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Biol 240 Lab Exam Summary Notes

Introduction to the Microscope Experiment 1 Simple Staining - A color compound stain is used to increase contrast between the specimen and the background - In a simple staining procedure, a single stain is used and all cells and structures generally stain the same colour, regardless of type - It allows you to distinguish bacterial cells of various morphological types such as cocci, rods, spirals and vibrios - Commonly used dyes for simple staining: methylene blue (blue), basic fuchsin (red), and crystal violet (purple) - Two types of staining procedures; positive and negative Positive staining procedures: for light microscopy, the stain is basic and has a positively charged (cationic) chromophore (colored portion of the stain molecule) Bacteria are slightly negatively charged on their outer surface causes an attraction between cationic chromophores and organism. Methylene blue has a blue chromophore (methylene +, chloride), resulting in positive blue staining of the microorganism Experiment: 1) Sterilize the inoculating loop - Hold the loop in the coolest part of the flame (yellow), then in the hot, blue cone until it glows red - Enter flame at approximately a 45 angle to the flame - Heat the entire inoculating wire and the lower portion of the handle, without letting it become too hot to hold - Cool 2) Transfer from a broth culture - Do not let the cap touch anything, keep your finger curled around this cap - Pass neck of open tube quickly through the burner flame two or three times Sterilizes air in and around the mouth of the tube and prevents cool air which may contain contaminants from entering the pure culture - Insert loop into the open tube (holding broth at a 45 angle) and remove a loopful of bacterial suspension - Reflame the mouth of the tube again and replace the cap 3) Place loopful of bacterial broth on a microscope slide 4) Repeat steps 1 3 to add a second loopful of same culture to the first loopful on the slide and spread the liquid into a dime-sized spot 5) Allow smears to air dry 6) Heat fix the organisms on the slide (smear side up) two or three times 7) Plaice slides on staining rack and cover the bacterial smears with 1-3 drops of Loefflers methylene blue stain 8) After 1 to 2 minutes pour off the exes stain into the sink and rinse slides with stream of tap water until the become colorless 9) Gently blot dry slides with paper towels 10) Examine under microscope Experiment 2 Operation of the Microscope - Light microscope consists of a lens system, a controllable light source, and a geared mechanism for adjusting the distance between the lens stream and the object observed - Magnification achieved by a compound microscope is the result of two systems of lenses: objective (near the specimen) and ocular (eyepiece lens) Objective lens system magnifies the specimen to produce a real image, which is projected up to into the focal plane of the ocular Then is magnified by the ocular to produce the virtual image which is seen by the eye

Parts: - Working parts of the microscope are set into a sturdy frame consisting of a base for support and an arm for carrying it - Stage has a hole in the center that permits light from below to pass upward into the lenses above Mechanical stages holds glass slides in place - Light source is located at the base with control switch (on/off) and a knob to adjust the intensity of the light source - Condenser contains lenses that collect and concentrate the light, directing it upward through any object on the stage (usually matches objective magnification) - Condenser can be lowered or raised by means of an adjustment knob under the stage (this decreases the amount of light that reaches the object) - Coarse (considerable movement) and fine (critical focusing) adjustment knobs are used to focus a microscope by altering the distance between the slide and objective lens - Total magnification of the image is achieved by the product of ocular (10X) and specific objective lens used - Oil is used to improve the resolution of the magnified image, providing sharpness of detail (100X objective) - Resolving power or resolution is the ability to distinguish two images as separate Experiment: - Microscopic examination of a slide preparation Also under an oil emersion Experiment 3 Basic Bacterial Shapes - Most bacteria exist in one of three general shapes: rod (bacillus), round (coccus) and corkscrew/spiral (spirochete or spirilum) - Other kinds of bacteria sometimes can be observed include stalk forms (Caulobacter), club-shaped (Corynebacterium), and comma-shaped (vibrio). - Morphological variation can be observed due to age or composition of the culture media Characteristic Coccus Bacillus Spirillium Shape of Cell Round Rod Spiral Motility Most nonmotile Most motile Most motile Arrangement Singly (micrococcus) Singly Singly Spirochetes Pairs (diplococcus) Pairs (diplobacillus) Chains (streptococcus) Chains (streptobacillus) Groups of four (Gaffkya) Groups (cubes) of eight (sarcina) Cluster or packets (staphylococcus) Spores None Produced by several None different species Experiment: - Observe each of your prepared slides using oil immersion objective Results: 1) Escherichia coli morphology: Bacillus 2) Bacillus subtilis morphology: Bacillus 3) Staphylococcus epidermidis morphology: Coccus 4) Rhodospirillum rubrum morphology: Spirillium

Staining Techniques Experiment 4 The Gram Stain - Invented by Christian Gram in 1884 - Used to differentiate types of bacteria depending on their abilities to retain a particular stain (hence the name differential staining technique) - Bacteria are divided into two groups: gram-positive and gram-negative - Gram-variable: both gram-positive and gram-negative cells of the same organism seen in the same culture Experiment: 1) Stain the fixed smear of organisms with a primary stain of crystal violet on a staining rack for a minute Should not be over heated during heat fixing as this may cause the cell wall to rupture causing Grampositive cells to release the primary stain and accept the counterstain 2) Pour any excess stain into the sink and gently rinse with tap water 3) Cover smears with Grams iodine stain (known as the mordant) which fixes prima ry stain in bacterial cells It reacts with the crystal violet and cell nucleic acid to form a complex which is not readily removed from the Gram-positive cells during the decolorization step 4) Pour any excess stain into the sink and gently rinse with tap water 5) Wash the stained smear with a decolorizing agent, 95% ethyl alcohol while holding slide at a 45 angle Avoid overdoing decolorization process, it may cause some Gram-positive cells to appear Gramnegative 6) Rinse off alcohol with water and rain off the water 7) Counterstain by covering smear with safranin for a minute * Note* Gram-positive organisms will not easily decolorize and will retain the purple stain of crystal violet Gram-negative organisms will be decolorized by the alcohol and are subsequently stained by the safranin and appear red/pink Older cultures of gram-positive organisms and those in acid medium frequently may appear either gramnegative or gram-variable Gram-negative organisms may also appear gram-variable due to factors like culture age or medium used Results: 1) Staphylococcus epidermidis gram reaction: Gram-positive 2) Escherichia coli gram reaction: Gram-negative Experiment 5 The Acid-Fast Stain (Kinyoun Method) - Differential stain used primarily in the identification of Mycobacterium tuberculosis (tuberculosis bacillus) and the Mycobacterium leprae (leprosy organism) These bacteria have waxy walls because the walls contain large amounts of lipoidal material (mycolic acids), that render the cell wall impermeable to most stains - Special staining procedures such as the Ziehl-Neelsen procedure must be employed Kinyoun modification (cold stain) the concentrations of phenol and carbolfuchsin are increased and a detergent is added so heating isnt necessary Experiment: 1) Prepare a heat-fixed smear of microorganism Smear is heated to facilitate penetration of the stain into the bacteria so it can retain the dye, even when treated with a decolorizing agent such as acid alcohol 2) The smear is flooded with carbolfuchsin (a dark, red dye containing 5% phenol) for 5 10 minutes Has high affinity for the lipid-rich material of the bacterial cell wall 3) Rinse slides with tap water, and drain off water 4) Decolorize the smears with acid alcohol applied drop wise until the wash draining from the slides is colorless (20-30 seconds) Acid-fast microbes are decolorized by acid alcohol and because of this property they are designated acid-fast organism 4) Rinse with tap water and counterstain with methylene blue for 2 minutes Methylene blue is then used as a counterstain to enable one to observe the decolorized, non-acid fast organism

* Note* Acid fast organisms are not decolorized and appear red Non-acid-fast organisms are decolorized and counterstained by the methylene blue and appear blue All acid-fast organisms are gram positive, but not all gram positive organism are acid-fast Results: 1) Staphylococcus epidermidis acid-alcohol fastness: non-acid fast 2) Mycobacterium tuberculosis acid-alcohol fastness: acid fast Experiment 6 The Spore Stain (Schaeffer-Fulton Method) - When environmental conditions become too harsh to permit further vegetative growth and reproduction, certain bacteria (such as Bacillus and Clostridium), are capable of condensing their vital cellular components into an endospore Vegetative cell slows down, loses moisture, and withdraws its substance into one area which it surrounds with a thick impermeable wall during the process of sporulation Size of endospore and its position in the cell are often distinctive characteristics of spore forming species - Nature of spores necessitates a vigorous treatment for staining Must use a specialized staining technique to drive a dye through the spore coat Experiment: 1) Prepare a heat-fixed smear of the microorganism This method has eliminated the use of moist heat in the form of steam to drive the stain into the spore In order to roughen up the spore wall to allow the dye to enter, we substitute moist heat with dry heat by over heat fixation (run slide through flame about 5 times) 2) Flood smear with the primary stain, malachite green for 30 minutes Stain is driven into the cell because of heat 3) Remove excess stain, gently rinse with tap water and drain off the water 4) Counterstain with safranin for 20 seconds to give vegetative cells a contrasting color (light red) 5) Wash again with tap water * Note* Spores will stain green Vegetative cells will stain red Results: 1) Bacillus megaterium presence of spores: yes 2) Clostridium sporogenes presence of spores: yes Experiment 7 The Negative Stain - Uses an acid dye Nigrosin (India ink) which has negative charges on its chromophore It will be repelled by the negatively charged other surface of the bacteria and form a deposit around the cell - As a result, cells remain unstained (clear) with the background being colored (gray to black) - Number of advantages Does not involve staining, little distortion of cells occurs Natural size and shape of the cells can be seen Method of choice for observing bacteria that are difficult to stain (such as Mycobacterium) Experiment: 1) Using a loop, place a small drop of Nigrosin near one end of a glass slide 2) Using an inoculating loop, remove one loopful of the culture and mix it in the drop 3) Using the edge of a slide, spread the drop out in a thin smear 4) Allow smear to air dry for 30 seconds (do not heat fix) 5) Examine the specimen under oil immersion objective Experiment 8 The Capsule Stain - Cell wall of certain species of bacteria is often surrounded by an envelope of mucilaginous substances which can be referred to as a capsule, slime layer, or glycocalyx - Capsule usually consists of polysaccharides, polypeptides and or carbohydrate material which accumulates on the cell surface giving the structure an enlarged appearance

Since polysaccharides are water soluble and uncharged, simple stains will not adhere to it - Alcian blue is a basic dye that is water soluble and is believed to form linkages with the acid groups of acidic mucopolysaccharides staining them blue (hence capsule will appear blue with this stain) - Among disease-producing bacteria, the presence of a heavy capsule is often indicative of a highly virulent form of the organism Presence of the capsule makes destruction of the microbe by phagocytic cells more difficult Diagnosis of pneumonia and other diseases is assisted by using capsule stain - Capsule may also be detected using a negative stain In a negative stain, the unstained halo-like material surrounding the cells would represent the capsule surrounded by a dark background Experiment: 1) Place a small drop of Nigrosin near one end of a slide and aseptically add a drop of the culture to the nigrosine drop 2) Spread the drop out into a very thing smear varying from opaque to black to grey 3) Allow the smear to air dry (do not heat fix) 4) Cover the slide with 1% crystal violet for 1 minutes 5) Gently flood the slide with tap water (add from the side, not directly onto the center of your slide) until all the stain removed and the water does not change colour Allow stain to float up and not precipitate on the slide 6) Using forceps place the long edge of the slide on the paper towel and allow the water to drain off 7) Air dry the slide making sure not to blot 8) Flood with Grams Safranin for 1 minutes 9) Float stain off using procedure outlined in step 5 10) Air dry the slide making sure not to blot 11) Examine the slide under an oil immersion lens * Note* Cells should appear pink to red while the capsule should appear as a clear zone surround the cells Culture Methods and Techniques Experiment 9 Culture Media Preparation And Sterilization - A culture medium is the material in which or on which bacteria are grown May be liquid, semisolid, or solid Chemically defined medium: mediums with known chemical constituents Complex medium: mediums with exceedingly complex materials whose compositions are not clearly defined (used for cultivation of nutritionally fastidious organisms) - Nutrient broth, or nutrient agar, is the medium most commonly used Medium supports the growth of a large number of organisms, and is called all purpose or general purpose medium Consists of beef extract and peptone (partially digested protein) dissolved in distilled water To obtain solid medium, add agar (a complex carbohydrate, galactan, extracted from marine alga of the genus Gelidium) Agar goes into solution or melts when heated to nearly 100 C and remains liquid until cooled to about 43C (must be reheated to 100C to cause liquefaction) - Sterile and Sterilized means free of all life, including viruses - Principal lethal agent in steam sterilization is heat Microorganisms are killed when their enzymes and cellular proteins are irreversibly destroyed - Autoclave is an instrument used to sterilize bacteriological media and surgical equipment Increases temperature by building up stem pressure, thus preventing boiling The contents in a bottle will expand during the sterilization with steam, therefore a bottle with a tight cap may not only sterilize improperly but may also explode - Normal steam sterilization keeps temperature at 121C and the pressure between 15 16 pounds per square inch for 15 to 20 minutes

Experiment 10 Selective, Differential, and Enriched Media - To help isolate organisms found in minority, various enrichment and selective culturing methods are available that either enhance the growth of some organisms or inhibit the growth of other organisms - Selective media contain specific chemicals which do not affect the growth of the organism you wish to isolate but will discourage the growth of other groups of microorganisms For example, incorporation of sodium azide isolates lactic acid bacteria (species of Streptococcus) that lack a cytochrome system and are unaffected by sodium azide which binds tightly to the iron of the porphyrin ring of organisms that do have a cytochrome system Many selective agents and among those more frequently used are dyes (such as crystal violet), high concentrations of NaCl (isolation of Halophilic bacteria), bile salts, antibiotics, specific sugars - Differential media contain dyes or chemicals which allow the observer to distinguish between types of bacterial colonies that have developed after incubation - Eosin Methylene Blue (EMB) agar maybe considered a selective as well as differential medium Contains lactose as an energy source, so that the medium allows the growth of organisms capable of producing the enzyme -galactosidase, which breaks down lactose to glucose and galactose Contains eosin Y and methylene blue (dyes) which suppress the growth of gram-positive organisms - Enriched media is used for microorganisms that require specific nutrients such as vitamins and other growth-promoting substances (stringent nutritional requirements have termed them fastidious) Addition of components such as blood, serum, or extracts of plant or animal tissue to nutrient broth or agar provides additional nutrients to support the growth of fastidious heterotrophs Experiment: 1) Streak selective, differential and enriched media plates with Escherichia coli, Enterobacter aerogenes, Staphylococcus epidermidis, and Streptococcus faecalis Results: 1) Tryptic Soy Agar (Enriched) - Escherichia coli and Enterobacter aerogenes have a yellow growth - Staphylococcus epidermidis and Streptococcus faecalis have a white growth 2) EMB Agar (Selective/Differential) - Escherichia coli, Enterobacter aerogenes and Staphylococcus epidermidis dont grow - Streptococcus faecalis has a pink mucoid growth 3) KF Streptococcal Agar - Escherichia coli has a metallic green growth - Enterobacter aerogenes has a pink mucoid growth - Staphylococcus epidermidis doesnt grow - Streptococcus faecalis has a green growth Experiment 11 Streak-Plate Method Isolation of Pure Cultures - All specimens of material obtained for natural environments contain a mixed population of microorganisms - Imperative that each species be isolated in pure culture - There are three dilution methods commonly used for the isolation of bacteria Streak-plate Spread plate Pour-plate - The streak-plate technique provides a simple procedure for this purpose Experiment: 1) Using a dilution technique spread a loopful of culture over the surface of an agar plate Results: 1) Staphylococcus aureus: white individual colonies; Gram-positive 2) Escherichia coli: white individual colonies, Gram-negative Experiment 12 The Pour Plate Method - The pour plate method is another way to obtain isolated colonies Experiment: 1) From a mixed population of bacteria dilute the specimen into a series of cooled (45-50C) fluid-agar medium and then pour into empty petri plates

Necessary to make several dilutions to ensure that at least one countable plate will be obtained on which distinct and separate colonies have formed on, or in, the agar medium 2) Before agar cools, rock the plate to disperse the inoculum 3) When the agar has solidified and the plate is incubated at 30C for 48 hours 4) After incubation, bacterial growth is visible as colonies in and on the agar of a pour plate Experiment 13 Aseptic Technique in Pipette Handling - Must handle cultures aseptically - Organisms must not be permitted to contaminate the worker or the environment with extraneous organisms - Pipettes are frequently used to transfer bacterial suspensions or to create serial dilutions At no time should the graduated portion of a pipette be allowed to come into contact with anything other than the diluent or culture Experiment: 1) Create a series of dilutions using sterile water 2) Transfer 1 ml of the sterile water from the last test tube to the nutrient broth, mix and incubate this tube at room temperature for a week Results: - Success of your sterile technique will measure by the absence or presence of bacterial growth in nutrient broth Experiment 14 Plate Count Method - Many bacterial studies require that we be able to determine the number of organism that are present in a given unit of volume - Several different methods are available to us for such population counts - Method used is determined by the purpose of the study - One method is to make an actual count of the number of organisms on a microscope slide Direct count of this type reveals the total number of bacteria including the dead and viable cells in a given volume - Use quantitate plating to determine the population cunt of a culture of organisms - The Standard Plate Count reveals information only as related to viable organisms; that is, colonies that are seen on the plates after incubation represent living organisms Used in determining organisms present in water, milk, and food - Combination of serial dilutions and the use of a suitable growth medium is used to detect colonies form aliquots of the dilutions - Based on assumption that each viable cell will develop into a colony Breaking up clumps into single cells is difficult; microbiologists use the term colony-forming units When number of viable bacteria in a sample if reported is expressed as the number of bacteria (colonies)/ml or number of CFU/ml of the sample CFU/ml = Number of colonies (plate) Dilution X Amount Plated Motility and Microbial Growth Experiment 15 Demonstration of Motility - Bacteria that possess flagella are able to move Flagella: long thing extension from the cell that create a current in water - Important to distinguish between true motility and Brownian motion True motility: cells move across microscopic field, usually zig zagging its way along Brownian motion: observed as organism stay in one place but vibrate or shake (due to water molecules bombarding the tiny organism and is not characteristic of true independent movement) Experiment: - Observe motile and nonmotile reactions in tubed culture media and by direct observation of living organisms 1) Motility-Test Agar Inoculate each tube of motility-test agar, a semisolid medium with Lactobacillus plantarum (incubated at 37C for 48h) and Pseudomonas fluorescens (incubated at room temperature)

2) Hang-Drop Preparation Transfer a loopful of water to the center of a clean coverslip Mix small amount of bacterial growth from the plate in the drop of water Ring edges of depression slide with petroleum jelly Place the well of depression slide over the culture droplet, gently press down to form a seal and invert the slide View under microscope with an oil immersion Results: 1) Lactobacillus plantarum: no-motility 2) Pseudomonas fluorescens: motility Experiment 16 Bacterial Flagella - The mode of insertion of bacterial flagella (either peritrichous or polar) is a characteristic of some taxonomic importance - Central to the use of this trait in routine identification of cultures is the existence of a reliable technique for measuring it - Bacterial flagella are beyond the limit of resolution of the light microscope Being typically about 15 nm in thickness - To observe flagella with the light microscope, the thickness of the flagella are increased by coating them with mordants such as tannic acid and potassium alum, and staining them with silver nitrate (West method) - In addition to increasing the diameter of the flagellum, the mordant also incorporates the stain into the precipitate and hence increases contrast Precipitation of mordant will also occur on any debris or cell bodies so clean slides and cultures are important This method will allow you to readily determine mode of flagellar insertion of any flagellate bacteria Experiment: - View prepared slide of Proteus vulgaris Results: 1) Proteus vulgaris: peritrichous flagella insertion Experiment 17 Nutrition of Microorganisms - Basic requirements of all organisms include: water, carbon, energy, nitrogen, minerals and growth factors - All organisms need a source of energy Most microscopic organisms utilize carbohydrates (sugars and starches) or amino acids as their energy sources Living things require nitrogen to manufacture protein molecules (many microorganisms use protein and amino acids as their nitrogen sources, while others use ammonium phosphate, potassium nitrate, and other inorganic nitrogen compounds) - All organisms require several metallic elements such as sodium, potassium, and calcium for growth - To determine requirements for certain microorganisms, the organism is supplied with culture media containing all the necessary dietary ingredients except one Experiment: 1) Streak Agar-Agar plate, Agar + Minerals plate, Agar + Minerals + Carbohydrates plate and Agar + Minerals + Carbohydrates + Organic Nitrogen plate Escherichia coli, Bacillus subtilis, Pseudomonas fluorescens and Lactobacillus plantarum Results: Escherichia coli, Bacillus subtilis, Pseudomonas fluorescens need dont require nitrogen (can function on Agar + Minerals + Carbohydrates plate) Lactobacillus plantarum need nitrogen (can only function on Agar + Minerals + Carbohydrates + Organic Nitrogen plate) Microbial Metabolism Experiment 18 Some Metabolic Activities of Bacteria - Specific metabolic activities can be used to distinguish species by cultivating them in media designed to support each processes

Biochemical reactions that take place in the culture can then be determined by relatively simple indicator regents built into the medium or added to the culture later - Fermentation of carbohydrates: many different species can be distinguished on the basis of the carbohydrates they do or do not utilize as well as by the nature of the products formed in the fermentation reaction Some bacteria can ferment simple carbohydrates, producing acidic, alcoholic, or gaseous end products in the process Media used for testing carbohydrate fermentation are often prepared as tubed broths, each tube containing a small inverted fermentation (or Durham) tube for trapping any gas formed when the broth is inoculated and cultured Acid-base indicator bromocresol purple is used to indicate a change in pH if acid is produced Organisms that grow in the broth but do not ferment the carbohydrate will produce no change in the color of the medium, and no gas will be found Some organisms may produce acid products in fermenting the sugar, but no gas, whereas others may form both acid and gas Some cases, organisms that do not ferment the carbohydrate may produce alkaline end products by utilizing the protein nutrients in the broth and as a result that is also detected by a change in indicator color - Production of indole: indole is by product of the metabolic breakdown of the amino acid tryptophan Production of indole can be identified by adding Kovacs reagent If indole is present it combines with the reagent to produce a brilliant red color - Activity of urease: some bacteria can split the urea molecule releasing carbon dioxide and ammonia (meditated by the enzyme urease, can be seen in Proteus species) Phenol red is added as a pH indicator When bacterial cells that produce urease are grown, degradation of the urease with release of ammonia can be detected as the pH becomes alkaline and the pH indicator becomes dark pink - Production of hydrogen sulfide: hydrogen sulfide is produced when amino acids containing sulfur are metabolized by microorganisms If the medium contains metallic ions (lead, bismuth, or iron in addition to an appropriate amino acid), the hydrogen sulfide formed during growth combines with the metallic ions to form a metal sulfide that blackens the medium SIM medium, a semisolid agar that contains iron is used to test for hydrogen sulfide production (exhibits the blackening in the form of ferrous sulfide) Experiment: 1) Glucose, lactose, sucrose, tryptone, urea broth and SIM agar is used to test Escherichia coli, Pseudomonas aeruginosa, Bacillus subtilis and Proteus vulgaris Results: Name of Organism Glucose Lactose Sucrose Indole Urease SIM Escherichia coli A/G A/G N(wk)/- + Pseudomonas aeruginosa N(wk)/- K/K/Bacillus subtilis A/N/N/Proteus vulgaris A/G N(wk)/- A/G + + + Experiment 19 Bacterial Enzymes - Some microorganisms produce enzymes capable of splitting the large complex molecules of polysaccharides, proteins or lipids These enzymes are extracellular and accomplish the breakdown of their respective substrates via hydrolysis - Carbohydrases such as amylase hydrolyze polysaccharides to sugars - Proteases hydrolyze proteins to peptides and amino acids - Lipases hydrolyze lipids (fats) to glycerol and fatty acids - Catalase and oxidase are examples of endoenzymes Catalase acts on hydrogen peroxide, formed as an oxidative end product of aerobic respiration, breaking it down to water and oxygen Bubbles of oxygen can be observed when a 3 percent solution of hydrogen peroxide is added to growth of a catalase positive organism

Oxidase activates the oxidation of reduced cytochrome c by molecular oxygen in aerobic organisms during electron transport Oxidized cytochrome c transfers molecular oxygen to tertramethyl-p-phenylenediamine when the reagent is added to growth of an oxidase positive organism Experiment: 1) Streak starch (carbohydrate) plate, tween 80 (fat) plate, and skim milk (protein) plate with Escherichia coli, Pseudomonas aeruginosa and Bacillus subtilis Results: 1) Starch plate grows Bacillus subtilis 2) Tween plate grows Pseudomonas aeruginosa and Bacillus subtilis (weak) 3) Skim milk plate grows Escherichia coli (weak), Pseudomonas aeruginosa and Bacillus subtilis Microorganisms and their Environments Experiment 20 Effect of Temperature on Microbial Growth - Physical factor that may critically affect microbial growth is temperature - Optimal temperature: temperature at which the specific biochemical reactions progress at maximum speed - Psychrophiles: bacteria that grow at a temperature of 0 C - Thermophiles: bacteria requiring high temperatures to grow (some of which grow at 75C) - Mesophiles: intermediate bacteria that grows between the temperatures of 20C and 45C Experiment: 1) Measure the effects of various temperatures on the growth rate and pigment production of different bacteria Microorganisms will be grown at a number of different temperatures to determine their optimal growth, as well as minimum and maximum temperatures for growth Results: 1) Staphylococcus aureus: Mesophile (optimal: 37 C) 2) Serratia marcescens: Mesophile (optimal: 37C, but can range from 5C-40C) 3) Pseudomonas fluorescens: Mesophile (optimal: 20C -25C) 4) Bacillus stearothermophilus: Thermophile (optimal: 55C) 5) Saccharomyces cerevisiae: Mesophile (optimal: 20C-37C) 6) Aspergillus niger: Mesophile (optimal: 20C-37C) Experiment 21 pH and Microbial Growth - Hydrogen ion concentration (pH) of an organisms environment exerts the greatest influence on its growth Limits the activity of enzymes with which an organism is able to synthesize new protoplasm - Each organism has an optimum concentration of hydrogen ions in which it grows best - pH values above and below which an organism fails to grow are respectively referred to as the maximum and minimum hydrogen ion concentrations - However theses values get altered when environmental factors change: If the composition of the medium, incubation temperature or osmotic pressure is varied, the hydrogen ion requirements become different Experiment: - Tests the degree of inhibition of organisms that result from media containing different concentrations of hydrogen ions 1) Inoculate different tubes of broth with different pH levels with specific microorganisms 2) Measure turbidity visually and using the spectrophotometer (optical density - O.D.) Results: 1) Saccharomyces cerevisiae: (optimal pH: 5) 2) Escherichia coli: (optimal pH: 6-7) 3) Lactobacillus plantarum: (optimal pH: 6) 4) Staphylococcus aureus: (optimal pH: 7) Experiment 22 Oxygen Requirements of Microorganisms - Obligate aerobes: cannot exist without oxygen - Obligate anaerobes: cannot grow in the presence of oxygen

- Facultative anaerobes: will grow in either in the presence or absence of oxygen but show much better growth when oxygen is available Capable of using oxygen or some alternative oxygen source - Microaerophiles: organisms that require oxygen but only in limited amounts - Indifferents: show no preference for either condition - Media containing reducing compounds such as cysteine and sodium thioglycollate lower the oxidation reduction potential to favor the growth of anaerobic bacteria - Fluid thioglycollate medium contains these ingredients plus a small amount of agar, resazurin, yeast extract, and protein - Helps localize the growth and favors anaerobiosis in the bottom of the tube - Dye resazurin indicates the presence of oxygen by turning pink Results: 1) Staphylococcus aureus: Facultative anaerobe 2) Escherichia coli: Facultative anaerobe 3) Lactobacillus plantarum: Aerotolerant anaerobe (indifferent) 4) Clostridium sp: Obligate anaerobes Inhibition of Killing Microorganisms Experiment 23 Effect of Ultraviolet Radiation on Microorganisms - Except for photosynthetic bacteria, most bacteria are harmed by ultraviolet radiation - Only the short ultraviolet wavelengths of 210-300 nanometers can cause damage 265 nanometers is the peak lethality for most microorganisms - Ultraviolet light has been used for sterilization purposes Causes peroxides to form in the exposed medium, which then acts as oxidizing agents - Produces germicidal effects which can be demonstrated - Exposure of nucleic acids can cause mutations as a result of abnormal linkages forming between nitrogenous bases As a result abnormal protein molecules may be produced or no protein molecule may be produced at all May prevent replication of the organism and therefore can be lethal unless repaired - The greater the amount of ultraviolet radiation absorbed, the greater the number of mutations that will occur Experiment: - Organisms that have been streaked on nutrient agar will be exposed to ultraviolet radiation for various lengths of time to determine the minimum amount of radiation exposure required to effect a 100 percent kill Bacillus megaterium (spore former) and Staphylococcus aureus (non-spore former) will be used to provide a comparison of relative resistance of vegetative and spore types Results: - Growth gradually decreases as exposure time increases - However UV radiation with a lid on shows the same growth as the control Experiment 24 Lethal Effects of Temperature - To compare the susceptibility of different organisms to elevated temperatures, two methods are available: the TDP and TDT Thermal death point (TDP) is the temperature at which an organism is killed in ten minutes Thermal death time (TDT) is the time required to kill as suspension of cells or spores at a given temperature - pH, moisture, composition of medium and age of cells will greatly influence these values, and therefore must be clearly stated Experiment: - We will subject cultures of two different organisms to temperature of 60 C, 80C and 100C - At intervals of 15 minutes organisms will be removed and plated out to test their viability Results: 1) Bacillus megaterium: no growth is shown after 15 minutes exposure to 80C water bath 2) Escherichia coli: no growth is seen after 45 minutes exposure to 80C water bath

Metals, Disinfectants, Antibiotics & Mutation Experiment 25 Effect of Osmatic Pressure on Microbial Growth - Growth of bacteria can be affected by the amount of water entering or leaving the cell - Medium surrounding a microorganism is hypotonic (low solute content) with a higher osmotic pressure in the cell Except for some marine bacteria is so strong and rigid that even slight cellular swelling is generally not evident - Hypertonic solution (high solute content): growth is considerably inhibited Degree of inhibition depends the type of solute and the nature of the organism - In media of growth inhibiting osmotic pressure, the cytoplasm becomes dehydrated and shrinks away from the cell wall - Plasmolyzed cells are often simply inhibited in the absence of sufficient cellular water and return to normal when placed in an isotonic solution Irreversibly affected due to permanent inactivation of enzyme systems Experiment: - Tests the degree of inhibition of organisms that result with media containing different concentrations of sodium chloride Results: 1) Escherichia coli: growth is only seen on sodium chloride concentrations of 0.5% and 5% 2) Staphylococcus aureus: growth is only seen on sodium chloride concentrations of 0.5%, 5% and 10% 3) Halobacterium salinarium: growth is only seen on sodium chloride concentrations of 25% Experiment 26 Oligodynamic Action - Oligodynamic action is the ability of heavy metals to exert a lethal effect on bacteria Effectiveness of small amounts of metal is probably due to the high affinity of cellular proteins for the metallic ions Cells die due to the cumulative effects of ions although concentration of ions in solution are miniscule Experiment: 1) Create bacterial lawns 2) Place discs (with 6mm diameter) containing metallic ions on this inoculated surface 3) After incubation observing zones of inhibition Results: 1) Barium chloride: Escherichia coli - 0mm / Staphylococcus aureus - 0mm 2) Cobalt: Escherichia coli - 20mm / Staphylococcus aureus - 23mm 3) Lead: Escherichia coli - 10mm / Staphylococcus aureus - 11mm 4) Penny: Escherichia coli - 25mm / Staphylococcus aureus - 40mm Experiment 27 Evaluation of Chemical Antimicrobial Agents - Disinfection is defined as the inhibition or killing of microorganisms that may cause disease - Process involving chemical interactions between a toxic antimicrobial substance and various enzymes or other constituents of microbial cells - Cidal: a disinfectant must be capable of killing pathogens while it is n contact with them, so that they cannot grow again when it is removed - Disinfectants are described according to the type of organism it kills; Bactericidal, virucidal, sporicidal, or simply germicidial - If antimicrobial substance merely inhibits the organisms when it is in contact with them, they may be able to multiply again when it is removed Static activity: it arrests growth - The choice of the disinfectant for a particular purpose is influenced by the type of microbial contamination - Practical factors must be considered when choosing a disinfectant Exposure time, germicide concentration required to kill microorganisms, the temperature, pH for optimal activity of the disinfectant, the concentration of microorganisms present, toxicity of the agent for skin or its effect on materials to be disinfected - Lysol acts as a disinfectant

Mixture of cresols (methylated phenols) Inhibits enzymes - Dettol acts as a disinfectant and antiseptic Mixture of cresols (methylated phenols) Inhibits enzymes - Iodine acts as a strong disinfectant but it can also be used on skin Composition consists of iodine, sodium iodine and alcohol Inhibits enzymes, disulphide bridges, and diiodotyrosine - Scope acts as an antiseptic Experiment: 1) Create bacterial lawns 2) Place discs (with 6mm diameter) containing various chemical agents on this inoculated surface 3) After incubation observing zones of inhibition Results: 1) 5% Lysol: Escherichia coli - 9mm / Staphylococcus aureus - 22mm 2) 10% Dettol: Escherichia coli - 7mm / Staphylococcus aureus - 8mm 3) 2% Iodine: Escherichia coli - 16mm / Staphylococcus aureus - 25mm 4) Full-Strength Scope: Escherichia coli - 6.5mm / Staphylococcus aureus - 16mm Experiment 28 Antibiotic Sensitivity Testing - Antibiotics are chemical agents that are produced by organisms, some of while are successful in the control of other organisms - Susceptibility of microorganisms to many different antibiotics can be determined by the disc-plate technique - Size of the zone of inhibition is influenced by a complex of factors Rate of diffusion of the drug through agar Size of the inoculum Rate of growth of the organism Susceptibility to the antibiotic Experiment: 1) Create bacterial lawn 2) Place discs (with 6mm diameter) containing antibiotics on this inoculated surface 3) After incubation observing zones of inhibition Results: 1) Tetracycline (30 mcg) - Escherichia coli - 15mm / Staphylococcus aureus - 23mm 2) Penicillin G (10 units) - Escherichia coli - 16mm / Staphylococcus aureus 6.5mm 3) Penicillin G (2 units) - Escherichia coli - 16mm / Staphylococcus aureus - 30mm 4) Erythromycin (15mcg) - Escherichia coli 6.5mm / Staphylococcus aureus - 30mm 5) Polymyxin B (300 units) - Escherichia coli - 0mm / Staphylococcus aureus - 35mm Experiment 29 Isolation of Bacterial Mutants - Bacterial mutants may arise spontaneously, or they may be produced through the use of mutagenic agents (mutagens) Such as ultraviolet rays, x-rays, nitrous acid, alkylating agents (nitrogen mustard and mitomycin), and base analogues - Mutants have an altered genetic code and are often expressed in a phenotype clearly different from that of the parent Difference depends upon how severely the genetic code was rearranged - One of the categories of mutations is resistance This type of mutant would be resistant to inhibitory agents such as antibiotics and bacteriocins - Possible to encounter either one-step or multi-step resistant mutants, depending on the culture and antibiotic chosen Low level of penicillin is added to a broth heavily inoculated wit a penicillin-susceptible strain of bacteria, a few bacteria will be resistant mutants and will grow in the presence of the penicillin. If their progeny are subsequently grown again in a somewhat higher amount of penicillin and this process is

continued using successively higher concentrations of penicillin, mutants with resistance to a high concentration can be isolated. In large population of bacteria, there may be mutants which are resistant to either low, medium or high concentrations of the antibiotic and can thus be obtained in one step Experiment: - Involves the isolation of antibiotic-resistant mutants