MCB1000L Applied MicrobiologyLaboratory Manual ByFrances DuncanWith contributions fromValerie Walker and Neil ClarkFourth Edition 2005 2 Table

of Contents TopicPageNumber Safety 3Aseptic Technique 8Microscopy 11Smears 17Staining 20Culturing and Isolation Techniques 25 Staphylococcus 33 Streptococcus 38Throat Culture 45Oxidase 48Urea 50Triple Sugar Iron Agar (TSI) 52Motility 56IMViC 58Disc Diffusion Susceptibility Methods 61Guidelines for Identification of Unknowns 67References 70 3 Safety Introduction In the laboratory individuals are exposed to hazards not found in a regularclassroom. It is essential that students follow all rules established by the lab instructor,lab manager, or lab assistant to ensure the safety of all individuals in the class. Failureto follow established rules may result in dismissal of the individual from the class.Laboratories have certain standard safety equipment. These typically include ageneral-purpose fire extinguisher, eyewash, safety shower and cut off switches forelectrical and gas outlets. It is the responsibility of the student to locate and know howto use the general safety equipment in the laboratory. Additionally, students should beaware of exits from the room in case of emergency, the location of the nearest fire callbox, how to summon Campus Security, and how to obtain emergency medicalassistance.The microbiology lab has some additional safety considerations. Sinceindividuals work with potentially pathogenic organisms care must be taken to preventpossible infection or transmission of the organisms from the laboratory. Students mustwear protective clothing (lab coats) while working the laboratory. Lab coats may not beworn outside the laboratory. Intact skin is an adequate barrier against microorganismsso gloves are not necessary in lab. Gloves will be provided and students may weargloves when handling cultures if they so desire. Tabletops must be disinfected beforeand after lab using the disinfectant provided. Instruction in aseptic technique will beprovided. Aseptic technique must be followed while working with microorganisms.Handwashing is a simple and effective way to prevent the transmission ofdisease. While antibacterial soap may provide some additional protection the majoreffect of handwashing is the mechanical removal of microbes from the skin. Frictionwhen washing hands is important to mechanically remove organisms from the surfaceof the skin. Using a paper towel to turn off the water prevents recontamination of thehands with microorganisms. Hands must be washed whenever the student leaves thelab.Two copies of the Laboratory Safety Rules are included. One must be signedand returned to the laboratory instructor at the end of class. The additional copy is foryour reference. 4 Microbiology Laboratory Safety Rules 1. All materials and clothes other than those needed for the laboratory are to be kept away from thework area.2. A lab coat or other protective clothing must be worn during lab. The lab clothing is not to be wornoutside of the laboratory.3. Clean the lab table before and after lab with the disinfectant solution provided4. Wash hands before leaving lab.5. Any item contaminated with bacteria or body fluids must be disposed of properly. Disposableitems are to be placed in the BIOHAZARD container. Reusable items are to be placed in thedesignated area for autoclaving prior to cleaning. Sharps are to be disposed of in the appropriatecontainer6. Reusable items should have all tape and marks removed by the student before being autoclaved.7. Because organisms used in this class are potentially pathogenic, aseptic technique must beobserved at all times. NO eating, drinking, application of cosmetics or smoking is allowed. Mouthpipetting is not allowed.8. Cuts and scratches must be covered with Band-Aids. Disposable gloves will be provided onrequest.9. Long hair should be tied back while in lab.10. All accidents, cuts, and any damaged glassware or equipment should be reported to the labinstructor immediately.11. Sterilization techniques will involve the use of Bacticinerators that are fire and burn hazards.Bacticinerators reach an internal temperature of 850 o C or 1500 o F. Keep all combustibles awayfrom the Bacticinerators. Do not leave inoculating loops or needles propped in the Bacticinerator.12. Microscopes and other instruments are to be cared for as directed by the instructor.13. It is the responsibility of the student to know the location and use of all safety equipment in the lab(eyewash, fire extinguisher, etc.) 14. Cultures may not be removed from the lab. Visitors are not allowed in the lab.15. Doors and windows are to be kept closed at all times.16. For the best lab experience, read labs before coming to class. Make notes as necessary. Wait for a laboratory

introduction by the instructor before starting work.I have read and understand the above rules and agree to follow them.Signed_____________________________________________ Date_________________ Name (Please print)________________________________________________________ 5 Microbiology Laboratory Safety Rules 1. All materials and clothes other than those needed for the laboratory are to be kept away from thework area.2. A lab coat or other protective clothing must be worn during lab. The lab clothing is not to be wornoutside of the laboratory.3. Clean the lab table before and after lab with the disinfectant solution provided4. Wash hands before leaving lab.5. Any item contaminated with bacteria or body fluids must be disposed of properly. Disposable itemsare to be placed in the BIOHAZARD container. Reusable items are to be placed in the designatedarea for autoclaving prior to cleaning. Sharps are to be disposed of in the appropriate container6. Reusable items should have all tape and marks removed by the student before being autoclaved.7. Because organisms used in this class are potentially pathogenic, aseptic technique must be observedat all times. NO eating, drinking, application of cosmetics or smoking is allowed. Mouth pipetting isnot allowed.8. Cuts and scratches must be covered with Band -Aids. Disposable gloves will be provided on request. 9. Long hair should be tied back while in lab.10. All accidents, cuts, and any damaged glassware or equipment should be reported to the lab instructorimmediately.11. Sterilization techniques will involve the use of Bacticinerators that are fire and burn hazards.Bacticinerators reach an internal temperature of 850 o C or 1500 o F. Keep all combustibles away fromthe Bacticinerators. Do not leave inoculating loops or needles propped in the Bacticinerator.12. Microscopes and other instruments are to be cared for as directed by the instructor.13. It is the responsibility of the student to know the location and use of all safety equipment in the lab(eyewash, fire extinguisher, etc.) 14. Cultures may not be removed from the lab. Visitors are not allowed in the lab.15. Doors and windows are to be kept closed at all times.16. For the best lab experience, read labs before coming to class. Make notes as necessary. Wait for alaboratory introduction by the instructor before starting work .I have read and understand the above rules and agree to follow them.Signed_____________________________________________ Date_________________ Name (Please print)________________________________________________________ 6 Safety Review Questions 1. List all emergency exits from the laboratory.2. Describe how you would reach Campus Security.3. Describe how you would obtain emergency medical assistance.4. What protective clothing must be worn during lab?5. What is a simple and effective way to prevent disease transmission?6. What general safety equipment is found in the laboratory?7. Where is the nearest fire call box?8. What do you need to bring to the lab table for lab?9. How do you dispose of material that may be contaminated with bacteria?10. How do you dispose of a broken slide? 7 11. You have just disinfected your lab table. Where do you dispose of the papertowels you used?12. After washing your hands, where do you dispose of your paper towels?13. When discarding reusable contaminated material where do you put it? Whatmust be done to it before it is discarded?14. It is the end of lab. What must you do before you leave lab? List the tasks inorder of performance. 8 Aseptic Technique Introduction When working with microorganisms it is desirable to work with a pure culture. Apure culture is composed of only one kind of microorganism. Occasionally a mixedculture is used. In a mixed culture there are two or more organisms that have distinctcharacteristics and can be separated easily. In either situation the organisms can beidentified. When unwanted organisms are introduced into the culture they are known ascontaminants.Aseptic technique is a method that prevents the introduction of unwantedorganisms into an environment. When changing wound dressings aseptic technique isused to prevent possible infection. When working with microbial cultures aseptictechnique is used to prevent introducing additional organisms into the culture.Microorganisms are everywhere in the environment. When dealing withmicrobial cultures it is necessary to handle them in such a way that environmentalorganisms do not get introduced into the culture. Microorganisms may be found onsurfaces and floating in air currents. They may fall from objects suspended over aculture or swim in fluids. Aseptic technique prevents environmental organisms fromentering a culture.Doors and windows are kept closed in the laboratory to prevent air currentswhich may cause microorganisms from surfaces

to become airborne. Once thesemicrobes are airborne they are more likely to get into cultures. Transfer loops andneedles are sterilized before and after use in the Bacticinerator to prevent introductionof unwanted organisms. Agar plates are held in a manner that minimizes the exposureof the surface to the environment. When removing lids from tubes, lids are held in thehand and not placed on the countertop during the transfer of materials from one tube toanother. These techniques are the basis of laboratory aseptic technique.In this laboratory exercise the location of environmental organisms will beexplored and how microorganisms can be transmitted through contact withcontaminated surfaces. 9 Laboratory Procedure General Instructions1. Students will work in groups.Materials/Equipment2 blood agar or nutrient agar plates per student plus one plate per groupMarkersInstructions1. Label one plate Open. Write on the agar containing side of the plate, not on thelid. Remove the lid from the plate and place in on the lab table, agar side up,until the end of lab.2. Obtain one agar plate per student and draw a line on the agar containing side ofthe plate to divide the plate in half. Label one side dirty and one side clean.Remove the lid and gently touch your fingertips to the agar on the dirty side.Replace the lid. Wash your hands or clean your hands with hand sanitizer andgently touch your fingertips to the agar on the clean side of the plate3. Obtain one agar plate per student and using a marker divide the plate intoquadrants. Label the quadrants 1, 2, 3, and 4.4. Put on gloves and try to touch as few surfaces as possible. The lab instructor willswab the left gloved palm of each student.5. Remove the lid from your agar plate and touch the first two fingers of your righthand to the agar in quadrant 1.6. Replace the lid on the agar plate and touch the first two fingers of your right handto the left palm of another student in your group.7. Remove the lid from your agar plate and touch the first two fingers of your righthand to the agar in quadrant 2.8. Repeat steps 6 and 7 with two other students in your group and inoculatequadrants 3 and 4.9. Carefully remove your gloves and place them in the biohazard container. Washyour hands.10. Replace the lid on the open plate. Stack all plates agar side up and incubatethem until the next lab period.11. During the next lab period examine plates for growth and record results on page10. Discard all plates in the biohazard container. 10 Conclusions 1. Describe the growth on the plate labeled Open.2. Are organisms found in the air? What results support your conclusions?3. Record your results from the first plate you inoculated with your hands in thechart below.Side of Plate Resultsdirtyclean4. What effect does hand washing have on microorganisms? Should you evertouch a sterile surface?5. Record the results from the second plate you inoculated with gloved hands in thechart below.Quadrant Results12346. One person in your group had microorganisms swabbed on their glove. Theothers did not. From you results can you determine who had the contaminatedglove?7. What conclusions can you draw from your data concerning where microbes arefound in the environment? 11 Microscopy Introduction Microorganisms are too small to be seen with the naked eye so a microscopemust be used to visualize these organisms. While a microscope is not difficult to use itdoes require some practice to develop the skills necessary to use the microscope to itsmaximum capabilities. Bacteria and other cellular microorganisms are measured inmicrometers ( m) or 1 x 10 -6 meters. Viruses are even smaller and are measured innanometers (nm) or 1 x 10 -9 m. When carrying a microscope always use both hands.One should be on the arm of the microscope and one should be under the base of themicroscope.DiscussionThere are several types of microscopes but the only one used in this laboratory isthe compound light or bright-field microscope. Individual microscopes will varydepending on the manufacturer but all microscopes have the same basic features. These microscopes are known as compound microscopes because there are twomagnifying lenses in the microscope. One magnifying lens is in the ocular and one is inthe objective. Each contributes to the magnification of the object on the stage. Thetotal magnification of any set of lenses is determined by multiplying the magnification ofthe objective by the magnification of the ocular. The nosepiece rotates allowing theobjectives to change and thus change the magnification of the microscope.The stage is where the slide is placed. The stage adjustment knobs allow theslide to be moved easily. Light provides the illumination for the specimen. To control NosepieceStage Adjustment KnobOcularObjectivesArmBaseStageIris DiaphragmCoarse AdjustmentKnobFine AdjustmentKnobLight Source 12

the amount of light reaching the eye the iris diaphragm may be opened or closed usingthe lever just under the stage. On low magnifications less light is need than on highermagnifications. Too much light on low magnification may mask the specimen,particularly something as small as a bacterial cell.The coarse and fine adjustment knobs are used to focus on the specimen. Whena slide is on the stage there is a space between the objective and the slide. This spaceis known as the working distance. The coarse adjustment knob will cause the workingdistance to visibly change while the fine adjustment knob is for final, fine focusing.The ability to see things using a microscope is limited by the resolving power ofthe microscope. The resolving power of a microscope is the distance two objects mustbe apart and still be seen as separate and distinct. For the light microscope this is 0.2 m. Objects closer together than 0.2 m will not be distinctly seen. Increasing themagnification will not make the objects more distinct, just bigger.Each objective has the magnification of the objective written on the objective.The magnification of the ocular is also inscribed on the ocular. Low magnifications areused for quickly examining the slide to find an appropriate area to examine. Highermagnifications allow the examination of a particular object on the slide. Examine yourmicroscope and complete the table below. Objective Magnification ofObjectiveMagnification ofOcularTotalMagnification ScanningLow PowerHigh PowerOil Immersion When you look through the ocular you will see a lighted circle. This is known asthe field of view or the field. While looking through the microscope move the irisdiaphragm lever and notice how the brightness of the light changes. As you move theobjectives to provide increased magnification you will look at a smaller section of theslide. Be sure you move the object you want to view into the center of the field beforemoving to the next objective.These microscopes are parfocal. Once you have focused on an object using oneobjective the object will be approximately in focus on the next objective. Use of the finefocus knob will sharpen the focus. 13 Procedure for Focusing 1. Obtain a slide.2. Use the coarse adjustment knob to obtain maximum working distance.3. Place the slide on the stage. The slide should fit into the slide holder but is notplaced under the slide holder. Use the stage adjustment knob to move the slideover the hole in the stage.4. Rotate the low power (10X) objective in place.5. Use the coarse adjustment knob to obtain the minimum working distance.Develop the habit of watching this process to be sure the objective does notcrash into the slide.6. Look through the ocular. Adjust the light with the iris diaphragm lever ifnecessary. Slowly turn the coarse adjustment knob until something comes intofocus. Use the fine adjustment knob to sharpen the focus.7. Using the stage adjustment knob move the slide around until you find an areayou wish to examine more closely. Move the slide until the object you wish toexamine is in the center of the field.8. Rotate the high power objective into place. Use the fine adjustment knob tosharpen the focus. Do not use the coarse adjustment knob. Adjust the lightusing the iris diaphragm lever if necessary.9. Rotate the high power object halfway to the next position. Place a drop ofimmersion oil on the slide, then rotate the oil immersion objective into place. Theobjective should be immersed in the oil on the slide. Use the fine adjustmentknob to sharpen the focus. Adjust the light using the iris diaphragm lever ifnecessary.10. When finished viewing the slide use the coarse adjustment knob to maximize theworking distance and remove the slide from the stage. If you want to look atanother slide, begin the process over. If you are finished with the microscopeclean the microscope and return it to storage. 14 Procedure for Cleaning a Microscope 1. Turn off the light and unplug the cord. Store the cord appropriately.2. Using the coarse adjustment knob to obtain maximum working distance andremove the slide from the stage.3. Using lens paper clean all the lenses starting with the cleanest firstocular, lowpower, high power and oil immersion. Use lens cleaner if necessary.4. Clean any oil off of the stage using Kimwipes or paper towels.5. Rotate the scanning objective into place. Use the coarse adjustment knob toobtain minimum working distance.6. Return the microscope to the appropriate storage area. 15 Review Questions Label the drawing of the microscope.Define:Resolving powerParfocalFieldWorking distanceTell the function of each of the following.Coarse adjustment knobFine adjustment knobIris diaphragmStage adjustment knobWhat unit of measurement is used for measuring bacteria?How do you determine the total magnification of a set of lenses? 16 Describe the process for focusing on a slide.Describe how to properly clean a microscope. 17 Smears Introduction

The microscopic examination of microorganisms is a valuable identificationtechnique. In order to view microbes it is necessary to prepare slides of the organisms.Microscopic preparations may be either wet mounts or smears. Wet mounts involveplacing cells in a drop of water, adding a coverslip and viewing the material under themicroscope. In microbiology most of the organisms viewed are bacteria which are smalland difficult to see without staining. Wet mounts are temporary preparations and theability to stain is limited. A smear is a thin preparation of cells allowed to dry on a slide.This material is then fixed to the slide using heat or a chemical. A smear is a morepermanent preparation and may be stained using a variety of techniques.Smears are made using plain tap water. While tap water is not sterile it has toofew organisms in it to interfere with a bacterial smear. At least 500,000 cells permilliliter must be present in order to see one cell per oil immersion field. Bacteria aremixed in water and allowed to dry on the slide to make a bacterial smear. This is thenfixed to the slide using heat. Heat fixing helps attach the cells to the slide so they arenot washed off during the staining process, kills the cells so the slide is not hazardous tohandle, and alters the cell wall for staining. The number of cells placed on the slide isimportant for viewing the cells. Too few organisms and it is hard to find them on theslide. Too many organisms and it is difficult to see individual cells to determine theirmorphology or shape. Laboratory Procedure General Instructions1. Students work individually.2. To sterilize an inoculating loop or needle insert the loop or needle into theBacticinerator and observe it. It must glow red for three seconds to be sterilized.Loops and needles should never be propped in the Bacticinerator. The handlesare aluminum and will melt. Also they conduct heat readily and can cause burnsif the handles heat up. A hot loop or needle must cool slightly before touching abacterial colony to prevent killing the cells.3. To aseptically remove a lid from a bottle or tube, grasp the lid with the little fingerof the dominant hand. Twist the bottle or tube to loosen and remove the lid. Donot put the lid on the table but keep it in your hand while removing material fromthe bottle or tube. Return the lid to the bottle or tube by turning the bottle or tubeto tighten the lid. 18 Materials/EquipmentClean glass slidesPrepared cultures of Staphylococcus aureus and E. coli Inoculating loopBacticineratorLaboratory markerInstructions1. Glass slides should be relatively clean and grease free. Slides that do notappear clean may be washed in soap and water and dried with a paper towel.Label two slides across one end Staph . and two slides E. coli. 2. Work with one slide at a time. Sterilize an inoculating loop. Aseptically removethe lid from the water bottle and remove a loopful of water from the bottle.Return the lid to the bottle.3. Tap the loopful of water onto the center of one of the labeled slides.4. Sterilize the loop.5. Obtain a slant culture of one of the organisms. Aseptically remove the lid. Insertthe sterile loop into the tube being careful not to touch the lip of the tube. Touchthe loop to the surface of the agar. DO NOT scrape or dig into the agar.Remove the loop and return the lid to the tube.6. Mix the material on the loop in the drop of water on the appropriately labeledslide. Spread the drop over the surface of the slide making a uniform preparationof bacteria and water. The thinner the smear the quicker it will dry.7. Allow the smear to air dry.8. Heat fix the slide by passing it 10 times over the top of the Bacticinerator.9. The slide is ready for staining. It may be stored until needed.10. Repeat Steps 2-8 to make two smears of Staphylococcus aureus and twosmears of E. coli . Store the slides in slide boxes for use in future lab exercises. 19 Smear Review Questions 1. Describe two preparations that may be used to observe microorganisms.2. What is the purpose of heat fixing?3. Outline the procedure for making a smear?4. Why must slides used in smear preparation be grease-free?5. Is it necessary to use sterile water when making a smear? Why or why not?6. List two reasons for not propping inoculating loops and needles in theBacticinerator during sterilization.7. How long does it take to sterilize an inoculating loop or needle?8. When removing a lid from a lid or a bottle using aseptic technique, what do youdo with the lid? 20 Staining Introduction Bacteria have almost the same refractive index as water. This means when youtry to view them using a microscope they appear as faint, gray shapes and are difficultto see. Staining cells makes them easier to see.Simple stains use only one dye that stains the cell wall of bacteria much likedying eggs at Easter. Differential stains use two or more stains and categorize cells intogroups. Both staining techniques allow the detection of cell

morphology, or shape, butthe differential stain provides additional information concerning the cell. The mostcommon differential stain used in microbiology is the Gram stain.Bacteria have three basic shapes or morphological types. Round cells areknown as cocci, rod-shaped cells are bacilli, and spiral-shaped cells are spirilla. Cocci Bacilli SpirillaPrincipleSimple Stain: The simple stain consists of one dye. The dye adheres to the cellwall and colors the cell making it easier to see. Gram Stain: The Gram stain is a differential stain. Four different reagents areused and the results are based on differences in the cell wall of bacteria. Somebacteria have relatively thick cell walls composed primarily of a carbohydrate known aspeptidoglycan. Other bacterial cells have thinner cell walls composed of peptidoglycanand lipopolysaccharides. Peptidoglycan is not soluble in non polar or organic solventssuch as alcohol or acetone, but lipopolysaccharides are nonpolar and will dissolve innonpolar organic solvents. 21 Crystal violet acts as the primary stain. This stain can also be used as a simplestain because it colors the cell wall of any bacteria. Grams iodine acts as a mordant.This reagent reacts with the crystal violet to make a large crystal that is not easilywashed out of the cell. At this point in the staining process all cells will be the samecolor. The difference in the cell wall structure is displayed by the use of the decolorizer.A solution of acetone and alcohol is used on the cells. The decolorizer does not affectthose cell walls composed primarily of peptidoglycan but those with the lipid componentwill have large holes develop in the cell wall where the lipid is dissolved away by theacetone and alcohol. These large holes will allow the crystal violet-iodine complex to bewashed out of the cell leaving the cell colorless. A counterstain, safranin, is applied tothe cells which will dye the colorless cells.The cells that retain the primary stain will appear blue or purple and are knownas Gram positive. Cells that stain with the counterstain will appear pink or red and areknown as Gram negative. The lipopolysaccharide of the Gram negative cell not onlyaccounts for the staining reaction of the cell but also acts as an endotoxin. Thisendotoxin is released when the cell dies and is responsible for the fever and generalfeeling of malaise that accompanies a Gram negative infection.When reporting a Gram stain you must indicate the stain used, the reaction, andthe morphology of the cell. Round, purple (blue) cells would be reported as Grampositive cocci and rod-shaped, purple (blue) cells would be reported as Gram positivebacilli. There are standard abbreviations that may be used for these reports.GPC Gram positive cocciGNC Gram negative cocciGPB Gram positive bacilliGNB Gram negative bacilliThe spiralshaped bacteria of medical importance do not Gram stain well and areusually demonstrated using a dark-field microscope. There are no standardabbreviations for Gram stain reactions for the spirilla. ProcedureSimple stain MaterialsHeat-fixed bacterial smearsMethylene blue, Crystal violet, or Safranin to act as simple stainBibulous paper or paper towelsMicroscope1. Cover the label on the slide with tape.2. Place the slide on the staining rack and flood the slide with stain for 1 minute. 22 3. Rinse the slide with tap water, tilting the slide slightly to rinse all the stain fromthe slide. Tap the slide gently to remove excess water.4. Place a piece of bibulous paper or paper towel on the lab table and put the slideon it. Fold the paper over the slide and gently blot the slide to remove the water.5. Examine the stained smear with the microscope and record your results inthe chart below. Organism Results Staphylococcus aureus E. coli Gram Stain MaterialsHeat-fixed bacterial smearsGram stain reagentsCrystal violetGrams iodineAcetone-alcohol decolorizerSafraninBibulous paper or paper towelsMicroscope1. Cover the label on the slide with tape.2. Place the slide on the staining rack and flood with crystal violet for 1minute.3. Rinse the slide with tap water, tilting the slide slightly to rinse all the stainfrom the slide.4. With the slide slightly tilted, drop a few drops of Grams iodine on the slideto rinse off the last of the rinse water. Place the slide flat and flood with Gramsiodine for 1 minute.5. Rinse the slide with water as in step 3.6. With the slide tilted slowly drop acetone-alcohol decolorizer on the slide.Blue color will run from the smear. Continue to apply decolorizer drop-by-dropuntil the blue stops running from the smear. 23 7. Immediately rinse with water.8. With the slide slightly tilted add safranin to the slide to replace the rinsewater then lay the slide flat and flood the slide with safranin for 30 seconds.9. Rinse safranin from the slide with tap water. Gently tap the slide toremove excess water.10. Place a piece of bibulous paper or paper towel on the lab table and putthe slide on it. Fold the paper over the slide and gently blot the slide to removethe water.11. Examine the stained smear with the microscope and record your results inthe chart below. Organism Results Staphylococcus aureus E. coli 24 Review Questions

What is the purpose of staining bacteria?List and describe the three basic bacterial shapes.What are the differences between a simple stain and a differential stain?What is the most common differential stain used in microbiology?What is the basis for Gram stain results between different bacteria?List the reagents used in the Gram stain and tell the function of each.What would be the proper way to report each of the following if they had been Gramstained?Purple (blue), round cellsPink (red), rod-shaped cellPink (red), round cellsPurple (blue), rod-shaped cells 25 Culturing and Isolation Techniques Introduction Microorganisms must have a constant nutrient supply if they are to survive.Free-living organisms acquire nutrients from the environment and parasitic organismsacquire nutrients from their host. When trying to grow microbes in the lab adequatenutrition must be provided using artificial media. Media may be liquid (broth) or solid(agar). Any desired nutrients may be incorporated into the broth or agar to growbacteria.Agar is the solidifying material used in solid media. It is an extract of seaweedthat melts at 100 o C and solidifies at about 42 o C. Most pathogenic bacteria prefer togrow at 37 o C so agar allows for a solid medium at incubator temperatures. Since agarremains solid until reaching 100 o C, thermophiles (heat-lovers) that prefer temperaturesabove 50 o C for growth can still be grown on solid media.Organisms grown in broth cultures cause turbidity, or cloudiness, in the broth.On agar, masses of cells, known as colonies, appear after a period of incubation.Certain techniques will allow bacterial cells to be widely separated on agar so that asthe cell divides and produces a visible mass (colony), the colony will be isolated fromother colonies. Since the colony came from a single bacterial cell, all cells in the colonyshould be the same species. Isolated colonies are assumed to be pure cultures. Principle A mixed culture contains two or more bacterial species that are known and canbe easily separated based on cultural or biochemical characteristics. Culturingtechniques provide a means for maintaining adequate nutrition for the organisms sothey can continue to survive. As organisms grow in a culture they consume theavailable nutrients and periodically need to be transferred to fresh media to continue togrow. Certain culturing techniques not only provide the organisms with a fresh supply ofnutrients but also allow for the separation of bacterial cells to obtain isolated colonies.These culturing procedures are known as isolation techniques.Streak plates allow for the growth of isolated colonies on the surface of the agar.An isolated colony is a colony that is not touching any other colonies and is assumed tobe a pure culture. These colonies are easily accessible for performing staining andidentification procedures. They also show colonial morphology that may be useful inidentifying the organism. Part of the identification of any organism includes adescription of colonial morphology. Since organisms may grow differently on differentmedia, the type of media used must be included as a part of any colonial morphology.Other elements of a colonial description include colony color, hemolysis (if grown onblood agar), form, elevation and margin. 26 Form refers to the overall appearance of the colony. Elevation is the height thecolony achieves on the surface of the agar. The appearance of the edge of the colonyis referred to as the margin. FR OM Circular > 1mm IrregularPunctiform < 1 mm ELEVATION ________________________________________________________ Flat Convex Umbonate MARGIN Entire Undulate CurledThe pour plate is used for counting organisms in a solution. A standard volumeof solution is mixed in the liquefied agar. Each organism in the solution is separatedfrom all others. When the agar solidifies the cells are trapped in the agar and developinto colonies. Each colony can be counted and represents a single cell in the originalsolution. If a milliliter of solution is mixed in the agar then the number of coloniesrepresents the number of organisms per milliliter of solution. Usually a portion of amilliliter is mixed in the agar so the number of colonies counted must be multiplied bythe dilution factor to determine the number of organisms in a milliliter of solution. Whencounting colonies in agar it is difficult to accurately count more than 300 colonies on aplate. Less than 30 colonies on a plate are considered statistically insignificant. Whenevaluating a solution for bacteria a series of dilutions is usually made and cultured. Theplate with 30-300 colonies is counted and the number multiplied by the dilution factor forthat plate to determine the number of bacteria

per milliliter in the original solution. Thismethod is used to evaluate the number of organisms in milk, drinking water, and eventhe water at the beach. While the cells grow and are isolated from each other in a pourplate, they will not develop typical colonial morphology and are not easily accessible forfurther testing. 27 ProcedureInoculation of a Broth Culture MaterialsMixed Culture in brothInoculating loopBacticineratorIncubatorSterile nutrient brothStudents work individually.1. Label the sterile nutrient broth with the source of the culture and your initials.2. Sterilize the loop.3. Using appropriate aseptic technique, remove a loopful of broth from the mixedculture tube.4. Insert the loop into the sterile broth tube and swirl gently. Sterilize the loop.5. Incubate the broth at 37 o C for 24-48 hours.6. Observe broth for turbidity. Record results in table at end of the proceduresection. Inoculating an Agar Slant MaterialsMixed Culture in brothInoculating loopBacticineratorIncubatorSterile nutrient agar slantStudents work individually.1. Label the sterile nutrient agar slant with the source of the culture and yourinitials.2. Sterilize the loop.3. Using appropriate aseptic technique, remove a loopful of broth from themixed culture tube. 28 4. Insert the loop into the sterile agar slant tube and starting at the base ofthe slant, draw the loop up the slant. Do not penetrate the agar. Sterilizethe loop.5. Incubate the slant at 37 o C for 24-48 hours.6. Observe the slant for growth. Record results in table at end of theprocedure section. Streak Plate MaterialsMixed Culture in brothInoculating loopBacticineratorIncubatorSterile nutrient agar plateStudents work individually.1. Label the sterile nutrient agar plate with the source of the culture and yourinitials.2. Sterilize the loop.3. Using appropriate aseptic technique, remove a loopful of broth from themixed culture tube.4. Lift the agar plate from the lid and streak about half of the plate. The loopshould be parallel to the agar surface to prevent digging into or gougingthe agar. 29 5. Return the plate to the lid. Sterilize the loop. Lift the agar plate and makeone streak into the inoculated portion of the plate. Finish by streaking about one-fourth of the uninoculated plate.6. Return the plate to the lid. Sterilize the loop. Lift the agar plate and makeone streak into the second inoculated portion of the plate. Finish bystreaking the remaining one-fourth of the uninoculated plate. Sterilize theloop.7. Place the plate in a 37 o C incubator for 24-48 hours. Observe for growthand record your results in the table provided at the end of the procedure section. 30 Pour Plate MaterialsMixed Culture in brothInoculating loopBacticineratorIncubatorNutrient agar deep, liquefiedSterile petri dishSterile pipetteStudents work in pairs.1. Label the bottom of the sterile petri plate with the source of the culture and yourinitials. Turn the plate so the lid is facing up.2. Obtain two tubes of liquefied nutrient agar, one for each student in the pair. Thenutrient agar was boiled (100 o C) to melt the agar. Agar at that temperaturewould kill bacteria, so the agar has been cooled to 60 o C and held in a water bathto maintain that temperature. This should not kill the bacteria when they areintroduced to the liquid agar and will also reduce the amount of condensation thatwill collect on the lid of the petri dish.3. Work quickly. The agar will solidify at 42 o C. One student of the pair shouldaseptically transfer two drops of the mixed culture broth to one of the agar tubes.The other student should aseptically transfer one drop of the mixed culture broththe second agar tube. Mix the tubes by rolling the tubes between your hands,then pour the inoculated liquid agar into a labeled sterile petri dish. Gently movethe dish in a figure eight to completely cover the bottom of the dish with agar.4. Allow the agar to solidify. Add to the labeling the amount of mixed culture usedin the agar. A milliliter contains approximately 20 drops. Two drops would beapproximately 0.1 ml and 1 drop would be approximately 0.05 ml.5. Incubate the plates at 37 o C for 24-48 hours. Examine the plates for growth andrecord the results in the table below. Culture Growth IsolationBrothSlantStreak PlatePour Plate 0.1 mlPour Plate 0.05 ml 31 Review Questions

How can you tell growth has occurred in a broth culture?What is the purpose of agar?At what temperature does agar liquefy?At what temperature does agar solidify?Why is liquefied agar cooled to 60 o C before adding organisms?List two methods for obtaining isolated colonies.What is the primary purpose of the streak plate?How many colony types did you observe on your streak plate?Describe each colony type observed using standard terms.What is the primary purpose of the pour plate? 32 How many colonies did you observe on the 0.1 ml pour plate?How many colonies did you observe on the 0.05 ml pour plate?If possible determine the number of organisms in the original mixed culture broth.The dilution factor for 0.1 ml is 10 and for 0.05 ml the dilution factor is 20.What is the general formula for determining the number of organisms in a solution usinga pour plate?Serial dilutions are made of milk. The following information is collected. Dilution Number of Colonies 1:100 3121:1000 2621:10000 22How many organisms are in a milliliter of the milk tested? 33 Identification of Gram Positive Cocci Staphylococcus Introduction The genus Staphylococcus contains both pathogenic and non-pathogenicorganisms. They do not produce endospores but are highly resistant to drying,especially when associated with organic matter such as blood, pus, and other tissuefluids. Most staphylococci are found routinely on the surface of the skin. Breaks in skinand mucous membranes allow entrance of these organisms into the body where theymay cause disease.The three major species include Staphylococcus aureus, Staphylococcus epidermidis, and Staphylococcus saprophyticus . The latter two are rarely implicated in disease, buthave been isolated in cases of endocarditis and urinary tract infections under certaincircumstances. Staph. aureus is considered the pathogenic strain, causing abscesses,boils, carbuncles, acne and impetigo. Less commonly, pneumonia, osteomyelitis,endocarditis, cystitis, pyelonephritis, and food poisoning have been attributed to thisorganism. These three strains of staphylococci can be distinguished from each other bya number of biochemical tests. Principle The identification of organisms is based on cellular, cultural and biochemicalcharacteristics. All species of Staphylococcus are Gram positive cocci. On nutrientagar they tend to be white, circular, entire, convex colonies. On blood agar Staphylococcus aureus may show hemolysis of the agar in the area around the colony.Additional biochemical tests that are useful in separating the Staphylococcus species include catalase, coagulase, growth and fermentation of mannitol salt, andresistance or susceptibility to the antibiotic novobiocin.The catalase test determines if the organism produces the enzyme catalase thatbreaks down hydrogen peroxide to water and oxygen.2 H 2 O 2 __catalase__ >2H 2 O+O 2 This enzyme allows organisms to breakdown harmful metabolites of aerobic respirationand may be seen in aerobic and facultatively anaerobic organisms. There are otherenzymes that some organisms produce to handle toxic endproducts of metabolism sonot all aerobes or facultative anaerobes produce catalase.Pathogenic organisms require mechanisms to help them overcome host defensemechanisms. One mechanism involves coating the bacterial cells in a body substance,such as fibrin, to fool the immune system. The coating of a natural body substance will 34

not trigger an immune response. The enzyme coagulase causes fibrin to be depositedon bacterial cells.Some organisms can not tolerate a high osmotic pressure. Media containinghigher than normal salt concentrations may inhibit the growth of these non-tolerantorganisms. Mannitol salt agar contains a high salt concentration so only salt tolerantorganisms will grow on it. Additionally, mannitol salt agar contains the sugar mannitol.Some organisms can utilize mannitol as a food source and will produce acidendproducts from this metabolism. Since this process is invisible an indicator is addedto the media to detect changes in pH. Phenol red is the indicator used in mannitol saltagar. It is red at a neutral pH but turns yellow if conditions in the media become acidic.Antibiotic susceptibility is another test that can be used to identify organisms. Afilter paper disc is impregnated with an antibiotic, in this case novobiocin. When thedisc is placed on agar, the antibiotic diffuses through the agar. An organism susceptibleto the antibiotic will be unable to grow on the media containing the antibiotic. A zone ofinhibition (no growth) will be seen around the disc. The size of the zone indicates theresistance or susceptibility of the organism to the antibiotic. ProcedureCatalase 1. Place a drop of 3% H 2 O 2 on a glass slide.2. Touch a sterile loop to a culture of the organism to be tested and pick up a visiblemass of cells.3. Mix the organism in the drop of hydrogen peroxide.4. Observe for immediate and vigorous bubbling.5. Dispose of slide in the contaminated slide container.Interpretation: Bubbling indicates a positive (+) test and scant or no bubbling indicates anegative (-) test. Coagulase 1. Dispense 1 drop of Test Latex onto one of the circles on the reaction card and 1drop of Control Latex onto another circle.2. Touch a sterile loop to a culture of the organism to be tested and pick up a visiblemass of cells. Mix the cells in the drop of Test Latex.3. Repeat Step 2 for the Control Latex. 35 4. Pick up and hand rock the card for up to 20 seconds and observe foragglutination or clumping of the latex particles.5. Dispose of the reaction card in the biohazard container.Interpretation: Agglutination of the Test Latex with no agglutination of the Control Latexis considered a positive (+) test for coagulase. No agglutination in either the Text Latexor Control Latex is considered negative (-) for coagulase. All reactions occurring after20 seconds should be ignored. If agglutination occurs in the Control Latex theagglutination is due to some factor other than the enzyme coagulase and the testresults are invalid. Mannitol Salt Agar 1. Label a tube of mannitol salt agar with the organism to be tested and your initials.2. Using a sterile loop transfer the organism to be tested to the surface of themannitol salt agar slant.3. Incubate the tube at 35 o C. for a minimum of 18 hours.4. Examine the tube for evidence of growth on the slant and for a color change fromred to yellow.6. Remove the markings from the tube using Grams decolorizer on a paper toweland place the tube in the designated area for disposal.Interpretation: Two different characteristics of the organism are determined with thisagar. The first is the organisms ability to tolerate a high salt environment. Evidence ofgrowth on the slant indicates the organism can grow in a high salt environment.Organisms that can ferment the sugar mannitol produce an acid end product thatchanges the red pH indicator in the media to yellow. Any yellow in the media isconsidered a positive test for mannitol fermentation. It is possible for organisms to growon the media and not ferment mannitol. Novobiocin Susceptibility 1. Divide a nutrient agar plate into three sections.2. Label a section with the name of the organism to be tested.3. Using a sterile loop transfer the test organism to the plate and streak the sectionfor confluent growth.4. Aseptically transfer a novobiocin antibiotic disc to the center of each streakedarea. Gently press the disc to the surface of the agar. 36 5. Invert the plate and place in the incubator for a minimum of 18 hours.6. Examine the plate for a zone of inhibition of growth around the antibiotic disc.7. Using a metric ruler, measure the diameter of the zone of inhibition and recordthe measurement in millimeters (mm).8. Discard the plate in the biohazard container.Interpretation: A zone of growth inhibition 17 mm or less in diameter indicatesresistance (R) to novobiocin. If the zone is greater than 17 mm the organism issusceptible (S) to novobiocin. LABORATORY INSTRUCTIONS Cultures provided: Staphylococcus aureus Staphylococcus epidermidis Staphylococcus saprophyticus Students work individually unless otherwise noted.1. Make a smear of one of the organisms provided. (See page 18) Complete theremainder of the laboratory work before heat fixing, staining and examining thesmear.2. Perform a

catalase test on all organisms.3. Select one of the three organisms and perform a coagulase test. Allow the othermembers of your group to observe your results. Observe the results of the other2 organisms.4. Select one of the three organisms and inoculate a mannitol salt agar slant. As instep 3, observe the results of all three organisms5. Test each organism for novobiocin susceptibility. Each person should test allthree organisms.6. Record all results on the Laboratory Record Sheet. (Page 37)7. As time permits, Gram stain the smear prepared in Step 1 (Page 22). 37 MCB 1000LIdentification of Staphylococcus Test Staph. aureus Staph. epidermidis Staph.saprophyticus Gram StainCatalaseCoagulaseGrowth on mannitolsaltMannitolfermentationNovobiocinsusceptibilityAll species of Staphylococcus are Gram ___________________ ____________________ and positive for the ___________________________ test. Also, all Staphylococcus species tolerate ___________________________ as indicated by their growth on mannitol salt agar.Which test differentiates Staph. aureus from the other species of Staphylococcus ?How can you differentiate Staph. epidermidis from Staph. saprophyticus ? 38 Identification of Gram Positive Cocci Streptococcus Introduction Members of the genus Streptococcus are responsible for disease as well asbeing part of the normal flora of humans. Among the diseases caused are bacterialpneumonia, meningitis, tonsillitis, endocarditis, scarlet fever, erysipelas, and urinarytract infections. Streptococcus species are also found normally in the mouth and on theskin surface.The streptococci are classified by two major methods: hemolytic activity andserologic classification of Lancefield. Classification Based on Hemolytic Activity When grown on sheep blood agar, streptococci display one of three types ofhemolysis of the red blood cells in the agar. Alpha hemolysis --The red blood cells in the media are partially digestedproducing a greening of the agar. Beta hemolysis --The red blood cells in the media are completely digestedproducing a clearing of the agar. Gamma hemolysis --No change is noted in the agar. The red blood cells are notaffected by the organism. ExpectedHemolysisalpha beta gamma Streptococcus pyogenes neveralwaysnever Streptococcus agalactiae never usually sometimes Streptococcus bovis sometimes sometimes usually Streptococcus pneumoniae alwaysnevernever Enterococcus faecalis sometimes sometimes usually 39 Classification Based on Lancefield Proteins

Rebecca Lancefield, working with various streptococcal species, discoveredproteins in the cell wall that were unique to certain organisms. These proteins werelabeled Group A, Group B, Group C, and so on through Group M. Currently threeLancefield Groups are of medical importance: Group A, Group B, and Group D. Of theorganisms used in this lab the following correlations apply:Group A Strep-Streptococcus pyogenes Group B Strep-Streptococcus agalactiae Group D Strep-Streptococcus bovis, Enterococcus (Streptococcus) faecalis Streptococcus pneumoniae does not possess Lancefield proteins and is not classified inone of the Lancefield groups. Viridans streptococci is the term applied to alphahemolytic Streptococcus species that lack Lancefield proteins. Principle All Streptococcus species are Gram positive cocci. Some will only grow on anenriched agar, such as 5% sheep blood agar. On sheep blood agar the colonies areusually gray, punctiform, convex, and entire. Various species display alpha, beta orgamma hemolysis. Important biochemical tests include catalase, bacitracinsusceptibility, optochin susceptibility, growth in high salt broth, hemolysis patterns seenwith the CAMP test, and the ability to hydrolyze esculin.The bacitracin and optochin susceptibility tests are similar to the novobiocinsusceptibility test used for the identification of Staphylococcus species. Filter paperdiscs impregnated with the appropriate chemical are placed on an agar surface. Thechemical diffuses through the agar. Organisms that are susceptible to the chemical willnot grow on the agar containing the chemical. The size of the zone of growth inhibitiondetermines the organisms susceptibility to the chemical.CAMP factor is a diffusable protein produced by certain species of Streptococcus . This factor will react with the beta toxin produces by Staphylococcus aureus to rapidly lyse sheep red blood cells. When a CAMP producing Streptococcus isgrown near a beta toxin producing strain of Staphylococcus aureus a definite hemolyticpattern is produced.Only a few organisms can tolerate a salt concentration of 6.5% NaCl. Those thatcan will grow in high salt broth.Bile esculin agar contains bile that inhibits the growth of many organisms. Someorganisms can hydrolyze esculin to esculetin and dextrose. Esculetin will react withferric citrate in the media to produce a black-brown product. 40 ProceduresBacitracin Susceptibility 1. Divide a sheep blood agar plate into four quadrants.2. Label a quadrant with the name of the organism to be tested.3. Using a sterile loop aseptically transfer the test organism to the plate and streakthe quadrant for confluent growth.4. Aseptically transfer a bacitracin disc (A disc) to the center of the quadrant.Forceps may be used to position the disc. Gently press the disc to the surface ofthe agar but do not embed the disc in the agar.5. Invert the plate and place in the incubator for a minimum of 18 hours.6. Examine the plate for a zone of inhibition of growth around the disc. Whenfinished discard the plate in the biohazard container.Interpretation: Any zone of inhibition of growth is considered positive (+) for this test. Ifa red ring can be seen around the disc this is considered a positive test. This testshould be done only on organisms that display beta hemolysis . Optochin Susceptibility 1. Divide a sheep blood agar plate into four quadrants.2. Label a quadrant with the name of the organism to be tested.3. Using a sterile loop aseptically transfer the test organism to the plate and streakthe quadrant for confluent growth.4. Aseptically transfer an optochin disc (P disc) to the center of the quadrant.Forceps may be used to position the disc. Gently press the disc to the surface ofthe agar but do not embed the disc in the agar.5. Invert the plate and place in the incubator for a minimum of 18 hours.6. Examine the plate for a zone of inhibition of growth around the disc. Using ametric ruler, measure the diameter of the zone of inhibition and record themeasurement in millimeters (mm). When finished discard the plate in thebiohazard container.Interpretation: A growth inhibition zone of 15-30 mm is considered a positive (+) test.Zone sizes of less than 15 mm are considered negative (-) for this test. This test shouldbe done only on organisms that display alpha hemolysis

. 41 CAMP Test 1. Obtain a sheep blood agar plate that has been prepared for a CAMP test byhaving Staphylococcus aureus streaked in a single line down the center of theplate.2. Lines have been drawn on the plate perpendicular to the Staph . streak. Thesewill act as guidelines for inoculating the plate. Label one of the lines on theCAMP plate with the organism to be tested.3. Using a sterile loop obtain a sample of the test organism. Using a single streakand moving from the outer edge of the CAMP plate toward the Staph. steak,inoculate the CAMP plate with the test organism. Do not allow the test organismto directly touch the Staph. streak or streak across the Staph. streak. The testorganism should be streaked using one of the perpendicular lines as a guide.4. Invert the plate and place it in the incubator for a minimum of 18 hours.5. Observe the plate for the development of a distinct arrowhead pattern ofhemolysis where the test organism and the Staph. almost touch.6. Discard the plate in the biohazard container.Interpretation: The arrowhead hemolysis pattern is considered positive (+) for this test.No hemolysis or indistinct hemolysis patterns are considered negative (-) for this test.This test should be done only on organisms that display beta or gamma hemolysis . Bile Esculin 1. Label a bile esculin slant with the organism to be tested and your initials.2. Using a sterile loop transfer the organism to be tested to the surface of the bileesculin slant.3. Incubate the tube for a minimum of 18 hours.4. Examine the tube for a definite blackening of the agar.5. Remove the markings from the tube using Grams decolorizer on a paper toweland place the tube in the designated area for disposal.Interpretation: Blackening of the agar is considered positive (+) for this test. No changein the color of the agar is considered negative (-) for this test. This test should be doneon all suspected streptococci. 42 High Salt 1. Label a high salt broth tube with the organism to be tested and your initials.2. Using a sterile loop transfer the organism to be tested to the broth.3. Incubate the tube for a minimum of 18 hours.4. Examine the tube for evidence of growth (turbidity). It may be helpful to comparethe tube to an uninoculated tube. Do not agitate the tubes before you examinethem.5. Remove the markings from the tube using Grams decolorizer on a paper toweland place the tube in the designated area for disposal.Interpretation: Organisms that can tolerate a high salt environment (6.5% NaCl) willgrow in this broth causing the broth to become cloudy or turbid. Turbidity is consideredpositive (+) for this test. Organisms that can not tolerate the high salt environment willnot grow and the broth will remain clear. Clear broth is considered negative (-) for thistest. This test should be done on all suspected streptococci. LABORATORY INSTRUCTIONS Cultures provided: Streptococcus pyogenes Streptococcus agalactiae Streptococcus pneumoniae Enterococcus (Streptococcus) faecalis Streptococcus bovis Students work individually unless otherwise noted.1. Make a smear of one of the organisms provided (See page 18). Complete theremainder of the laboratory work before heat fixing, staining and examining thesmear.2. Perform a catalase test (Page 34) on all organisms and record your results onthe Laboratory Worksheet (Page 44).3. Examine all cultures for hemolysis and record your observations on theLaboratory Worksheet (Page 44).4. Refer to your Laboratory Worksheet (Page 44) and on all beta hemolyticorganisms set up a Bacitracin Susceptibility test. 43 5. Refer to your Laboratory Worksheet (Page 44) and on all alpha

hemolyticorganisms set up an Optochin Susceptibility test.6. Refer to your Laboratory Worksheet (Page 44) and on all beta and gammahemolytic organisms set up a CAMP Test. Work in lab groups to get all requiredorganisms tested, but be sure each member of the group sets up one test.Organisms may be used more than once in your group if necessary.7. Working in groups, set up a bile esculin slant on all organisms. Each member ofthe group must set up at least one test.8. Working in groups, set up a high salt broth on all organisms. Each member ofthe group must set up at least one test.9. After appropriate incubation, examine all tests and record results on theLaboratory Worksheet (Page 44).10. As time permits, Gram stain the smear prepared in Step 1 (Page 22). 44 MCB 1000LIdentification of Streptococcus Test* Strep.pyogenes Strep.agalactiae Strep.pneumoniae Enterococcus faecalis Strep.bovis Gram StainCatalaseHemolysisBacitracinOptochinCAMP TestBile EsculinHigh Salt*If a test is not done on an organism because it is an inappropriate test for thatorganism, mark the results box with a large X.What characteristic do Staphylococcus and Streptococcus share?What test would distinguish Staphylococcus from Streptococcus ?An organism is GPC, catalase negative, and alpha hemolytic. List all appropriatetests for identification of this organism.An organism is GPC, catalase negative, and beta hemolytic. List all appropriatetests for identification of this organism.An organism is GPC, catalase negative, and gamma hemolytic. List allappropriate tests for identification of this organism.Once you know an organism is GPC, what test should you do next? 45 Throat Culture Introduction The human mouth has numerous and varied organisms as part of itsnormal flora. Both aerobes and anaerobes flourish is this warm, moistenvironment. Virtually every type of microorganism can be found in the mouth.The most prevalent are the viridans streptococci. These alpha hemolyticorganisms account for most of the organisms that grow aerobically in a throatculture. In addition to these Gram positive cocci, numerous species of Staphylococcus may also be found. Neisseria, Branhamella and the anaerobic Veillonella comprise the majority of the Gram negative cocci found in the mouth.Various Gram negative bacilli, such as Haemophilus species and Klebsiella pneumoniae, are also present. The nonpathogenic Corynebacterium, ordiphtheroids, are also alpha hemolytic. Diphtheroids are pleomorphic Grampositive bacilli. Spirochetes, a few yeasts and occasional protozoa round out thenormal mouth flora. These organisms are commensals that probably protect usfrom other organisms that may enter our mouths. The presence of our normalflora prevents other organisms from finding space or nutrients to support theirgrowth.While our normal flora potentially protects us from certain diseases, theydo contribute to one. The organisms of the mouth contribute to the developmentof dental caries. Certain organisms adhere to the teeth forming a network forothers to adhere. These organisms produce the plaque found on your teeth.Some of the organisms involved in plaque metabolize sugars found in the mouthto acids that etch the tooth enamel and weaken it. If the tooth enamel isdamaged, organisms can penetrate to the pulp of the tooth damaging it. Regularremoval of these organisms and plaque helps prevent tooth decay. Principal The one organism responsible for disease in the throat is

Streptococcus pyogenes or Group A Strep. This organism is beta hemolytic and not part of thenormal throat flora. Sheep blood agar provides the enrichment necessary forgrowing many of the Streptococcus species and also acts as a differential media.The hemolysis produced on sheep blood agar helps separate the normal alphahemolytic organisms from the pathogenic, beta-hemolytic Streptococcus pyogenes .Organisms that grow in the throat also need special atmosphericconditions to grow. These organisms are exposed to the higher carbon dioxidecontent in exhaled breath. To successfully grow these organisms this carbondioxide rich atmosphere must be reproduced. Organisms that require lessoxygen are known as micoraerophiles. In the laboratory this atmosphere may beproduced by placing the plates in a large jar, lighting a candle in the jar and 46 replacing the lid. As the candle burns, some of the oxygen in the jar is convertedto carbon dioxide.Typically pharyngitis would cause redness and possibly pockets of pus onthe back of the throat. When culturing a throat these areas indicatinginflammation should be swabbed to provide the specimen. Usually a swab in aprotective plastic sleeve (Culturette) is used to take a throat culture. Once thespecimen has been taken the swab is returned to its protective sleeve and anampule of transport media is broken in the bottom of the sleeve. Transportmedia is a special purpose media that contains balanced salts to protect thespecimen from pH changes and keeps the swab moist while in transit to thelaboratory for culturing. Nutrients are not provided so growth does not occur butthe organisms can survive for several hours in the transport media, particularly ifrefrigerated. Procedure 1. Obtain a sheep blood agar plate, sterile swab and tongue depressor.2. Label the agar plate with your patients name.3. Using the tongue depressor, flatten the patients tongue. Having thepatient say Ahhhh helps flatten the tongue. Being careful not to touchany other parts of the mouth, use the sterile swab to firmly swab the backof the patients throat. Use care. Some people have a very strong gagresponse and this may induce vomiting.4. Gently roll the swab across the surface of the blood agar plate. Using asterile loop, streak the plate for isolation. First streak through the areawhere you rolled the swab and cover approximately half of the plate.Sterilize the loop and streak one quarter of the plate streaking into theoriginal streaking only once. Repeat the procedure for the remainingquarter of the plate streaking into the second streaking only once. Discardthe swab and tongue depressor in the biohazard container.5. Place the plate in a candle jar. The jar will be incubated at 35-37 o C for aminimum of 18 hours.6. Following incubation, examine the plate for the presence of beta hemolyticcolonies. A predominance of beta hemolytic colonies would indicate apossible throat infection with Streptococcus pyogenes .7. When finished examining the plate, discard in the biohazard container. 47 Review Questions 1. List 3 organisms that are considered normal throat flora.2. Why is sheep blood agar used for throat cultures?3. What organism is pathogenic in the throat?4. What incubation conditions are required for throat cultures?5. What is the purpose of the candle jar?6. Define microaerophile.7. What are viridans streptococci?8. What are the most predominant aerobic organisms in the throat?9. Complete the table below. Colony ColonialMorphologyGram StainResultsColony 1Colony 2 48 Identification of Gram Negative BacilliOxidase Introduction The oxidase test can be used in the identification of GNB to distinguishnon-fermenters (oxidase positive) from fermenters (oxidase negative). Principle The oxidase test checks for the presence of the enzyme indophenoloxidase. Tetramethyl-para-phenylenediamine (oxidase reagent) will be oxidizedin the presence of atmospheric oxygen by indophenol oxidase causing theformation of a dark-purple compound known as indophenol. Procedure Organisms used Pseudomonas aeruginosa E. coli Proteus vulgaris 1. Students work in groups to complete this lab.2. Obtain a sterile swab. Touch the swab to the organism being tested.3. Place one drop of oxidase reagent on the organism on the swab. Usingmore than one drop of reagent may dilute the color reaction and result in afalse negative.4. Observe the swab for 10-30 seconds for the development of a dark-purplecolor around the edge of the organism. This is interpreted as a positivetest. No color change or a color change after 30 seconds is interpreted asa negative test.5. Share your

results with the other members of your group.6. Record all results in the chart below.7. Dispose of the swabs in the biohazard container. The reagent droppersmay be discarded is the regular trash can. Results PseudomonasaeruginosaE. coli Proteus vulgaris Oxidase 49 Review Questions 1. Based on your results, which organism(s) could be classified as a non-fermenter?2. E. coli and Proteus vulgaris are members of the family Enterobacteriaceaeso their reactions are representative of the entire family (all members ofthe family behave the same way). Klebsiella pneumonia is also a memberof the family Enterobacteriaceae. What would its oxidase test result be?Is it a fermenter or a nonfermenter? 50 Identification of Gram Negative BacilliUrea Introduction The urea test can be used in the identification of GNB, particularly those inthe family Enterobacteriaceae. Principle If an organism produces the enzyme urease it will break down urea toammonia and carbon dioxide.urea urease > ammonia + carbon dioxideAmmonia will increase the pH of the media to 8.0 or higher. The mediacontains phenol red as a pH indicator. At a pH 8.0 or higher the indicator is abright pink color. If urea is split to ammonia and carbon dioxide the pH changewill cause the media to turn bright pink and the test will be considered positive forurease. Procedure Organisms used Pseudomonas aeruginosa E. coli Proteus vulgaris 1. Work in groups to complete this lab.2. Urea media may be either a broth or a slant. Obtain urea media andinoculate tubes with the three organisms listed above. Use appropriateaseptic technique when inoculating the tubes.3. Incubate the tubes for a minimum of 18 hours at 35-37 o C.4. Examine the tubes for a color change. Tubes that are bright pink areconsidered positive for the test. Any other color change is considerednegative. Remove labels from the tubes and discard in the designatedarea.5. Record all results in the table below. Result PseudomonasaeruginosaE. coli Proteus vulgaris Urea 51 Review questions 1. What enzyme is produced by organisms that can split urea?2. What is the indicator used in urea media?3. Why does the media turn pink when the test is positive? 52 Identification of Gram Negative BacilliTSI Introduction The TSI (Triple Sugar Iron) agar provides information concerning glucosefermentation, utilization of the sugars lactose and sucrose, and the anaerobicrespiratory process that uses sulfur as the final electron acceptor to producehydrogen sulfide. This information is useful in the identification of Gram negativebacilli. Principle TSI Agar contains three sugars: glucose (0.1%), lactose (1.0%), andsucrose (1.0%). It also contains phenol red to indicate a change in pH andferrous sulfate to demonstrate H 2 S production. Sugar Fermentation Fermentation is an anaerobic process. When sugar is fermented an acidendproduct is produced and sometimes gas. Phenol red turns yellow under acidconditions and red under alkaline conditions.Yellow agar acid production

 sugar fermentationThe enzymes for glucose fermentation are constitutive enzymes soglucose is the first choice of an organism for fermentation. The acid producedwill turn the agar in the tube yellow. Some organisms also produce gas fromglucose fermentation. This gas may be trapped in the agar pushing the agar upin the tube or causing cracks or bubbles in the tube. It is possible for the gas toescape around the agar and not be detected.If only glucose can be used the organism quickly uses the availableglucose in the tube. In order to survive the organism begins using the protein inthe agar as a carbon source. The first step of protein utilization is deamination(forming ammoniaalkaline). Deamination is an aerobic process and onlyoccurs on the slant. Those organisms that can ferment only glucose deaminateproteins to continue to survive and the slant reverts to red due to the alkalineconditions produced.Organisms that can use either sucrose or lactose (or both) will begin toferment these sugars once the glucose has been consumed. The enzymesrequired for utilization of these sugars are inducible so the presence of sucroseand lactose in the media will activate the necessary operons. Acid will continueto be produced as a result of the metabolism of the lactose and/or sucrose andthe tube will remain yellow. There is a sufficient quantity of either sugar in themedia to support the organism for at least 2-3 days. Hydrogen Sulfide (H 2 S) Production Anaerobic respiration does not require oxygen since an inorganic salt actsas the final electron acceptor instead of oxygen. Sulfur is one of the anaerobicelectron acceptors used by some facultative and obligate anaerobes. Sulfur isreadily available in the environment and in media in both organic (amino acids)and inorganic (sulfates) molecules.Hydrogen sulfide is a colorless, volatile liquid. In order to detect itspresence in the media, ferrous sulfate is used as an indicator.H 2 S + ferrous sulfate ferrous sulfide (Black precipitate)H 2 S production is an anaerobic process so the black precipitate willappear only in the butt of the TSI tube. Reporting Results and Interpretation Black buttH2S + Yellow agaracidANo black in buttH2S Red agaralkalineKGas producedGReport slant/butt Report Interpretation A/A Glucose and sucrose and/or lactose fermentedA/AG Glucose fermented with gas production,sucrose and/or lactose fermentedK/A Glucose only fermentedK/AG Glucose only fermented with gas producedH 2S + Hydrogen sulfide producedH 2S -- Hydrogen sulfide negativeK/K No sugars fermentedK/H 2S Lactose and sucrose not fermentedH 2/S production (black butt) is all that can beseenA/ H 2S Lactose and/or sucrose fermentedH 2/S production (black butt) is all that can beseen Procedure Organisms used Pseudomonas aeruginosa E. coli Proteus vulgaris 1. Students work in groups to complete this exercise2. Label a TSI slant with one of the organisms to be tested.3. All tests in this media rely on anaerobic conditions. To provide this theorganism must be introduced into the media, not on the surface. Using asterile inoculating needle, touch the organism to be tested and stab theTSI media penetrating to the bottom of the tube. When removing theneedle, streak the slant.4. Incubate all tubes for at least 18 hours at 35-37oC.5. After incubation examine the tubes for color changes. Record all results inthe table below.6. When finished examining the tubes, remove all labels and markings andplace in the designated contaminated area. Organism Fermentation H2S Production Pseudomonas aeruginosaE. coli Proteus vulgaris Review Questions Why is the media stabbed when inoculating it?Why does the slant turn red if only glucose is fermented?Which organism(s) produced gas? How could you tell?Which organism(s) produced H2S? How could you tell?What is the interpretation of the results forPseudomonas aeruginosa ?What is the interpretation of the results for E. coli ?What is the interpretation of the results forProteus vulgaris ?Do the TSI fermentation results match the oxidase results? Identification of Gram Negative BacilliMotility Introduction

Procaryotic cells (bacteria) have a single strand of protein for a flagellum.The flagellum is the only organelle for motility in the procaryotic cell. Eucaryoticcells may move by using flagella, cilia, or pseudopods. Motility in bacteriaindicates that the organism has flagella. Principle Bacterial flagella may be stained using a special flagellar stain todemonstrate their presence. This procedure is somewhat tedious. An alternateway to show bacteria have flagella is to demonstrate their ability to move. Sincethe only organelle for motility in bacteria is the flagellum, movement indicates thepresence of flagella. Media that contains half the agar content as usual is semi-solid. It does not pour but is too soft to produce a slant. This consistency willallow bacteria to swim through the agar from the initial inoculation point if theyposses flagella. Cells will be distributed along the migration route and will causethe media to become cloudy so the trail will be visible. If a tetrazolium salt(triphenyltetrazolium chloride or TTC) is added to the medium the bacterialpresence in the media will be much easier to see. TTC is colorless and solublein the oxidized form but becomes insoluble and turns red when reduced. Sinceany metabolic process involves the oxidation of molecules to produce energy, thedye is readily reduced by microbial growth and other microbial activities, such asmotility. When TTC is present in the media the cloudy trail left by bacteriaswimming through the media is red. The semi-solid agar technique is the mostcommon test for motility in the clinical laboratory.Motility may also be demonstrated using the hanging drop method. Adrop containing bacteria is suspended from a cover slip using a depression slide.The slide is then examined to see if the organisms are moving directionally for adistance of 2-3 cell lengths. This is considered true motility. Due to the size ofthe bacterial cell it is possible to see Brownian movement if the cells are nonmotile. This movement is the result of molecular bombardment of the cells and isnot due to the presence of flagella. Procedure Organisms used E. coli Klebsiella pneumoniae Enterobacter cloacae 1. Students work in groups to complete this exercise.2. Obtain a tube of motility media. Using a sterile inoculating needleinoculate the motility media by stabbing halfway into the agar.3. Incubate for a minimum of 18 hours at 35oC.4. After incubation, examine the initial stab line. If the line is sharp theorganisms did not move and there is no motility. If the line is fuzzy, showsa cloud of growth around it, or is indistinct in any way the organism wasable to move away from the initial stab line and is motile.5. Record all results in the table below. Remove all marks from the tube anddiscard in the designated area.6. OPTIONAL: If available, observe the demonstration of the hanging droptechnique. Organism E. coli Klebsiella pneumoniae Enterobacter cloacae Results Review Questions What is Brownian movement? Is it motility?What organelle(s) for motility do bacteria posses?List three methods that may be used to demonstrate flagella in bacteria. Identification of Gram Negative BacilliIMViC Introduction IMViC is a mnemonic to remember the four biochemical tests being used:Indole, Methyl red, Voges-Proskauer, and Citrate. These four tests help dividethe Enterobacteriaceae into two major groupsthe E. coli group and the Enterobacter-Klebsiella group. PrincipleIndole Organisms that posses the enzyme tryptophanase can break down theamino acid tryptophan to indole. When indole reacts with para-dimethyl-aminobenzaldehye (Kovacs reagent) a pink-colored complex is produced.Tryptophan is plentiful in most media, but growth on blood agar or chocolate agarproduces the best effects. Methyl Red Some organisms produce acid from the metabolism of glucose in asufficient quantity to produce a pH of 4.4 in the media. These acids are notfurther metabolized and are said to be stable acids. At a pH of 4.4 or less the pHindicator methyl red is a bright cherry red. Voges-Proskauer Some organisms initially produce acid from glucose metabolism butfurther metabolize the acid produced to neutral end products, such as acetoin,and 2,3-butanediol. Initially the pH may drop to 4.4 but the neutral end productsraise the pH so the methyl red test will be negative. Acetoin and 2,3-butanediolunder alkaline conditions will react with alpha-naphthol (1-naphthol) to produce amahogany red color. Citrate Citrate contains carbon. If an organism can use citrate as its only sourceof carbon the citrate in the media will be metabolized. Bromthymol blue isincorporated into the media as an indicator. Under alkaline conditions

thisindicator turns from green to blue. The utilization of citrate in the media releasesalkaline bicarbonate ions that cause the media pH to increase above 7.4. Procedure Organisms used E. coli Klebsiella pneumoniae Enterobacter cloacae 1. Students work in groups to complete this exercise.2. Obtain a DrySlide indole test card. Using a sterile loop transfer cells froman agar plate or slant to the test area on the card. Observe for thedevelopment of a pink color within 30 seconds. Record your results in thetable provided (Page 60) and discard the test card in the biohazardcontainer.3. Obtain an MRVP broth and using aseptic technique inoculate the tube. Itis important to inoculate this test heavily. Incubate for at least 24 hours at35oC.4. Obtain a citrate slant. Aseptically inoculate the slant and incubate for atleast 24 hours at 35oC.5. After incubation of the MRVP broth obtain a spot plate and a steriledropper. Observe the MRVP for turbidity. If turbidity is not noted the testresults are not reliable. The tube may be reincubated until growth isevident. Place 3 drops of turbid broth into two of the wells on the spotplate. Methyl Red Test To one well add 1-2 drops of methyl red reagent.Observe for an immediate cherry red color that indicates a positive test.Orange or yellow is considered negative. Record your results in the tableprovided (Page 60). Voges-Proskauer Test To the remaining well add 2 drops of alpha-naphthol and 1 drop of potassium hydroxide (KOH). Observe for thedevelopment of a mahogany red color. The color development takes 20minutes or longer. Be extremely careful with the KOH. It is caustic andmay cause burns if it gets on your skin. The mahogany red color isconsidered positive. Record your results in the table provided (Page 60).Remove all marks from the MRVP tube and discard in the designatedarea. Clean the spot plate by covering the surface with disinfectant. Allowthe disinfectant to sit for a few minutes, then rinse with water, wash anddry. Return the spot plates to their original location.6. Observe the citrate for a change from green to blue. Blue is consideredpositive for this test. Record your results in the table provided (Page 60).Remove all marks from the tube and discard in the designated area. Organism Indole Methyl Red Voges-ProskauerCitrate E. coli Enterobacter cloacae Klebsiella pneumoniae Review Questions What is the IMViC pattern for the E. coli group?What is the IMViC pattern for the Enterobacter-Klebsiella group?What is the difference between a reagent and an indicator?Complete the following table. Test Substrate Reagent Indicator PositiveResultIndoleMethyl RedVoges-ProskauerCitrate Is it possible for an organism to be Methyl red and Voges-Proskauer positive?Explain your answer.What might happen if the MRVP tests are read too soon? 61 Disc Diffusion Susceptibility Methods Introduction When a filter paper disc impregnated with a chemical is placed on agarthe chemical will diffuse from the disc into the agar. This diffusion will place thechemical in the agar only around the disc. The solubility of the chemical and itsmolecular size will determine the size of the area of chemical infiltration aroundthe disc. If an organism is placed on the agar it will not grow in the area aroundthe disc if it is susceptible to the chemical. This area of no growth around thedisc is known as a zone of inhibition. Principle Antiseptics, disinfectants and antibiotics are used in different ways tocombat microbial growth. Antiseptics are used on living tissue to removepathogens. Disinfectants are similar in use but are used on inanimate objects.Antibiotics are substances produced by living organisms, such as Penicillium or Bacillus , that kill or inhibit the growth of other organisms, primarily bacteria.Many antibiotics are chemically altered to reduce toxicity, increase solubility, orgive them some other desirable characteristic that they lack in their natural form.Other substances have been developed from plants or dyes and are used likeantibiotics. A better term for these substances is antimicrobials, but the termantibiotic is widely used to mean all types of antimicrobial chemotherapy.Many conditions can affect a disc diffusion susceptibility test. Whenperforming these tests certain things are held constant so only the size of thezone of inhibition is variable. Conditions that must be constant from test to testinclude the agar used, the amount of organism used, the concentration ofchemical used, and incubation conditions (time, temperature, and atmosphere).The amount of organism used is standardized using a turbidity standard. Thismay be a visual approximation using a McFarland standard 0.5 or turbidity maybe determined by using a

spectrophotometer (optical density of 1.0 at 600 nm).For antibiotic susceptibility testing the antibiotic concentrations arepredetermined and commercially available. Each test method has a prescribedmedia to be used and incubation is to be at 35-37 o C in ambient air for 18-24hours.The disc diffusion method for antibiotic susceptibility testing is the KirbyBauer method. The agar used is Meuller-Hinton agar that is rigorously tested forcomposition and pH. Further the depth of the agar in the plate is a factor to beconsidered in the disc diffusion method. This method is well documented andstandard zones of inhibition have been determined for susceptible and resistantvalues. There is also a zone of intermediate resistance indicating that someinhibition occurs using this antimicrobial but it may not be sufficient inhibition toeradicate the organism from the body. The standardized methods for antiseptic and disinfectant testing are morerigorous and more difficult to reproduce in a student laboratory. Two commontests are the Phenol Coefficient Test (a comparison of the effect of the chemicaland phenol on several organisms) and the Use Dilution Test (testing thechemical under actual conditions of use). A disc diffusion test can be used toapproximate the Use Dilution Test. The chemical under consideration is used tosaturate a filter paper disc. This disc is then used to introduce the chemical tothe agar for testing. The actual zone sizes have not been standardized as in theKirby-Bauer method, but a comparison of zone sizes for the same chemicalamong organisms will provide an approximate effectiveness of the chemical. ProcedureKirby-Bauer Antimicrobial Susceptibility Test Organisms to be tested: Staphylococcus aureus E. coli Procedure1. Students will work independently in the laboratory exercise.2. Obtain a plate culture of one of the organisms to be tested.3. Using a sterile loop, emulsify a colony from the plate in the sterile salinesolution. Mix thoroughly making sure that no solid material from the colony isvisible.4. Repeat this procedure until the turbidity of the saline solution matches that ofthe standard available for your class.5. Dip the swab into the broth culture of the organism. Gently squeeze the swabagainst the inside of the tube to remove excess fluid. Use the swab to streaka Mueller-Hinton agar plate or a nutrient agar plate for a lawn of growth. Thisis best accomplished by streaking the plate in one direction, then streaking atright angles to the first streaking, and finally streaking diagonally. End byusing the swab to streak the outside diameter of the agar.6. Allow the plates to dry for about 5 minutes. 7. Antibiotic disks can be placed on the surface of the agar using a dispenserthat dispenses multiple disks at the correct distance apart, or by obtainingindividual disks and placing them on the surface of the agar using flamesterilized forceps.a. Dispenser method:1. Obtain the dispenser containing the correct antibiotic disks for theorganism you are using.2. Place the dispenser over the surface of the plate and using thelever/plunger dispense the disks.3. Using sterile forceps or a loop, gently press the disks onto thesurface of the agar, taking care not to press them into the agar.b. Dispensing individual disks:1. Obtain 6 of the appropriate individual disk dispensers.2. Using the levers, dispense the disks at equal distances apart on thesurface of the agar.3. Using flame sterilize forceps or a sterile loop gently press the disksonto the surface of the agar.4. 6 disks may also be individually placed onto the surface of the agarusing sterile forceps.8. Invert the plates and incubate for 24 hours at 37 C.9. Using a metric ruler measure the diameter of the zone of inhibition (if present)for each antibiotic used.10. Compare the measurement obtained from the individual antibiotics to thetable of standards to determine if the bacterial species tested is resistant orsensitive to the antibiotic.11. Use the data you collected and that of the rest of the class to fill in the tablebelow. Discard the plates in the biohazard container. Antiseptic/Disinfectant Susceptibility Test Organisms used Staphylococcus aureus E. coli Bacillus cereus Pseudomonas aeruginosa 1. Students work individually on this laboratory exercise.2. Obtain one of the organisms to be tested, 5 nutrient agar plates, and asterile swab.3. Dip the swab into the broth culture of the organism. Gently squeeze theswab against the inside of the tube to remove excess fluid. Use the swabto streak a nutrient agar plate for a lawn of growth. This is bestaccomplished by streaking the plate in one direction, then streaking atright angles to the first streaking, and finally streaking diagonally. End byusing the swab to streak the outside diameter of the agar. Repeat thisprocedure for the remaining plates.4. Place a disc soaked in an antiseptic or disinfectant in the center of eachplate. Be sure to label the plates with the organism and chemical used.5. Incubate the plates in the standard upside down position until the next labperiod.6. Measure the diameter of the zone of inhibition for each chemical. Theclass will share data so you can fill in the table provided.7. Discard the plates in the biohazard container. Chemical Staph.aureusE. coli Bacillus cereus Ps.aeruginosa 66 Review Questions

What conditions must be held constant when doing disc diffusion procedures?DefineAntisepticDisinfectantAntibioticZone of inhibitionAccording to your results, which chemical is the most effective? On what do youbase this conclusion?What are the standard tests used for determining the effectiveness of antisepticsand disinfectants?What is the standard method used for antimicrobial susceptibility testing? Guidelines for the Identification of Unknown Organisms Unknown organisms will be grown on 5% Sheep blood agar or Nutrient agar andwill be distributed as streak plates with isolated colonies. Each plate willrepresent a pure culture.Each student is responsible for doing their own work but may ask other studentsfor opinions, advice, or instructions on what to do.The Lab Instructor may provide assistance on all but the last two unknowns.(Unknown 3 and Unknown 4)On any of the unknowns, students are allowed to refer to class notes, texts, andany other source of information they may have developed during the course.An Unknown Report Form will be provided for each unknown. Students mayhave only one form for each unknown. The form must be filled out neatly andcompletely using blue or black ink. Spelling must be correct and all test notationsmust be appropriate. This report should be treated as though it were a part of apatient chart. Erasures, liquid paper, and any obliteration of information recordedon the form are not allowed (loss of points). Should errors occur the studentmust make a single mark through the error, initial, date, and report the correctedresult. + fd m/d/y ? The steps for identifying an unknown are Gram stain Colonial morphology Biochemical testing Only the biochemical tests necessary for identification of the organism areappropriate. Unnecessary tests will result in a deduction of points from the finalgrade. Tests are to be ordered from the lab instructor using the appropriate form.This form must also be filled out completely. A separate order form is used foreach unknown. More than one order form may be used for one unknown. If atest is ordered the results must appear on the report form or a brief explanationas to why the test was not done should be made. Failure to do so will result in aloss of points from the final grade.Gram staining and initial spot testing will give the student a general ideaconcerning the identity of the unknown. Only tests useful for identifying thesuspected organism should be performed. A sufficient number of tests should beperformed so that the organism may be identified at the next laboratory session. 68 Test results must be reported appropriately. Failure to do so may result ina loss of points from the final grade. Most results can be reported with a + or -.That is sufficient. A detailed description of the results is inappropriate.The identity of the unknown should be written appropriately. The genusname should be capitalized but not the species name. If you are in the habit ofprinting in all upper case letters, be sure to differentiate the first letter of thegenus name as larger than the others. Failure to do so will lead to a loss ofpoints on the final grade.Below is an example of the test request form. The top should becompletely filled out and the appropriate tests checked. This will be retained bythe Laboratory Instructor and attached to your completed Unknown Report Form.It is considered in grading your unknown. MCB 1000LMedia/Test Request FormName___________________________________Date___________________Unknown Number________Requested Media/Test MaterialsCoagulase_______________ TSI_________________Mannitol Salt____________ Urea________________Novobiocin_______________ Indole_______________Optochin________________ MRVP______________Bacitracin_______________ Citrate______________CAMP__________________ Motility_____________High Salt________________Bile Esculin______________ 69Name____________________________Date Started_______________________ MCB 1000LUnknown Report Form Unknown Number______________Gram Stain Results______________ Date________________Colony Morphology (include media used) Date________________ Test Performed Results Date Identity of Unknown ___________________________________________Date completed__________________

References Howard, Barbara J. (ed.) 1987. Clinical and pathogenic microbiology,2 nd ed. Mosby, Baltimore.Finegold, Sidney M., William J. Martin, and Elvyn G. Scott. 1978.Diagnostic microbiology, 5 th ed. Mosby, Baltimore.BBL TM DrySlide

TM Indole. 1998. Technical Insert, Becton DickinsonMicrobiology Systems, Sparks, Maryland.BBL Oxidase. 1995. Technical Insert, Becton DickinsonMicrobiology Systems, Sparks, Maryland. 5,059 Reads Info and Rating Category: Research > Science Rating: Upload Date: 03/10/2010 Copyright: Attribution Non-commercial Tags: This document has no tags. Flag document for inapproriate content