Exercise 1. Microscope: Parts of the microscope: 1.

. Oculars (eye piece): lenses that magnify 10X, on our microscopes used in class 2. Ocular width adjustment: adjust for your eye span. For the Zeiss microscope, width adjustment is made by separating oculars to correct distance. Once this separation is made the distance between the oculars should be set on each collar to make the microscope parfocal. This adjustment must be done each time you use your scope. The leica and leitz microscopes do not need this adjustment. 3. Revolving nosepiece: can be rotated to change from one object to another. 4. Objectives: three or four magnifications. 4X (very low power), 10X (low power), 40X (high power), and 100X (oil immersion). Immersion oil used with the 100X objective has a refractive index approximately the same as glass- light passes through without being lost or bent. 5. Stage adjustment controls: moves the slide on two horizontal planes- either back and forth or side to side 6. Mechanical stage: provides movable platform for slide, enables to change field of view. 7. Iris diaphragm or aperture diaphragm: opens and closes diaphragm to control the amount of light striking the object. Improper illumination is common problem, as the light must be increased as magnification increases. 8. Condenser: condenses light waves into a pencil shaped cone, thereby preventing the escape of light waves. Its ideal position is just beneath the stage. 9. Condenser knob: raises and lowers condenser. For this course the condenser knob should be all the way up. 10. Light control: turns light source on and off. The degree it is turned on determines the intensity of light. 11. Light source: uses visible light to provide illumination for sample. 12. Coarse adjustment: moves stage up and down rapidly for purposes of approximate focusing. Should only be used when focusing with low power objectives. 13. Fine adjustment: moves the stage up and down very slowly for purpose of definitive focusing. Compound microscope: have 2 types of lenses- the ocular and objective lenses. These lenses provide a vital function for the microscopes which is magnification (enlargement of objects) and resolution (being able to distinguish structures that are separated short distances. Resolution is more important that magnification. Resolution is dependent on the length of the light waves used for illumination and the quality of lenses. Resolution is inversely proportional to wavelength. The ordinary light microscope with oil immersion objective can distinguish or resolve 2 points approximately 0.2 microns apart- defined as limit of resolution. Parfocal: refers to the microscopes ability to remain in approximate focus when switching from one objective lens to another. Working distance: refers to the distance between the specimen and objective lens (when going from 10X to 100X, the working distance shortens from 8.3mm to 0.14mm. Refractive index: refers to how light will travel through a certain medium. The main effect of immersion oil is to collect aberrant light rays and allow them to enter the objective. Aberrant light rays are those that would otherwise be lost due to diffraction. Without oil, light rays are diffracted as it passes through the glass slide into the air and not all light enters the objective, so oil is used because it has the same refractive index as glass so diffraction of light will not occur. Total magnification: is the product of the objective lens and of the ocular lens. (either 4X, 10X, 40X, or 100X multiplied by the ocular 10X) Wet mount: made by suspending microbes in liquid and covering the drop with a cover slip. ADVANTAGES: allows the viewer to study live microorganisms. DISADVANTAGES: it dries

out after several minutes, so it can only be used to short periods of time, and only observed using the 10X or 40X objectives. Fixed smear: fixing and staining bacteria to slide (permanent- with cover slip/ semipermanent- without cover slip) ADVANTAGES: use for longer periods of time, and use oil immersion. DISADVANTAGES: not live bacteria. Advantages of phase contrast microscope: it is a type of light compound microscope that separates two lights into two phases that cancel each other to form as image. This allows viewers to observe details in umpigmented living organisms. How to take care of microscope: clean all oil off the 100X with lens paper ONLY and lens cleaner. If you got oil on 40X, contact instructor, clean off dirt or oil on stage, leave low power objective in place, wrap cord around base, place in cabinet, sign and date sheet in cabinet. Never go back to 40x objective once oil has been added to the slide. Exercise 2. Simple stains: Simple stain: one in which one stains is used to create a contrast between the specimen and its background. Used to give info about shape, size, and arrangement. The simple stain is positive if the specimen is stained and background isnt. Different morphologies of bacteria: bacillus, coccus, or spiral. Cell arrangments for cocci or spheres: 1. Pair of cocci stuck together- diplococci 2. Chains of four or more organisms- streptococcus arrangement 3. Irregeular groups of microorganisms resembling grape clusters- staphylococci arrangement 4. Packets of 4 cells- tetrads Rod shaped bacteria known as bacilli can be found in chains or very short rods. These cell arrangements are not as constant as cocci arrangments and therefore shouldnt be considered a characteristic of a particular species. Spiral shaped: 1. Spirochetes- rods with one or more several curves 2. Spirilla- are rigid rods possessing one or several curves 3. Vibrios- are short, curved bacteria with one curve (coma looking) Variations in shapes and sizes of a bacterial species are frequently seen and the term pleomorphism is the term used to denote these variations within a species. Exercise 3. Transfer of bacteria: Aseptic technique: is the transfer of culture and media without contamination by unwanted organisms. 1. Flame inoculating loops and needs to a cherry red to sterilize and prevent contamination from the environment. Flame the entire part of the needle or loop that will be in contact with culture and medium. 2. Flame mouth of all glass tubes and bottles immediately after lid is removed and before the lid is replaced. This sterilizes the mouth of the tube/bottle and creates an updraft of warm air that will prevent contaminants from falling into the open container. 3. Never turn a lid upside down or set on the tabletop since this will allow debris and microorganisms to fall into the lid and possibly the culture. Lids should be held in your hand with the inside facing down. 4. Hold all tubes at an angle to prevent debris and microorganisms from falling into the tubes. 5. Inoculating loops, needles, swabs, pipettes, and syringes that are employed in the mechanical transfer of the cultures should be sterile to prevent environmental contamination. 6. Sterile pipettes should not be touched at the end or come in contact with any surface to prevent environmental contamination. Discard items in lab: 1. Used slides- cleaned with detergent and returned to slide container. 2. Used cover slips- go in cardboard box for glass waste. 3. Used razor blades/pins- go in metal can for contaminated glass waste. 4. Plastic petri plates- go into autoclave bag on discard table. 5. Plastic swabs, pipettes- go into autoclave bag on discard table. 6. Glass pipettes- should be placed in a plastic containers located on lab bench. 7. Culture tubes- go in wire baskets on discard table 8. Glass bottles- go into metal containers on discard table. 9. Glass petri plates- go into metal container at discard table. 10. Uncontaminated paper towels- in metal container on lab bench 11. Black wire racks- returned to lab shelves, colored racks remain on lab bench. To properly label an agar plate, you put your initials and seat number on the agar side of the plate and place in the incubator agar side up. This will help prevent the lid from falling off as

well as keep track of whose agar plate is whose. If you label the lid, and it comes off, no one will know which agar side belongs to you. And agar side up so no contaminants on lid will fall into agar. Exercise 4. Environmental sampling: RODAC plate: used to obtain a direct environmental sample. It is a special plate in which the agar has been slightly overfilled. The agar side is pressed lightly on the surface being samples, the lid is put back on and the plate is incubated. Organisms on the surface sampled adhere to the agar and grow as discrete colonies that are counted. RODAC stand for Replicate Organism Detection and Counting. The limitation of the RODAC plate is that is can only be used successfully on flat, impervious surfaces that are free of crevices. Swab technique: a sterile swab is moistened with a special phosphate buffer diluent solution and is then used to wipe irregular surfaces. The swab is then returned to the tube with the buffer solution and transported back to lab. The solution keeps the organisms in an osmotically correct environment so that they remain viable during transportation, however the nutrients are not available so growth will not occur. So the liquid must be plated, incubated and counted. How to interpret the results: a plate with less than 100 colonies indiciates a surface with acceptable sanitation. For the swab and buffer, you count the colonies as well but you must account for standarization by multiplying the number of colonies on the plate by 10. Exercise 5. Differential stains: Steps to making a bacterial smear: 1. Clean slide with detergent and dry with paper towels, then heat dry over Bunsen burner 2. Prepare a smear by taking a loop full of broth culture, using aseptic technique, and spread it on the slide. 3. First allow the smear to air dry, and then briefly wave the slide over the Bunsen burner, with the bacteria side up. (if the bacterial smear is not completely dry when passed through the flame, the bacteria will boil and explode). 4. Gram stain in following order: cover smear with crystal violet for 1 minute, thoroughly rinse with water, then cover bacteria with iodine for 1 minute and rinse, decolorize the smear by holding the slide at an angle over the sink and apply acetone-alcohol drop by drop. Broth cultures take about 5-8 drops and if made from colony, it will take more. Counterstain smear with safranin for 1 minute. Differential stain: employs the use of two or more dyes to distinguish organisms based on cell characteristics or structures. A gram stain is a differential stain. Gram stain: by using crystal violet and iodine (mordant) together they combine to form an insoluble colored compound in the microbes being stained. This compound is called crystalviolet iodine complex. acetone-alcohol is used to decolorize the microbe. Organisms that resist decolorizing and retain the crystal-iodine complex appear purple and are gram positive. If the cell does decolorize or give up the crystal-iodine complex more quickly and accept the safranin will appear pink or red. These are gram negative. Gram positive bacteria have a thick homogenous layer of peptidoglycan while gram negative have a thin layer of peptidoglycan plus an additional outer membrane. The peptidoglycan acts as a barrier act as a barrier, preventing the loss of crystal violet. Once the iodine is applied, which acts as a mordant by chemically combining with the crystal to form a large complex. this encourages retention of the crystal violet. The third step, action alcohol, causes the wall of gram positive bacteria to shrink and lock in the crystal violet. As for gram negative, the acetone dissolves some proteins in the membrane, thus making pores for the crystal violet to escape. The last step only affects gram negative, in that it now stains the clear bacteria pink so they can be seen. Possibly reasons for variable gram stains: 1. Improper heat fixing of the smear- if a smear is heated too much, the cells can rupture. 2. Cell density of smear- an extremely thick smear may not colorize or decolorize as rapidly as one of ordinary density, leading to inconclusive results. 3. Length and thoroughness of washing after crystal violet- if Crystal violet remains on the slide after iodine is assed, a false gram negative will result can be obtained because crystal violet will combine with iodine and precipitate off slide. 4. Age of bacteria culturegram reactions are only reliable for cultures that are 24 hours old or less. Gram + cultures

are older than 24 hours will have increased breaks in their cell wall and thus may convert from an original gram + to gram reaction. Gram variable reaction: a mixture of gram + and gram bacteria on a slide. Mechanism of different differential stains: 1. Flagella- uses mordant to thicken the flagella so they can be seen with the microscope. The mordant is mixed with the basic dye fuchsin and then a counter stain- crystal violet is used. 2. Capsule: is a thick polysaccharide layer located outside the cell wall. They are hard to stain and are usually observed with negative stain, where the background is stained with an acidic stain ad the cell is stained wit a basic stain. The acidic stain (called congo red) stains the background because it is repulsed by the negative charges of the cell wall. The basic stain (fuchsin) stains the cell because its attracted to the negative charge on the cell wall. The capsule remains unstained and is seen as a bright translucent areas around cell. 3. Endospore: are heat and chemical resistant structures. Heat is used to expand the endospore coat, allowing stain (malachite green) to enter the endospore. The cells are then cooled, trapping the dye in the endospore. Cells are rinsed and counter stained by safranin. 4. Fat body: are inclusions inside bacteria. Fat bodies have a big affinity for basic dyes and appear darker when stained than the other parts of the cell. Exercise 6. Pure cultures: streak plates: Purpose of a streak plate: to obtain a pure culture. It is used to isolate a pure culture when more tan one culture is present and also to verify that a particular culture is pure. Pure culture: is a group of bacteria that is of one species. Steps to streak plating: 1. Pick up bacteria using a sterilized loop, streak them thickly on the top 1/3 of your plate. 2. Flame the loop, touch the hot loop to agar to cool before going across the previous streak, then streak section 2 in a zig-zag fashion. 3. Flame the loop. Cool ad streak from section 2 to 3, only going across your second streak once and streak in section 3 in a discrete zig-zag fashion Exercise 7. Quantification of bacteria: How to set up dilutions for bacterial quantification: in order to set up proper dilutions, a dilution scheme must first be made. Once you reach an appropriate dilution concentration, the dilution is plates onto PCA plates with sterile pipettes and spread with hockey sticks, incubated at 37C, and then the number of colonies are counted. The most common dilutions are 1/10 (where 1ml of sample goes into 9 ml of dilutent) or 1/100 (1 ml of samples goes into 99ml of dilutent). These are used to make CFU/mL calculations less formidable. In order to calculate CFU/ml: multiply the number of colonies counted by (1/dilution). For example, 50 colonies counted in 10E-7 dilution= 50X10(7)= 5.0x10(8) CFU/mL Petrifilm: contains dehydrated medium that needs to be inoculated with one full milliliter of sample to rehydrate the medium. The advantages of are its convenience, ease of use, small size, and stability. The disadvantages are its cost, its restriction to 1 mL of sample and lack of significant colony morphology. There are different varieties of of petrifilm each designed to grow a particular group of organisms. Colonies appearance on aerobic plate count and coliform (used in class): the APC had more colonies that were small and pinpoint. The CC with the pink background had little red dots that were thicker and irregular Exercise 8. Scientific writing: Sections of scientific writing: 1. Title- should be brief, but must clearly and sufficiently reflect its content. The title may state the subject of the article or it may give the articles major conclusions. 2. Abstract/summary- is informative. It must allow the reader to understand the essence of he authors research without having to refer to the article. Specific detail of data are given, but methodology is no unless its unique. An abstract should be brief but have major conclusions. 3. Introduction- includes brief background of the relevant published literature. It usually ends with a statement of the hypothesis the research was designed to solve. 4. Materials and methods- should be written in enough detail to allow another investigator to duplicate the experiments. New methods are described completely and sources of unique chemicals and equipment are stated. Standard methodologies are not

explained. 5. Results- is presented in a sequence that logically supports or rejects the hypothesis. Illustrations and tables should be accompanied by a title and an informative legend. Extensive interpretation of data is not given in this section. 6. Discussion- of the results and the conclusions drawn from the data includes interpretation of its meaning. It should also address any discrepancies between these results and other papers. 7. References- that are cited in the text are listed in a style dictated by the journal. In this class, we will use the style of the American society for microbiology. Where references are cited within the paper, only the number is used. References are numbered in alphabetical. Reference: citations are arranged in alphabetical order by first author (last name, first initial) and numbered consecutively. The name of all authors are provided, but subsequent authors are listed by first initial, last name. all authors should be in bold. Next is the year the journal is published, followed by entire title of the reference. The journal name is after the title, abbreviated according to an acceptable style. Volume number is bolded, followed by issue number (if present) in parentheses, colon, page span (starting page- ending page). The page reference includes all inclusive pages. Example: journal article- lowry, J., and J. Hansen. 1965. Electron microscope studies of bacterial flagella. J. Mol. Biol. 11: 293-313. Exercise 9. Culturing the unculturable: Purpose of winogradsky column: is to model ecosystems that are used to study soil and sediment microbiology. Most of the organisms in column cannot be grown on ordinary lab medium. The core of soil is mixed with Na2SO4, Na2Co3 and cellulose and water. It is incubated in the light to allow the growth of photosynthetic organisms. Types of organisms found in column are clostridium that are anaerobic and degrade cellulose to fermentation products. Desulfovibrio uses the fermentation products as an energy source and sulfate as a final electron acceptor and produces hydrogen sulfide that will produce a black color. The H2S diffuses to the aerobic zone where it is used by chemolithotrophs such as buggiotoa and thiobacillus as an energy source. Thiobacillus can also oxidize ferrous ion to ferric ion and produce ferric oxide (rust color). Green algae and cyanobacteria undergo oxygenic photosynthesis and purple and green photosynthetic bacteria undergo anoxygenic photosynthesis. Exercise 10. Environmental adaptation: Nostoc: is a group of filamentous, heterosystic cyanobacterium. Cyanobacteria gain their energy by oxygenic photosynthesis using chlorophyll a which absorbs red and blue light and reflects all other wavelengths. This gives photosynthetic organisms their green color. Cyanobacteria also have light harvesting pigments called phycobiliproteins (2 formsphycocyanin (625nm) and phycoerythrin (550nm)). Each pigment has different absorption spectra. The relative amounts of pigments formed depends on wavelength of light illuminating the organism. Nutritional requirements of nostoc: uses light as its energy source, carbon dioxide as its carbon source and ammonium , nitrate, or nitrogen gas as its nitrogen source. Phosphate, sulfur, and other trace elements are obtained from inorganic comounds. No growth factors required. Known as obligate photoautotrophs. Heterocyst: differentiated structures formed when Nostoc needs to do nitrogen fixation. The nitrogenase enzyme is extremely oxygen sensitve so only cyclic photosynthesis (requires no O2) occurs in heterocyst. It also contains a thick capsule, which is thought to slow down the diffusion of oxygen into these cells. Nitrogen fixation: the fixing of nitrogen gas into ammonia. Heterocyst and nitrogen fixation only occurs if the culture if deprived of a nitrogen source other than nitrogen gas. If ammonia sulfate was absent, nostoc will perform nitrogen fixation in a heterocyst, which causes some cells to look larger and more round under a microscope and by using a heterocyst, the cell experiences slower growth. Exercise 11. Culturing bacteria: All living organisms require the same elements to grow and divide. CHONPS are needed in marco amounts and K, Mg, Ca, Na, and Fe are needed in micro amounts. Some bacteria require

organic forms of carbon for both energy and carbon source (chemoorganoheterotrophs). Some use light energy in photosynthesis (phototrophs). Some use carbon from CO2 (autotroph). Some use light energy and organic forms of carbon (autoheterotroph). Chemolithoautotrophs obtain energy from inorganic compounds such as H2,NH3, H2S and use CO2 as carbon source. Types of media: 1. Defined media: are composed of designated amounts of specific compounds, the exact contents are known. 2. Complex media: contain nutrient rich substances such as yeast extract, peptone, or typtone and therefore precise chemical constituents are unknown and will vary. 3. Selective: contains particular ingredients to prevent growth of particular microbes (dyes, salts, etc) or lacks ingredients that most microbes require. 4. All-purpose: contains all general ingredients required for growth of non-fastidious microbes and doesnt specifically exclude growth in any way. 5. Differential: microbes will appear differently on the medium, depending on growth. Diff can be in colony color or change in medium color. Basic requirements for growth: water, sources of energy, sources of carbon, nitrogen, and other essential elements, required growth factors (organic compounds like amino acids, vitamins that microbe cannot make on own), correct physical environment (temp, pH, osmotic conditions, oxygen concentration). Affecting environmental conditions can alter growth by either increasing growth or decreasing growth. You can alter environmental conditions by changing temp or medium used. Purpose of medium: 1. TSA- complex, all-purpose, not differential. Intended use: for the cultivation, isolation, and maintenance of a wide variety of fastidious and non-fastidious chemoorganoheterotrophs. 2. MSA- complex, selective, differential. Isolation and differentiation of staphylococci and closely related organisms based on mannitol fermentation. 3. Minimal salts medium-defined, selective, not differential. Cultivation of chemoorganoheterotrophs that are not nutritionally fastidious. 4. EMB- complex, selective, differential. Isolation, cultivation and differentiation of gram negative enteric bacteria based on lactose fermentation. 5. Photosynthetic mineral salts broth- complex, selective, not differential. For the cultivation and enrichment of the purple nonsulfur photosynthetic bacteria. The medium must be inoculated anaerobically in the light. If the containers are not sealed, aerobic organisms that can grow in low nutrients will grow, utilizing the succinate, a nonfermentable carbohydrate. Some oligotrophoc chemoorganoheterotrophs that grow by anaerobic respiration can grow in this medium Exercise 12. Spectrophotometer: Spectrophotomemter: is an instrument used for measuring the effectve transmission of monochromatic light through a sample liquid. A beam of light is passed through a suspension and the amount of light transmitted by the suspension is measured. The amount of light transmitted is inversely proportional to the amount of light absorbing or scattering particles in the solution. A spectrophotometer contains a source of white light and an optical system which separates this light into its component wavelengths, collectively called its spectrum. The selected wavelength passes through the sample and strikes a photo-sensitive vacuum tube. The resulting electronic signal is amplified and displayed on the meter, indicating the percent transmittance (how much light passes through the sample) or absorbance (how much light is absorbed by the sample). The spectrophotometer can be used to determine the concentration of a wide variety of lightabsorbing molecules and is widely used for enzyme assays and for determining the mass of bacteria in a culture.

Beer-Lambert low of solutes: states that there is a straight line relationship between absorbance (aka optical density) of a solution and the concentration of a solute. The optical density is a function of the original intensity o the light beam and the intensity of the beam passing through the solution. How to perform sample measurement: 1. Power- flip power switch to on, let warm up for 5 minutes 2. Wavelength selection- adjust wavelength control to the desired wavelength setting. 3. Blanking for measurement- fill a spectrophotometer tube about full with the solution, but not the bacteria, tubes should be wiped with kimwipes before inserting into the spectronic 21, aligning the mark of the test tube with the line sample holder. Close the cover. Adjust knob to zero absorbance or 100% transmittance 4. Sample measurement- fill a second tube with at least 3 ml of sample, align test tube in the same manner as the blank, close cover and read absorbance of each sample. 5. It is necessary to re-blank the machine every time a different wavelength is used. When operating with a fixed wavelength it is necessary to re-blank every 15 minutes to correct drift. Determine molarity of a sample, given mol mass and concentration: Exercise 13. Growth Curve of E. coli: Growth curve phases: 1. Lag phase- refers to the fact that cell division does not occur immediately; the microbe must adjust to their new medium. The length of this will depend on the condition of the cells at the time of inoculation and the type medium into which they were inoculated. 2. Exponential phase- microbes are growing and dividing at the maximal rate possible given their genetic potential, the nature of the medium and environmental conditions. The population ismost uniform in terms of chemical and physiological properties. During this stage the generation time can be determined directly from a graph of log cell number vs. time. 3. Stationary phase- growth ceases. Normally this occurs once the population level has reached 10^9 cells/ml. This stage is reached because an essential nutrient is depletes, a toxic waste product has accumulated and/ or quorum sensing has occurred. 4. Death phase- if incubation continues, the culture will reach this stage. Growth curves are normally determined using viable plate count methods. The number of viable microbes in the sample is calculated form the number of colonies formed multiplied by the dilution factor. Bacterial cells numbers can also be approximated by measuring cell mass by means of OD or turbidity. Determination of CFU/mL from plate counts: add all the colonies counts together (25250), dividing each by their respective dilution. Then divide by the number of statistically significant plates to get an average CFU/mL. Dilution schemes: for an OD measurement at any time point- if OD is below 0.5, dilution A. if its over 0.5, use dilution B. Dilution A- 1ml of culture to 9.0 ml, then 0.1 ml to 9.9, then 0.1 to 9.9 (plate), then 1 ml to 9.0 (plate). Dilution B- 0.1 ml of culture to 9.9, then 0.1 to 9.9, then 0.1 to 9.9 (plate), and 1 to 9.0 (plate). Know how to graph on semi-log paper Be able to determine generation time: take a concentration point during exponential phase, double it and find at what time that concentration occurs (difference in time points is the generation time) Possible impacts of temp, nutrient level, oxygen level, and presence of antibiotics on a bacteria culture: it will alter growth and generation time Exercise 14. Identification of unknown:

Be able to prepare a dichotomous key/flow chart clearly separating possible bacteria species. Use of motility agar: is an excellent way to determine the motility of any microbe. It contains 1/5 the agar normally found in solid medium and also contains gelatin- this allows the bacteria to move out into the medium if they are motile. Nonmotile bacteria: will grow in stab line only, sometimes resembling a knife-like appearance. Motile bacteria: will move away from the stab line, and it very motile will makes the entire medium turbid. If the organism only grows on the very surface of the medium: this test is inconclusive for motility. Oxygen requirement: bacter show considerable variation as to their requirement for and tolerance of oxygen. 1. Aerobe- an organism that grows in the presence of oxygen and is able to use oxygen as a final electron acceptor. An aerobe will only gorwh on the very surface of the motility agar, and not where else in the tube. 2. Anaerobe- an organism that can grow in the absence of oxygen. Strict or obligate anaerobe- an anaerobe, which is killed by the presence of oxygen. It can only grow below the penetration point of the oxygen, typically the bottom of the tube. Aerotolerant anaerobe- an anaerobe that does not utilize oxygen as an electron acceptor but can grow and tolerate the presence of oxygen, with equal growth throughout the tube. Facultative anaerobes- prefers to grow in the presence of oxygen utilizing oxygen as final electron acceptor, but in absence of oxygen, it can ferment or undergo anaerobic respiration. There will be increased growth where oxygen is present. 3. Microaerophiles: organism that cannot grow in the normal 20% oxygen concentration found in the atm, but instead grows in a limited oxygen environment of 2-10%. It will form a thin band of growth in a motility tube, a short distance below the agar surface. You need to subculture on a new slant at each lab period, as it is vital to have a fresh culture for setting up various biochemical tests. Exercise 15. Respiration/fermentation/anaerobic respiration: Respiration: the presence of a c-type cytochrome correlates with a positive oxidase test. This test can be used to separate the pseudonomads from the enterics. 1. Oxidase test: determines the presence of the cytochrome c oxidase enzyme, using isolated colonies on a TSA plate or filter paper method. Place 1 drop of oxidase reagent (tetra-methyl- parathemylene diamine HCl) on an isolated colony. Oxidase positive colonies typically exhibit a color change from pink to maroon to black within 5 minutes. Oxidase negative colonies do not change color. A filter paper test will be used on unknown cultures that are naturally red or orange. A color change to pink/magenta is a positive reaction, indicating the presence of cytochrome c oxidase enzyme. No color change indicates the absence of the enzyme. Oxidative-fermentative: OF media is used to determine whether an organism metabolizes carbohydrates strictly by oxidative respiration, by fermentation or both. The carbs to be tested is added to the basal media, which also contains peptone. 2. The OF medium contains bromthymol blue, a pH indicator. It is green at a neutral pH, yellow at an acidic pH, and blue at a basic pH. If the organism can break down glucose under the given conditions (with or without oxygen), an acid is produced and the tube will change from green to yellow. If acid is produced in both the open and closed (covered in oil), the metabolism of the organism is described as oxidative fermentative. If the acid is produced only in open tube, the organism is oxidative and only grows by respiration. If the acid is produced in the closed tube, the organism is fermentative and anaerobic. If no growth, cannot utilize glucose or other required nutrients are required. Anaerobic respiration: Nitrate broth is used to determine a bacteriums ability to reduce nitrate. Nitrite is detected by addition of dimethyl-alpha-naphthylamine (nitrate B) and sufanilic acid (Nitrate A) to inoculate nitrate broth.

After incubation, add 4 drops of nitrate A, then immediately 4 drops of nitrate B. in the presence of nitrite, these reagents cause the culture to turn red, indicating that nitrate reduction had occurred. If nitrate had been reduced to nitrite during anaerobic respiration, a red color is seen within 20 minutes, while a pink color means a slight nitrate reduction occurred. If the medium does not change color after 20 minutes, ass a small amount of zinc dust. Zinc causes nitrate to be converted to nitrite and due to prior addition of nitrate A and B, a red color will result, if nitrate is still present in the medium and the organism could not reduce it. If no color change occurs after the addition of zinc, the bacteria reduced nitrate all the way to nitrogen gas. Exercise 16. Additional gram-positive test: Catalase test: determines the presence of the catalase enzyme. Place 1 drop of 3% H2O2 reagent on an isolated colony. Immediate bubbling indicates a positive test for the catalase enzyme, due to the enzymatic conversion of hydrogen peroxide to oxygen and water. No bubbling indicates a catalase negative organism. Casein agar: determines the presence of a caseinase enzyme. A clear zone on the plate, typically adjacent to the growth of the organism, indicates the presence of the caseinase enzyme. This is best seen by holding the plate up to the light and looking through the agar. If the agar remains cloudy then the organism lacks the caseinase enzyme. Caseinase breaks peptide bond between amino acids and casein is a protein found in milk. Exercise 17. Additional gram-negative tests: Decarboxylation: the media for decarboxylase reactions consists of glucose, nutrient broth, the pH indicator bromcresol purple, and the specific amino acid. Glucose is broken down by many of these organisms with the production of acid. Those organisms capable of producing decarboxylases are induced by these acidic conditions. The decarboxylase will break downthe specific amino acids into CO2 and an amine. Amine are very basic so the pH will shift back to neutral. Thus these enzymes allow these bacteria to continue growing instead of being inhibited by the acid. Interpreting the results: if the organism can utilize the glucose in the medium an acid shift occurs, resulting in the medium becoming yellow in color. When acid is produced, an organism that produces the specific decarboxylase will decarboxylate the amino acid and the pH will be shifted to alkaline causing the media to return to a purple color. The media will remain yellow id the organism does not produce decarboxylase. No change in color will result if organism does not produce acid from glucose or uses the ingredients from the broth for energy. Organisms will only produce the decarboxylase under following conditions: the organism must have the gene for decarboxylase, environment is acidic, amino acid substrate for the decarboxylase must be present. Urea test: urea is a waste product of protein metabolism. Some bacteria have an enzyme urease which breaks down urea into ammonia and CO2. pH on uninoculated medium is 6.8. during incubation, bacteria that make urease will breakdown urea, producing ammonia that causes pH to rise. At an alkaline pH the indicator turn a dark pink. Lactose fermentation test: some enteric bacteria can utilize lactose. The ability to ferment lactose will be tested by looking for acid production in tubes that only contain lactose as a carbon and energy source. Bromcresol purple is the indicator. It is purple at neutral and yellow at acidic pH. Indole test: determines the presence of tryptophanase enzyme. Add 5 drops of Kovacs reagent. A cherry red color on top layer of tube is positive test for production of indole. Indole is produced from the breakdown of the amino acid tryptophan by the enzyme tryptophanase. If indole is not produced a yellow ring will form on the top layer of the tube. Methyl red test: determines the presence of acid end products. Add 10 drops of methyl red to tube. The development of red color indicates acid is produced with a pH below 4.5. This means that an abundance of acidic fermentation products are produced when pyruvte is reduced, de to a mixed acid fermentation. The development of a yellow color indicates no acid is produced, orange indicates little acid was produced. Voges-proskauer test: determines the presence of acetoin. Add 5 drops of VP I reagent (alpha-napthol) and 5 drops of VPII (40% KOH). Put tube in 37C water bath for 30 minutes.

3.

A pink or red color ring at top indicates a positive test for acetoin. Acetoin is a fermentation product of pyruvate reduction in some enterics. Can get a negative test for Mr and VP indicating that the organism is not breaking down glucose or using a different form of metabolism, but cannot have both positive. Citrate slant: determines the presence of the citrate permease enzyme. A color change of the medium from green to blue denotes a positive for utilization of citrate as a sole carbon source. A positive test indicates that the organism produces a citrate permease enzyme to transport citrate into the cell. Once inside the cell the citrate is utilized and the excess Na produces a basic change in the medium (blue color). If the slant remains green it indicates the absence of the citrate permeas enzyme.