PH 151: Principles of Microbiology Exercise 1 MICROSCOPY Objectives: At the end of the exercise, the student must be able to:

1. know and execute the proper use and care of the compound microscope 2. be familiar with the different parts of the compound microscope, and the functions of each parts 3. visualize cellular morphology from stained slide preparations. Materials: Microscope, Lens paper/Cotton, Cleansing Solution, Prepared slides of the ff: blood smear; fungi, bacteria, and protozoa. Procedure: 1. Get the microscope assigned to you from the cabinet by taking the arm with one hand and supporting the instrument at the base with the other. 2. Place the microscope at least six inches away from the edge of the working table with the microscope arm facing you. 3. All unnecessary materials such as books, papers, and bags should be removed from the working table. 4. All the objective lenses should be cleaned whenever necessary, to maintain the efficiency of the microscope. 5. Examine the different prepared slides under the different objective lenses. 6. Place the prepared slide with the specimen within the stage clips of the fixed stage. Move the slide to center the specimen over the opening in the stage directly over the light source. 7. Rotate the scanning lens or low power lens into position. A click could be heard once the objective lens is in its proper position. Lower the body tube with the coarse adjustment knob to bring the specimen into focus. Sharpen the focus by rotating the fine adjustment knob. The intensity of light can be regulated by opening or closing the iris diaphragm. 8. To use the high power objective, swing t to place. Since it has a smaller aperture than the low power objective, the iris diaphragm must be opened to fill the objective aperture with light. Sharpen the focus with the fine adjustment knob. 9. To focus the oil immersion lens, swing the oil immerse objective halfway towards the specimen to focus. Place a drop of immersion oil on the slide and swing the lens in place. Lower the objective until it touches the oil. Adjust the distance of the lens to bring the specimen into focus. 10. Illustrate each prepared slide as it is seen in the different objective 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 and remove the slide from the stage. 3. Using lens paper clean all the lenses starting with the cleanest firstocular, low power, high power and oil immersion. Use lens cleaner if necessary. 4. Clean any oil off of the stage using wipes or paper towels. 5. Rotate the scanning objective into place. Use the coarse adjustment knob to obtain minimum working distance. 6. Return the microscope to the appropriate storage area. Study Question: 1. What are the parts of the microscope and the function of each part? 2. What are the different types of microscope, their distinguishing features, and practical uses? Tabulate your answers. 3. What is meant by resolving power and how is it related to magnification? What factor(s) limit (s) the resolving power of the light microscope? How would you enhance it?

PH 151: Principles of Microbiology Exercise 2: Aseptic Transfer Techniques

Objectives: 1. To perform the different methods of aseptic transfer techniques Materials: sterile distilled water, sterile tryptic soy broth, test tube rack, 1ml pipette, alcohol lamp, tryptic soy agar slant, beaker, alcohol, vial w/ sterile disks, and forceps Procedure: A. Sterile Pipetting with Pipettors 1. Obtain one tube of sterile TSB and one tube of sterile water. Place in a tube rack. 2. Label the TSB tube 0.5 mL H2O then write your initials & the date. 3. Loosen the lids on the 2 tubes. 4. Open a prepackaged, sterilized 1 mL pipette and attach it to the blue pipettor. 5. Remove the lid from the water tube, flame the lip of the tube, insert the 1 mL pipet and withdraw 0.5 mL of water. Re-flame the lip and recap. 6. Remove the lid from the TSB tube, flame the lip of the tube, deposit 0.5 mL of water into the TSB, reflame the lip and recap. 7. Return the pipet to the package and discard in the biohazard bag. Place the tube with the water in the rack designated for autoclaving. 8. Incubate the TSB tube at 35C for 48 hours until next lab session 9. Return the stock culture to the lab. B. Sterile Transfer of Bacteria from Broth to TSA Slant 1. Obtain a tube of TSB containing E. coli or S. aureus and 1 TSA slant. Place all tubes in tube rack, keeping the rack near a lit alcohol lamp. 2. Label the sterile TSA slant with the appropriate organism name, procedure #, your initials & the date. 3. Flame the mouth of the TSB tube and the sterile slant. Use the inoculating loop to remove 1 loop full of bacteria from the stock TSB tube and carefully streak the sterile slant from bottom to top. ONE STREAK ONLY!!!! Be careful to not gouge the agar. Re-flame the mouths of both tubes. 4. Flame the inoculating loop and place tube in incubator at 35C for 48 hours. 5. Return the stock culture to the lab. C. Sterile Transfer of Bacteria from TSA Slants to Broth with a Stab/Needle 1. Obtain a slant inoculated with S. aureus or E. coli. 2. Obtain a sterile tube of TSB. 3. Label the sterile TSB tube with the appropriate organism name, procedure #, your initials & the date. 4. Flame the inoculating needle and use the needle to remove a small amount of growth from the stock slant. A light touch is suggested, it does not take a large amount of inoculum to obtain copious amounts of organisms. Deposit the inoculum in the TSB tube. BE SURE to flame the lip of the TSB tube and the TSA slant before and after inoculation. 5. Roll the TSB tube between the hands to thoroughly mix the organisms. (This is called subbing out the organism. It is a procedure to keep the original culture fresh.) 6. Incubate at 35C for 48 hours. 7. Return the stock culture to the lab. D. Sterile Transfer Using Alcohol-Flamed Forceps 1. Obtain 1 tube TSB, forceps, small beaker of alcohol, and vial of sterile disks. 2. Label the TSB tube Sterile Disk, initial and date. Loosen the lid of the tube and place in the tube rack.

3. Dip the tips of the forceps into the alcohol and, holding the forceps HORIZONTALLY, place them in the flame. Be careful not to let the alcohol run towards the hand. 4. Open the sterile disk tube and flame the lip. Remove 1 sterile disk with the sterilized forceps. 5. Open TSB tube, flame the lip, insert the sterile disk into the tube. 6. Re-flame and re-cap both the sterile tube and the TSB tube. 7. Incubate the tube at 35C for 48 hours. *Check all incubated tubes for growth indicated by turbidity of the medium. If the media are turbid, aseptic transfer techniques are not properly executed.

Exercise 3: MICROSCOPIC EXAMINATION OF BACTERIA (Unstained Smears) Objectives: 1. To perform and observe wet mount and hanging drop slides 2. To observe the activities of living cells. 3. To recognize different types of microbes in unstained preparations. Materials: Canal water, Mixed culture of Pseudomonas aeruginosa, Bacillus subtilis, and Staphylococcus aureus, Glass Slides, Depression slide, Cover Slips, Wire Loops, Alcohol Lamp, Petroleum jelly, Dropper, Microscope Procedure: A. Wet Mount Method 1. Place a drop of the canal water on the center of the slide. Place one end of the cover slip on the slide and slowly lower the end over the drop with the use of a toothpick to avoid formation of bubbles. 2. The water should just fill the space between the slide and the cover slip. Remove excess water by holding the edge of a tissue paper at the edge of the cover slip. If there is too little water and some of the space in the slide is dry, add more water by placing a drop at the edge of the cover slip. 3. Examine the slide under the microscope and observe the movements present. Draw your observations. 4. Prepare and examine a wet mount slide of the bacterial mixture. B. Hanging Drop Method 1. Spread a ring of petroleum jelly around the concavity of the depression slide. 2. Using a sterile technique, fish a loopful of bacterial culture from the mixture and place it in the center of the cover slip. 3. Place the depression slide, with the concavity facing down, over the cover slip so that the depression covers the drop of culture. Press the slide gently to form a seal between the slide and the cover slip. 4. Quickly turn the slide right side up so that the drop continues to adhere to the inner surface of the cover slip. Examine the slide under a microscope and draw the observations. Oil immersion lens may be used for detailed observation. 5. Repeat the procedures using canal water as a sample. Study Questions: 1. Why are living, unstained, bacterial preparations more difficult to observe microscopically than stained preparations? 2. For what purposes is it essential that living specimens be observed? 3. What method is most reliable in studying bacterial motility? Why?

Exercise 3 MICROSCOPIC EXAMINATION OF BACTERIA (Stained Bacterial Smears) Objectives: At the end of the experiment, the student must be able: 1. To make a bacterial smear 2. To enumerate the advantages of staining microorganisms 3. To explain the basic principles of staining 4. To be able to perform the different staining techniques. Materials: Bacterial cultures of Staphylococcus aureus, Escherichia coli, Mycobacterium phlei, Bacillus subtilis, and Salmonella sp.; Methylene blue, Crystal violet, Safranin, Carbolfuchsin, Malachite Green, Congo Red, Flagella Mordant, Grams Iodine, Ethyl Alcohol, Acid Alohol, Filter paper, water bath, inoculating loops/needles, alcohol lamp, staining racks, microscope Procedures: Preparation of a Smear 1. If the culture is taken from an agar medium: a. Properly label the slides as indicated by the instructor. Sterilized the slides by passing it over the flame atleast five times. b. Sterilized the inoculating loop by holding it (in about 45O angle) at the hottest part of the flame, until it is red hot. Allow the loop to cool (without touching it) in a vertical position to minimize contamination. c. Place drop or a loopful of sterile saline solution on a clean slide. d. Aseptically remove a small amount of culture from the agar medium and touch the drop of saline solution several times until it turns cloudy. e. Burn the remaining bacterial culture off the loop. Morphology of individual bacteria will not be observed if the smear is too thick. f. Using a sterile loop, spread the suspension in an outward motion to form a thin film, approximately the diameter of a 25-centavo coin. g. Air dry the smear. When it is completely dry, fix the smear by passing the slide (smear side up) over the flame three to five times. 2. If the culture is taken from a broth medium: a. Properly label the slides. Sterilized the slides and the loops. b. Uncap the culture tube and aseptically fish a loopful of bacteria from the culture tube. Spread it over the slide (like procedure 1f) c. Air dry the smear, then heat fix the slides 3. Throat Swab: a. Obtain a throat swab by gently rolling a sterile cotton swab on the sides of the throat. b. Roll the swab back and forth on a slide to make a smear. c. Air dry the smear and heat fix the slide. d. Burn the cotton swab until it is charred. A. Simple Staining 1. Prepare a smear from a given bacterial culture. 2. Place the slides on a staining rack. Cover the entire smear with methylene blue. Do not flood the smear with the dye to avoid over staining. Leave the dye to stain for one minute. 3. Wash the dye off the slide with distilled water. Wash off the stains that got on the bottom of the slides. 4. Air dry the slide or blot it dry with paper towels. 5. Examine the slide uner oil immersion objective.

B. Negative Staining 1. Properly label the slides. Sterilized the slides and the loops. 2. Place a loopful of nigrosin dye on the slide. 3. Aseptically fish a loopful of bacterial suspension from a broth and mix it gently with nigrosin. 4. Spread the mixture using the edge of another slide. Spread with varying pressure across the slide so that there are alternating light and dark areas. Make sure that the dye is not so thick or the bacteria will not be seen. The mixture can also be spread by pressing another slide on the suspension and pulling it apart. 5. Let the smear air dry completely. Do not heat fix nor wash the stain off. 6. Observe the smear under oil immersion objective. Examine on the area that appears light purple. C. Gram Stain 1. Obtain a throat swab. Make another smear of Staphylococcus aureus and Escherichia coli. 2. Place the slides on the staining rack and cover the smear with crystal violet. Leave the stain for one minute. 3. Wash the stain off with distilled water and cover the smear with grams iodine. Leave it for one minute. 4. Flood the smear with ethyl alcohol until no violet color washes off. This usually takes about 10 seconds. Rinse with water. 5. Cover the smear with safranin for one minute. Rinse the slides. 6. Air dry the slides and examine under oil immersion objectives. 7. Record the morphology, arrangement and gram reaction of the bacteria. D. Acid-Fast Stain (Ziehl-Neelsen Method) 1. Prepare a smear from a sputum sample. Air dry and heat fix the smear. 2. Cover the smear with a filter paper and flood with carbolfuchsin. Place the slides on a hot plate or over a boiling water for 5 minutes to let the stain steam. Make sure that the stain will not dry up by continuously adding carbolfuchsin. 3. Rinse the smear with tap water. Decolorize with acid-alcohol, drop by drop until carbolfuchsin fails to wash from the smear. 4. Flood the smear with methylene blue for two minutes and rinse with water. 5. Air dry the smears and examine under oil immersion. E. Special Stains 1. Endospore Stain ( Schaeffer-Fulton Method) Be careful: Malachite Green has a messy habit of ending up everywhere but no matter how careful you are, it will most likely end up n your fingers a. Make two smears of Bacillus subtilis culture. Air dry and heat fix the smears. b. Place a small piece of paper towel or filter paper on the top of the slide to reduce the evaporation of the stain. The paper towel should be smaller than the slide. c. Flood the smear and paper with malachite green and steam for 5 minutes. Add more stain if needed. Keep it wet. d. Remove the paper and discard carefully. Do not put it in the sink. Wash the stained smears well with distilled water. e. Counterstain with safranin for 30 seconds. Wash with distilled water and air dry. f. Examine under the microscope with the oil immersion objective and record your observations. 2. Capsule Stain a. Prepare a thick smear of bacteria in a loopful of congo red. Let the smear air dry. b. Fix the smear with acid alcohol for 15 seconds. c. Wash the smear with distilled water and cover with Ziels carbolfuchsin for 1 minute.

d. Wash with distilled water, air dry, and examine microscopically under the oil immersion objective. The bacteria will stain red and the capsules will be colorless against a dark blue background. 3. Flagella Stain Flagella stains require special precautions to avoid damaging the flagella. Scrupulously clean slides are essential, and the culture must be handled carefully to prevent flagella from coming off the cells. a. Make a smear of Salmonella sp. culture. Allow the organism to adhere to the slides and air dry the smear. Do not heat fix. b. Cover the slide with flagella mordant and allow it to stand for 10 minutes. c. Gently rinse off the stain with distilled water. d. Cover the slide with Ziels carbolfuchsin for 5 minutes. Rinse gently with distilled water. e. Allow the smear to air dry and examine microscopically under the oil immersion objective. Study Questions: 1. What is the purpose of heat fixation? What happens when too much heat is applied? 2. What is more effective in staining, acidic or basic dyes? Why? 3. Enumerate the advantages and disadvantages or negative and simple staining. 4. Compare and contrast gram positive and gram negative bacteria. Tabulate your answers 5. What is the determining factor or the crucial step in gram staining? 6. What is the importance of flooding the smear with grams iodine after staining with crystal violet? 7. Compare the reactions of gram negative with gram positive bacteria in every step of the staining process. Tabulate your answer. 8. What is the purpose of heating in the acid fast and endospore staining? 9. What is the diagnostic value of the acid-fast procedure? 10. Why is acid alcohol used instead of ethyl alcohol, as a decolorizer? 11. Are acid-fast bacteria gram positive or gram negative? Explain. 12. What was the color of the spores and the position in the cells they occupy? Why were the spores colored differently from the other parts of the cell? 13. What advantage does spore formation gives the members of the genera Bacillus and Clostridium? 14. What is the chemical nature of the capsules? What is their importance in the bacteria themselves? 15. What is the Quellung reaction? What are some of its practical applications? 16. What is the importance of flagella in a bacterium?

Exercise 4 STERILIZATION PROCEDURES Objectives: 1.) To be able to learn the principle, procedure, and application of autoclaving. 2.) To be able to perform proper sterilization techniques. Background: Sterilization is the process of eliminating viable microorganisms. This is through removal or destruction of these microorganisms. Sterilization methods are only applicable to inanimate objects. Application of these methods to living tissues is very dangerous and impractical. Sterilization is a very vital procedure in microbiology and other sciences. It employs two kinds of methods: physical and chemical. Physical methods involves mechanical methods (scrubbing, filtration, sedimentation), moist heat (boiling, use of steam, pasteurization, inspissations, tyndallization), dry heat (burning, baking), radiation, and the use of fluorescent dyes. Chemical means of sterilization usually involves the use of antiseptics and disinfectants, chemotherapeutic agents, antibiotics, gases, and dyes. In microbiological procedures, the most important equipment for sterilization is the autoclave. The Autoclave is a vertical or horizontal cylinder provided with an airtight lid which is fastened by means of screws. It provides the most rapid and most efficient method of sterilization by heat. The steam may be introduced directly or by boiling water at the bottom of the chamber, first driving out cold air, after which the chamber is filled with live steam under pressure. The steam pressure should be reduced after autoclaving by opening the valve to let out the steam before opening the chamber door. Most culture media are sterilized in the autoclave 15 lbs. psi for 1520 minutes at a temperature of 121.3 OC. Autoclaving temperatures and time may be however varied depending on the amount and type of culture media. Large flask of media and glasswares are autoclaved for a match longer period of time. Materials: Used paper or old newspapers, Petri dishes (2 per person), 10ml Culture tubes (4 per person), alcohol lamp, Media bottles, Nutrient Agar, Nutrient Broth, Eosin-Methylene Blue (EMB) Agar, 10mL pipettes, masking tapes/autoclave tapes, Gummed labels. Procedure: OPERATION OF THE AUTOCLAVE 1. Fill the autoclave with distilled water up to the desired mark (at least 3 inches). 2. Place the materials to be sterilized on the shelf on the autoclave. As much as possible, leave a space between objects on the shelf so that the steam flow or evacuation is not impeded. 3. Close the door of the autoclave and tightened it by moving the knob on the right side. 4. Adjust the time knob to the 15 minute mark. 5. After 15 minutes, wait for the temperature to decrease up to the 60 mark before opening the autoclave to make sure that the pressure inside autoclave is decreased. 6. All materials that will be autoclaved should be wrapped in a used paper or old newspaper so they could be stored for future use. Study Questions: 1. Define sterilization, disinfection, antisepsis, and bacteriostasis. 2. Give the temperature used in the moist heat and dry heat sterilization. 3. What materials are ideally sterilized in the above methods of sterilization? 4. Why is moist heat not recommended in sterilization of metallic apparatuses, bladed instruments, and oils? 5. What is the difference between a chemotherapeutic agent and an antibiotic? 6. Give the principles of action of the different antiseptics and disinfectants. 7. Why do we need to place distilled water instead of ordinary tap water?

Exercise 5 PREPARATION OF CULTURE MEDIA Objectives: 1.) To be able to know the different methods of media preparation. 2.) To be able to prepare different culture media. Background: Culture Media are very important in the cultivation and growth of microorganisms. Microrganisms are usually cultivated when they are difficult to find in a direct smear or when their identity is doubtful. Their growth characteristics and behavior in a culture media are important in their identification and classification. Direct smears and culture methods are complementary- the first giving a rough idea on the possibilities, and the second, giving a more refined detail and information. Bacteria have different physical and nutritional requirements thus selection of proper media is essential. Culture media contain the nutritional requirement of the microbes. They are prepared primarily to support the growth of microorganisms but they can also be used to identify the biochemical and physical properties of the microorganisms. Some of the media may promote the growth of all microorganisms while others could inhibit it. Each kind of culture media is formulated to suit their function. Materials: Used paper or old newspapers, Petri dishes (2 per person), 10ml Culture tubes (4 per person), alcohol lamp, Media bottles, Nutrient Agar, Nutrient Broth, Eosin-Methylene Blue (EMB) Agar, 10mL pipettes, masking tapes/autoclave tapes, Gummed labels. Procedure: GENERAL RULES IN THE PREPARATION OF CULTURE MEDIA 1. Follow the directions found in the media container exactly. Weigh the ingredients carefully and keep the scales in good condition. 2. Since most of the stock media are in the dehydrated form, avoid too much exposure of the media to the environmental moisture. Keep the media container tightly closed. Always use a dry spatula in obtaining the media. 3. Dissolve the media in enough eater (to avoid changes in osmotic pressure) and gradually bring to a boil. DO NOT USE EXCESSIVE AND SUDDEN RISE IN TEMPERATURE TO AVOID CHARRING OF THE MEDIUM. Also, use minimum amount of heat during sterilization. To avoid over boiling during sterilization, do not fill the vessel with more than 2/3 full of liquid. 4. Cover the flask with autoclavable cap, non-absorbent cotton wrapped in gauze, or aluminum foil. 5. Generally, unless otherwise specified, media are sterilized at 15-20 lbs. psi for 15-20 minutes. The media containing high carbohydrate content are autoclaved at a shorter period of time. Those containing high protein content such as egg yolk, or serum are sterilized by inspissations. Heat-labile urea is sterilized by filtration, with the use of special filters. 6. Prepare the culture media in small aliquots to use a shorter period of time, and to avoid repeated heating and refrigeration of the medium. 7. Store the media in a cool, dark place and in a tightly stoppered container (to avoid contamination) in the refrigerator and not near the freezer area. Plate-dispensed media are only good for 1 week provided they are free of contamination. PREPARATION OF AGAR PLATES 1. Rehydrate a suitable base medium according to label instructions. Dissolve the agar properly and heat it to boiling at medium temperatures so that the particles are properly dispersed and dissolved. 2. Sterilized the medium in an autoclave. 3. The media should be cool enough (not too cool or it will solidify in the media bottle) when poured in the plates to avoid moisture droplets to be accumulated on the cover of the plate. 4. Pour the medium into plates to about 4mm thick, or approximately 18-20 ml in a 90-mm diameter plate. A small opening in the plate should be done during pouring to avoid contamination. 5. Always heat the opening of the plates and the media bottle before and after pouring.

6. Allow the media to cool and solidify at room temperature on an undisturbed flat surface. 7. Invert the plates once the medium has completely solidified. 8. If the plates will not be used immediately, it is recommended to wrap the lid with a parafilm to avoid contamination. Place it in a ziplock or plastic container and store in the refrigerator. Always label your containers with the name of the media and the date when the media was prepared. It is highly recommended the media is used within five days from preparation. PREPARATION OF A BROTH MEDIUM 1. Dissolved the dehydrated broth medium in distilled water (follow label instructions). 2. Heat to boiling on a medium temperature to facilitate proper dissolution and dispersion of the medium particles. 3. Allow the medium to cool a bit and dispense 5ml into culture tubes. Loosen the cap during sterilization. 4. Sterilized the medium in an autoclave. Tighten the cap after sterilization. 5. If the media are not used immediately; store them in a ziplock or plastic container and place in the refrigerator. Always label the containers with the name of the media and the date of preparation. PREPARATION OF AGAR SLANT AND BUTTS 1. Dissolved the dehydrated medium in distilled water (follow label instructions). 2. Heat to boiling on a medium temperature to facilitate proper dissolution and dispersion of the medium particles. 3. Allow the medium to cool a bit and dispense 5ml into culture tubes. Loosen the cap during sterilization. 4. Sterilized the medium in an autoclave. Tighten the cap after sterilization. 5. SLANTS are prepared by solidifying the agar with the tube in a slanting position 9the size and the angle of the slant will determine whether it is a regular or a special slant). 6. BUTS are prepared by solidifying the tube in an upright position. 7. If the media are not used immediately; store them in a ziplock or plastic container and place in the refrigerator. Always label the containers with the name of the media and the date of preparation.

Study Questions: 1. List the factors that factors that will guide you in choosing what media to prepare. 2. Why do we always use distilled water instead of tap water in dissolving our media? 3. What are the problems encountered in media sterilization? 4. Classify the different culture media according to: a. composition b. consistency c. manner of preparation d. function

Exercise 6 PURE CULTURE TECHNIQUES

Objectives: 1. To learn the principles and purposes of isolating microorganisms 2. To be able to perform different pure culture methods 3. To be able to isolate a microorganism in a mixed culture. Background: Microorganisms in the environment do not segregate themselves into species; instead they exist in mixtures of any other cell types. These microorganisms can be separated into pure cultures in the laboratory. This culture contains only one type of organism and is suitable for the study of their cultural, morphological, and biochemical properties. Pure culture techniques are designed to produce discrete colonies. Colonies are individual, macroscopically, visible masses of microbial growth on a solid medium surface, each representing the multiplication of a single organism. Once these colonies are obtained, aseptic transfer will be made on to nutrient agar slants for isolation of pure cultures. Materials: 2 EMB Plates, Inoculating loops, Inoculating Needles, Alcohol Lamps, Pure Broth Culture of Escherichia coli, Mix Culture of Gram Negative Bacteria, Nutrient Broth, Agar Slant, Agar Butt, Butt-slant (special slant) Procedure: Reminder: REFRIGERATED CULTURE MEDIUM SHOULD BE THAWED BEFORE INOCULATION. Remove formed moistures at the plate cover using a sterile cotton swab. ALWAYS APPLY ASEPTIC TECHNIQUES!!! Heat your loops, tubes, and plates before and after inoculation. STREAK PLATE METHOD 1. Divide an EMB plate into two portions. You can draw a line on the petri dish with a pen. 2. Sterilized the loop. 3. Fish a colony from a plate medium, or a loopful from a broth medium. 4. Streak on the left part in an S-shape pattern. Streak the right part in a zigzag pattern. 5. Close the lid of the plate and heat it with the flame. Flame the loops used. 6. Label the plate with the source of the culture and your name. 7. Place the plate in a 37 oC incubator for 24-48 hours. CLOCK STREAK METHOD 1. Label the sterile NA plate with the source of the culture and your name. 2. Sterilize the loop. 3. Using appropriate aseptic technique, remove a loopful of broth from the mixed culture tube. 4. Flame the lid of the lid before inoculation. Lift the agar plate from the lid and streak about half of the plate. The loop should be parallel to the agar surface to prevent digging into or gouging the agar. 5. Return the plate to the lid. Sterilize the loop. Lift the agar plate and make one 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 make one streak into the second inoculated portion of the plate. Finish by streaking the remaining one-fourth of the uninoculated plate. Sterilize the loop. 7. Place the plate in a 37 oC incubator for 24-48 hours.

4. INOCULATION OF A BROTH CULTURE

5.

6.

1. Label the sterile nutrient broth with the source of the culture and your name. 2. Sterilize the loop. 3. Using appropriate aseptic technique, remove a loopful of broth from the mixed culture tube. 4. Insert the loop into the sterile broth tube and swirl gently. Sterilize the loop. 5. Incubate the broth at 37 oC for 24-48 hours. INOCULATION IN AGAR BUTTS 1. Label the sterile agar butt with the source of the culture and your name. 2. Sterilize the loop. 3. Using appropriate aseptic technique, touch the needle on the surface of a colony on a plate. Fish a small portion of this colony with the inoculating needle. 4. Stab the butt with the needle up to the middle portion. Remove the needle and flame it. Flame the mouth of the tube before recapping. 5. Incubate the broth at 37 oC for 24-48 hours. INOCULATING IN AGAR SLANTS 1. Label the sterile nutrient agar slant with the source of the culture and your name. 2. Sterilize the loop. 3. Using appropriate aseptic technique, remove a loopful of broth from the mixed culture tube. 4. Insert the loop into the sterile agar slant tube and starting at the base of the slant, draw the loop up the slant in a streaking manner. Do not penetrate the agar. Sterilize the loop. 5. Agar butts are inoculated by stabbing the butt portion and streaking the slant portion using an inoculating needle. 5. Incubate the slant at 37 oC for 24-48 hours. Draw the appearance of your Streak plate and Clock streak after incubation in your results. Study Questions: 1. 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 oC before adding organisms? 2. What are the different methods of isolating microbes? 3. Define inoculation, fishing, culture, imoculum, and colony. 4. What is the difference between isolation and inoculation? 5. Is it possible to have a mixture of bacteria in a single colony? Explain. 6. What is a pure culture? Differentiate axenic culture from pure culture.

Exercise 7 CULTURAL CHARACTERIZATION OF BACTERIA

Objectives: 1. To observe the growth patterns of cultured microorganism 2. To classify the growth behavior of these microorganisms Background: Microbes (not just bacteria) require appropriate conditions of incubation. These may include the correct nutrients, chemicals, temperatures, etc. When the conditions are right, microbes are able to multiply and will form colonies. A colony is the only way to see microorganisms with the unaided eye and it should be remembered that colonies are large groups of microbes, not individuals. Using nutrients, chemical, pH, temperature, salinity, and other conditions of incubation, a microbiologist is provided with the first clue to what a particular organism might be. For accurate reporting, the microbiology student must learn the descriptive vocabulary of the microbiologist. Descriptions of colonies must be concise and be phrased in such a manner that other scientists will know what is being described. These descriptions are very important in the identification and classification of the bacteria. Materials: Results of the culture medium in exercise 9 after incubation Procedure: 1. Research and print a copy on the cultural characteristics of the bacteria. Attach this copy on your lab report. 2. Draw the following in your results: a. Clock Streak b. Nutrient Broth c. Agar Butt d. Agar Slant e. Special Slant 3. Classify the colonies in your plate according to: size, form, elevation, margin, pigmentation, and optical features. Record these classifications under your drawing. 4. Classify your slants according to: amount, form, consistency, and chromogenesis. Record these classifications under your drawing. 5. Classify your broth medium according to: surface growth, subsurface growth, amount, and sediment. Record these classifications under your drawing. 6. Classify your Agar butt according to the growth along the line of stabbing and liquefaction of the medium. Record these classifications under your drawing.

Exercise 8 PHYSICAL AND CHEMICAL FACTORS AFFECTING GRWOTH

Objectives: 1. To determine the effects of several chemical and physical factors on the growth of different microorganisms. 2. To acquaint the students with the diverse growth temperature, pH, oxygen, and osmolarity requirements of bacteria. Background: Microorganisms have different requirements in order to grow. Their growth can be affected by physical and chemical factors. Physical factors include temperature, pH, and osmolarity. Chemical factors on the other hand include all the nutrient requirements of a microorganism like carbon, nitrogen, oxygen, and trace minerals sources. In this exercise, we will examine the effects of most of each factor on the growth of several bacterial species. Materials: Cultures of E. coli, Bacillus subtilis, S. aureus, and Vibrio cholera, alcohol lamps, loops, can/jar, candle, Agar Slants, EMB Plates, Agar Plates Procedure: A. Effect of Temperature 1. Inoculate 4 agar slants with E. coli, and another 4 with Bacillus subtilis. 2. Incubate the first tube at the refrigerator, the second at room temperature, the third at the incubator, and the last one at the oven. 3. Examine the slants for growth after 24 hours of incubation. Note the amount of growth in each slant and tabulate your results. B. Effect of pH 1. Inoculate E. coli, Vibrio cholerae, and S. aureus into agar slants of different pH (pH 3, 5, 7, 9, 10). 2. Incubate the tubes at 35 oC for 24 hours. 3. Examine the slants for growth after incubation and record your observations. Determine the minimum, optimum, and maximum pH requirement for growth of each organism. Tabulate your results. C. Effects of Osmolarity of the Environment 1. Inoculate E. coli, Vibrio cholerae, and S. aureus into agar slants of different salt (0%, 0.5%, 5% and 10%), and glucose (0%, 10%, 25%, 50%) concentrations. 2. Incubate the tubes at 35 oC for 24 hours. 3. Examine the slants for growth after incubation and record your observations. Determine the minimum, optimum, and maximum glucose and salt concentration required for growth of each organism. D. Effect of Oxygen Availability 1. Streak the entire agar plate with Bacillus subtilis, and the entire EMB plate with E. coli. 2. Incubate the agar plates in the incubator for 24 hours. 3. Place the EMB plates inside a big can or jar. 4. Light a candle and place it inside the can/jar, at the top of the plates. 5. Cover the can/jar and let it stay at room temperatures for 24 hours. The candle will stop burning when the oxygen level inside the can/jar is greatly decreased. This will cause anaerobic environment inside the can/jar. 6. Observe for growth after 24 hours and record your observations.

Study Questions. 1. What everyday practices in the control of microbial growth illustrate the conditions taken up in this experiment? 2. How does osmolarity of the environment affect growth? 3. What are buffers and why are they added to culture media? Give atleast 3 examples of some buffers used in culture media. 4. What inhibits bacterial growth at nonoptimal pHs? 5. What is the pH tolerance of bacteria compared to yeast? 6. How do microorganisms change the pH or their own environment? 7. Compare isotonic, hypotonic, and hypertonic solutions and the effects they have on bacterial cells. Tabulate your answers. 8. How is it possible for a bacterium to grow in a hypertonic environment? In a hypotonic environment? 9. Give atleast 5 examples of most common pathogenic anaerobe that affects humans and their location in the body.

Exercise 9: BIOCHEMICAL ACTIVITIES OF BACTERIA

Objectives: To determine and compare the biochemical action of several bacterial species to different substances. Background: Microorganisms are remarkable for the versatility of their biochemical activities considering that they are unicellular organisms. They can break up a variety of materials ranging from a simple to complex substances. Their synthetic abilities are also amazing, as can be seen on their growth on chemical defined media. They can build their own structures from the nutrients present in the media. These activities are attributed to the enzymes that they produce. The action of enzymes in a specific substrate is very useful in many ways. They enable scientist to study cellular metabolism in general. The relationship of enzymes to genes, enable us to study genes through enzyme-mediated reactions. This is very important in addressing the problems in metabolism and heredity. Also, many industrial processes are dependent on these actions of bacteria.

I. ACTION OF PROTEINS AND OTHER NITROGENOUS COMPOUNDS A. Hydrolysis of Gelatin Materials: Cultures of E. coli and Salmonella spp.; Nutrient Gelatin Tubes Procedure: 1. Inoculate each of the above organisms into a separate gelatin tubes by a stab down to the butt. 2. Incubate at 34 oC for 48 hours. 3. Observe the liquefaction of the media by placing the tubes including an uninocualted tube in a refrigerator (or in a beaker with ice) for 30 minutes (or after the uninoculated tube solidify). An organism that can produce an enzyme gelatinase will be able to hydrolyze the gelatin in the medium. Hydrolyzed gelatin will not solidify while unhydrolyzed gelatin will. 4. Draw and record you results and determine which bacteria produces the enzyme gelatinase.

B. Hydrolysis of Casein Materials: Cultures of E. coli and B. subtilis; Solid Casein Medium Procedure: 1. Divide the plate into two sectors and point inoculate each section with the organisms. 2. Incubate at 34 oC for 24-48 hours. 3. Examine the plate for hydrolysis of casein indicated by a clear halo around the colony. 4. Draw and record you results.

C. The Production of Indole

Materials: Cultures of E. coli and E. aerogenes; Tubes of SIM medium, Ehrlichs Reagent Procedure: 1. Inoculate each of the organisms in separate SIM medium by stabbing with the needle to the bottom of the medium. 2. Incubate at 34 oC for 48 hours. 3. Lysine deaminase, Lysine decarboxylase and motility reactions are read before adding the indole reagent. 4. Examine the tubes for turbidity, indicating motility of the organism. 5. Lysine deaminase is indicated by a red or red-brown color reaction in the top centimeter of the medium. 6. Lysine decarboxylase is indicated by a purple color throughout the medium. Negative cultures produce a yellow tube. 7. Allow 4-5 drops of the Ehrlichs reagent to run down the side of the tube. 8. Note the development of a purplish-red color in the reagent layer of the medium, indicating the production of indole. Indole positive organisms are capable of hydrolyzing the amino acid tryptophan.

D. Reduction of Nitrates to Nitrites Materials: Cultures of Micrococcus luteus, Pseudomonas aeruginosa, and Bacillus subtilis; Nitrate broth tubes Procedure: 1. Inoculate each of the organisms in separate nitrate broth tubes. Leave 1 tube uninoculated. 2. Incubate at 34 oC for 48 hours. 3. Add approximately 1.0 ml (20 drops) of sulfanilic acid solution (Solution A) and 1.0 ml of dimethyl-alphanapthylamine (Solution B) to each of the tubes including the control. Observe for the development of red, purple, or maroon color and record your observations in a table. 4. To the cultures in which no red color developed, add a minute amount of zinc. Observe for the development of red, purple, or maroon color and record your observations in a table. 5. Determine whether the organism are capable of reducing nitrates. E. Decomposition of Urea Materials: Cultures of E. coli and Salmonella spp; Urea broth Tubes Procedure: 1. Inoculate each organism in separate urea broth tubes. 2. Incubate at 34 oC for 48 hours.

3. Examine the tube for the development of red (cerise) color and determine if the organism is capable of hydrolyzing urea.

Study Questions: 1. What is the benefit of gelatin hydrolysis among certain bacteria? 2. What is gelatin? What is unique about gelatin at 35C versus 5C? 4. Why did you refrigerate the gelatin cultures before observing them for liquefaction? 5. Can gelatin hydrolysis be correlated with the pathogenicity of a bacterium? Explain your answer. 6. Why is gelatin liquefied in the presence of gelatinase? 7. Of what use to bacteria is the ability to produce H2S? 8. What substrates are acted on in SIM medium in order for H2S to be produced? 9. How does a black precipitate of FeS indicate the production of H2S? 10. How is SIM medium used to detect motility? 11. In addition to H2S production and motility, for what other test can SIM medium be used? 12. What does cysteine desulfurase catalyze? Show the reaction. 13. What does thiosulfate reductase catalyze? Show the reaction. 14. What is the component in the SIM deep tubes that makes this medium suitable to detect the production of indole by bacteria? 15. In the nitrate reduction test, why is the development of a red color a negative test when zinc is added? 16. What are the end products that may result from the action of bacteria with nitrate-reducing enzymes? 17. What is the purpose of the phenol red in the urea broth medium? 18. When would you use the urease test?

Exercise 9: BIOCHEMICAL ACTIVITIES OF BACTERIA

Objectives: To determine and compare the biochemical action of several bacterial species to different substances.

II. ACTION ON CARBOHYDRATES A. Starch Hydrolysis Materials: Cultures of Bacillus subtilis and Escherichia coli; Starch Agar Plates; Lugols iodine Procedure: 1. Expose one starch agar plate in the environment for 5 minutes. Incubate at room temperature. 2. At the end of 48 hour period, flood the surface of the plate with Lugols iodine. Allow the solution to be absorbed for 3-5 minutes and pour off the excess. 3. Repeat the procedure by inoculating the starch agar plate with the organisms listed above. 4. Examine the plates and look for a clear zone around isolated colonies. The clear zone results from the breakdown of starch. The purple to black areas are places where starch has not bee hydrolyze. 5. Record and illustrate your results.

B. Carbohydrate Fermentation Materials: Cultures of Salmonella spp. Escherichia coli, and Staphylococcus aureus; Set of fermentation broth tubes containing glucose, lactose, maltose, mannitol and sucrose Procedure: 1. Inoculate each of the organisms in different fermentation set. 2. Incubate at 35-37 oC for 48 hours. 3. Examine the tubes for color and presence of gas bubble. 4. Record whether acid (yellow color of the medium) and/or gas (bubble formation) is produced by the organism in each of the sugars. Record your result as A= acid production, A/G= acid and gas production, - = negative for both acid and gas production.

C. TSI Reaction Materials: Cultures of E. coli and Salmonella spp; Tubes of Triple Sugar Iron (TSI) Agar Procedure:

1. Inoculate each of the organisms above into the TSI tube by stabbing through the butt and streaking the surface of the slant. 2. Incubate at 34 oC for 24 hours. 3. Examine the tubes for the presence or absence of the blackening within the medium. Determine whether the organism is capable of producing hydrogen sulfide or not. 4. Examine the color of the slant and the butt and determine the kind of reaction that has taken place: Acid (A) = yellow; Alkaline (K)= red; None (NC)= no color change. For example a yellow slant and a red butt has an A/K reaction (slant/butt). 5. Examine the tubes for the presence or absence of bubbles or cracks in the agar, indicating the formation of carbon dioxide or hydrogen gas. 6. Record all your observations in a table.

D. Production of acetyl-methyl-carbinol (Voges-Proskauer Test) and mixed acids (Methyl Red Test) from Glucose (MR-VP Test) Materials: Cultures of Escherichia coli, and Ps. aeruginosa; MR-VP broth tubes; methyl red, 5% alpha-naphthol in absolute ethyl alcohol; 40% KOH Procedure: 1. Inoculate each of the organisms above in 2 tubes with MR-VP broth. 2. Incubate at 34 OC for 48 hours. 3. Separate each tubes into two set in which one set is a replicate of the other. 4. To one set, perform the methyl red test by adding 5 drops of methyl red solution. The presence of acid is indicated by the development of red color of the medium. A negative reaction is indicated by a yellow color. 5. To the other set, test for the production of acetyl-methyl-carbinol by adding 0.6ml (12 drops) of 5% alpha naphthol in absolute ethyl alcohol and 0.2 ml (4 drops) of 40% KOH into the tubes. No change in the color of the medium indicates a negative result.

III. OTHER TESTS A. Action on Red Blood Cells Materials: Cultures of Streptococcus faecalis, Bacillus subtilis and Escherichia coli; Blood Agar Plates Procedure: 1. Divide the blood agar plate into three sections and label each with the name of the organisms above. 2. Point inoculate each of the organism on their corresponding sectors and incubate the plate at 34 OC for 24 hours.

3. Study the growth characteristics of each organism and observe the changes in the blood agar medium at the edge of the growth of each organism. Hemolysis of red blood cells is indicated by a clear halo surrounding the bacterial colonies. 4. Record and illustrate your observations.

B. Citrate Utilization Materials: Cultures of E.coli, and Ps. aeruginosa; Simmons Citrate Medium slant Procedure: 1. Inoculate each of the organisms above into two separate citrate slant by streaking the surface of the medium. 2. Incubate at 34 OC for 48 hours. 3. Examine the tube for the growth and for the changes in the color of the medium. A positive result is indicated by the change in color of the medium from green to blue.

Study Questions: 1. What is/are the enzyme/s involve in starch hydrolysis? What is/ are the end products of these enzymes? 2. How is it possible that bacteria may grow heavily on starch agar but not necessarily produce -amylase? 3. What are the ingredients in each fermentation tube and what is the purpose of these ingredients? 4. What products did the three bacteria formed in each fermentation tube? 5. What are the three sugar components of TSI Agar? Why do you obtain different reactions in the slant and in the butt part of the agar? 6. Why is there more lactose and sucrose in TSI agar than glucose? 7. What is the pH indicator in TSI agar? 8. Why must TSI test observations be made between 18 to 24 hours after inoculation? 9. What is the purpose of the thiosulfate in the TSI Agar? 10. What is the main purpose of MR-VP reaction? What are the products detected in the methyl red and voguesproskauer reaction? 11. What is the purpose of citrate utilization test? What are the products of this reaction and how are these products detected? 12. What is the main purpose of haemolytic tests? What is the main substance responsible for this reaction? 13. Why is there different types of haemolytic reactions?

Exercise 10 ANTIMICROBIAL SUSCEPTIBILITY TESTING (Kirby-Bauer Procedure) Objectives: 1. To perform the agar-disk diffusion method of antimicrobial susceptibility testing; 2. To determine the sensitivity of the different types of bacteria to an array of antibiotics Background: The antimicrobial activities of several chemotherapeutic agents vary in their scope. Some are only effective to a specific kind of microorganism, while others have broad-spectrum activities, being effective on a wide range of microorganisms. The sensitivities of several pathogenic bacteria are known, however it is important to know the susceptibility of microorganisms to determine the drug of choice to be administered to a patient. This is also important in determining whether a certain microorganism has developed a resistance to an antibiotic it was previously susceptible to. The most common method used in laboratories to test the sensitivity of various microorganisms is the Kirby-Bauer agar-disk diffusion method. This method uses a filter paper impregnated with various chemotherapeutic agents of specified concentrations, and Mueller-Hinton agar as the medium of choice. A standardized inoculum of an organism to be tested is spread over the Mueller-Hinton Agar by means of a cotton swab. The filter-paper disks are then placed on the surface of the agar. The chemotherapeutic agent diffuses from the disk from an area of a higher concentration to an area of a lower concentration. The growth inhibition, indicated by a clear zone around the antibitotic disk, is measured after incubation. The size of this clear zone, or the zone of inhibition, determines the susceptibility of the microorganism. Materials: Mueller-Hinton agar plates, Cultures of: Escherichia coli, Pseudomonas aeruginosa, Salmonella typhi, Staphylococcus aureus, Sterptococcus faecalis, and Mycobacterium phlei , Penicillin G, Gentamicin, Streptomycin, Chloramphenicol, Tetracyclin, Nalidixic Acid, and Rifampin, alcohol lamps, cotton swab. Procedure: 1. Using a sterile technique, inoculate the organisms into the Mueller-Hinton agar plate as follows: a. Dip a sterile cotton swab into a well-mixed saline solution of test culture and remove the excess inoculums by pressing the swab against the inner side of the test tube. b. Streak the entire plate first on a horizontal direction, then vertically to ensure a heavy growth on the entire surface. 2. Allow all the plates to dry for about 5 minutes. 3. Using forceps obtain a disk from the antibiotic cartridge and place it on the surface of the agar. Place the disks at equal distances from each other. Gently press the disks with the forceps or with an applicator stick to ensure adherence of the disk into the agar. Always sterilize the forceps before obtaining an antibiotic disk and after placing the disk in the agar plate, by dipping it in an alcohol and flaming it over the alcohol lamp. 4. Incubate the plates at 35 OC for 24 hours. 5. Measure the diameter of the zone of inhibition after incubation and determine the susceptibility of the organisms using the ff. criteria: Antibiotic Symbol Disc Concentration 10 g 10 g 10 g 30 g 30 g 30 g Diameter of Zone of Inhibition (mm) Resistant 20 or less 11 or less 12 or less 14 or less 13 or less 12 or less Intermediate 21-28 12-14 13-14 15-18 14-18 13-17 Sensitive 29 or more 15 or more 15 or more 19 or more 19 or more 18 or more

Penicillin G Streptomycin Gentamicin Tetracycline Nalidixic Acid Chloramphenicol

P S GM Te NA C

Rifampin Trimethoprin/Sulfamethoxazole

RA SXT

5 g 10 g

10 or less

11-15

16 or more

Study Questions: 1. What factors affect the zone of inhibition? 2. What is the action of each chemotherapeutic agent? Which chemotherapeutic agent is the most effective? The least effective? 3. What other factors are considered before using the chemotherapeutic agent in vivo? 4. In which growth phase is the organism most sensitive to the antibiotic? 5. What are other methods of measuring the sensitivity of organisms to antibiotic? 6. What is MIC and MBC? How are they measured? 7. What is being measured in the agar-disk diffusion method, bactericidal or bacteriostatic activity of the organism? 8. Why is the agar-disk diffusion method not a perfect indication on how well the chemotherapeutic agent will perform in vivo? 9. What is McFarlands Standards? How is it useful to antibiotic sensitivity test? 10. Why is Mueller Hinton Agar used in this test and not other ordinary agar media?

Exercise 11 ISOLATION AND CHARACTERIZATION OF ORGANISMS IN THE ENVIRONMENT (Fungi) Objectives: 1. To be able to isolate, characterize, and identify the fungal species commonly found in the vicinity of the school; 2. To be able to perform the laboratory techniques employed in making and examining fungal cultures; 3. To enumerate the diseases associated with infection of identified fungal species. Background: Fungi are heterotrophic, ekaryotic microorganisms that are capable of metabolizing a wide variety of organic substrates. Like bacteria, they can also be beneficial and detrimental to humans. They are very important in the decomposition of organic matter thus making the soil fertile. Their fermentative ability is very useful in producing beer, wine, cheese, and other products. Some of them has an antimicrobial property and thus used in manufacturing antibiotics. On the other hand, they can also cause spoilage of food, produce toxins, and cause diseases to humans. The fungi have two morphological characteristics: the yeast and the molds. The molds are the major fungal organisms that can be seen with the naked eye. The thallus or the body of the molds is composed of long filaments of cells joined together. These filaments are called hyphae. When the environemnet of the fungus is suitable for growth, these hyphae could form into a filamentous mass called mycelium, which is visible to the unaided eye. The hyphae of the molds can be septate or coenocytic. Septate hyphae are those that are divided into distinct, uninucleate cell-like units. Coenocytic hyphae are those that appear as long, continuous cells with many nuclei and no visible division. The portion of the hyphae that obtains nutrient is called vegetative hypha, while those that are concerned with reproduction are called reproductive or aerial hypha. This aerial hypha projects above the medium where the fungus is growing, and often contains reproductive spores. The yeasts are unicellular and about 5-10 times larger than bacteria. Morphologically, they may be ellipsoidal, spherical, and in some cases cylindrical. The yeast reproduce asexual through a process called budding. During buding, some yeast could produce buds which fail to detach. These buds form short chains of cells called pseudohypha. Some fungi exhibit dimorphism-they can grow as yeast or molds depending on the temperature. At 37 OC, fungus is in yeast form, while at 25 OC, it is in mold form. The fungi has a characteristic spores which they use to reproduced either sexual or asexually. These spores are the major useful tool in classification and identification of Fungal species. Asexual spores are formed through mitosis and subsequent cell division. The following are examples of asexual spores: 1. Sporangiospores - these single-celled spores are formed within sacs, called sporangia and supported by sporangiophore, at the end of special hyphae. 2. Conidium - produces by conidiogenous cell (ex: phialide), attached to a specialized hyphae, called a conidiophore. a. Microconidium - small, single-celled conidium b. Macroconidium - large, multi-celled conidium which are very important for distinguishing 3. Thallospores - asexual spores that directly formed by hyphal cells 4. Arthroconidia - single-celled conidia formed by disjointing of hyphal cells 5. Chlamydoconidia - thick-walled, single-celled conidia highly resistant to adverse conditions. They are formed from cells of vegetative hyphae. 6. Blastoconidia - conidia formed by budding

Sexual reproduction of the fungi is similar to the fertilization process. The haploid nucleus of a dnor cell penetrates the cytoplasm of the recipient cell. The nucleus of both cells fuse to form a diploid zygote and this zygote then divides by meiosis. The sexual spores produce by each fungus characterize the phyla where the fungus belong. True Fungi are classified into the following four classess on the basis of their sexual mode of reproduction. 1. Phycomycetes water, bread, and terrestrial mold; reproductive spores are external and uncovered. 2. Ascomycetes yeast and molds, sexual spores called ascospores, are produced in a sac-like structure called ascus. 3. Basidiomycetes fleshy fungi, toadstools, mushrooms, puffballs, and bracket fungi; reproductive spores basidiospores, separate from specialized sacs called basidia. 4. Deuteromycetes- no sexual reproductive phase has been described

Materials: Potato-Dextrose Agar (PDA) , Wire Needles, Alcohol Lamps, Lactophenol-cotton-blue, Potassium hydroxide, Glass slides and cover slips. Procedure: 1. Expose a plate of PDA to the environment for 3o minutes. Incubate the plate for 3-5 days. 2. Examine the plate for fungal colonies. 3. Subculture each colony into another PDA plate by obtaining a small sample from each colony using a wire needle and point inoculate it to another plate of PDA. Incubate the plate for 3-5 days. 4. If a pure culture is obtained (only one type of colony can be seen) characterize the morphological appearance of the culture on the media. Take note of the shape, color, and texture of the colony. ( If there are two or more types of formed colonies, continue subculture process until a pure culture is obtained.) 5. After examining the colonial morphology of the fungi, prepare a wet mount by suspending some of the cultures in a few drops of lactophenol-coton-blue solution. This should be done gently to avoid damaging the fungal cultures. If you want to have a detailed characterization, the following website would be of great help: http://www.mycology.adelaide.edu.au/Keys/Key%20to%20Medically%20Important%20Conidial%20Moulds.htm l 6. Cover with cover slip and examine under low power and high power. 7. Take note of the microscopic characteristics of the fungi. 8. Identify the possible genus or genera of the fungi base on colonial morphology and microscopic characteristics. (Use the dichotomous scheme presented below) 9. Draw the appearance of the fungal culture in the PDA plate and the microscopic features accurately.

A DICHOTOMOUS KEY TO SOME COMMON AIRBORNE FUNGI

To use this key, start at point one and select the most appropriate option. Record your choice. At the right hand margin is a number or name. If a number, then go to that line and again select the most appropriate option. Again, record your choice. Continue until you have an answer. Click on the link and check you answer against the diagrams provided. If the diagram differs from your choice, you may have another fungus, or your fungus has been incorrectly identified. Check back each of your option and determine whether you have recorded the correct option or whether the information might be incorrect 1 2 Hyphae absent. Colony small, rounded and shiny, commonly white to cream Hyphae present, colony cottony, may be coloured Colony with small cells, 0.5 to 2m diam, single cells cannot be seen under dissecting microscope Colony with cells 3 - 10m diam, single cells can just be seen in smear on slide under dissecting microscope Small cells (spores) on stalks can be seen above or in hyphae using the dissecting or compound microscope. Spores may be in a sac-like or round structure. Spores are invisible, in colony using the dissecting microscope, and on stained mycelia on slide under the compound microscope Hyphae lack cross walls (examine stained tips of young hyphae on a slide under the compound microscope). Spores in sac held above mycelia (sporangium, diagram over page). Hyphae have cross walls. Spores commonly held away from hyphae, may be in thickwalled sac (pycnidium) Spores held in sporangium, or released from sporangium, hyphae with short darkened "roots" on agar. Spores held in sporangium, hyphae lack "roots" into agar Spores produced in compound pycnidium (diagram over page) Spores formed on free hyphae Spores consist of a single cell, not internal walls Most spores have cross walls, immature spores lack cross walls Spores in dry chains when undisturbed Spores in clumps or clusters, sometimes wet looking Chains of spores are unbranched Chains of spores are branched Chains of spores held in a brush - like dry cluster, each chain arises from a bottle-like phialide Chains of spores emerge from phialides which radiate from a swollen vesicle at the top of a specialized coarse hypha Colonies a deep olive to almost black colour, dry spores are generally rounded, lemon shaped or sometimes irregular Colonies fawn, spores uniform in shape and size Colonies flat, creamy, shiny, when young, turning dark with age Colonies fluffy to flat, usually grey to green Green masses of spores, white when immature, common in soil Grey masses of spores, colony raised and open Spores with both vertical and horizontal walls, dark to black Spores with walls in one direction only, may be pale or dark Spores rounded, with walls radiating from centre of spore, held in clusters on short hyphae, culture often red in the agar Spores with longitudinal and lateral walls when mature Elongate spores formed in branched chains, youngest at tip Go to 2 Go to 3 Bacteria Yeast 4 Sterile fungus 5

6 Rhizopus Mucor Phoma 7 8 14 9 12 10 11 Penicillium Aspergillus Cladosporium Monilia Aureobasidium 13 Trichoderma Botrytis 15 17 Epicoccum 16 Alternaria

6 7 8 9 10

11

12 13 14 15

16

17 18

Rounded spores formed singly on the sides of short dark hyphae Spores curved, may be dark or pale Spores cylindrical to rounded, dark, one to many cross walls Colonies fluffy, white, with curved spores that have one to many cross walls Colonies dark, spores short, three celled, with central cell larger than the termini

Stemphylium 18 Helminthosporium Fusarium Curvularia

Illustration of Microscopic Characteristics: Alternaria Aspergillus

Branched chains of spores with septa in vertical and horizontal positions.

Bear chains of conidia arising from phialides that are spread over the surface of a vesicle.

Penicillium

Cladosporium

Forms masses of conidia from phialides. The youngest cells are at the base of the chain.

Conidia form at the tip of the hyphae. The fragments are commonly lemon-shaped but may also be irregular to branched.

Monilia

Aureobasidium

Monilia is a common pathogen of fresh peaches and plums. Spores form in branched chains. The chains fragment in the wind and spread rapidly.

This dimorphic fungus is seen as both hyphae and yeast cells at once. Black hyphae for lateral buds which appear as pink ooze on the agar

Trichoderma

Botrytis

Spores arise from phialides held on hypha. The spores remain in wet clusters until disturbed.

Tall, slender hyphae hold clusters of spores that emerge from the tup of the hyphae. The spores are single cells. The appearance on strawberries is usually grey.

Epicoccum

Stemphylium

Dark spores are held in clusters on short hyphae. The cluster is crunchy when you press the cover slip over the mount.

Single spores are borne at the ends of the short hyphae.

Helminthosporium

Fusarium

Single dark hyphae bear numerous septate spores on the sides and at the top.

Best identified from the banana shaped spores that have two or more cross walls.

Curvularia

Rhizophus

This fungus is clearly identified from the spores. The central cell is the larger giving the cell a curved appearance.

Forms a root-like attachment in the agar.

Mucor

Phoma

Spores held in a sporangium, which when broken, reveals a projection into the sporangium.

Single-celled spores are formed inside a pycnidium. On maturity they ooze from the opening at the top.

Exercise 12 MICROBIAL ANALYSIS OF FOOD AND WATER Objectives: 1. To quantify the organisms present in an water or food sample 2. To be able to perform various methods of quantifying bacteria 3. To determine whether a food sample is contaminated by fecal coliforms 4. To determine whether the water is safe for drinking or not.

Background: Studies involving the analysis of materials such as food, milk, and water require quantitative enumeration of microorganisms in the substances. Many methods have been devised to accomplish this. These includes: direct microscopic count, breed smears, electronic cell counters, chemical methods for enumerating cell mass or cellular constituents, turbidimetric measurements for increases in cell mass, and serial dilution-agar plate method. All of these methods can be used to enumerate bacterial cells; however these cells include both living and dead bacteria. Studies that usually involve the determination of the number of viable or living cells are usually accomplish through serial dilution-agar method This methods involves serial dilution of a bacterial solution in a sterile water blanks, which serve as a diluents of known volume. Once diluted, the suspension is plated on a suitable nutrient media. Another method used to quantify bacteria is the Mosta Probable Number, or MPN method. This method is an estimation of the number of colifoms like Enterobacter, Klebsiella,Citrobacter, Escherichia. This test involves a multiple series of Durham fermentation tubes and is divided into three parts: the presumptive, confirmed, and completed tests. The number of positive tubes is then correlated to the MPN index to determine the most probable number of the fecal coliforms in a sample. Foods serve as important inanimate vectors in the transmission of diseases since they contain organic nutrients which provide a medium for bacterial multiplication and growth under favorable conditions. Food and dairy products may be contaminated in a variety of ways and from a variety of sources. The quality of a particular food sample can be determined by enumerating the number of microorganisms present. The presence of the high number of microorganisms suggests a good possibility that pathogens may be present. Although a sample has a low bacterial count, it could still transmit infection. Several microorganisms are important in the processing of foods such as cheese, pickles, saukerat, yogurt, and sausages. However, the presence of other microorganisms can result to food spoilage and food poisoning. In milk and water, the presence of coliform and enteric bacteria indicates fecal contamination and may suggest the pathogens are present. Materials: Water/ Food samples, Agar plates, sterile petri dishes, Molten Agar, sterile distilled water blanks, Hockey Sticks, alcohol lamps, 1.0 ml pipette, Rubber aspirator, Double strength lactose broth, Single strength lactose broth, durnham tubes, wire loops, EMB plates, MRVP broth, SIM agar butt, Citrate slants. Procedure: I. HETEROTROPHIC PLATE COUNT A. Serial Dilution: 1. Prepare three 15ml-test tubes and label them 101, 102, and 103. 2. Place 9 ml of distilled water in each tube and cover the tubes with a cotton plug. 3. Sterilized the tubes in the autoclave. After sterilization, let the tubes cool down or place them in a water bath for fast cooling. 4. From a bacterial suspension, pipette 1.0ml sample and place it on the tube labeled 101. Mix the sample thoroughly by aspiration. 5. Using another sterilized pipette, obtain 1.0 ml sample from the tube labeled 10 1 and place this on the tube labeled 102. Mix the sample thoroughly by aspiration.

6. Using another sterilized pipette, obtain 1.0 ml sample from the tube labeled 10 1 and place this on the tube labeled 102. Mix the sample thoroughly by aspiration.

Diagram of Serial Dilution and Agar-Plating Method

Spread Plate Method 1. Make three agar plates and label them 101, 102, and 103. 2. From the tube labeled 101, pipette 1.0 ml sample and place it on the plate labeled 101. 3. Spread the sample evenly using a hockey stick. Let the plate stand for a few minutes to let sample dry. 4. Repeat procedures 2 and 3 for the samples in tubes 102 and 103. 5. Incubate the plates at 35 oC for 24 hours. Count the colonies formed in the colony counter, after incubation. 6. Ideal plates that should be counted should contain 30-300 colonies. Plates containing more than 300 colonies should be reported as TNTC or Too Numerous To Count. 7. Determine the number of viable organisms by multiplying the number of colonies by the dilution factor. Pour Plate Method 1. Sterilize three empty petri dishes and label them 101, 102, and 103. 2. From the tube labeled 101, pipette 1.0 ml sample and place it on the plate labeled 101. 3. Add 20 ml of molten agar cooled to 45 oC. 4. Spread the sample evenly by slowly shaking the plate in a circular manner or by using the vortex mixer. Let the plate stand for a few minutes to let sample dry. 5. Repeat procedures 2 and 3 for the samples in tubes 102 and 103. 6. Incubate the plates at 35 oC for 24 hours. Count the colonies formed in the colony counter, after incubation. 7. Ideal plates that should be counted should contain 30-300 colonies. Plates containing more than 300 colonies should be reported as TNTC or Too Numerous To Count. 8. Determine the number of viable organisms by multiplying the number of colonies by the dilution factor. * For food samples: Weigh 10.0 gram of the food sample and place it on a blender. Place 90.0 ml of distilled water on the blender and blend the mixture until the food sample is thoroughly shredded and mixed with the water. This is the 101 dilution. Follow the procedures in the heterotrophic plate count starting from the second dilution.

II. MPN METHOD A. Presumptive Test 1. Mix the bottle of water to be tested 25 times. Inoculate five of the double-strength lactose (or lauryl tryptose) broth tubes with 10 ml of the water sample; five single-strength tubes with 1 ml of the water sample; and five single-strength tubes with 0.1 ml of the water sample. Carefully mix the contents of each tube without spilling any of the broth by rolling the tubes between the palms of your hands. 2. Incubate the three sets of tubes for 24 to 48 hours at 35C. 3. Observe after 24 2 and 48 3 hours. The presence of gas in any tube after 24 hours is a positive presumptive test. The formation of gas during the next 24 hours is a doubtful test. The absence of gas after 48 hours is a negative test. 4. Determine the number of coliforms per 100 ml of water sample. For example, if gas was present in all five of the 10-ml tubes, only in one of the 1-ml series, and none in the 0.1-ml series, your test results would read 510. Table 46.1 indicates that the MPN for this reading would be 33 coliforms per 100 ml of water sample. B. Confirmed Test 1. Record your results of the presumptive test. 2. Using an inoculating loop, from the tube that has the highest dilution of water sample and shows gas production transfer one loopful of culture to the brilliant-green lactose bile broth (or EC broth) tube. 3. Incubate for 48 3 hours at 35C. The formation of gas at any time within 48 hours constitutes a positive confirmed test. C. Completed Test 1. Record your results of the confirmed test. 2. From the broth tubes of the previous procedure, streak a sample in an EMB plate. 3. Incubate the plate inverted for 24 hours at 35C. 4. If coliforms are present, select a well-isolated colony and inoculate a single-strength, brilliant green lactose bile broth tube and streak a nutrient agar slant. 5. Gram stain any bacteria found on the slant. 6. The formation of gas in the lactose broth and the demonstration of gram-negative, nonsporing rods in the agar culture is a satisfactorily completed test revealing the presence of coliforms and indicating that the water sample was polluted. This is a positive completed test.

DETERMINATION OF E. Coli 1. If the coliform contaminant is E. coli, the EMB plate cultures would show a metallic green sheen. If this is present, inoculate a sample from these plates the the following media: SIM agar butt, two MRVP broth tubes, and citrate slant. 2. Determine the biochemical reactions in these media after 48 hours of incubation. A typical E. coli would be positive for indole and methyl red production and negative for the rest.

The Most Probable Number (MPN) Procedure for Water Examination for the Presence of Coliforms by the Presumptive, Confirmed, and Completed Tests.

Study Questions: 1. What are the advantages and disadvantages of the serial dilution-agar plating method?

2. What is the major disadvantage of microbial counts performed by methods other the serial dilution-agar plating method? 3. What are the other methods of quantitatively enumerating microorganisms? 4. What are the other uses of pour plate technique aside from quantifying microorganisms? 5. Aside from quantitative enumeration, what are other methods to enumerate microorganisms? 6. Why are coliforms selected as the indicator of water potability? 7. Does a positive presumptive test indicate that water is potable? 8. Why is the MPN test qualitative rather than quantitative? 9. What is the function of the following in the MPN test? a. lactose broth b. EMB Agar c. nutrient agar slant d. Gram stain 10. What does a metallic green sheen indicate on an EMB plate?