Lab manual 2009

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Dr. Lu lab

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  • 1. 0
  • 2. 1 Exercise No.____ Instrumentation Microbiology as a science includes the study of bacteria (bacteriology),viruses (virology), fungi (Mycology), and animal parasites (Parasitology). Afterthe discovery of microscopic life forms by Leeuwenhoek, other fields such asImmunology & Genetics are also classified as branches of Microbiology. In the study of Microbiology, the single most important apparatus used inroutine examinations is the compound microscope. The microscope is not onlyessential , it is also a must to master the proper usage and handling of this. Asidefrom the microscope comes many other laboratory equipment and glasswarewhich are also needed in performing the daily tasks in a microbiology laboratory.This is the main goal of the initial chapter: To introduce to the dental students thebasic instrumentation in the microbiology laboratory.Objectives:1. To familiarize the dental student with the common glassware & apparatus usedin the microbiology laboratory.2. To be able to differentiate the functions of some of the common instruments &tools used in the performance of laboratory exercises in microbiology.Part 1. The Compound Microscope:Objectives: a. To train the dental student in the use of the compound microscope b. To enforce proper operation of the microscope specially with the Oil Immersion objectiveMaterials: a. Compound microscope b. Prepared slide specimen c. Cedar wood oil d. Xylol
  • 3. 2Part 2. Apparatus & GlasswareObjectives: a. To acquaint the student with the various apparatus used in microbiology b. To know the different functions & uses of the different glassware in microbiology.Materials: a. All apparatus & glassware checked & distributed to the students b. Apparatus in the Micro Lab ( GDLSC Stockroom )
  • 4. 3Illustration:(1) Draw and label properly all functional parts of the compound microscope.
  • 5. 4(2) Draw & give a use for each of the following : 1. Plain slide: _______________________ 2. Depression slide: _____________________ _________________________________ ____________________________________ 3. Candle jar:________________________ 4. Gas Pak: _____________________________ _________________________________ _____________________________________ 5. Colony counter:____________________ 6. Wire basket: _______________________ _________________________________ __________________________________ 7. Inoculating loop:___________________ 8. Inoculating needle:___________________ _________________________________ _________________________________
  • 6. 59 Petridish : _______________________ 10. Coplin jar : _________________________ _________________________________ ____________________________________11. Bunsen burner:___________________ 12. Waterbath: _________________________ __________________________________ _____________________________________13.Dilution bottle :____________________ 14. Dropping bottle: _____________________ __________________________________ ____________________________________15.Petridish can: ___________________ 16. Pipette can:_________________________ _________________________________ ____________________________________
  • 7. 6Review Questions:1. Differentiate magnification , resolution & resolving power:________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________2. Why is there a need for cedar wood oil & Xylol in the use of the Oil ImmersionObjective?_______________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________3. Briefly discuss the following: a.Electron microscope: __________________________________________________ ___________________________________________________________________ __________________________________________________________________b. Fluorescence Microscope: ______________________________________________ ___________________________________________________________________ ___________________________________________________________________ c. Phase Contrast Microscope: ____________________________________________ ___________________________________________________________________ ___________________________________________________________________ d. Inverted Microscope: _________________________________________________ ___________________________________________________________________ ___________________________________________________________________ e. Darkfield Microscope: _______________________________________________ ___________________________________________________________________ ___________________________________________________________________
  • 8. 74. Differentiate an autoclave from a hot air sterilizer. Which is more commonly used inthe microbiology laboratory? Which is usually used in the dental clinic?________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________5. Why do you think is it necessary to take precautionary measures in handling yourspecimen and for yourselves prior to coming in and out the laboratory as dental students?________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________
  • 9. 8 Exercise No.____ Bacterial Cytology & Physiology Bacteria are characteristically smaller than the more advanced microscopiceukaryotes and range in size from 0.2 um, in width , which is barely visible withthe compound microscope, to some spiral-shaped species that reach 400-500 umin length. Generally speaking, most bacteria are 1 to 6 um in length withdiameters between 0.2 to 1.5 um. A typical bacterial cell taken apart could consist of a capsule, cell wall ,cytoplasmic membrane, some organelles, nucleus, fimbriae & flagella plus thepresence of spores. They could be classified according to shape into: spherical,rod shaped, curved or spiral and square, with variations in most types which isknown as pleomorphism. They also have the ability to exhibit reproduction and insome cases motility and cellular interactions.Objectives:1. To study the basic structure of the bacterial cell.2. To enable to student to distinguish the different morphological forms of thebacterial cell.3. To fully understand the functions of the fundamental parts and the specializedstructures contained in a bacterial cell.Materials:1. Compound microscope2. Prepared microscopic slides of the following : a. Staphylococcus aureus b. Bacillus subtilis c. Vibrio cholera3. Cedar wood oil4. Xylol
  • 10. 9Illustrations:(1) Draw a typical bacterial cell and label all its functional parts and structures:(2) In the space provided , illustrate the different specialized structures whichcould be present in a bacterial cell and give their classifications:
  • 11. 10 (3) On the space provided , illustrate all pleomorphic forms of the cocci, bacilli &spirilli: a. Cocci - diplococci, staphylococci, streptococci, sarcinae, tetrads b. Bacilli - single, diplococci, streptobacilli, palisade c. Spirilli - spiral, comma-shaped
  • 12. 11Review Questions:(1) Give a specific function for each of the basic structures of the bacterial cell:__________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________(2) Give the chemical composition of the cell wall and cell membrane of thebacterial cell:______________________________________________________________________________________________________________________________________________________________________________________________________(3) Name the specialized structures of the bacterial cell and give their functions:____________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________(4) Enumerate the different types of flagella based on number and location:__________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________
  • 13. 12(5) List down the different types of spores based on its location within thebacterial cell:______________________________________________________________________________________________________________________________________________________________________________________________________(6) Aside from the three morphological forms ( cocci, bacilli & spirilli) , are thereother special morphological forms?________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________
  • 14. 13 Exercise No._______ Microscopic Study of Protozoans & Observation of True Motility Protozoans are unicellular, free living, in some cases parasiticmicroorganisms. They belong to the Kingdom Protista. Protozoa are excellenttools for research because they are easily cultivated in the laboratory. Theyreproduce asexually and clones are easily generated with the same genotype of thedesired “parent”. The effects of environmental factors, such as radiation are easilyanalyzed in these organisms. Protozoans are classified into the Sarcomastigophora, Sporozoa,Myxospora and the Ciliophora. Specific examples of protozoans include:Amoeba, Mastigamoeba, Euglena, Paramecia, Volvox, Bicoeca, Ochromonas,Opalina, Stentor, Hymenostomina & Pleuromona. Some of the morphologicalstructures present on them are the pellicle, shell, mucocyst, cytosome, oral groove,cytopharynx, contractile & food vacuole, cytopyge, extrusomes & Uroid. One of the most common characteristic of protozoans is the ability ofmovement or locomotion. This is brought about by organs of locomotion whichare flagella and pili or fimbriae. True motility could easily be observed with asample from any canal waterObjectives:(1) To observe the actual living microorganisms present in the samples obtainedfrom our present surroundings.(2) To demonstrate and observe true motility as exhibited by the protozoans at thesame time also study the organs for locomotion.Materials:1. Specimen from any canal water2. Vials3. Hanging - drop slide or depression slide4. cover slip5. Vaseline6. medicine dropper
  • 15. 14Procedure:1. Clean the concave slide and coverslip thoroughly.2. Take a drop of the specimen and carefully drop it on the center of thecoverslip. Ring the coverslip with a thin layer of Vaseline.3. Carefully invert the prepared coverslip over the concavity of the depressionslide, making sure that the drop of specimen in the center is hanging into theconcavity without touching the slide.4. Study the preparation under low power objective with the diaphragm partlyopen and the condenser slightly lowered.5. Switch the objective to high power and observe the specimen. The slide maybe moved to have a wider field of coverage in searching for motile organisms.6. Make a note of all the different types of motile organisms observed.Note: After the exercise, remember to immerse the dropper used and thedepression slide together with the coverslip into the disinfectant solution beforewashing with soap & water.Observations:________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________Illustration : ( Set-up)
  • 16. 15Review Questions:(1) What do you think is the purpose of ringing the coverslip with Vaseline beforeinverting it onto the depression slide?________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________(2) What are the other methods of testing for bacterial motility?________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________
  • 17. 16(3) Could there be other types of movement aside from true motility? What do youattribute this to?________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________
  • 18. 17 Exercise No._______ Staining Techniques Staining is a technique used in the microbiology laboratory with the help ofcoloring dyes, indicator and reagents to enhance or distinguish a specific organismor a morphological structure to facilitate their study under the microscope. Themost commonly used dyes in microbiological work are known as cationic dyes.The color containing portion of the molecule is known as chromatophore while thenonpigment containing part is known as auxochrome. Before staining, one must first prepare a smear of the specimen byspreading a thin film of the bacterial suspension on a clean slide with the use ofan inoculating loop. The smear is then air dried, heat fixed r immersed inchemical solutions. Stains could also be used in combination to producecontrasting colors and effects to distinguish 2 separate group of organisms in onesample.Objectives:1. To introduce the different types of staining procedures.2. To differentiate and distinguish the functions of the individual reagentsincluded in each staining procedure.3. To have a realistic experience in the actual staining of microorganisms.Materials:1. Specimen of microorganisms to be provided in the lab.2. Different staining solutions3. Clean glass slides4. Inoculating loop5. Bunsen burner6. Dropping bottle7. Staining Rack
  • 19. 18A. Simple Staining:Procedure: Note : Inoculating loop must be flamed red hot before & after use. 1. Prepare 2 smears with the inoculating loop for each of the microorganisms provided. 2. Heat fix and allow the specimen to cool. 3. Flood one set of the slides with Carbol fuchsin & the other set with Methylene Blue. 4. Allow the stain to remain for one minute. 5. Wash with tap water and blot dry. 6. Examine all specimen under oil immersion objective. 7. Note down all observations.Observations:B. Differential Staining:I. Gram Stain:Procedure: 1. Prepare a thin smear for all specimen provided. 2. Fix & allow to cool. 3. Flood the smear/s with a few drops of Crystal or Gentian Violet. 4. Let the slide stand for 60 seconds then wash with tap water. 5. Add a few drops of Gram’s Iodine.
  • 20. 19 6. Let the slide stand for 60 seconds then rinse with tap water. 7. Add 95% alcohol to the smear until no more color comes off from the alcohol (this may take 30 seconds to 2 minutes) 8. Wash again with tap water. 9. Add a few drops of Safranin 10. Allow to stand for 30 seconds 11. Wash off the excess stain. 12. Blot dry and examine the specimen under Oil Immersion Objective.Observations:II. Acid Fast Staining:Procedure: 1. Prepare a thin smear of the specimen provided. 2. Heat fix and cool. 3. Flood the slide with aqueous Carbol Fuchsin solution. Steam the slide for 5 minutes over a flame. Do not allow the preparation to dry up by adding Carbol Fuchsin from time to time. 4. Wash off the excess stain with tap water. 5. Decolorize the smear with acid alcohol until no excess stain comes off. 6. Counterstain with Methylene Blue for 30 seconds to one minute. 7. Wash off excess stain with tap water. 8. Blot dry and examine under Oil Immersion objective.
  • 21. 20Observations:III. Negative Staining:Procedure: 1. Obtain gingival scrapings from crevices by means of a toothpick or a periodontal scaler. 2. Spread the scrapings on a glass slide and apply a drop of distilled water. 3. Mix Nigrosin with the smear and allow to dry. 4. Wash the excess dye off , dry and examine under Oil Immersion objective. Use lead pencil in illustrating results.Observations:
  • 22. 21Review Questions:(1) Enumerate and give the functions of the reagents used in Gram’s & Acid FastStain:___________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________(2) What is the purpose of heating the bacterial smear before staining?________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________(3) What is the factor behind an organism being gram (+) or gram (-)?________________________________________________________________________________________________________________________________________________________________________________________________________________________(4) What is the purpose of steaming the smear in acid fast staining? ___________________________________________________________________________________________________________________________________________________________________________________________________________________________________
  • 23. 22(5) Give examples of Acid fast bacteria:________________________________________________________________________________________________________________________________________________________________________________________________________________________(6) Give the (+) and (-) color results of both the Gram’s stain & Acid fast stain:________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________(7) Explain the reason why sometimes the color of the background of thespecimen is in contrast to that of the microorganism?________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________(8) Give the importance and the circumstances when Negative staining is used:________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________
  • 24. 23(9) What could be the possible explanation for you obtaining a result of havingboth Gram positive and Gram negative results on one slide:________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________
  • 25. 24 Exercise No._____ Media Preparation Culture media refers to any artificial or natural substance or nutrientsprovided to the microorganism for it to obtain nourisment in order to sustaingrowth and reproduction. An optimum balance of nutrients and a suitable growth environment areneeded to culture a microbe in the laboratory. The culture media should containall the necessary materials for the microbe to move quickly into the log phase ofgrowth. Water, Carbon, Nitrogen, minerals and growth factors must be availablein a usable form. Many growth media can be prepared by the dental student toisolate , grow and identify a particular specie of microbe in the laboratory. In media preparation, the reagents and materials required may vary fromone to the other but there a standard set of procedure to follow: 1. Gathering all the materials needed. 2. Dissolving the ingredients in the proper solvents 3. Adjusting the pH of the media 4. Filtering the media 5. Dispensing the media in their appropriate containers 6. Cotton plugging the tubes 7. Sterilizing the media 8. Testing the sterility 9. Storing the media.Objectives:1. To develop the ability to prepare the different types of culture media based onthe standard procedures and the requirements of the individual microorganisms.2. To know the various nutrient requirements of various individualmicroorganisms
  • 26. 25Materials:1. Beef Extract 0.3 gm2. Peptone 2.0 gm3. Sodium Chloride 0.5 gm4. Distilled water 100 ml5. Erlenmeyer flask6. Pipettes, funnel, test tube7. Graduated cylinder8. Indicators9. Comparator block & pH standardsA. Nutrient Broth:Procedure: 1. Mix the ingredients in a 100 ml flask. Add aliquot portion of distilled water. 2. Dissolve by heating over flame. 3. Adjust the pH to 7.4 - 7.5 using comparator block method. 4. Filter the media if necessary. 5. Dispense into 10 ml amount in test tubes, Plug with cotton plugs. 6. Autoclave at 25 psi for 15 to 20 minutes 7. Store in refrigerator for future use.B. Nutrient Agar:Procedure: 1. Same as the above preparation but add 2 gram agar. 2. Boil until all agar is completely dissolved. 3. Dispense 10 cc into each test tube. 4. Plug the test tube with non-absorbent cotton. 5. Autoclave the tubes at 15 psi for 15 to 20 minutes. 6. Slant 5 tubes and allow the other tubes to solidify in the upward position. 7. Store the media in the refrigerator for future use.Review Questions:
  • 27. 26(1) What constituent in nutrient broth satisfy the essential requirements for energy,cellular growth & repair?________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________(2) Why is agar used in solid media preparation?________________________________________________________________________________________________________________________________________________________________________________________________________________________(3) How would you adjust the pH of solid media ?________________________________________________________________________________________________________________________________________________________________________________________________________________________(4) What are the different sterilizers available in the laboratory?________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________(5) Is there danger or harmful effects in oversterilizing the media? _____ Explain:________________________________________________________________________________________________________________________________________________
  • 28. 27________________________________________________________________________________________________________________________________________________(6) What types of media are sterilized in the autoclave ?_______________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________(7) Can all microorganisms grow in nutrient broth and media ? ________ Why orwhy not?________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________(8) Name the other types of media and give examples of organisms which grow inthem :________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________(9) Give the differences between a butt culture and the slant culture aside fromtheir physical appearance:___________________________________________________________________
  • 29. 28______________________________________________________________________________________________________________________________________________________________________________________________________________________(10) Differentiate pure, stock & mixed culture. Give their uses and examples foreach:________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________
  • 30. 29 Exercise No. ______ Aseptic Technique & Methods of Inoculation One of the most important laboratory procedures in microbiology is toobtain pure cultures for the identification of organisms. When the samples fromsoil, water, food, or body are collected into nutrient containing petridishes, it isclear that each sample contains a variety of microbes. This is particularly true ofspecimens collected from the skin or the mucous membranes of patients withinfection. The clinical specimens contain both the harmless microbes ( normalflora ) of the body and the microbes causing the disease ( pathogens ) . In order toaccurately identify which microorganism is responsible for the illness, the manydifferent types of microbes must be separated. After separation the individualmicrobes must be placed on the medium that will allow them to reproduce into aclearly visible population or colony containing only one specie. Samples from thiscolony can then be picked out and transferred to a separate culture for furthergrowth and identification. The microbiologist will then be able to study suchcharacteristics such as morphology, metabolism and antibiotic sensitivity. Special wires known as the inoculating loop and needles are used in thetransfer of specimen. Aseptic technique is followed to prevent both the mixing ofcultures and contamination of the student or working area with pathogenicmicrobes or their toxins.Objectives:1. To develop the skill of proper inoculation of different kinds of culture media.2. To be able to practice asepsis in the laboratory which will serve as a training ground for the dental students future practice in the clinic & actual practice.3. To study bacterial growth in different types of culture media.Materials:1. Culture media a. plain broth tube b. plain agar butt c. plain agar slant d. agar plates2. Inoculating loop and needle
  • 31. 303. Bunsen burner4. Organism/specimenProcedure:A. Inoculation of Liquid media: 1. Hold the tube gently on the left hand and the loop or needle ( which must be previously heated ) with the right hand. 2. With the pinkie ( little finger) of the right hand, pull out the cotton plug of the tube. 3. Heat the mouth of the test tube and proceed with the actual inoculation by slightly rubbing the loop with the inoculum on the side of the tube or simple just by twirling the loop to dislodge the inoculum. 4. Heat the mouth of the tube , put back the cotton plug onto the test tube before setting it down and reflame the loop or needle before setting it down on the table.B.Inoculation of Butt culture: 1. Repeat steps 1 & 2 from the previous instructions but this time use the inoculating needle. 2. Heat the mouth of the test tube and plunge the needle into the agar in one swift downward motion stabbing about 2/3 of the media. Pull out the needle, reheat the mouth of the tube and replace the cotton plug onto the test tube. 3. Flame the inoculating needle until red hot and cool before finally setting it down on the table.C. Inoculation of Slant culture:
  • 32. 31 1. Repeat steps 1 & 2 of procedure A, but this time heat the inoculating loop. 2. With the little finger remove the cotton plug and heat the mouth of the test tube. 3. Starting from the butt end of the slant draw the loop over the surface in a straight line towards the end of the slant. 4. Starting again from the butt end, trace a zigzag course from side to side, at the same time slowly drawing the loop towards the end of the slant. 5. Heat the mouth of the test tube , replace the plug, heat the loop, cool and set it down on the table.D. Inoculation of Plated Media:I Interrupted Streak: 1. Flame the loop and pick up the specimen from the test tube after flaming the mouth of the tube. Reflame the mouth and replace the cotton plug. Set down the test tube. 2. Hold the petridish with your left hand, with the cover side up. With the thumb and little finger, raise the lid up and proceed with the inoculation. 3. Start inoculation at the farther side of the plate tracing a zigzag course from side to side until you reach the middle of the plate or the widest part of the petridish. 4. Close the lid and rotate the plate 180 degrees. Repeat the procedure from the other end of the plate in the same manner.II. Multiple Inoculation: -This is done with the same steps as Procedure A except that this is a procedure done when the available media is insufficient for the number of
  • 33. 32 specimen. The plate is then divided into about 5 to 8 quadrants as that of a pizza pie, and each organism is streaked into each quadrant.III. Overlap streak Method: -This procedure is carried out in the same manner as above except that in the overlap method, we depend on the overlap zones to provide the isolated colonies in the second & the third areas of inoculation.IV. Radial Streak Method: 1. Follow all the preliminary steps as in procedure A. 2. Place a loopful of the broth culture near the edge of the agar plate. 3. From here make radial straight lines streaking towards the other side. Start from one side of the plate until the whole plate has been inoculated. This method is usually used in culturing fungi.V. Pour plate method: 1. Prepare 3 sterile test tubes & petridishes . 2. Put 9 cc of sterile saline to each of the 3 test tubes. 3. To the first tube add 1 ml of the broth culture of Specimen A and mix. 4. Transfer 1 ml from the contents of the first tube into the second tube and mix. You now have a dilution of 1:10 5. Transfer 1 ml from the contents of the 2nd tube into the 3rd tube and mix. You now have a dilution of 1:100 6. From each of the test tubes pipette out 1 ml of the contents separately and transfer them into a corresponding petridish. 7. Add about 10 - 12 cc of melted agar to each of the petridish. 8. Allow the agar to solidify then incubate.Illustrations:(1) Graphically present the different types of culture media used in inoculation:
  • 34. 33
  • 35. 34(2) On the space provided , graphically illustrate and label the 7 different methodsof inoculation
  • 36. 35
  • 37. 36Review Questions:(1) Why are the inoculated plates incubated in an inverted position ?________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________(2) Differentiate a pure culture, mixed culture and a stock culture : _____________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________(3) What are the other ways of identifying microorganisms? ___________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________(4) Assuming that the same serial dilution was carried out in a laboratoryexperiment in 10 tubes with 4 ml of saline in each . Pour plate method ofinoculation was performed. It was later found out that on the 7th petridishcultured there was a total of 16 individual microorganisms found. Deduce a waybased on the given and compute the total number of microorganisms present intube one neglecting all chances of human error in the transferring process.______________________
  • 38. 37Show complete solution in the space provided:
  • 39. 38 Exercise No. _____ Biochemical Activities of Bacteria Bacteria are capable of performing several biochemical activities, one ofwhich is fermentation of many molecules including carbohydrates, amino acids &organic acids. These properties could serve as tools of identification since theydemonstrates some metabolic capability of the microbe. The set-up used in the laboratory to detect biochemical activity is with theuse of Durham tubes inverted in test tubes containing different carbohydrates. TheDurham tube would collect the gases such as Carbon Dioxide, Methane orHydrogen that might be produced in the fermentation process. If gas is produced ,a visible bubble will form clearly in the Durham tube. Some bacteria produce acid& gas as fermentation products and in some instances only one of the two isproduced. Differential media is also used to differentiate microbes. One of the mostimportant sets of tests is the IMViC Test. It is a series of biochemical test used toidentify members of the Enterobacter family. Many members of this familyproduce diseases such as : E. coli - Infantile diarrhea Enterobacter - Urinary tract infection Klebsiella - Pneumonia Salmonella - Typhoid fever & Gastroenteritis Shigella - Bacillary Dysentery An example would be two of the abovementioned organisms exhibits thesame properties. E.coli & Enterobacter are both found in the colon of humans,they are both Gram negative rods, and also exhibit the same symptoms in parasiticrelationships. The IMViC test would be the easiest way to distinguish them apart.Objective :1. To understand the biochemical activity & metabolic capabilities of someclosely related microbes.
  • 40. 392. To differentiate two groups of microorganisms based on their biochemicalproperties. (Escherichia coli & Enterobacter aerogenes)3. To have an actual demonstration of the most common biochemical tests used inthe microbiology laboratory.Materials:1. Broth culture of both E. coli & E. aerogenes2. Erlich’s reagent3. Clark & Lub’s media4. Ether5. Methyl red indicator6. Simon Citrate media7. 40 % KOH8. Test tubesA. Indole Test: - demonstrates the production of indole based on the presence or absence of tryptophanProcedure : 1. Prepare 2 test tubes containing Specimen A ( E. coli) & Specimen B ( E. aerogenes ) in broth culture. 2. Add 1 ml of ether ( note: do not aspirate by mouth) to 48 hour broth culture of both specimen A & B 3. Shake well and allow to stand until ether rises to the surface. 4. Gently add about 0.5 ml of Ehrlich’s reagent drop by drop down the side of the test tube so that it forms a ring between the medium and the ether layer. The presence of indole is indicated by a cherry red or deep red color in the reagent layer at the top.B. Methyl Red Test - a pH indicator ( Methyl Red ) is used to determine whether the microbe has produced acid or not.Procedure:
  • 41. 40 1. Prepare 2 tubes containing Clark & Lub’s broth 2. Inoculate specimen A & B into the tubes 3. Incubate the tubes for 72 hours at 37 degrees 4. After incubation, add 2 to 3 drops of Methyl Red. 5. Red Color would be an indication for the production of acid and yellow color would be indicative of alkaline or negative resultsC. Citrate Test - indicates whether or not the organism can utilize citrate as a sole source of Carbon.Procedure: 1. Prepare two tubes with Simon Citrate media. 2. Inoculate by streaking on the slant portion of the media. 3. Incubate the tubes at 37 degrees for 24 hours. 4. Blue color would be indicative of positive results and green color would be negative.D. Voges-Proskauer Test - indicates the production of an organic molecule called acetyl- methylcarbinol or acetoin from glucose in microorganisms.Procedure: 1. Prepare 2 tubes of Clark & Lubs media 2. Inoculate specimen A & B into the tubes 3. Incubate the tubes at 37 degrees for 72 hours. 4. After incubation add an equal amount of 40% KOH and shakevigorously for 2-5 min. 5. Allow the tubes to stand and note the color reaction. 6. Production of an Eosin like color up to 2 hours would be positive. Color would depend on the oxidation of acetoin.Observations & Results: In the space provided, draw a 3 column table and tabulate your results:
  • 42. 41Review Questions:1. Give the positive results & at least 2 organisms identified by the following tests: a. Catalase test: ____________________________________________________ _________________________________________________________________ __________________________________________________________________ b. Coagulase Test: __________________________________________________ __________________________________________________________________ __________________________________________________________________ c. Oxidase Test ____________________________________________________ _________________________________________________________________ _________________________________________________________________ d. Urease Test _____________________________________________________ _________________________________________________________________ e. Hemolysis Test __________________________________________________ _________________________________________________________________ _________________________________________________________________
  • 43. 42 f. Hydrogen Sulfide Test _____________________________________________ _________________________________________________________________ _________________________________________________________________ g. Greig’s Test _____________________________________________________ _________________________________________________________________ _________________________________________________________________2. What is the API 20 system?________________________________________________________________________________________________________________________________________________________________________________________________________________________
  • 44. 43 Exercise No. _____ Factors Influencing Bacterial Growth Aside from the nutritional requirements of the microorganisms which areprovided for in the culture media, different microbes require differentenvironmental conditions for them to exist, grow and reproduce. Environmentalfactors such as temperature, pH, Osmotic pressure, salt concentration, Oxygen &Carbon Dioxide concentration and presence of moisture are all essential & shouldbe taken into consideration as part of the whole living environment. This set ofexercise is designed at altering and adjusting some of the variables and observingthe effects of these variants on the behavior and growth of specific microbes.Objectives:1. To know the different factors behind the growth of microorganisms.2. To differentiate microorganisms based on their ability to exist under different environmental conditions.Materials:1. Broth culture of different microorganisms2. 20 test tubes3. Candle jar or anaerobic jar4. Dessication chamber5. Mild acid & base6. Water bathProcedure:A. Temperature Requirement: 1. For every specimen provided , inoculate each microorganism into 4 tubes and label them A, B, C, D.
  • 45. 44 2. Incubate all tube A’s at 37 degrees. 3. Store all tube B’s in the refrigerator ( 4 - 12 degrees ) 4. Incubate all tube C’s in the incubator at 50 degrees. 5. Store all tube D’s in the locker ( 25 degrees ) 6. After 24 hours check and tabulate results.B. Oxygen Requirement : 1. For every specimen provided, inoculate each into 3 sets of tubes 2. Store the first set of tubes in the incubator. 3. Store the second set of tubes in a candle jar or anaerobic jar 4. Store the third set of tubes in the cabinet. 5. Incubate for 24 hours. Check and tabulate the resultsC. pH Requirement:
  • 46. 45 1. To each of the specimen provided, inoculate each into three tubes. 2. To the first set of tubes , add 1 ml of mild acid. 3. To the second set of tubes, add 1 ml of mild base 4. Use the third set of tubes as control. 5. Incubate the tubes for 24 hours. 6. Check and tabulate your results.D. Moisture Requirement: 1. Inoculate the specimens provided in a set of tubes and store them in a desiccated chamber. 2. Incubate for 24 hours. 3. Observe and record the results.Review Questions:
  • 47. 461. Differentiate the following : Optimum growth temperature, minimum growthtemperature & maximum growth temperature:________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________2. Name the different types of microorganisms based on their oxygen requirement:________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________3. Different psychrophiles, mesophiles & thermophiles: _______________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________4. Aside from the factors discussed in the laboratory exercise, what other factorsdo you think influence bacterial growth? ___________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________(5) Is there an actual theoretical value for optimum growth temperature? Why orwhy not? Justify your answer:
  • 48. 47______________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________
  • 49. 48 Exercise No. _____ Microbial Control Microbes present in the environment which are harmful to us need to becontrolled or eliminated. This is carried out by inhibiting their growth and modeof actions, destroying the microbes thereby eliminating them from theenvironment. This is carried out by elements known as antimicrobial agents.These agents could be classified into physical, chemical and through the processof filtration. The choice of a particular agent depends on the type of material tobe treated, specific kind of microbe to be controlled and the environmentalconditions present at the time of use.Objective:1. To have a general overview on the different methods of inhibiting & controllingmicrobes.2. To distinguish the different physical & chemical agents used in microbialcontrol3. To be able to understand the bacteriostatic & bactericidal effects of somecommon agent used in microbial controlMaterials:1. Inoculating loops ( 3 )2. Nutrient broth3. Water bath4. Tripod5. Bunsen burner6. Broth culture of Staphylococcus aureus7. Hydrogen peroxide8. Phenol9. Clean test tubes
  • 50. 49Procedure:A. Effect of heat on Microorganisms: 1. Boil water in a water bath 2. Label three test tubes nutrient broth as: A - control B - dry heat C - moist heat 3. Expose the three tubes to the same environment ( to be given) 4. Dip one inoculating loop directly into tube A 5. Flame another inoculating loop directly for 10 minutes then dip into tube B. 6. Place the 3rd inoculating loop in boiling water for 10 minutes then dip intube C. 7. Incubate the three tubes for 24 hours.Tabulate your results using the following symbols: ( - ) no growth ( +- ) slight growth or trace ( + ) moderate growth ( ++ ) heavy growthObservations:Using the symbols provided from the previous page draw a table & tabulate yourresults in the space provided below:B. Effect of Chemical Agents on Microorganisms :
  • 51. 50 1. Make dilutions of the following disinfectants: a. Phenol b. Hydrogen Peroxide Prepare dilutions of 1:10, 1:100, 1:1000 2. Place all the tubes in a water bath at 20 degrees for 5 minutes 3. Transfer 0.5 ml of the culture in each of the tubes 4. Incubate the nutrient broth for 24 hours.Observations: Using the same symbols given in part A, tabulate your results in the spaceprovided below:Review questions:1. Name the dental uses of phenol :________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________
  • 52. 512. What is phenol coefficient? ____________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________3. If results obtained were all ( ++ ) in all dilutions, what would be the mostsensible alternative or approach?________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________4. Another method of microbial control is filtration. List down at least 5 kinds offilters which could be used for this method? _________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________5. Define the following terms: a. Antiseptic: ______________________________________________________ __________________________________________________________________ __________________________________________________________________ b. Asepsis:_________________________________________________________ _________________________________________________________________ __________________________________________________________________ c. Disinfectant : ____________________________________________________ _________________________________________________________________ d. Sterilization : ____________________________________________________
  • 53. 52____________________________________________________________________________________________________________________________________e. Bactericidal :_________________________________________________________________________________________________________________________________________________________________________________________f. Bacteriostatic : _____________________________________________________________________________________________________________________________________________________________________________________
  • 54. 53 Exercise No. _____ Bacterial Growth Curve Large population of bacterial cells are routinely used in the laboratoryexperiments in which bacterial growth and related processes are measured. Asmall inoculum of cells is usually introduced into the culture media and after ashort period of time, when the cells have reached confluency, harvested. It istherefore of utmost importance to determine the time it takes for the organism toreach confluency and the stages of growth that the culture is in. Cultures after being inoculated undergo several stages: A. Lag phase is a phase wherein cells are given ample time to react to their new environment and there is only increase in size, B. Log phase or the exponential phase is a stage of rapid growth and cell multiplication, C. Stationary phase is achieved after a period of time and the number of organisms remains constant due to the depletion of nutrients, pH change from waste accumulation or simply overcrowding, D. Death or decline phase occurs when the organism starts to die off due to the increasingly irreversible effects from the stationary phase.Objective:1. To study the development of a bacterial culture from inoculation to its decline.2. To be able to quantitatively measure the bacterial population at given pointsduring culture.3. To graphically illustrate the different stages of the bacterial growth curve basedon the results obtained in the exercise.Materials:1. Broth culture of Staphylococcus aureus & Escherichia coli2. Spectrophotometer3. Cuvette4. Graphing paper
  • 55. 54Procedure: 1. Prepare the broth culture of the different specimen provided in serial dilutions of 1:10, 1:100 and 1:1000 labeled as tube A, B, & C. 2. Prepare a control by storing broth in a tube without any specimen. 3. Incubate the tubes at 37 degrees. 4. Take the readings of the tubes with a spectrophotometer at the given intervals: 0 minutes, 30 minutes, 2 hours, 4 hours , 8 hours, 12 hours, 24 hours , 48 hours 5. Take the reading of the control and subtract the value from the values obtained in all tubes at all times. 6. Tabulate all results and plot with a graphing paper.Review Questions:1. What are the stages of the bacterial growth curve? Discuss Briefly. ___________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________2.. What are the factors influencing the decline of a bacterial culture? ___________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________
  • 56. 553. Would all microorganisms exhibit the same bacterial growth curve? ______Why?__________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________4. Why is there an existence of a stationary phase? Is it really because themicroorganisms “stop” growing ?_____________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________5. What are the other methods of determining cell population? _________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________
  • 57. 56 Exercise No. _____ Antibiotic Susceptibility Test The single most important clinical test performed by many microbiologistsis the culture and susceptibility test because the results would provide thephysician, dentist or any medical personnel with significant information regardingthe pathogen and a possible means of controlling it. A culture and susceptibility test is the growth of a known pathogen in pureculture and its exposure to known antibiotics of different concentration todetermine which drug will kill or inhibit the growth of the microorganism.Objectives:1. To understand the principle behind a culture and sensitivity test.2. To illustrate the effects of different antibiotics on a given microorganism3. To quantitatively measure the delimiting power of a specific antibiotic to agiven microbe.Materials:1. Broth cultures of specimen provided2. Agar plates3. Antibiotic paper discs4. Sterile swabs5. Sterile forceps6. Transparent rulerProcedure: 1. Prepare one agar plate for each specimen provided. Label properly. 2. Inoculate the agar plates. 3. Using flamed forceps, place the antibiotic discs on the agar surface . Gently press down the discs to ensure contact with the agar. 4. Incubate the plates in an inverted manner at 37 degrees for 48 hours. 5. Measure the clearing zone or zone of inhibition with your rulers. 6. Record and tabulate your results in millimeters.
  • 58. 57Observations:Review Questions:1. What is the Kirby-Bauer method in susceptibility testing? ___________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________2. What are the three ranges of inhibition zones? How are they interpreted? _____________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________
  • 59. 583. How do you differentiate a broad from a narrow spectrum antibiotic?________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________4. What is MIC in relation to antibiotic susceptibility? How does it affect thechoice of antibiotic prescription? __________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________5. Would the same procedure done above be applicable in determining allergies inman? _______________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________6. Pick out any one microorganism which you have studied and try to classify it interms of susceptibility to any 10 common antibiotics used in Microbiology orPharmacology:________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________
  • 60. 59________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________7. What is the basis of determining a microorganism in being susceptible orresistant to a specific antibiotic? _____________________________________________________________________________________________________________________________________________________________________________________________________________________________________________
  • 61. 60 Exercise No. _____ Normal Flora of the Oral Cavity There are more than 30 species of microorganisms that have been isolatedfrom the oral cavity. From shortly after birth till death, the individual supports arelatively stable oral microbiota. Its composition depends upon the introduction ofvarious microorganisms from the external environment and more importantlyupon the various inherent and acquired factors that control the environment withinthe oral cavity. Although there are many factors which make it difficult to obtain validinformation concerning the kinds and number of microorganisms in the oral cavityat a given time, this exercise is designed to only identify indigenous organismspresent. Some examples of these organisms are lactobacilli, streptococci,veillonella, spirochetes, filamentous forms, fusiform bacilli & vibrios. They areconstantly present in large numbers.Objectives:1. To confirm the presence of microorganisms in the normal flora of the oralcavity2. To identify the organisms present in the oral cavity by studying the saliva.3. To practically apply the different laboratory procedures introduced in thelaboratory to be used in the identification of the microorganisms.Materials:1. Broth culture of salivary secretions.2. Agar plates3. Methyl violet4. Gram’s Stain5. Nigrosin Dye6. Inoculating loop and needle7. Bunsen burner8. Glass slides
  • 62. 61Procedure: 1. Using clean glass slides perform the following staining procedures: a. Direct methyl violet staining b. Gram’s Stain c. Negative staining 2. Inoculate the agar plate with the broth culture of saliva 3. Invert the plate and incubate for 48 hours. 4. Observe and record your results for all procedures performed.Observations:Review Questions:1. What are the microorganisms most commonly found in the healthy oral cavity?________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________2. What is the nature of the oral environment of the infant at birth ? _____________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________
  • 63. 623. Name the factors that regulate the oral microbiota: _________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________4. What is commonly known as the indicator microorganism in the oral cavity?Why?__________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________
  • 64. 63 Exercise No. _____ Microbiota of Dental Caries The teeth, because of their composition, inertness and environment areparticularly susceptible to destruction. Caries is a destructive disease of the teethof man and all other forms of life that possess calcified dentition, the etiology ofwhich is intimately associated with the oral microbial flora. Dental caries begins most often in areas of coronal enamel where salivamay stagnate, food debris may impact, and the oral microbial flora may find asuitable environment for growth such as enamel pits & fissures, and near the pointof interproximal areas. Microbial activity initiates the destruction of enamel byconverting carbohydrates to organic acids which demineralize the enamel or bychelation of enamel calcium by a variety of bacterial metabolites.Objectives:1. To confirm the presence of specific microorganism present in dental caries2. To compare the microbiota of patients with rampant dental caries from thosewithout..Materials:1. Saliva from a patient with rampant dental caries2. Saliva from a patient without caries3. Rogosa’s agar slant4. Sterile test tubes5. Clean slides6. Reagents for gram and negative stainingProcedure: 1. Collect a small amount of saliva from both types of patients and store in sterile tubes
  • 65. 64 2. Inoculate the Rogosa;s agar slant. Incubate for 48 hours at 37 degrees. 3. Perform Gram and negative staining procedures. 4. Examine under oil immersion objective.Observations::Review Questions:1. What are the microorganism which are predominant in the occurrence of dentalcaries?________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________2. Discuss the factors that mediate dental caries: _____________________________
  • 66. 65________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________3. What is Snyder’s colorimetric test? ______________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________4. What is the DMF index ? _______________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________
  • 67. 66 Exercise No. _____ Microbiota of Dental Plaque Dental plaque is the mass of bacteria in a matrix of organic material whichadheres tenaciously to the tooth surface. It is made up largely of protein andpolysaccharide which is partly derived from saliva and partly produced by theorganism . Plaque does not include food debris or exfoliated epithelial cells whichare only transient features. Dental plaques are composed of about 20% precipitated salivary mucoid ormucin and about 80% microorganisms.Objectives:1. To identify the presence of microorganisms in dental plaque2. To know the different types of plaque.Materials:1. Clean glass slides2. Reagents for Gram’s and Negative staining procedure3. ToothpickProcedure: 1. Collect plaque from the enamel and dentin with the use of a toothpick. 2. Prepare a bacterial smear from the collected sample. 3. Perform Gram’s and negative staining procedure. 4. Wash and dry stained smears. 5. Examine specimen under OIO. 6. Record your results and observations.
  • 68. 67Observations:Review Questions:1. What organisms make up the considerable bulk of the microbial flora of thedental plaque?________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________2. .How are plaque formed? ______________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________
  • 69. 68________________________________________________________________________________________________________________________________________________3. Name the bacterial species which are predominant in superficial enamel decay.________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________4. What are the three types of dental plaque? ________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________
  • 70. 69 Exercise No. _____ Microbiota of the Periodontal Pocket & Root Canal The tissues that surround and support the teeth like the gingival tissues,alveolar bone, periodontal membrane are subject to various diseases, collectivelyknown as periodontal diseases. In these pathological processes, the oral microbialflora is greatly involved. Bacteria do not produce disease simply by their physical presence, althoughtheir adequate multiplication is essential to infection. To produce a disease, someconstituents or products of bacteria like toxins and enzymes must react with thetissues or cells to destroy them or interfere with their normal functions.Objectives:1. To distinguish the microorganisms present in the periodontal pocket and theroot canal.2. To identify the predominating organisms generally causing periodontaldiseases.Materials:1. Sample from a periodontal pocket & infected root canal2. Brain Heart Infusion broth ( BHI )3. Reagents for gram’s and negative staining .4. Clean glass slidesProcedure: 1. Inoculate the collected specimen in BHI 2. Incubate at 37 degrees for 48 hours 3. Perform Gram’s and negative staining procedures. 4. Examine under OIO. 5. Record your results and observations.
  • 71. 70Observations:Review Questions:1. What factors influence the initiation & progression of periodontal diseases? __________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________2. What is Vincent’s angina? Ludwig’s angina? ______________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________
  • 72. 713. Name the enzymes produced by the members of the oral microbial flora? ______________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________4. Why is BHI commonly used in culturing root canal specimen? _______________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________5. What is the microbiota of an infected root canal? ___________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________
  • 73. 72 Exercise No. ______ The Study of Fungi Mycology is the study of fungi which are non-photosyntheticmicroorganisms that grow typically in filaments and reproduce by sporulation.Example of this group of organisms would include the lichens, mushrooms,toadstools, puffballs and the microscopic plants known as molds and yeast. Someof them have evolved into true pathogens and are able to actively infect, causeharm and be transmitted from one suitable host to another. Infections caused by fungi is known as mycoses. The types of clinicalmycoses could be divided into cutaneous, subcutaneous & systemic. Thesedifferent types of fungi that are clinically significant could be acquired from soilor are opportunistic normal flora of the body.Objectives:1. To study the microscopic appearance of a common fungi.2. To enumerate the cultural characteristics and mode of reproduction of fungiMaterials:1. An old piece of bread2. Hand lens3. Clean glass slideProcedure: 1. Moisten a little piece of old bread and incubate 2. Study the bread from the incubator. 3. With the hand lens, examine the bread and identify the colonies. 4. Prepare a wet mount using the fresh state, and focus under the microscope. 5. Draw and label all parts observed
  • 74. 73Observations:Review Questions:(1) Why are fungi considered plant-like organism and yet studied undermicrobiology?________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________(2) Give the different types of mycoses and examples of each: _________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________
  • 75. 74(3) Name some of the members of the fungi family which are beneficial tomankind & give their uses: _______________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________