1. Large numbers of actively growing bacteria in milk will create a lowered oxidation-reduction potential due to the exhaustion of dissolved oxygen. Rubber stoppers on the tubes are used because if oxygen from the atmosphere is introduced into the milk, it will alter the test results (false negative). 2. No, milk with a short reduction time is safe to drink if no pathogens are present. 3. Resazurin, tetrazolium salts, neutral red 4. The reductase test takes less time and requires less skill. 5. Psychrophiles, thermophiles and thermodurics would not give a positive reductase test. Exercise 64 (SV 50) Microbial Spoilage of Canned Food A. Results 1. Tabulation of observations 2. Microscopic drawings B. Short Answer Questions 1. B. coagulans and B. stearothermophilus 2. C. thermosaccharolyticum 3. C. sporogenes 4. No 5. Endospores germinate in canned or plastic sealed food. Bacteria produce exotoxin (botulinum toxin) in the food under anaerobic conditions. 6. Spoilage is more likely with home canning because of a lack of knowledge about the causes of spoilage and how to prevent them, carelessness in handling raw materials prior to canning, equipment malfunction that leads to undetected underprocessing, and defective containers that allow entry of microbes after processing. Exercise 65 (SV 51) Microbiology of Alcohol Fermentation A. Results B. Short Answer Questions 1. Simple sugars in grape juice are fermented. Fermentation produces ethanol and carbon dioxide. 2. Expansion of the balloon demonstrates production of carbon dioxide gas. The rubber stopper would trap gas, cause the formation of gas bubbles in the wine, and yield sparkling wine or champagne. 3. Acetobacter contamination would result in acetic acid production, which would impart a sour taste to the wine. This change can be measured as a lowered pH of the wine. This process is performed in the production of vinegar (dilute acetic acid). 4. Hydrogen sulfide production would impart an undesirable flavor and odor (rotten eggs). 5. Starches from the flour in bread dough are used for fermentation. Ethanol and carbon dioxide are produced. Carbon dioxide gas causes the bread dough to rise before baking and ethanol evaporates during baking. Lactobacillus sanfrancisco bacteria ferment sugars in the bread dough to produce acid, which is responsible for the taste of sour dough bread. 6. Fermentation contributes to food preservation by creating an acidic and reduced environment that inhibits the growth of spoilage microbes. Exercise 66 (CV only)

Mutant Isolation by Replica Plating A. Tabulation of Results B. Results 1. Variable 2. Variable 3. Variable C. Short Answer Questions 1. The ratio of resistant bacterial colonies to susceptible ones is representative of the rate of spontaneous mutations in bacteria that result in resistance. Exposure of bacteria to low levels of mutagens (e.g. UV light) will increase the mutation rate. Higher levels of mutagen can be detrimental by causing too many mutations. 2. Bacteria can acquire antibiotic resistance genes through natural genetic transfers such as transformation, transduction, and conjugation. 3. By overusing and misusing antibiotics (e.g. antibiotics for viral infections, not completing a course of antibiotics, continuous antibiotic prophylaxis, antibiotics in animal feed), humans have contributed to the evolution of resistant bacterial populations. 4. A mutation in one of the bacterial ribosomal protein genes is likely to protect against streptomycin, a protein synthesis inhibitor. Exercise 67 (CV only) Bacterial Transformation A. Results B. Short Answer Questions 1. Plating ten times more cells should yield ten times more Apr colonies. 2. The no plasmid sample as a negative control should yield no Apr colonies. If Apr colonies do appear, it is likely that the student accidentally plated plasmid transformed cultures on no plasmid plates or cross-contamination occurred during the experiment between the no plasmid and plasmid samples as a result of student using the same pipet tips when adding competent cells to the tubes containing plasmid vs. no plasmid, or the student accidentally contaminated the samples during plating of the cells on plates by forgetting to sterilize the cell spreader between plates. 3. Transformation efficiency is influenced by the concentrations of DNA and cells, the plasmid-tocell ratio, the method used to achieve competence (calcium chloride vs. electroporation), the size of the plasmid, and the age of the cells. 4. a. Transformation is the transfer of naked DNA into the cell. b. Transduction is the bacteriophage-mediated transfer of DNA. c. Conjugation is the transfer of plasmid DNA from one cell to another through a sex pilus. 5. Competence is the ability to take up naked DNA from the environment. Bacteria such as Streptococcus pneumoniae are naturally competent and pick up DNA from the environment without assistance. Other bacteria such as E. coli, which cannot pick up DNA on their own, can be made competent artificially by chemical or electrical means. 6. By overusing and misusing antibiotics (e.g. antibiotics for viral infections, not completing a course of antibiotics, continuous antibiotic prophylaxis), humans have contributed to the evolution of resistant bacterial populations among their own normal flora. Because pneumococci are naturally competent, they may pick up resistance factors from normal flora bacteria. If these transformed pneumococci cause infection, they will be more difficult to treat with antibiotics. 7. Transformation of plasmid DNA is useful for gene cloning in bacteria. Exercises 6869 (CV only) Polymerase Chain Reaction for Amplifying DNA, Plasmid Isolation

A. Results B. Short Answer Questions 1. The most likely explanation is that this bacterium has the ampicillin resistance gene integrated in the chromosome and is not maintained on a plasmid. Thus, the gene could be amplified in a PCR reaction but plasmid cannot be isolated. 2. The strength of hydrogen bonds between base pairs of double-stranded DNA is influenced by temperature. When temperature is increased to 95C, hydrogen bonds will be broken and complementary DNA strands will separate in a process called denaturation. When the temperature is reduced to 56C, hydrogen bonds will form between primers and their complementary DNA sequences in a process called annealing. 3. Because the high denaturation temperature would inactivate normal DNA Polymerase, new enzyme would have to be added to the reaction tube after annealing of primers during each cycle of amplification. This process would be time and enzyme consuming. 4. Primers serve as a starting point for DNA polymerization by the Taq Polymerase. Primers must be included at a high enough concentration to drive the annealing of primers to template rather than the rehybridization of the template strands. 5. Precautions must be taken not to introduce to your PCR tubes outside sources of DNA that might be amplified or interfere with amplification of the proper target. 6. Forensic analyses often involve small amounts of evidence (e.g. a small drop of blood or a single hair follicle) and little DNA with which to work. PCR can be used to amplify the available DNA for analysis. DNA contamination is an issue because the contaminant may be amplified in addition to the target and produce unreliable results. 7. DNA is a negatively charged molecule that will migrate to the positive pole of the gel. Because the agarose gel has pores that allow movement of the DNA through the solid matrix, the migration of larger molecules will be impeded whereas smaller molecules will migrate more freely. Therefore, DNA fragments will separate according to their relative sizes. 8. Different forms (e.g. supercoiled, open circle, or linear) of a plasmid migrate differently and will show up as multiple bands for a single plasmid following gel electrophoresis. 9. Ethidium bromide intercalates or inserts between DNA bases. When exposed to UV light, ethidium bromide fluoresces, which facilitates DNA detection in an agarose gel. This agent is also a carcinogen because insertion into cellular DNA results in replication errors and introduces mutations that may transform human cells into cancer cells. Exercise 70 (SV 52) The Staphylococci: Isolation and Identification A. Results 1. Tabulation 2. Microscopy 3. Tabulation 4. Tabulation 5. Final determination B. Short Answer Questions 1. As a selective medium, MSA has a high concentration of salt (7.5% NaCl), which is inhibitory to most bacteria other than the staphylococci. As a differential medium, MSA contains the sugar mannitol as a substrate for fermentation and phenol red as a pH indicator to detect the production of acid. S. aureus ferments mannitol whereas most other staphylococci do not. 2. Virtually all strains of S. aureus are coagulase positive whereas all other species are negative. 3. S. aureus produces coagulase during infection to surround itself in a coat of clotted blood protein, which protects it against host defenses as the bacterium multiplies in the host. 4. Alpha toxin is an exotoxin that destroys red blood cells to release nutrients and growth factors that increase S. aureus multiplication in the host bloodstream. 5. Nosocomial infections are infections acquired in a hospital or healthcare setting. 6. S. aureus is an opportunistic pathogen that causes infections of compromised hosts (e.g. surgical

patients, burn and trauma patients) in the hospital. Thirty percent of the normal population carries S. aureus so endogenous infections in patients and transmission from healthcare worker to patient are common. 7. Numerous strains of S. aureus has developed multiple resistances to common antibiotics including penicillins and aminoglycosides and the resulting infections have become difficult to treat effectively. 8. Proper hand washing, personal protective equipment (PPE), antisepsis, disinfection, sterile environments, and isolation are employed to prevent staphylococcal infection in the hospital. 9. At-risk patients are tested for S. aureus nasal carriage because of the risk for endogenous infection. 10. Hospital workers are tested for S. aureus nasal carriage because of the risk for transmission to vulnerable patients or to track down the source of an outbreak. Exercise 71 (SV 53) The Streptococci: Isolation and Identification A. Results 1. Tabulation 2. Microscopy 3. Tabulation 4. Tabulation 5. Final determination B. Short Answer Questions 1. Stabbing of the blood agar creates an anaerobic environment for the streptococci and improves the development of hemolysis. 2. Alpha hemolysis is partial lysis of red blood cells and will create a halo of greenish discoloration around colonies on the blood agar. Beta hemolysis is complete lysis of red blood cells and will create a clear halo around colonies on the blood agar. 3. Lancefield classified streptococci into several major groups (e.g. group A, B, and C) based upon surface carbohydrate antigens (Lancefield antigens). 4. a. Beta hemolysis b. S. pyogenes (group A strep) c. Strep throat is treated with antibiotics; however, viruses, which will not respond to antibiotic therapy, cause most cases of pharyngitis. 5. a. Both are beta-hemolytic, gram-positive cocci b. Differentiation can be achieved with use of the catalase test (streptococci are catalasenegative), gram staining (streptococci form chains), immunological tests, and/or a salt tolerance test (streptococci are less tolerant). 6. Lancefield-antigen testing (S. pyogenes is group A, S. agalactiae is group B), bacitracin susceptibility (S. pyogenes is susceptible, S. agalactiae is resistant), and CAMP reaction (S. pyogenes is negative, S. agalactiae is positive) 7. Optochin susceptibility (S. pneumoniae is susceptible, oral viridans streptococci are resistant) and bile solubility (S. pneumoniae is soluble, oral viridans streptococci are insouluble) 8. Enterococci are tolerant to 6.5% NaCl whereas other group D streptococci are not. 9. SXT sensitivity (group A strep are resistant, group C strep are sensitive) 10. S. pneumoniae 11. S. agalactiae (group B strep) 12. Viridans streptococci, such as S. mutans, create dental caries by fermenting sugars in the oral cavity and producing acids that erode tooth enamel. Exercise 72 (SV 54) Gram-Negative Intestinal Pathogens A. Results

1. 2. 3.

Variable Variable Species determination can be made with a multitest system, such as the API 20E (Exercise 42, SV 39) or Enterotube II (Exercise 43, SV 40) systems.

B. Short Answer Questions 1. Salmonella, Shigella, and pathogenic strains of E. coli 2. To prevent infection and illness by enteric pathogens, water sanitation, proper food handling and preparation, and proper personal hygiene are important. 3. Bile salts and/or desoxycholate are added to media to select against gram-positive growth. 4. Salmonella and Shigella do not ferment lactose. MacConkey agar, Hektoen-Enteric (HE) agar, and Xylose Lysine Desoxycholate (XLD) agar all differentiate between lactose fermentors and non-fermentors. 5. Salmonella are motile and produce hydrogen sulfide. Shigella are non-motile and do not produce sulfide. SIM medium is ideal for differentiation because it simultaneously tests for both characteristics. HE agar, XLD agar, and Kliglers iron agar include a sulfide test but not one for motility. 6. Proteus is the most similar to the SS pathogens. Urea hydrolysis is used for differentiation. Proteus is positive and Salmonella is negative. 7. RDS and Kliglers iron agar slants contain glucose and lactose. The slant is stabbed and then streaked with the isolate. The streak is an aerobic environment and the bacteria preferentially ferment lactose. The stab is an anaerobic environment and the bacteria preferentially ferment glucose. 8. Alkaline reversion occurs when bacteria ferment sugars in differential media (e.g. RDS agar) to produce acid, which results in a color change, and then after the sugar is consumed, the bacteria metabolize proteins and produce ammonia, which raises the pH of the medium and reverses the color change. In order to read results accurately, the media cannot be incubated too long; otherwise, a false-negative result will be produced. Exercise 73 (SV 55) Slide Agglutination Test: Serological Typing Results and Questions are located at the end of Exercise 74 (SV 56). A. Results 1. Variable 2. Variable B. Short Answer Questions 1. Proteins, polysaccharides, lipoproteins and nucleoproteins. 2. Antibodies. 3. A group of bacteria possessing a common set of antigens. Serotypes of a particular bacterial species may be physiologically identical but differ in their antigenic makeup. 4. The formation of visible clumps which appear when antibodies react with the antigens present on a bacterial cell. 5. The negative control is a mixture of antigen and saline (antibody is missing) and so will not agglutinate. The positive control contains both antibodies and antigens and will agglutinate. 6. Controls allow the comparison of an experimental result to a fully agglutinated result (positive control) and a non-agglutinated result (negative control). Exercise 74 (SV 56) Slide Agglutination (Latex) Test: For S. aureus Identification A. Results 1. Variable 2. Variable

B. Short Answer Questions 1. Coagulase and protein A 2. S. aureus is the only staphylococcal species positive for the coagulase tube test, which is demonstrated by the clotting of plasma. There is 97% correlation with the agglutination test. 3. The agglutination test is more rapid and also tests for a second antigen of S. aureus. 4. Results occur more rapidly and are much easier to visualize than ordinary precipitin-like reactions that demonstrate the presence of soluble antigen. Exercise 75 (SV 57) Tube Agglutination Test: The Heterophile Antibody Test A. Results 1. Variable 2. If the titer is 320 or higher, the result is considered significant. B. Short Answer Questions 1. Infectious mononucleosis (IM) 2. Epstein-Barr virus (EBV) 3. 80%90% of all adults possess antibodies for EBV. 4. Heterophile antibodies are produced against one antigen but also react with tissues of other organisms. The basis for this test is the fact that EBV antibodies agglutinate sheep red blood cells. 5. Microscopic analysis of blood would reveal moderate leukocytosis with a marked increase in the number of lymphocytes (50% to 90%). Exercise 76 (SV 58) White Blood Cell Study: The Differential WBC Count A. Results 1. Tabulation B. Short Answer Questions 1. Variable 2. Mistakes in differentiating the types of leukocytes, particularly the neutrophils and eosinophils, and accidentally retracing the path used to count cells might cause tabulation errors. 3. Cellular immunity involves the action of the T lymphocytes, which directly target infected cells for destruction. Humoral immunity involves the action of antibodies produced by B lymphocytes. 4. Cellular and humoral immunity act in concert, where helper T cells stimulate and coordinate the functions of both B cells and cytotoxic T cells. 5. a. Neutrophilia is an increase in neutrophils and indicates the occurrence of localized infections. b. Neutropenia is a decrease in neutrophils and occurs in typhoid fever, undulant fever, and influenza. c. Eosinophilia is an increase in eosinophils and indicates allergic conditions or invasions by parasitic roundworms. d. Lymphocytosis is an increase in lymphocytes that accompanies whooping cough and some viral infections. e. Monocytosis is an increase in monocytes that accompanies infectious mononucleosis. Exercise 77 (SV 59) Blood Grouping A. Results 15. Variable answers dependent upon results B. Short Answer Questions 1. Antibodies against the antigens of the incompatible blood cells will cause agglutination and the

2. 3. 4.

cells will be targeted for destruction. Individuals with type AB blood are known as universal recipients because they do not produce antibodies to the A and B antigens so they can accept any blood type (AB, A, B, or O). Individuals with type O blood are universal donors because their RBCs have neither the A nor B antigen so individuals of any blood type (AB, A, B, or O) will not reject type-O blood. An Rh-negative mother will begin to produce antibodies against the Rh antigen if she is carrying a Rh-positive fetus. If she becomes pregnant a second time with a Rh-positive child, her antibodies will target the fetus RBCs for destruction. This condition is known as erythroblastosis fetalis. There is a treatment available (RhoGAM) that suppresses this phenomenon.

Exercise 78 (SV 60) A Synthetic Epidemic A. Results 1. Tabulation B. Short Answer Questions 1. Variable 2. Variable 3. Transmission of respiratory viruses, in addition to direct contact transmission (e.g. hand shaking), may include droplet transmission and indirect transmission by contact with fomites. 4. Washing hands 5. a. Abstinence or barrier methods of prophylaxis (e.g. condoms) will limit transmission of gonorrhea, a sexually transmitted disease. b. Limiting contact with infected individuals (e.g. kissing) and fomites (e.g. drinking glasses) will limit transmission of infectious mononucleosis, which is spread through saliva and oral secretions. c. Proper handling and preparation of meats, particularly poultry, and other food items will limit transmission of salmonellosis, a foodborne illness. d. Isolation, facemasks, and monitoring for exposure (e.g. PPD skin test) will limit transmission of tuberculosis, which is spread by respiratory droplets. e. Controlling mosquito populations by removing standing water and spraying pesticides and limiting contact with mosquitoes with use of bug repellant or long clothing will limit transmission of West Nile encephalitis, a vector-borne disease.